Re: autophagy related Gene(ATG) ... 2020 Cell

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Cell

. Author manuscript; available in PMC: 2020 Jan 10.

Published in final edited form as: Cell. 2019 Jan 10;176(1-2):11–42. doi: 10.1016/j.cell.2018.09.048

Biological Functions of Autophagy Genes: A Disease Perspective

Beth Levine 1,2,3, Guido Kroemer 4,5,6,7,8,9,10

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PMCID: PMC6347410  NIHMSID: NIHMS1517910  PMID: 30633901

The publisher's version of this article is available at Cell

Abstract

The lysosomal degradation pathway of autophagy plays a fundamental role in cellular, tissue and organismal homeostasis and is mediated by evolutionarily conserved autophagy-related (ATG) genes. Definitive etiological links exist between mutations in genes that control autophagy and human disease, especially neurodegenerative, inflammatory disorders and cancer. Autophagy selectively targets dysfunctional organelles, intracellular microbes and pathogenic proteins, and deficiencies in these processes may lead to disease. Moreover, ATG genes have diverse physiologically important roles in other membrane trafficking and signalling pathways. This review discusses the biological functions of autophagy genes from the perspective of understanding – and potentially reversing – the pathophysiology of human disease and aging.

요약

자가포식의 리소좀 분해 경로는

세포, 조직 및 유기체의 항상성 유지에 근본적인 역할을 하며,

진화적으로 보존된 자가포식 관련 (ATG) 유전자에 의해 매개됩니다.

자가포식을 제어하는 유전자의 돌연변이와 인간 질병,

특히 신경 퇴행성 질환, 염증성 질환 및 암 사이에는

명확한 인과 관계가 있습니다.

자가포식은

기능 장애를 일으킨 세포 기관,

세포 내 미생물 및 병원성 단백질을 선택적으로 표적으로 하며,

이러한 과정에 결함이 있으면 질병으로 이어질 수 있습니다.

또한 ATG 유전자는

다른 막 수송 및 신호 전달 경로에서 다양한 생리학적으로 중요한 역할을 합니다.

이 리뷰는

인간 질병 및 노화의 병리 생리학을 이해하고 잠재적으로 역전시키는 관점에서

자가포식 유전자의 생물학적 기능을 논의합니다.

Introduction

A decade has elapsed since our review in 2008 in Cell on “Autophagy in the Pathogenesis of Disease” (Levine and Kroemer, 2008). During this period, more than 33,000 new articles related to autophagy were published; a Nobel prize was awarded for the discovery of the molecular mechanisms of autophagy (Levine and Klionsky, 2017; Mizushima, 2018); and considerable interest has emerged in autophagy modulation as a potential target in clinical medicine (Galluzzi et al., 2017a).

The fundamental concepts discussed in our 2008 review (Levine and Kroemer, 2008) remain unchanged. The lysosomal degradation pathway of macroautophagy (herein referred to as autophagy) plays a crucial role in cellular physiology, including adaptation to metabolic stress, removal of dangerous cargo (e.g. protein aggregates, damaged organelles, intracellular pathogens), renovation during differentiation and development, and prevention of genomic damage. Generally, these and other functions protect against numerous diseases, including infections, cancer, neurodegeneration, cardiovascular disorders, and aging (Mizushima and Komatsu, 2011). Under certain circumstances, autophagy may be detrimental either via its pro-survival effects (such as in cancer progression (Amaravadi et al., 2016)) or via possible cell death-promoting effects (Marino et al., 2014a).

Over the past ten years, significant progress has been made in understanding the molecular mechanisms of autophagy, the regulation of autophagy, and the effects of autophagy on physiology and pathophysiology (Dikic and Elazar, 2018; Galluzzi et al., 2014; Mizushima, 2018). New major conceptual advances underscore the plurality of functions of the autophagic core machinery in various membrane trafficking and signaling events (Cadwell and Debnath, 2018) and delineate the exquisite specificity with which autophagy targets selected cargo for degradation (Gatica et al., 2018). These advances, together with discoveries in human genetics linking ATG gene mutations to specific diseases (Jiang and Mizushima, 2014; van Beek et al., 2018), provide a multidimensional perspective of mechanisms by which ATG gene-dependent pathways protect against mammalian disease.

Herein we review selected highlights of the past decade of research on the biological functions of autophagy genes, primarily from a perspective of understanding and treating human disease.

소개

2008년에 Cell에 발표된

“질병의 발병 기전에서 자가포식” (Levine and Kroemer, 2008)에 대한 리뷰가 발표된 지

10년이 지났습니다.

https://pmc.ncbi.nlm.nih.gov/articles/PMC2696814/

자가포식은 생존, 분화, 발달 및 항상성 유지에 필수적인 리소좀 분해 경로입니다. 자가포식은 주로 감염, 암, 신경 퇴행, 노화, 심장 질환 등 다양한 병리학에서 유기체를 보호하는 적응적 역할을 합니다. 그러나 특정 실험적 질병 환경에서는 자가포식의 자기 식인적 기능이나, 역설적으로 생존을 촉진하는 기능조차도 해로울 수 있습니다. 이 리뷰는 자가포식의 생리적 기능과 인간 질병의 원인과 예방에 대한 가능한 역할에 대한 최근의 연구 성과를 요약합니다.

이 기간 동안 자가포식 관련 새로운 논문 33,000여 편이 발표되었고,

자가포식의 분자 메커니즘이 발견되어

노벨상(Levine and Klionsky, 2017; Mizushima, 2018)이 수여되었으며,

임상 의학에서 잠재적 표적으로 자가포식 조절에 대한 관심이 크게 높아졌습니다(Galluzzi et al., 2017a).

자가포식은 생리학적 및 병리학적 상황에서 유기체의 항상성 유지에 핵심적인 역할을 합니다. 따라서, 자가포식의 변화는 암, 신경 퇴행 및 심장 질환과 같은 다양한 임상적 상태와 관련이 있는 것으로 알려져 있습니다. 지난 10년 동안, 자가포식은 새로운 치료제 개발의 표적으로 많은 주목을 받아 왔습니다. 그러나, 이러한 노력은 아직 임상적으로 실행 가능한 치료법으로 이어지지는 않았습니다. 이 리뷰에서는 자가포식 조절제의 치료적 잠재력을 논의하고, 그 개발을 제한한 장애물을 분석하며, 임상에서 자가포식 조절의 치료적 잠재력을 최대한 발휘할 수 있는 전략을 제안합니다.

2008년 리뷰 (Levine and Kroemer, 2008)에서 논의된 기본 개념은 변함없이 유지됩니다.

거대자가포식 (이하 자가포식)의 리소좀 분해 경로는

대사 스트레스에 대한 적응,

위험한 물질 (예: 단백질 응집체, 손상된 세포 기관, 세포 내 병원체)의 제거, 분화 및 발달 과정의 재생,

유전적 손상의 예방 등

세포 생리학에서 중요한 역할을 합니다.

일반적으로 이러한 기능들은

감염, 암, 신경퇴행성 질환, 심혈관 질환, 노화 등

다양한 질병으로부터 보호하는 역할을 합니다(Mizushima and Komatsu, 2011).

특정 상황에서,

자가포식은 생존 촉진 효과(예: 암 진행 (Amaravadi 외, 2016)) 또는

세포 사멸 촉진 효과 (Marino 외, 2014a)를

통해 해로울 수 있습니다.

지난 10년 동안,

자가포식의 분자적 메커니즘,

자가포식의 조절,

그리고 생리학 및 병리생리학에 대한 자가포식의 영향에 대한 이해가 크게 진전되었습니다

(Dikic and Elazar, 2018; Galluzzi et al., 2014; Mizushima, 2018).

자가포식은 세포 스트레스의 다양한 조건에서 유도되는 고도로 보존된 이화 작용으로, 에너지나 영양분이 부족할 때 세포 손상을 방지하고 생존을 촉진하며 다양한 세포 독성 자극에 반응합니다. 따라서 자가포식은 주로 세포 보호 기능을 하며, 세포가 경험하는 다양한 자극에 올바르게 반응하기 위해 엄격하게 조절되어야 하며, 이를 통해 끊임없이 변화하는 환경에 적응할 수 있게 합니다. 현재 자가포식은 암과 신경 퇴행 등 다양한 인간 질병에서 조절이 무질서한 것으로 밝혀졌으며, 그 조절은 치료적 접근법으로서 상당한 잠재력을 가지고 있습니다.

새로운 주요 개념적 발전은

다양한 막 수송 및 신호 전달 사건에서 자가포식 핵심 기전의 다중 기능을 강조하고 (Cadwell and Debnath, 2018),

자가포식이 분해할 대상을 선택하는 정교한 특이성을 설명합니다 (Gatica et al., 2018).

이러한 발전과 ATG 유전자 돌연변이와

특정 질병을 연결하는 인간 유전학의 발견 (Jiang and Mizushima, 2014; van Beek et al., 2018)은

ATG 유전자 의존적 경로가 포유류 질병을 예방하는 메커니즘에 대한

다차원적인 관점을 제공합니다.

여기에서는 주로 인간 질병의 이해와 치료의 관점에서,

지난 10년간의 오토파지 유전자의 생물학적 기능에 대한 연구의

주요 내용을 검토합니다.

Autophagy and other Autophagy Gene-Dependent Pathways

The original scientific definition of autophagy (Greek, “self-eating”) is the delivery of cytoplasmic cargo to the lysosome for degradation. There are at least three distinct forms of autophagy — chaperone-mediated autophagy, microautophagy and macroautophagy — which differ in terms of mode of cargo delivery to the lysosome. Macroautophagy is the major catabolic mechanism used by eukaryotic cells to maintain nutrient homeostasis and organellar quality control. It is mediated by a set of evolutionarily conserved genes, the autophagy-related (ATG) genes (Klionsky et al., 2003), originally discovered in yeast genetic screens (Mizushima, 2018). With a few exceptions, all ATG genes are required for the efficient formation of sealed autophagosomes that proceed to fuse with lysosomes.

In higher eukaryotes, many ATG genes functionally diversified to facilitate delivery of extracellular cargo to the lysosome, to promote the plasma membrane localization or extracellular release of intracellular cargo, and to coordinate intracellular communication with various cell signaling pathways (Figure 1). These other functions are not, sensu stricto, autophagy and accordingly, will be referred to as ATG gene-dependent pathways. There are broad implications of ATG gene functions in different membrane trafficking and signaling pathways for mammalian cell biology, physiology and disease.

자가포식 및 기타 자가포식 유전자 의존 경로

자가포식(그리스어, “자식 먹기”)의 원래 과학적 정의는

세포질 내의 물질을

리소좀으로 전달하여 분해하는 것입니다.

자가포식에는 리소좀으로 물질을 전달하는 방식에 따라

최소 세 가지의 뚜렷한 형태,

즉 샤페론 매개 자가포식, 미세 자가포식, 거대 자가포식이 있습니다.

거대자가포식은

영양소 항상성 및 세포 기관의 품질 관리를 유지하기 위해

진핵 세포가 사용하는 주요 이화 작용 메커니즘입니다.

이 메커니즘은 진화적으로 보존된 일련의 유전자,

즉 자가포식 관련 (ATG) 유전자에 의해 매개되며 (Klionsky 외, 2003),

원래는 효모 유전자 스크리닝에서 발견되었습니다 (Mizushima, 2018).

몇 가지 예외를 제외하고,

모든 ATG 유전자는 리소좀과 융합하는

밀폐된 오토파고솜의 효율적인 형성에 필요합니다.

고등 진핵생물에서는

많은 ATG 유전자가 기능적으로 다양화되어

세포외 화합물을 리소좀으로 전달하고,

세포막에 세포내 화합물을 국한하거나 세포외로 방출하며,

다양한 세포 신호 전달 경로를 통해 세포내 통신을 협응하는 역할을 합니다 (그림 1).

이러한 다른 기능은 엄밀한 의미에서 자가포식이 아니므로,

ATG 유전자 의존적 경로로 지칭될 것입니다.

ATG 유전자의 기능은

포유류 세포 생물학, 생리학 및 질병에 대한

다양한 막 수송 및 신호 전달 경로에 광범위한 영향을 미칩니다.

Figure 1. Autophagy gene-dependent membrane trafficking pathways.

Shown are schematic illustrations of different membrane trafficking pathways that involve autophagy (ATG) proteins (green ovals). See text for explanations of each pathway and a discussion of their physiological functions. See Table 1 for examples of genetic mutations that impair autophagy-related pathways which are associated with human disease. The major type of autophagy, macroautophagy, is labeled as “classical degradative autophagy” to distinguish it from other trafficking pathways that utilize overlapping ATG proteins. Due to space limitations, not all ATG proteins, proteins involved in vesicle fusion, or secretary cargo are depicted. PM, plasma membrane. LC3-II (green circle) is the phosphatidyl-ethanolamine-conjugated form of the autophagy protein, LC3.

자가포식 (ATG) 단백질 (녹색 타원형)과 관련된 다양한 막 수송 경로의 개략도가 표시되어 있습니다. 각 경로의 설명과 생리적 기능에 대한 설명은 본문을 참조하십시오. 인간 질병과 관련된 자가포식 관련 경로를 손상시키는 유전적 돌연변이의 예는 표 1을 참조하십시오. 자가포식의 주요 유형인 거대자가포식은, 중복되는 ATG 단백질을 이용하는 다른 수송 경로와 구별하기 위해 “고전적 분해 자가포식”으로 표시되어 있습니다. 공간 제한으로 인해 모든 ATG 단백질, 소포 융합에 관여하는 단백질 또는 분비 물질을 모두 묘사하지는 않았습니다. PM, 혈장 막. LC3-II (녹색 원)는 자가포식 단백질 LC3의 포스파티딜-에탄올아민 결합형입니다.

Degradative Autophagy: The “raison d’être” of Autophagy Genes

The originally discovered function of ATG genes is to orchestrate and mediate the formation of double-membraned structures that deliver intracytoplasmic contents to the lysosome for degradation. This process is conserved in all eukaryotic organisms, occurs at basal levels in nearly all cell types, and is increased by diverse intracellular and extracellular cues. It is essential for cellular homeostasis, cellular protein and organelle quality control, and organismal adaptation to environmental stress. These principles are firmly supported by nearly two decades of studies involving genetic ablation of the autophagy machinery in diverse eukaryotic species (Levine and Kroemer, 2008; Mizushima and Komatsu, 2011).

This lysosomal degradation pathway is usually described as involving a set of ~16–20 core conserved ATG genes. The ATG proteins encoded by these genes are traditionally classified into distinct biochemical and functional groups that act at specific stages of autophagosome initiation or formation. In this scheme (see other recent reviews for details (Dikic and Elazar, 2018; Yu et al., 2018)), the ULK1 serine threonine kinase complex (involving ULK1, FIP200, ATG13 and ATG101) plays a major role in autophagy initiation, phosphorylating multiple downstream factors. Two distinct Beclin 1/class III phosphatidylinositol 3-kinase (PI3KC3) complexes generate phosphatidylinositol 3-phosphate (PI3P) to act in autophagosome nucleation (PI3KC3–C1 involving Beclin 1, VPS34, VPS15 and ATG14) or endolysosomal and autophagolysosomal maturation (PI3KC3–C2 involving Beclin 1, VPS34, VPS15 and UVRAG). Vesicles containing ATG9A, the only transmembrane core ATG protein, supply membrane to autophagosomes. WIPI (WD repeat domain phosphoinositide-interacting) proteins and their binding partners, ATG2A or ATG2B, function in early stages of membrane elongation at the site of PI3P generation. Autophagosome membrane expansion and completion involves two ubiquitin-like protein conjugation systems: the Ub-like ATG12 conjugates with ATG5 and ATL16L1 and the Ub-like LC3 subfamily (ATG8 in yeast) conjugates with membrane-resident phosphatidylethanoloamine (PE). Unlike in yeast, the ubiquitin-like protein conjugation systems are not essential for autophagosomal membrane completion in mammalian cells, although they determine the efficiency of the process (Tsuboyama et al., 2016).

분해성 자가포식: 자가포식 유전자의 존재 이유

ATG 유전자의 원래 발견된 기능은

세포질 내 내용을 리소좀으로 전달하여 분해하는

이중막 구조의 형성을 조정하고 매개하는 것입니다.

이 과정은 모든 진핵생물에서 보존되어 있으며,

거의 모든 세포 유형에서 기초 수준에서 발생하며,

다양한 세포 내외 신호에 의해 증가됩니다.

이는

세포 내 환경 균형,

세포 단백질 및 소기관 품질 관리,

환경 스트레스에 대한 유기체 적응에 필수적입니다.

이러한 원리는 다양한 진핵 생물 종에서

자가포식 기전의 유전적 제거를 포함한

약 20년간의 연구에 의해 확고하게 뒷받침되고 있습니다 (Levine and Kroemer, 2008; Mizushima and Komatsu, 2011).

이 리소좀 분해 경로는

일반적으로 약 16~20개의 핵심 보존 ATG 유전자가 관여하는 것으로 설명됩니다.

이 유전자에 의해 암호화되는 ATG 단백질은

전통적으로 오토파고좀의 개시 또는 형성의 특정 단계에서 작용하는

별개의 생화학적 및 기능적 그룹으로 분류됩니다.

이 체계에서 (자세한 내용은 다른 최근 리뷰를 참조하십시오 (Dikic 및 Elazar, 2018; Yu et al., 2018)),

ULK1 세린 트레오닌 키나아제 복합체 (ULK1, FIP200, ATG13 및 ATG101 포함)는

여러 하류 인자를 인산화하여 자가포식 개시에 중요한 역할을 합니다.

두 개의 서로 다른 Beclin 1/class III 인산화인오스톨리노스 3-키나제(PI3KC3) 복합체는

자포소체 핵형성에 작용하는 인산화인오스톨리노스 3-인산(PI3P)을 생성합니다

(PI3KC3–C1 복합체는 Beclin 1, VPS34, VPS15 및 ATG14를 포함).

또는 내소체 및 자포소체 성숙에 작용하는 PI3KC3–C2 복합체는

Beclin 1, VPS34, VPS15 및 UVRAG를 포함합니다).

ATG9A를 유일한 막을 관통하는 핵심 ATG 단백질로 포함하는 소포는

자포소체에 막을 공급합니다.

WIPI(WD 반복 도메인 인산인오시톨 상호작용) 단백질과 그 결합 파트너인 ATG2A 또는 ATG2B는

PI3P 생성 부위에서 막 연장 초기 단계에 기능합니다.

자율포식소막의 확장 및 완성에는 두 가지 유비퀴틴

유사 단백질 결합 시스템이 관여합니다:

유비퀴틴 유사 ATG12는 ATG5 및 ATL16L1과 결합하며,

유비퀴틴 유사 LC3 하위 가족(효모에서는 ATG8)은

막에 존재하는 포스파티딜에탄올아민(PE)과 결합합니다.

효모와 달리, 유비퀴틴과 유사한 단백질 접합 시스템은

포유류 세포에서 오토파고소좀 막의 완성에 필수적인 것은 아니지만,

그 과정의 효율성을 결정합니다 (Tsuboyama et al., 2016).

This classification of the ATG proteins has provided a useful framework for studying and understanding autophagy. However, its apparent simplicity is at variance with extensive data indicating a highly complex level of interconnectivity among the ATG proteins and newly described functions of ATG proteins at different stages of autophagy. Based on unbiased proteomic analyses, most ATG proteins interact with other ATG proteins that reside outside of their “classic” functional complex (Behrends et al., 2010). Experimentally, some of these interactions are known to be important for autophagosome formation. For example, FIP200 (a member of the ULK1 kinase complex) interacts with ATG16L1 to properly target it to the isolation membrane (also known as the phagophore) of the nascent autophagosome (Nishimura et al., 2013). ATG14 (a component of the autophagy-specific PI3KC3–C1 complex) also functions in SNARE-driven membrane fusion (Diao et al., 2015). Similarly, Atg13 (a component of the yeast Atg1/mammalian ULK1 kinase complex) interacts with Atg9 to recruit Atg9 vesicles to the pre-autophagosomal structure (Suzuki et al., 2015). The broader interconnectivity and functional multiplicity of core autophagy proteins in autophagosomal biogenesis requires further elucidation. Moreover, as indicated by a recent conditional genetic interactions study using diverse yeast–omics datasets (Kramer et al. 2017), new systems biology approaches will likely identify additional genes required for autophagy, especially those that may function in a stimulus-dependent, cell type-dependent or species-specific manner.

The core ATG proteins, conserved from yeast to humans, are necessary but not sufficient for degradative autophagy. The degradation of autophagosomal cargo cannot proceed without successful fusion to an available and functional lysosome. Research in the past decade has unmasked some of the key factors required for lysosomal biogenesis (Settembre et al., 2013b), autophagolysosomal fusion (Yu et al., 2018), lysosomal function during autophagy (Shen and Mizushima, 2014) and autophagic lysosome reformation (Chen and Yu, 2017). Adenoviral-mediated gene delivery of TFEB, a master transcriptional regulator of lysosomal biogenesis, improves outcomes in various rodent disease models, including Parkinson’s disease, lysosomal storage disorders, tauopathies, α1-antitrypsin deficiency, and hepatic hyperammonemia (Napolitano and Ballabio 2016; Soria et al., 2018). Autophagolysosomal fusion requires changes in lysosomal pH, certain cytoskeleton motor proteins (dynein), tethering factors (the HOPS complex, the Rab GTPase, RAB7), SNARE proteins (the Q-SNARE, syntaxin 17 on autophagosomes which interacts with R-SNARE proteins, SNAP29 and VAMP8 on endosomes/lysosomes), phospholipids, and members of the LC3/GABARAP family (Kriegenburg et al., 2018) that are bridged to tethering factors or SNARES by adaptor proteins. Screens in C. elegans identified novel metazoan-specific genes required for fusion steps in degradative autophagy (Tian et al., 2010). One example relevant to human disease is EPG5, which encodes a RAB7 effector. Autosomal recessive mutation of EPG5 results in Vici syndrome, a neurodevelopmental and multisystem disorder (Table 1). Mutations in genes that regulate lysosomal acidification such as ATP6AP2 and presenilin 1 are associated with X-linked Parkinsonism and Alzheimer’s disease (Table 1). Thus, we must consider regulators of lysosomal biogenesis, the fusion machinery, and determinants of lysosomal function in our efforts to decipher how deficient autophagy leads to disease and how autophagy can be regulated to prevent or treat disease.

ATG 단백질의 이러한 분류는

오토파지 연구 및 이해에 유용한 틀을 제공했습니다.

그러나,

그 명백한 단순성은 ATG 단백질들 간의 고도로 복잡한 상호 연결성과 오토파지 각 단계에서

ATG 단백질의 새로 밝혀진 기능들을 나타내는 방대한 데이터와 상반됩니다.

편견 없는 단백질체 분석에 따르면,

대부분의 ATG 단백질은 “고전적인” 기능 복합체 외부에 존재하는

다른 ATG 단백질과 상호 작용합니다 (Behrends et al., 2010).

실험적으로, 이러한 상호작용 중 일부는 오토파고소체 형성에 중요함이 알려져 있습니다. 예를 들어, ULK1 키나제 복합체의 구성원인 FIP200은 ATG16L1과 상호작용하여 이를 신생 오토파고소체의 격리막(파고포어라고도 함)으로 적절히 표적화합니다(Nishimura et al., 2013). ATG14 (자가포식 특이적 PI3KC3–C1 복합체의 구성 요소)도 SNARE에 의한 막 융합에서 기능합니다 (Diao et al., 2015). 同様に, Atg13 (효모 Atg1/포유류 ULK1 키나제 복합체의 구성 요소)은 Atg9와 상호작용하여 Atg9 소포를 전자포소체 구조로 모집합니다 (Suzuki et al., 2015). 자가포식 소체 생성에 있어 핵심 자가포식 단백질의 광범위한 상호 연결성과 기능적 다중성은 더 자세한 연구가 필요합니다. 또한, 다양한 효모 오믹스 데이터 세트를 사용한 최근의 조건부 유전적 상호 작용 연구 (Kramer et al. 2017)에서 알 수 있듯이, 새로운 시스템 생물학 접근법을 통해 자가포식에 필요한 추가적인 유전자, 특히 자극에 의존적, 세포 유형에 의존적 또는 종에 특정한 방식으로 기능할 가능성이 있는 유전자가 확인될 가능성이 높습니다.

효모에서 인간에 이르기까지 보존되어 있는 핵심 ATG 단백질은

분해성 자가포식에 필요하지만,

그것으로만 충분하지는 않습니다.

자가포식 소포의 화물은

사용 가능한 기능적인 리소좀과 성공적으로 융합되지 않으면

분해될 수 없습니다.

지난 10년간의 연구를 통해

리소좀 생성에 필요한 몇 가지 핵심 요소가 밝혀졌습니다 (Settembre et al., 2013b),

토파고리소좀 융합 (Yu et al., 2018),

오토파지 동안의 리소좀 기능 (Shen and Mizushima, 2014) 및

오토파지 리소좀 재형성 (Chen and Yu, 2017).

리소좀 생성의 주요 전사 조절인자인 TFEB의 아데노바이러스 매개 유전자 전달은

파킨슨병, 리소좀 저장 장애, 타우병, α1-항트립신 결핍, 간성 고암모니아혈증 등

다양한 설치류 질환 모델에서 치료 효과를 개선합니다(Napolitano and Ballabio 2016; Soria et al., 2018).

자율포식체-리소좀 융합은

리소좀 pH 변화,

특정 세포골격 모터 단백질(다이닌),

결합 인자(HOPS 복합체, Rab GTPase RAB7),

SNARE 단백질(자율포식체에 존재하는 Q-SNARE,

syntaxin 17은 엔도좀/리소좀에 존재하는 R-SNARE 단백질 SNAP29 및 VAMP8과 상호작용),

포스포리피드, LC3/GABARAP 가족의 구성원(Kriegenburg et al., 2018)이 필요합니다.

C. elegans의 스크린을 통해 분해 자가포식 단계의 융합에 필요한 새로운 다세포 동물 특유의 유전자가 확인되었습니다 (Tian et al., 2010). 인간 질병과 관련된 한 예로 RAB7 효과 인자를 암호화하는 EPG5가 있습니다. EPG5의 상염색체 열성 돌연변이는 신경 발달 및 다기관 장애인 Vici 증후군을 유발합니다 (표 1). ATP6AP2 및 프레세닐린 1과 같은 리소좀 산성화를 조절하는 유전자의 돌연변이는 X-연관 파킨슨증 및 알츠하이머병과 관련이 있습니다 (표 1).

따라서,

우리는 자가포식이 어떻게 질병을 유발하는지, 그리고

질병을 예방하거나 치료하기 위해 자가포식을 어떻게 조절할 수 있는지 해명하기 위해

리소좀 생성의 조절 인자, 융합 기전, 리소좀 기능의 결정 인자를 고려해야 합니다.

Table 1.

Examples of genetic mutations in human disease that impair autophagy

GeneDiseaseMechanismReference

Mutations in genes required for autophagy and lysosomal function
ATG16L1	Crohn’s Disease (CD)	ATGL16L1 T300A is a major risk allele for CD. The T300A polymorphism has a caspase 3 cleavage site that decreases protein levels. T300A knock-in or hypomorphic or intestinal knockout mice show decreased intestinal bacterial clearance; increased cytokine responses; reduced Paneth cell lysozyme secretion and clearance of IRE la protein aggregates during ER stress; enhanced enterocyte TNFa-induced necroptosis; dendritic cell defects in regulatory T cell induction and suppression of mucosal inflammation.	(Jiang and Mizushima, 2014) (Murthy et al., 2014) (Lassen et al., 2014) (Chu et al., 2016) (Bel et al., 2017) (Tschurtschenthaler et al., 2017) (Matsuzawa-Ishimoto et al., 2017)
ATG16L2	Systemic lupus erythematosus (SLE)	ATG16L2 R114W allele is a disease susceptibility gene	(Molineros et al., 2017)
ATG5	Childhood ataxia Systemic sclerosis SLE	Loss-of-function mutation reduces autophagy and causes ataxia Intronic variants are associated with susceptibility to systemic sclerosis Polymorphisms associated with SLE susceptibility; mouse studies suggest mechanism may involve deficient LC3-associated phagocytosis	(Kim et al., 2016) (Martinez et al., 2016)
ATP6AP2	X-linked Parkinsonism with spasticity Multisystem disorder	Exon skipping mutations linked to Parkinsonism Missense mutations associated with immunodeficiency, liver disease, and psychomotor impairment lead to defective lysosomal acidification due to impaired v-ATPase assembly, resulting in defects in autophagy	(Korvatska et al., 2013) (Rujano et al., 2017)
BECN1	Breast and ovarian cancer	Monoallelic deletion associated with risk and poor prognosis of sporadic breast and ovarian cancer; monoallelic deletion in mice leads to decreased autophagy and increased tumors, including basal-like breast cancer	(Qu et al., 2003) (Yue et al., 2003) (Valente et al., 2014) (Tang et al., 2015)
CLEC16A	Diabetes Multiple sclerosis	CLEC16A variants associated with multiple autoimmune diseases. Mice deficient in Clecl6a have autophagy defects associated with Purkinje degeneration and ataxia, impaired β-cell mitophagy, and autoimmunity	(Soleimanpour et al., 2014) (Schuster et al., 2015) (Bronson et al., 2016) (Redmann et al., 2016)
CTNS	Cystinosis	Recessive loss-of-function mutations in CTNS, a gene encoding a proton transporter that exports cysteine from lysosomes associated with renal lysosomal storage disease; gene deletion in mice results in deficient autophagy, altered lysosomal dynamics and accumulation of dysfunctional ROS overproducing mitochondria	(Festa et al., 2018)
EPG5	Vici syndrome	Recessive mutations in EPG5, a gene required for autophagolysosomal fusion results in a neurodevelopmental disorder with multisystem involvement	(Cullup et al., 2013) (Hori et al., 2017)
GBA	Gaucher’s disease Parkinson’s disease (PD)	GBA1 encodes the lysosomal enzyme glucocerebrosidase. Homozygous GBA defects cause Gaucher’s disease and heterozygous defects predispose to PD	(Afiaki et al., 2017)
GRN	Frontotemporal dementia (heterozygous) or Neuronal ceroid lipofuscinosis (homozygous)	Loss-of-function mutations compromise lysosomal function and autophagic flux	(Chang et al., 2017)
LAMP2	Danon’s cardiomyopathy	X-linked deletion results in vacuolar cardiomyopathy and myopathy; Lamp2 deletion in mice results in autophagosome accumulation and cardiomyopathy	(Nishino et al., 2000) (Tanaka et al., 2000)
PIK3R4 (VPSI 5)	Cortical atrophy and epilepsy	Mutations in PIK3R4, a component of the Beclin 1 complex required for endosomal-lysosomal trafficking and autophagy, associated with human neurodevelopmental disease	(Gstrein et al., 2018)
SNX14	Autosomal recessive spinocerebellar ataxia	SNX14 binds lysosomal phosphatidylinositol (3,5)-bisphosphate and is required for autophagosomal clearance	(Akizu et al., 2015)
SPG11, SPG15 (ZFYVE26), SPG49 (TECPR)	Hereditary spastic paraplegia	SPG15 binds phosphatidylinositol 3-phosphate and SPG49 binds LC3 to function in autophagolysosomal trafficking	(Ebrahimi-Fakhari et al., 2016)
WDR45 (WIPI4)	Beta-propeller protein-associated neurodegeneration	WDR45 (WIPI4) binds to phosphoinositide 3-phosphate and interacts with ATG2 and ATG9; disease-associated mutations impair autophagy	(Haack et al., 2012) (Saitsu et al., 2013) (Ebrahimi-Fakhari et al., 2016)
Mutations in genes that regulate autophagy and lysosomal function
APP	Alzheimer’s disease	Mutant amyloid precursor protein expressed in mouse hippocampal neurons inhibits mitophagy and autophagy	(Reddy et al., 2018)
AT-1 (SLC33A1)	Spastic paraplegia, Developmental delay Autism spectrum disorders	AT-1 translocates cytosolic acetyl coA into ER lumen; mutations and duplications associated with a variety of CNS phenotypes in humans; in mice overexpression‎ blocks Atg9a-Faml34b-LC3 interactions, leading to defective ER-phagy and progeria	(Peng et al., 2018)
C9orf72	Amyotrophic lateral sclerosis (ALS) Frontotemporal dementia (FTD)	Hexanucleotide repeat expansion in C9orf72 gene is most common genetic cause of ALS and FTD. Regulates autophagy and lysosomal homeostasis through interactions with SMCR8, ULK1 and Rab-GTPases	(Nassif et al., 2017) (Corrionero and Horvitz, 2018)
ERBB2	Breast cancer	Amplification of ERBB2 and consequent overexpression‎ of ERBB2 (HER2) interacts with Beclin 1 and inhibits autophagy	(Vega-Rubin-de-Celis et al., 2018)
GBA1	Gaucher’s disese Parkinson’s disease	Mutations in GBA1 decrease glucocerebrosidase activity, leading to defects in autophagic-lysosomal function and a-synuclein aggregate accumulation	(Schapira, 2015) (Afiaki et al., 2017)
GPR65	Inflammatory bowel disease	GPR65 I231L risk variant of this proton-sensing G protein-coupled receptor impairs lysosomal acidification, decreases intracellular bacterial clearance and alters lipid droplet turnover	(Lassen et al., 2016)
HTT (Huntingtin)	Huntington’s disease	PolyQ extension in HTT competitively disrupts interaction between the deubiquitinase ataxin 3 and Beclin 1, leading to enhanced Beclin 1 proteasomal degradation and reduced autophagy	(Ashkenazi et al., 2017)
IRGM	Non-alcoholic fatty liver disease (NAFLD) Crohn’s disease Tuberculosis	IRGM functions in assembly and activation of autophagy machinery; a synonymous variant reduces protein expression‎ leading to reduced autophagy and lipophagy in NAFLD; polymorphisms are associated with risk of Crohn’s disease and tuberculosis	(Jiang and Mizushima, 2014) (Lin et al., 2016) (Chauhan et al., 2016b)
LRRK2	Crohn’s disease (CD) Parkinson’s disease (PD)	Risk alleles for CD and PD increase kinase activity of leucine-rich repeat kinase 2 and reduce autophagic flux; a protective allele increases flux	(Cooper et al., 2012) (Hui et al., 2018)
MeCP2	Rett syndrome (X-linked neuro-developmental disorder)	Deficiency of methyl-CpG-binding protein-2 (MeCP2), a transcriptional regulator, results in defective autophagy in patient fibroblasts and knockout mouse cerebellum, and mitochondrial retention in erythrocytes	(Sbardella et al., 2017)
MTMR3	Inflammatory Bowel Disease (IBD)	MMTR is a PI3P phosphatase that decreases autophagy. Macrophages from carriers of the risk allele express higher MTMR3 protein levels and have increased pathogen recognition receptor-induced caspase-1 activation and IL-1β secretion	(Lahiri et al., 2015)
PLEKHM1	Osteopetrosis	Disease associated mutants impair binding to RAB7A and secretory lysosome trafficking in osteoclasts	(Stenbeck and Coxon, 2014)
RAB7A	Charcot-Marie-Tooth type 2B disease	Disease-associated RAB7A mutants reduce autophagic flux in HeLa cells and patient-derived fibroblasts are autophagy-deficient	(Colecchia et al., 2018)
PS1	Alzheimer’s disease	Mutations in presenilin 1 that disrupt v-ATPase assembly, lysosomal acidification and autophagy cause early onset Alzheimer’s disease	(Lee et al., 2010)
PTPN2	IBD Type 1 Diabetes Juvenile arthritis	Disease-associated SNP in PTPN2, a gene encoding protein tyrosine phosphatase nonreceptor type 2 causes impaired autophagosome formation and defective bacterial handling in macrophages and intestinal epithelial cells	(Scharl et al., 2012)
SMS	Snyder-Robinson syndrome (SRS)	Loss-of-function mutations in spermine synthase (SMS) cause SRS, an X-linked intellectual disability syndrome; deficiency in SMS generates toxic metabolites that impair lysosomal function and autophagic flux	(Li et al., 2017a)
TMEM230	Parkinson’s disease	Transmembrane protein involved in retromer function; loss reduces autophagic cargo degradation and secretory autophagy	(Kim et al., 2017)
v-ATPase	Autosomal Recessive Osteoporosis	Mutations in the α3 subunit encoded by TCIRG1 impair lysosomal acidification at the ruffled border of osteoclasts, leading to defects in bone resorption	(Ochotny et al., 2013)
WASP	Wiskott-Aldrich syndrome	Deficiency of the actin cytoskeleton-regulatory WASP protein impairs formation of autophagosomes, resulting in deficient xenophagy and excessive inflammasome activation and pyroptosis	(Lee et al., 2017b)
Mutations in genes required for cargo delivery in selective autophagy
ALFY	Primary microcephaly	Dominant mutation in this autophagy scaffold protein causes human microcephaly	(Kadir et al., 2016)
CALC0C02 (NDP52)	Crohn’s disease	Missense mutation of this autophagy adaptor reduces its function and enhances NF-kB activation of inflammatory genes	(Ellinghaus et al., 2013)
FAM134B	Hereditary sensory and autonomic neuropathy type II	Mutations disrupt the interaction of this ER protein with LC3 and GABARP to impair ER-phagy	(Khaminets et al., 2015)
FANC genes	Fanconi anemia (FA) congenital syndrome Hereditary breast and ovarian cancer Sporadic cancers	FA pathway genes required for clearing damaged mitochondria (mitophagy) and preventing aberrant inflammasome activation	(Sumpter et al., 2016)
OPTN1	Amyotrophic lateral sclerosis (ALS) Primary open angle glaucoma (POAG) Paget’s disease of the bone (PGD)	Mutations in ALS reduce interaction of this autophagy adaptor with TBK1 and reduce Parkin-dependent mitophagy; mutations in POAG increase interaction with TBK1, activate Bax-dependent apoptosis, and are associated with mitochondrial dysfunction. Truncated protein mutation associated with PGD	(Wong and Holzbaur, 2014) (Li et al., 2016) (Shim et al., 2016) (Silva et al., 2018)
PARK2/Parkin	Autosomal recessive and sporadic early onset Parkinson’s disease Colon, lung, and brain cancer	Parkin is an E3 ligase that functions in mitophagy and xenophagy; mutations are associated with Parkinson’s disease and cancer risk; polymorphisms associated with increased susceptibility to intracellular bacterial infections	(Kitada et al., 1998) (Xu et al., 2014) (Mira et al., 2004)
PARK6/PINK1	Autosomal recessive and sporadic early onset Parkinson’s disease	PINK1 is a serine-threonine kinase that translocates to the outer mitochondrial membrane upon damage, mediating Parkin recruitment and mitophagy	(Jiang and Mizushima, 2014)
PEX13	Zellweger syndrome spectrum disorders	Disease-associated mutations impair mitophagy and patients with mutations have accumulation of abnormal mitochondria	(Lee et al., 2017a)
SQSTM1 (p62)	ALS FTD Paget’s disease Distal myopathy	SQSTM1 is an autophagy adaptor that binds ubiquitin and LC3; mutations in the ubiquitin-binding association domain result in spectrum of multisystem proteinopathies.	(Goode et al., 2014) (Lee et al., 2018)
SMURF1	Ulcerative colitis	SMURF1, a susceptibility gene for ulcerative colitis, encodes an E3 ligase that functions in mitophagy, virophagy and xenophagy of intracellular bacteria	(Franco et al., 2017)
TBK1	ALS Frontotemporal dementia Other neurodegenerative phenotypes POAG	TBK1 kinase phosphorylates the autophagy receptor OPTN1, increasing its interaction with ATG8 proteins and polyubiquitinated proteins	(Cirulli et al., 2015) (van Beek et al., 2018)
TRIM20	Familial Mediterranean fever	Disease-associated TRIM20 mutants fail to interact with inflammasome components and target them for autophagic destruction	(Kimura et al., 2015)
VPS13D	Ataxia with spasticity	Recessively inherited defects in this ubiquitin-binding protein cause failure in mitophagy and mitochondrial dysfunction	(Seong et al., 2018)

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Beyond Self-Eating: Autophagy Genes Function in Phagocytosis

Several core ATG genes function in a process that shares some similarities with autophagy but involves digestion of unwanted extracellular (rather than intracellular) material. During this process, termed LC3-associated phagocytosis (LAP), single-membraned macroendocytic vacuoles (macropinosomes, phagosomes and entotic vacuoles) engulf extracellular cargo (such as bacteria, dead cells or live cells), become decorated by lipidated LC3, and are directed to the lysosome for degradation (Cadwell and Debnath, 2018; Heckmann et al., 2017). LAP is distinguished from autophagy by four main features: (1) the origin of the vacuolar contents (extracellular versus intracellular), (2) the requirement of cargo engagement of an extracellular receptor for activation, (3) the type of membrane that fuses with the lysosome (single membrane versus double membrane), and (4) the utilization of a subset versus all of the core ATG proteins. LAP requires NADPH-oxidase (NOX2) to generate reactive oxygen species (ROS), certain components of the Beclin 1/VPS34 complexes, PI3P generation, LC3-conjugation to the single membrane of the phagosome, and all components of the LC3 conjugation machinery (Martinez et al., 2011; Martinez et al., 2015; Sanjuan et al., 2007). However, it does not require other core ATG proteins, such as components of the ULK1 complex or the autophagy-specific Beclin 1/VPS34 complex component, ATG14. Somewhat enigmatically, LAP requires Rubicon, an inhibitory component of the autophagy-specific Beclin 1/VPS34 complex. The precise effects of LC3 decoration of phagosomes on their fusion with lysosomes and on lysosomal function are unknown. The presence of LC3 on phagosomes may enhance efficiency of phagolysosomal maturation, perhaps through a mechanism similar to that of LC3/GABARAP family members in autophagolysosomal maturation.

자가포식 그 이상: 식세포 작용에서 자가포식 유전자의 기능

여러 핵심 ATG 유전자는 자가포식과 일부 유사점을 공유하지만,

세포 내 물질이 아닌 세포 외의 불필요한 물질을 소화하는 과정에서 기능합니다.

LC3 관련 식세포 작용(LAP)이라고 하는 이 과정에서,

단일 막을 가진 거대 세포 내 소포(거대 세포 소포, 식세포 소포 및 내포 소포)가

세포외 화물(세균, 사체 세포 또는 살아있는 세포 등)을 포식하고,

지질화된 LC3로 장식되며,

분해 위해 리소좀으로 이동됩니다(Cadwell and Debnath, 2018; Heckmann et al., 2017).

LC3-associated phagocytosis (LAP)

https://pmc.ncbi.nlm.nih.gov/articles/PMC5839790/

세포질 물질의 대량 분해를 매개하는 보존된 자가포식 관련 단백질(ATG)의 확인은 자가포식과 일련의 생리학적 과정 및 질병 상태를 연결하는 획기적인 발견의 토대가 되었습니다. 최근의 연구 결과, 이러한 ATG가 고전적인 자가포식과 관련이 있지만 명확하게 구별되는 과정을 조율한다는 사실이 밝혀지고 있습니다. 자가포식 관련 기능에는 분비, 식세포 물질의 이동, 바이러스 입자의 복제 및 배출, 염증 및 면역 신호 전달의 조절이 포함됩니다. 여기에서는 ATG에 의존하는 공통적인 과정을 정의하고, 이러한 관련 경로를 기계적으로 분리하는 데 따르는 과제를 논의합니다. 개별 ATG의 기능을 구별하는 분자적 사건을 밝히는 것은 자가면역에서 암에 이르기까지 다양한 질병의 기원을 이해하는 데 도움이 될 것입니다.

LAP는 네 가지 주요 특징으로 자가포식(autophagy)과 구별됩니다.

(1) 액포 내용물의 기원(세포외 대 세포내),

(2) 활성화에 필요한 화물의 세포외 수용체 결합,

(3) 리소좀과 융합하는 막의 유형(단일 막 대 이중 막),

(4) 모든 핵심 ATG 단백질 대신 일부의 사용.

LAP는 활성산소종(ROS)을 생성하기 위해 NADPH-oxidase (NOX2), Beclin 1/VPS34 복합체의 특정 구성 요소, PI3P 생성, LC3의 단일 막과의 결합, 그리고 LC3 결합 기계 장치의 모든 구성 요소가 필요합니다 (Martinez et al., 2011; Martinez et al., 2015; Sanjuan et al., 2007). 그러나 ULK1 복합체의 구성 요소나 자가포식 특이적인 Beclin 1/VPS34 복합체의 구성 요소인 ATG14와 같은 다른 핵심 ATG 단백질은 필요하지 않습니다. 다소 수수께끼처럼, LAP는 자가포식 특이적인 Beclin 1/VPS34 복합체의 억제 성분인 Rubicon을 필요로 합니다.

파고소좀의 LC3 장식물이 리소좀과의 융합 및 리소좀 기능에 미치는 정확한 영향은 알려지지 않았습니다. 파고소좀에 LC3가 존재하면 파고리소좀 성숙 효율을 향상시킬 수 있으며, 이는 자식소체 성숙에서 LC3/GABARAP 가족 구성원의 메커니즘과 유사한 방식으로 작용할 수 있습니다.

LAP was originally described in murine macrophages during phagocytosis of particles that engage Toll-like receptors (TLRs) (Sanjuan et al., 2007) and is involved in type I interferon (IFN) secretion in response to DNA immune complexes and other TLR9 ligands (Hayashi et al., 2018; Henault et al., 2012). Physiologically important functions of LAP have been identified by comparing phenotypes of mice with myeloid-specific deletion of LAP-specific genes (e.g. Rubicon or NOX2) and autophagy-specific ATG genes (e.g. FIP200 or Atg14). LAP is required for degradation of photoreceptor outer segments by retinal pigment epithelium (RPE), a process necessary for intact vision (Kim et al., 2013b). LAP is induced by the fungus, A. fumigatus, and the intracellular bacterium, L. monocytogenes, and enhances host defense against these pathogens (Gluschko et al., 2018; Martinez et al., 2015). Mice lacking several components of the LAP pathway develop an autoimmune systemic lupus erythematosus (SLE)-like disease, perhaps due to a defect in the clearance of dying cells that triggers enhanced proinflammatory signaling and autoantibody production (Martinez et al., 2016).

Given the crucial roles of receptor-activated phagocytosis in human physiology, it is likely that LAP, like classical autophagy, will emerge as an important pathway in human disease. While the two pathways utilize overlapping genetic machinery, a critical distinction renders them antagonistic. Specifically, Rubicon is required for LAP but suppresses autophagy, and recent studies confirm‎ a mutually inhibitory relationship between LAP and autophagy in photoreceptor degradation in RPE cells (Muniz-Feliciano et al., 2017). It is not clear why these two pathways are counter-regulated; possibly, cells may need to shut off the alternative pathway during stress to avoid competition for overlapping resources (Muniz-Feliciano et al., 2017). At a mechanistic level, it is uncertain how Rubicon functions to promote Beclin 1/VPS34 kinase activity at the phagosome (Martinez et al., 2015), but inhibit it at other organellar sites (Matsunaga et al., 2009). Interestingly, the WD repeat-containing C-terminal domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes in LAP, but dispensable for canonical autophagy (Fletcher et al., 2018), illustrating another difference in the molecular roles of an ATG protein in autophagy and LAP.

The genetic overlap and mutual antagonism of LAP and autophagy have practical implications for autophagy-targeted therapies. Theoretically, specificity in autophagy induction might be enhanced by activating the ULK1 complex rather than downstream shared nodes in autophagy and LAP (although the ULK1 kinase complex may have substrates outside of autophagy). The appeal of targeting Rubicon — a negative regulator of autophagy whose knockout in mice has beneficial effects (e.g. improved high-fat diet-induced hepatic steatosis (Tanaka et al., 2016) and increased cardiac protection during lipopolysaccharide-induced sepsis (Zi et al., 2015)) — may be tempered by potential adverse effects of LAP inhibition, such as increased susceptibility to fungal diseases and autoimmunity. Furthermore, treatments that target shared ATG proteins may result in unpredictable effects on each pathway, assuming these proteins are rate-limiting and the two pathways compete for access to these shared core ATG proteins.

The identification of LAP as a lysosomal degradation pathway that utilizes certain core ATG genes requires us to adopt a wider interpretative lens for studies of mice with deletions of these genes. Does deficient LAP versus deficient autophagy contribute to pathological phenotypes in mice with whole body or tissue-specific deficiency of genes such as beclin 1, ATG5, ATG7, or ATG16L1? To what extent does increased autophagy versus decreased LAP contribute to Rubicon knockout phenotypes? Do the GWAS associations between polymorphisms in some of these genes and diseases that involve disordered immune regulation (such as asthma, SLE, and inflammatory bowel disease) (see Table 1) suggest a role for altered LAP in their pathogenesis? The observation that a deficiency of LAP-associated (but not of non-LAP-associated) ATG genes results in a SLE-like syndrome in mice (Martinez et al., 2016) underscores the importance of this question. Specific molecular markers to distinguish LAP from autophagy in both animal models and human disease are needed.

LAP는 톨 유사 수용체(TLR)와 상호작용하는 입자의 식작용 과정에서 마우스 대식세포에서 처음 발견되었으며(Sanjuan et al., 2007), DNA 면역 복합체 및 기타 TLR9 리간드에 대한 반응으로 유형 I 인터페론(IFN) 분비에 관여합니다(Hayashi et al., 2018; Henault et al., 2012). LAP의 생리학적으로 중요한 기능은 골수 특이적 LAP 특이적 유전자(예: Rubicon 또는 NOX2) 및 자가포식 특이적 ATG 유전자(예: FIP200 또는 Atg14)가 결손된 마우스의 표현형을 비교하여 확인되었습니다. LAP는 망막 색소 상피(RPE)에 의한 광수용체 외단층의 분해에 필수적이며, 이는 정상적인 시력 유지에 필요한 과정입니다(Kim et al., 2013b). LAP는 곰팡이 A. fumigatus와 세포 내 세균 L. monocytogenes에 의해 유도되며, 이러한 병원체에 대한 호스트 방어를 강화합니다(Gluschko et al., 2018; Martinez et al., 2015). LAP 경로의 여러 구성 요소가 결핍된 마우스는 죽어가는 세포의 제거 결함으로 인해 염증 신호 전달과 자가항체 생산이 증가하여 자가면역성 전신성 홍반성 루푸스(SLE) 유사 질환을 발병합니다(Martinez et al., 2016).

인간 생리학에서 수용체 활성화 식세포의 중요한 역할을 고려할 때, LAP는 고전적인 자가포식과 마찬가지로 인간 질병에서 중요한 경로로 부상할 가능성이 높습니다. 두 경로는 겹치는 유전적 기전을 이용하지만, 중요한 차이점으로 인해 서로 상반된 역할을 합니다. 구체적으로, 루비콘은 LAP에 필요하지만 자가포식을 억제하며, 최근 연구에서는 RPE 세포의 광 수용체 분해에서 LAP와 자가포식 사이에 상호 억제 관계가 있음을 확인했습니다 (Muniz-Feliciano 외, 2017). 이 두 경로가 서로 억제되는 이유는 명확하지 않습니다. 아마도 세포는 스트레스 상황에서 중복 자원의 경쟁을 피하기 위해 대체 경로를 차단해야 할 수 있습니다(Muniz-Feliciano et al., 2017). 기전적 수준에서 Rubicon이 파고소체에서 Beclin 1/VPS34 키나아제 활성을 촉진하는 방식(Martinez et al., 2015)과 다른 세포 소기관 부위에서 이를 억제하는 방식(Matsunaga et al., 2009)은 불확실합니다. 흥미롭게도, ATG16L1의 WD 반복을 포함하는 C-말단 도메인은 LAP에서 LC3를 내소체 막으로 모집하는 데 필수적이지만, 일반적인 자가포식에는 필요하지 않습니다 (Fletcher et al., 2018). 이는 자가포식과 LAP에서 ATG 단백질의 분자적 역할의 또 다른 차이를 보여줍니다.

LAP와 자가포식의 유전적 중복과 상호 적대적 관계는 자가포식을 표적으로 하는 치료에 실용적인 의미를 지닙니다. 이론적으로, 오토파지 유도에서의 특이성은 오토파지와 LAP에서 하류에 있는 공유 노드보다 ULK1 복합체를 활성화함으로써 강화될 수 있습니다 (ULK1 키나아제 복합체는 오토파지 외부에 기질을 가질 수도 있지만). 루비콘을 표적으로 하는 것의 매력은 오토파지의 부정적 조절인자로, 쥐에서 루비콘을 제거하면 유익한 효과 (예: 고지방 식이 유발 간 지방증 개선(Tanaka et al., 2016) 및 리포폴리사카라이드 유발 패혈증 시 심장 보호 효과 증가(Zi et al., 2015)) —는 LAP 억제의 잠재적 부작용(예: 곰팡이 질환에 대한 감수성 증가 및 자가면역 반응)으로 인해 완화될 수 있습니다. 또한, 공유 ATG 단백질을 표적으로 하는 치료법은 이러한 단백질이 속도 제한 인자이며 두 경로가 공유된 핵심 ATG 단백질에 대한 접근을 경쟁한다고 가정할 때, 각 경로에 대한 예측 불가능한 영향을 초래할 수 있습니다.

특정 핵심 ATG 유전자를 이용하는 리소좀 분해 경로로 LAP가 확인됨에 따라, 이러한 유전자가 결손된 마우스를 연구할 때 더 폭넓은 해석의 관점을 채택해야 합니다.

베클린 1, ATG5, ATG7 또는 ATG16L1과 같은 유전자가 전신 또는 조직 특이적으로 결핍된 마우스에서 LAP 결핍이 자가포식 결핍에 비해 병리학적 표현형에 기여하는 정도는 어느 정도일까요?

자가포식 증가와 LAP 감소가 루비콘 녹아웃 표현형에 어느 정도 기여할까요?

이러한 유전자 중 일부의 다형성과 면역 조절 장애(천식, SLE, 염증성 장 질환 등)를 수반하는 질병(표 1 참조) 사이의 GWAS 연관성은 병인의 기전에서 LAP의 변화가 중요한 역할을 한다는 것을 시사하는 것일까요?

LAP 관련 (LAP 비관련은 제외) ATG 유전자의 결핍이 쥐에서 SLE와 유사한 증후군을 유발한다는 관찰 결과 (Martinez et al., 2016)는 이 질문의 중요성을 강조합니다. 동물 모델과 인간 질병에서 LAP와 자가포식을 구별하기 위한 특정 분자 마커가 필요합니다.

Beyond Lysosomal Degradation: Autophagy Genes Function in Secretion and Exocytosis

ATG genes are used not only for targeting intracellular cargo to the lysosome for degradation, but also for pathways that involve the targeting of intracellular cargo to either the plasma membrane or extracellular environment (Figure 1). Generally, these pathways have been grouped under the umbrella term “secretory autophagy”; however, as the “phagy” part is missing from the process, we prefer the more linguistically precise term of ATG gene-dependent secretion. There are many different types of ATG gene-dependent secretion (reviewed elsewhere from a cell biology perspective [Cadwell and Debnath, 2018]) but the mechanisms governing most of these processes are not well understood. Here, we focus on these pathways as they may relate to mammalian physiology and disease.

Unconventional secretion involves the extracellular release of proteins that lack amino-terminal signal peptide leader sequences and bypass “conventional” transit through the endoplasmic-reticulum (ER)-Golgi apparatus to reach the plasma membrane. A role for ATG proteins (Atg1/5/6/7/8/9/12/17) in this process was first discovered in yeast secretion of the acyl-CoA-binding protein, Acb1 (Duran et al., 2010; Manjithaya et al., 2010). In mammalian cells, unconventional secretion of leaderless proteins, such as the pro-inflammatory cytokines processed by the inflammasome, IL-1β and IL-18, also require the autophagy protein, ATG5 (Dupont et al., 2011). The precise mechanisms underlying ATG gene-dependent unconventional secretion remain unclear. It is not certain whether targets are captured in an autophagosomal lumen (Dupont et al., 2011) and/or the intermembrane space between the double membrane of the autophagosome (Zhang et al., 2015b); nor is it certain how targets are delivered to and released from the plasma membrane. Autophagosome-like vesicles containing IL-1β bypass syntaxin 17-dependent fusion with lysosomes and instead use specific SNAREs and syntaxins involved in vesicle fusion with the plasma membrane for cargo secretion (Kimura et al., 2017).

리소좀 분해 그 이상: 분비 및 세포외 분비에서 자가포식 유전자의 기능

ATG 유전자는

세포 내 화물을 리소좀으로 보내 분해하는 데만 사용되는 것이 아니라,

세포 내 화물을 세포막이나 세포 외 환경으로 보내는 경로에도 사용됩니다 (그림 1).

일반적으로 이러한 경로는 “분비 자가포식”이라는 포괄적인 용어로 분류되어 왔지만,

이 과정에서 “포식” 부분이 빠져 있기 때문에,

언어적으로 더 정확한 ATG 유전자 의존적 분비라는 용어를 사용하는 것이 더 적절할 것입니다.

“secretory autophagy

ATG 유전자 의존적 분비에는 다양한 유형이 존재하며(세포 생물학 관점에서 다른 문헌에서 검토됨 [Cadwell and Debnath, 2018]), 이러한 과정의 대부분을 조절하는 메커니즘은 아직 잘 이해되지 않고 있습니다. 본 연구에서는 이러한 경로가 포유류 생리학과 질병과 관련될 수 있다는 점에서 초점을 맞춥니다.

비전형적 분비는

아미노말단 신호 펩티드 리더 시퀀스가 결여된 단백질이 내소체-골지체(ER-Golgi) 장치를 우회하여

세포막으로 직접 분비되는 과정을 포함합니다.

이 과정에서의 ATG 단백질(Atg1/5/6/7/8/9/12/17)의 역할은 효모의 아실-코엔자임 A 결합 단백질 Acb1 분비 과정에서 처음 발견되었습니다(Duran et al., 2010; Manjithaya et al., 2010). 포유류 세포에서, 인플라마좀에 의해 처리되는 전염성 사이토카인, IL-1β 및 IL-18과 같은 리더가 없는 단백질의 비전통적인 분비도 자가포식 단백질 ATG5를 필요로 합니다 (Dupont et al., 2011).

ATG 유전자에 의존하는 비전형적 분비 메커니즘의 정확한 기전은 아직 명확하지 않습니다. 표적 단백질이 오토파고소 내강(Dupont et al., 2011)과/또는 오토파고소 이중 막 사이의 간막 공간(Zhang et al., 2015b)에 포획되는지, 그리고 표적 단백질이 세포막으로 전달되고 방출되는 메커니즘도 명확하지 않습니다. IL-1β를 포함하는 자포소체 유사 소포는 syntaxin 17에 의존하는 리소좀과의 융합을 우회하며, 대신 소포와 세포막의 융합에 관여하는 특정 SNAREs와 syntaxins를 사용하여 화물 분비를 수행합니다 (Kimura et al., 2017).

A function of ATG genes in secretion of pro-inflammatory mediators (and more broadly, other leaderless proteins) could have vast importance for inflammatory disorders and a wide range of other diseases. However, it is currently difficult to assess the physiological importance of ATG gene-dependent secretion of IL-1β and IL-18 in vivo, as macrophage (or hematopoietic cell)-specific deletion of Atg5, Atg16l1, and Atg7 in mice is associated with increased, rather than decreased, levels of IL-1β and IL-18 production (Kimmey et al., 2015; Martinez et al., 2016; Saitoh et al., 2008). These findings may reflect basal functions of ATG genes in the negative control of inflammasome activation (Zhou et al., 2011), whereas the ATG gene-dependent secretion of pro-inflammatory mediators may be unmasked during certain stress conditions, such as inflammasome activation triggered by lysosomal membrane damage (Kimura et al., 2017). The possibility of an autophagy-dependent secretome in vivo warrants further investigation and may lead to the identification of proteomic signatures of autophagy activation as clinically useful serum biomarkers. Theoretically, autophagy-inducing therapies might lead to untoward effects via the unconventional secretion of pro-inflammatory mediators or other pathogenic proteins.

Perhaps the best-established link between ATG gene-dependent secretion and mammalian physiology and disease relates to the exocytosis of secretory granules and lysosomes. Notably, human genome-wide association studies (GWAS) that revealed a polymorphism in a core ATG gene, ATG16L1T300A, as a major risk allele for Crohn’s disease (Barrett et al., 2008) spurred the discovery of a fundamental role for the ATG protein conjugation machinery in secretory granule exocytosis (Cadwell et al., 2008). In mice, hypomorphic expression‎ of Atg16l1, Atg16l1T300A knock-in mutation, Paneth cell-specific deletion of Atg16l1, Atg5, or Atg7, or whole-body deletion of Atg4b results in abnormal granule morphology and a defect in granule exocytosis and lysozyme secretion by Paneth cells (Bel et al., 2017; Cabrera et al., 2013; Cadwell et al., 2008; Lassen et al., 2014), a specialized ileal epithelial cell type that controls the intestinal microbiota by secreting lysozyme and antimicrobial peptides. Similar defects in Paneth cell morphology are observed in patients with (but not those without) the ATG16L1T300A Crohn’s disease risk allele (Cadwell et al., 2008). The precise membrane trafficking mechanisms by which ATG proteins facilitate secretory granule exocytosis in Paneth cells or other cell types remain unknown. However, a recent study indicates that lysozyme is localized in large LC3-positive vesicles in Paneth cells from wild-type but not Atg16l1T300A mice (Bel et al., 2017). Thus, in a manner similar to autophagosome-like vesicles involved in unconventional protein secretion, secretory granules may be earmarked for exocytosis by the presence of LC3 on their surface.

A related, but topologically distinct, link between autophagy and secretory lysosome exocytosis was uncovered in another specialized type of secretory cell, the osteoclast. Osteoclasts resorb bone by a mechanism that involves secretory lysosome fusion with bone-apposed plasma membrane composed of ruffled borders, with the discharge of matrix-degrading molecules into the site of osteal degradation. In mice, the ATG protein conjugation machinery and the Rab GTPase, Rab7, are essential for generating an LC3-labeled ruffled border, cathepsin K release and normal bone resorption (DeSelm et al., 2011), thus indicating a role for ATG genes in mediating polarized secretion of lysosomal contents to the extracellular space. In this scenario, the plasma membrane, not the secretory lysosome, is labeled by LC3. Thus, during secretion, the ATG protein conjugation machinery and resulting lipidated LC3 can function either in the formation of normal secretory granules that properly fuse with the plasma membrane or in the creation of a specialized plasma membrane that fuses with secretory lysosomes.

The predicted clinical outcome of defects in ATG gene-dependent osteoclast functions would be osteopetrosis, a disease marked by abnormally dense bone. Consistent with this prediction, mutations in PLEKHM1, a Rab7 effector, and the v-ATPase α3 subunit involved in lysosomal acidification, are each associated with osteopetrosis in patients (Stenbeck and Coxon, 2014). In contrast, aging, which is associated with reduced autophagy in most cell types (Hansen et al., 2018), is accompanied by osteopenia and osteoporosis in mice and humans. This may reflect the roles of ATG genes in other cell types in the bone that favor bone growth and normal bone density, including protection against endoplasmic reticulum (ER) and oxidative stress in osteoblasts and osteocytes (Li et al., 2018b; Liu et al., 2013a; Onal et al., 2013) and maintenance of the proper function of bone mesenchymal stem cells (Ma et al., 2018). Thus, studies in bone represent an elegant example of how the autophagic machinery can exert different specialized functions in distinct cell types within a given organ – functions that may have opposite effects (such as bone resorption and bone formation) – to orchestrate overall tissue homeostasis. As osteopenia/osteoporosis and associated skeletal fractures are a major cause of morbidity and mortality in aging humans, this area warrants further investigation as a potential clinical target for autophagy upregulation.

Numerous other defects in protein secretion in ATG gene knockout mice have been described, although it is unclear whether they reflect a direct role for ATG genes in autophagy-independent trafficking or more indirect consequences of autophagy deficiency on secretory processes. These include defects in the assembly and secretion of octogonial core proteins which leads to abnormalities in vestibular development (Marino et al., 2010) and defects in pancreatic β-cell insulin granule morphology and secretion (Watada and Fujitani, 2015), melanogenesis and pigmentation (Ganesan et al., 2008), and mucus secretion of airway epithelial cells and intestinal goblet cells (Patel et al., 2013).

Accumulating evidence suggests that ATG proteins also have pleiotropic effects on the cellular release of exosomes, a process that is mediated by fusion of the multivesicular body (MVB) with the plasma membrane (Baixauli et al., 2014; Hessvik and Llorente, 2018). Autophagy induction can prevent – whereas ATG gene silencing or pharmacological inhibition can increase – extracellular release of exosomes, including those containing pathogenic protein cargoes, such as α-synuclein (Fussi et al., 2018), prions (Abdulrahman et al., 2018) and amyloid precursor protein (Miranda et al., 2018). This regulatory mechanism is presumed to involve MVB fusion with autophagosomes, thereby diverting MVB transport away from the plasma membrane. Increased exosome release in the setting of impaired autophagy may function as an alternative quality control pathway to maintain cellular homeostasis and prevent cell death due to proteotoxicity. However, there are also examples in which ATG genes stimulate exosome production. Atg5, but not Atg7, has been shown to decrease late endosome acidification by disrupting the v-ATPase, thereby promoting the production of exosomes that enhance tumor metastasis (Guo et al., 2017). Similarly, the ATG3-ATG12 conjugate which is required for LC3 lipidation during basal (but not starvation) conditions interacts with the ESCRT protein, Alix, and positively controls Alix-dependent exosome biogenesis (Murrow et al., 2015). Given the expanding repertoire of exosome-dependent processes (including neurodegeneration, immune signaling, metabolism, tumor metastasis and viral infection), the effects of the autophagic machinery on the fate of the MVB – lysosomal degradation or exocytosis – may partly underlie the pathophysiological effects of ATG gene mutation.

The ATG machinery modulates retromer function to control the endosome-to-cell-surface recycling pathway (Roy et al., 2017). During metabolic stress, LC3 on autophagic structures binds to the RabGAP protein TBC1D5 to sequester it away from an inhibitory interaction with the retromer complex. This sequestration allows retromers to associate with endosomal membranes and mediate plasma membrane translocation of the glucose transporter, GLUT1, a facilitator of glucose uptake. GLUT1 is required for the low levels of basal glucose uptake required to sustain cellular respiration, and its plasma membrane localization normally increases when cells are exposed to low glucose. Perturbation in ATG protein conjugation may significantly cripple this metabolic homeostatic mechanism involving GLUT1 trafficking and, in addition, affect the cell surface localization of other as-of-yet-unidentified receptors.

ATG 유전자들이 프로염증 매개체(그리고 더 넓게는 리더 단백질이 없는 단백질)의 분비에 관여하는 기능은 염증성 질환 및 다양한 다른 질환에 대한 광범위한 중요성을 가질 수 있습니다. 그러나 현재 ATG 유전자 의존적 IL-1β 및 IL-18 분비의 생리적 중요성을 in vivo에서 평가하는 것은 어렵습니다. 마우스에서 대식세포(또는 혈액 생성 세포) 특이적 Atg5, Atg16l1, 및 Atg7 유전자 결손은 IL-1β 및 IL-18 생산이 감소하는 대신 증가하는 것과 연관되어 있습니다(Kimmey et al., 2015; Martinez et al., 2016; Saitoh et al., 2008). 이러한 결과는 ATG 유전자의 염증체 활성화에 대한 음성 조절의 기초적 기능을 반영할 수 있습니다(Zhou et al., 2011), 반면 ATG 유전자에 의존적인 염증 매개체 분비는 특정 스트레스 조건, 예를 들어 리소좀 막 손상으로 유발된 염증체 활성화 시에 드러날 수 있습니다(Kimura et al., 2017). 생체 내에서 자가포식 의존적 분비체(secretome)가 존재할 가능성은 추가적인 연구가 필요하며, 임상적으로 유용한 혈청 바이오마커로 자가포식 활성화의 단백질 서명(proteomic signature)을 확인하는 데 이어질 수 있습니다.

이론적으로,

자가포식 유도 요법은 전염성 매개체 또는 기타 병원성 단백질의 비정상적인 분비를 통해

바람직하지 않은 효과를 초래할 수 있습니다.

ATG 유전자 의존적 분비와 포유류 생리학 및 질환 간의 가장 잘 확립된 연관성은 분비 소체와 리소좀의 분비와 관련됩니다. 특히, 인간 유전체 연관 연구(GWAS)에서 크론병의 주요 위험 알레일로 ATG16L1T300A 유전자의 다형성을 밝혀낸 연구(Barrett et al., 2008)는 ATG 단백질 결합 기계가 분비 소체 분비에 근본적인 역할을 한다는 발견을 촉진했습니다(Cadwell et al., 2008). 쥐에서 Atg16l1의 저형성 발현, Atg16l1T300A 노크인 돌연변이, Paneth 세포 특이적 Atg16l1, Atg5, 또는 Atg7 삭제, 또는 전체 신체 Atg4b 삭제는 Paneth 세포의 비정상적인 소체 형태, 소체 분비 장애, 및 리소자임 분비 결함을 유발합니다(Bel et al., 2017; Cabrera et al., 2013; Cadwell et al., 2008; Lassen et al., 2014), 장내 미생물군집을 조절하는 특수한 소장 상피 세포 유형으로, 리소자임과 항균 펩타이드를 분비합니다. ATG16L1T300A 크론병 위험 알레르를 가진 환자(하지만 그렇지 않은 환자에서는 아니며)에서 Paneth 세포의 형태학적 결함이 관찰되었습니다(Cadwell et al., 2008). ATG 단백질이 Paneth 세포나 다른 세포 유형에서 분비 소체 분비를 촉진하는 정확한 막 수송 메커니즘은 아직 알려지지 않았습니다. 그러나 최근 연구에서 리소자임이 야생형 마우스의 Paneth 세포에서 LC3 양성 대형 소체에 국한되어 있지만 Atg16l1T300A 마우스에서는 그렇지 않다는 것이 밝혀졌습니다(Bel et al., 2017). 따라서, 비전형적 단백질 분비에 관여하는 자포소체 유사 소체와 유사한 방식으로, 분비 소체는 표면에 LC3가 존재함으로써 분비에 지정될 수 있습니다.

자가포식과 분비 리소좀 세포외 분비 사이의 관련이 있지만, 위상학적으로는 다른 연관성이 또 다른 특수한 유형의 분비 세포인 파골세포에서 밝혀졌습니다. 파골세포는 낡은 경계로 구성된 뼈에 인접한 혈장 막과 분비 리소좀이 융합되는 메커니즘을 통해 뼈를 재흡수하며, 뼈 분해 부위로 매트릭스 분해 분자를 방출합니다. 쥐에서 ATG 단백질 결합 기계와 Rab GTPase인 Rab7은 LC3로 표시된 주름진 경계 형성, 카테프신 K 방출 및 정상적인 뼈 흡수(DeSelm et al., 2011)에 필수적이며, 이는 ATG 유전자가 리소좀 내용물의 극성 분비를 세포외 공간으로 매개하는 역할을 함을 시사합니다. 이 시나리오에서 LC3로 표지되는 것은 분비 리소좀이 아닌 세포막입니다. 따라서 분비 과정에서 ATG 단백질 결합 기계 장치와 결과적으로 지질화된 LC3는 정상적인 분비 소체 형성과 세포막과의 적절한 융합에 기능하거나, 분비 리소좀과 융합하는 특수화된 세포막 형성에 기능할 수 있습니다.

ATG 유전자 의존적 골용해세포 기능 결손의 예측된 임상적 결과는 골밀도가 비정상적으로 높은 질환인 골다공증입니다. 이 예측과 일치하게, Rab7 효과자인 PLEKHM1과 리소좀 산성화에 관여하는 v-ATPase α3 서브유닛의 돌연변이는 각각 환자에서 골다공증과 연관되어 있습니다 (Stenbeck and Coxon, 2014). 대조적으로, 대부분의 세포 유형에서 자가포식 감소와 관련된 노화 (Hansen et al., 2018)는 쥐와 인간에서 골감소증 및 골다공증을 동반합니다. 이는 골격 내 다른 세포 유형에서 ATG 유전자가 골 성장과 정상적인 골밀도를 촉진하는 역할을 반영할 수 있습니다. 이는 골아세포와 골세포에서 내소기관(ER) 및 산화 스트레스로부터의 보호를 포함합니다(Li et al., 2018b; Liu et al., 2013a; Onal et al., 2013) 그리고 골간엽 줄기세포의 적절한 기능 유지(Ma et al., 2018)와 관련될 수 있습니다. 따라서 뼈에 대한 연구는 자가포식 기전이 특정 기관 내의 서로 다른 세포 유형에서 서로 반대되는 효과(예: 뼈의 재흡수와 뼈의 형성)를 발휘하여 전체적인 조직의 항상성을 조율하는 방법을 보여주는 훌륭한 예입니다. 골감소증/골다공증 및 관련 골절은 노화 인구의 사망 및 질병의 주요 원인인 만큼, 이 분야는 자가포식 조절의 잠재적 임상적 표적으로서 추가적인 연구가 필요합니다.

ATG 유전자 결손 마우스에서 단백질 분비에 대한 수많은 다른 결함이 보고되었지만, 이러한 결함이 ATG 유전자가 자가포식 독립적 수송에 직접적인 역할을 하는지, 아니면 자가포식 결핍이 분비 과정에 미치는 더 간접적인 결과인지는 아직 명확하지 않습니다. 이러한 결함에는 전정 발달 이상으로 이어지는 팔각형 핵심 단백질의 조립 및 분비 결함 (Marino et al., 2010) 췌장 β-세포 인슐린 그레인 형태 및 분비 결함(Watada and Fujitani, 2015), 멜라닌 생성 및 색소 침착(Ganesan et al., 2008), 기도 상피 세포 및 장 점액 세포의 점액 분비 결함(Patel et al., 2013) 등이 포함됩니다.

증가하는 증거는 ATG 단백질이 세포 내 엑소좀 방출 과정에 다중 효과를 미친다는 것을 시사합니다. 이 과정은 다중 소포체(MVB)와 세포막의 융합을 통해 매개됩니다(Baixauli et al., 2014; Hessvik and Llorente, 2018). 자가포식 유도는 α-시누클레인 (Fussi et al., 2018), 프리온 (Abdulrahman et al., 2018) 및 아밀로이드 전구체 단백질 (Miranda et al., 2018)과 같은 병원성 단백질 화물을 포함하는 엑소좀의 세포외 방출을 방지할 수 있으며, ATG 유전자 침묵화 또는 약리학적 억제는 이러한 방출을 증가시킬 수 있습니다. 이 조절 메커니즘은 MVB가 자가포식 소체와 융합하여 MVB의 수송을 세포막에서 다른 곳으로 전환시키는 것과 관련이 있는 것으로 추정됩니다. 자가포식 장애에서 엑소좀의 방출이 증가하는 것은 세포의 항상성을 유지하고 단백질 독성에 의한 세포 사멸을 방지하기 위한 대체적인 품질 관리 경로로 기능할 수 있습니다. 그러나 ATG 유전자가 엑소좀 생성을 자극하는 사례도 있습니다. Atg5는 Atg7과 달리 v-ATPase를 방해하여 후기 엔도좀 산성화 감소로 이어지며, 이는 종양 전이를 촉진하는 엑소좀 생성을 촉진합니다 (Guo et al., 2017). 同様に, 기초 조건(하지만 굶주림 조건에서는 아니)에서 LC3 지질화(lipidation)에 필요한 ATG3-ATG12 복합체는 ESCRT 단백질 Alix와 상호작용하여 Alix 의존적 엑소좀 생성을 긍정적으로 조절합니다(Murrow et al., 2015). 엑소좀 의존적 과정의 확장된 범주(신경퇴행, 면역 신호전달, 대사, 종양 전이, 바이러스 감염 등)를 고려할 때, 자식작용 기계장치가 MVB의 운명(리소좀 분해 또는 엑소토시시스)에 미치는 영향은 ATG 유전자 변이의 병리생리학적 효과의 일부를 설명할 수 있습니다.

ATG 기계는 레트로머 기능을 조절하여 엔도좀에서 세포 표면으로의 재순환 경로를 제어합니다(Roy et al., 2017). 대사 스트레스 시 자포성 구조물에 존재하는 LC3는 RabGAP 단백질 TBC1D5와 결합하여 이를 레트로머 복합체와의 억제적 상호작용에서 격리시킵니다. 이 격리 과정은 레트로머가 엔도좀 막과 결합하여 포도당 운반체 GLUT1의 세포막 이동을 매개합니다. GLUT1은 세포 호흡을 유지하기 위해 필요한 기초 포도당 흡수 수준을 유지하는 데 필수적이며, 세포가 저포도당 환경에 노출될 때 세포막 국소화가 증가합니다. ATG 단백질 접합에 장애가 발생하면 GLUT1의 이동과 관련된 이 대사 항상성 메커니즘이 크게 손상될 수 있으며, 또한 아직 확인되지 않은 다른 수용체의 세포 표면 국소화에도 영향을 미칠 수 있습니다.

Autophagy Genes in Other Dynamic Membrane Events

Non-autophagic functions of ATG genes in membrane trafficking modulate the infection of host cells by viruses, bacteria and other pathogens. These include processes described above such as LAP (which may be partially antagonized by virulent micro-organisms that enter professional phagocytes) and LC3-regulated exocytosis (which is involved in the egress of viruses that either reside inside autophagosomes or whose envelopes become decorated with LC3) (reviewed in [Cadwell and Debnath, 2018]). Many additional ATG gene-dependent membrane reorganization events – or interference with such events – also regulate infection. For example, several ATG genes are required for the formation of intracytoplasmic membrane-associated replication factories of certain medically important RNA viruses, such as hepatitis C virus (Dreux et al., 2009). Similarly, the formation of multi-membranous vacuoles that support replication of the bacterium, B. abortus, involves ATG genes required for class III PI3K activity but not those required for LC3 conjugation (Starr et al., 2012). In contrast, IFN-γ inhibits T. gondii replication by a process involving LC3/GABARAP lipidation and recruitment of IFN-γ-inducible GTPases to the parasitophorous vacuole, where they disrupt the membrane and destroy the parasite’s replicative niche (Choi et al., 2014). Similarly, IFN-γ mediated control of murine norovirus (a model for human epidemics of gastroenteritis) involves labeling membrane-associated viral replication complexes with lipidated LC3 and recruitment of IFN-γ-inducible GTPases (Biering et al., 2017). Thus, marking replication-associated membrane structures by LC3 conjugation may represent a conserved mechanism underlying IFN-γ-mediated control of intracellular pathogen replication. Further understanding of the precise processes by which different subsets of ATG proteins provide or destroy host membranes necessary for different stages of pathogen replication may lead to the development of new anti-infective strategies.

다른 동적 막 사건에서 자가포식 유전자

막 수송에서 ATG 유전자의 비자가포식 기능은

바이러스, 박테리아 및 기타 병원체에 의한

숙주 세포의 감염을 조절합니다.

이에는 위에서 설명된 과정들이 포함됩니다.

예를 들어,

LAP(전문 식세포에 침입하는 병원성 미생물에 의해 부분적으로 억제될 수 있음)와

LC3 조절 분비(자율포식체 내부에 존재하거나 LC3로 장식된 외피를 가진 바이러스의 배출에 관여함) 등이

있습니다(Cadwell and Debnath, 2018에서 검토됨).

ATG 유전자에 의존하는 추가적인 막 재편성 사건 또는 이러한 사건의 방해도 감염을 조절합니다. 예를 들어, 특정 의학적 중요성을 가진 RNA 바이러스(예: C형 간염 바이러스)의 세포질 내막과 연관된 복제 공장의 형성에 여러 ATG 유전자가 필요합니다(Dreux et al., 2009). 同様に, 세균 B. abortus의 복제를 지원하는 다중막 소포의 형성은 LC3 결합에 필요한 ATG 유전자보다는 class III PI3K 활성에 필요한 ATG 유전자에 의존합니다 (Starr et al., 2012).

반면, IFN-γ는 LC3/GABARAP 지질화 및 IFN-γ 유도성 GTPase의 기생체 소포체로의 모집을 통해 T. gondii의 복제를 억제합니다. 이 과정에서 GTPase는 기생체 소포체의 막을 파괴하여 기생체의 복제 환경을 파괴합니다 (Choi et al., 2014). 유사하게, IFN-γ에 의한 인간 위장염 유행의 모델인 마우스 노로바이러스의 조절은 지질화된 LC3로 막 관련 바이러스 복제 복합체를 표지하고 IFN-γ 유도성 GTPase를 모집하는 과정을 포함합니다 (Biering et al., 2017).

따라서

LC3 접합을 통해 복제 관련 막 구조를 표시하는 것은

IFN-γ 매개 세포 내 병원체 복제 제어의 기본이 되는 보존된 메커니즘을 나타낼 수 있습니다.

병원체 복제의 여러 단계에 필요한 숙주 막을 ATG 단백질의 여러 하위 집합이 제공하거나 파괴하는

정확한 과정을 더 자세히 이해하면

새로운 항감염 전략이 개발될 수 있습니다.

Beyond Membrane Trafficking: Autophagy Proteins Have Other Functions

The autophagy proteins not only help orchestrate the cross-talk of diverse vesicular trafficking pathways, but also interface with multiple other cellular pathways, including (but not limited to) cell death pathways, cell cycle regulation, and innate immune signaling. The interaction of FIP200 with Atg13 is essential for autophagy in vivo and neonatal survival in mice, but the non-autophagic function is sufficient to maintain embryogenesis through a mechanism involving protection against TNFα-induced apoptosis (Chen et al., 2016). Atg7, independently of its E1-like enzymatic activity and function in autophagy, regulates p53-dependent cell cycle arrest and apoptosis, and the neonatal lethality of Atg7 knockout mice is partially rescued by inhibition of the DNA damage response through deletion of the protein kinase Chk2 (Lee et al., 2012). In mice, deletion of Atg9a, but not Atg5, results in a defect in necrosis at the bone surface during developmental morphogenesis (Imagawa et al., 2016). The precise mechanisms underlying these (and additional) functions of individual ATG proteins in cell death and cell cycle regulation are not well understood.

ATG proteins regulate inflammatory and immune signaling both through autophagy-dependent mechanisms (such as by the autophagic removal of damaged mitochondria that produce ROS and activate RIG-I signaling and the NLRP3 inflammasome) and autophagy-independent mechanisms that generally involve ATG protein interactions with immune signaling molecules. For example, the ATG5-ATG12 conjugate inhibits type I IFN signaling in response to viral infection by binding to the CARDs (caspase activation and recruitment domains) of RNA recognition molecules such as RIG-I and MAVs (Jounai et al., 2007). Similarly, the cytosolic DNA sensing innate immunity pathway mediated by cGAS (cyclic GMP-AMP [cGAMP] synthase) and STING (Stimulator of interferon genes) is regulated by autophagy proteins. The generation of cGAMP by cGAS activates ULK1, which phosphorylates and inhibits STING-dependent cytokine production (Konno et al., 2013). As unrestrained STING signaling (either via inherited mutations in the ADAR and ribonuclease H2 complex or gain-of-function mutations in STING) causes human autoinflammatory diseases, ULK1 activating agents have been proposed as potential treatments for such disorders (Konno et al., 2018). Beclin 1 binds cGAS to suppress cGAMP synthesis and halt interferon production (Liang et al., 2014). Atg9a may also function as a negative regulator of innate immune signaling by decreasing the assembly of STING and TBK1 in the presence of double-stranded DNA (Saitoh et al., 2009). Of note, these same RNA-sensing and cytosolic DNA-sensing signaling pathways are activators of autophagy, which is itself an important innate immune effector pathway (Deretic and Levine, 2018). Thus, ATG proteins play a crucial role in both mediating innate immunity and in providing feedback inhibition to fine-tune inflammatory signaling so as to avoid deleterious consequences.

막 수송을 넘어: 자가포식 단백질의 다른 기능

자가포식 단백질은

다양한 소포체 수송 경로의 교차 통신을 조율할 뿐만 아니라,

세포 사멸 경로, 세포주기 조절, 선천적 면역 신호 전달 등

여러 다른 세포 경로와도 상호 작용합니다.

FIP200과 Atg13의 상호 작용은 생체 내 자가포식 및 쥐의 신생아 생존에 필수적이지만, 비자가포식 기능만으로도 TNFα에 의한 세포 사멸을 방지하는 메커니즘을 통해 배아 발생을 유지할 수 있습니다 (Chen et al., 2016). Atg7은 E1과 유사한 효소 활성 및 자가포식 기능과 별개로 p53 의존성 세포주기 정지 및 세포사멸을 조절하며, Atg7 녹아웃 마우스의 신생아 사망률은 단백질 키나아제 Chk2의 결손을 통한 DNA 손상 반응의 억제에 의해 부분적으로 회복됩니다 (Lee et al., 2012). 쥐에서 Atg9a를 삭제하면 Atg5를 삭제했을 때와는 달리 발달적 형태 형성 과정에서 뼈 표면에서의 괴사 결함이 발생합니다(Imagawa et al., 2016). 개별 ATG 단백질의 세포 사멸 및 세포 주기 조절에 대한 이러한(및 추가적인) 기능의 정확한 메커니즘은 잘 이해되지 않고 있습니다.

ATG 단백질은

자가포식 의존적 메커니즘(예: ROS를 생성하고 RIG-I 신호 전달 및 NLRP3 인플람소좀을 활성화하는 손상된 미토콘드리아의 자가포식적 제거)과 자가포식 독립적 메커니즘(일반적으로 ATG 단백질과 면역 신호 전달 분자의 상호 작용을 포함)을 통해 염증 및 면역 신호 전달을 조절합니다.

예를 들어, ATG5-ATG12 복합체는 RIG-I 및 MAVs와 같은 RNA 인식 분자의 CARDs(caspase 활성화 및 모집 도메인)에 결합하여 바이러스 감염에 대한 유형 I IFN 신호전달을 억제합니다(Jounai et al., 2007). 마찬가지로, cGAS (사이클릭 GMP-AMP [cGAMP] 신타제) 및 STING (인터페론 유전자 자극제)에 의해 매개되는 세포질 DNA 감지 선천적 면역 경로는 자가포식 단백질에 의해 조절됩니다. cGAS에 의한 cGAMP의 생성은 ULK1을 활성화하여 STING 의존성 사이토카인 생성을 인산화 및 억제합니다 (Konno et al., 2013). STING 신호전달의 무제한 활성화(ADAR 및 리보뉴클레아제 H2 복합체의 유전적 변이 또는 STING의 기능 획득 변이)가 인간 자가염증 질환을 유발하기 때문에, ULK1 활성화제는 이러한 질환의 잠재적 치료제로 제안되었습니다(Konno et al., 2018). Beclin 1은 cGAS에 결합하여 cGAMP 합성을 억제하고 인터페론 생성을 중단시킵니다(Liang et al., 2014). Atg9a는 이중 가닥 DNA 존재 시 STING과 TBK1의 조립을 감소시켜 선천 면역 신호전달의 음성 조절자로 기능할 수 있습니다(Saitoh et al., 2009). 주목할 점은, 이러한 동일한 RNA 감지 및 세포질 DNA 감지 신호 전달 경로는 자가포식(자가포식)의 활성화제이며, 자가포식은 그 자체로 중요한 선천적 면역 효과기 경로입니다 (Deretic and Levine, 2018). 따라서 ATG 단백질은 선천적 면역을 매개하고 염증 신호 전달을 미세 조정하여 해로운 결과를 방지하기 위해 피드백 억제를 제공하는 데 중요한 역할을 합니다.

The Selectivity of Autophagy: a Guardian of Cellular Homeostasis

For nearly half a century, the process of macroautophagy was believed to lack cargo specificity. In fact, the morphological identification of an autophagosome required visualizing the simultaneous presence of diverse cytoplasmic contents, such as ER, ribosomes and mitochondria inside a double-membraned vacuole. However, a transformative body of work over the past decade has fully dispelled this notion. We now know that there can be extreme specificity in governing the choice of cargo that is degraded by the autophagosome and an intricate system for earmarking and capturing such cargo. This process, termed selective autophagy, may be more crucial in protection against most mammalian diseases than “bulk autophagy”, which is primarily a homeostatic mechanism during nutrient stress.

Many parts of the cell can be “selected” for degradation by autophagy (Figure 2 and Table 2). Numerous studies have reported the selective autophagy of various organelles, including mitochondria, ER, peroxisomes, lipid droplets, ribosomes, midbody rings and the nucleus. Autophagy selectively degrades aggregation-prone misfolded proteins such as those involved in the pathogenesis of certain neurodegenerative, skeletal and cardiac muscle, and liver diseases. In addition, it degrades the individual proteins that serve as adaptors to bridge cargo with the nascent phagophore as well as specific inflammatory and immune signaling molecules. Moreover, selective autophagy can target pathogens that reside inside vacuoles or directly inside the cytosol for lysosomal degradation. Once captured, cargo degradation proceeds through a route that involves the same molecular machinery as bulk autophagy. Different forms of selective autophagy are often named by a term comprising a prefix derived from the cargo (e.g. mito-, ER-, ribo-, nucleo-, pexo-, lipo-) and the suffix “phagy”. For selective autophagy of microbial invaders, the term xenophagy is commonly used.

자가포식의 선택성: 세포의 항상성 유지자

거의 반세기 동안, 거대자가포식 과정은 화물 특이성이 없는 것으로 여겨져 왔습니다. 사실, 오토파고좀을 형태학적으로 확인하려면 이중막 소포체 내부에 ER, 리보솜, 미토콘드리아 등 다양한 세포질 내용물이 동시에 존재하는 것을 시각화해야 했습니다. 그러나 지난 10년 동안의 획기적인 연구 결과로 이러한 개념은 완전히 사라졌습니다. 이제 우리는 오토파고솜에 의해 분해되는 화물의 선택을 제어하는 극도의 특이성과 그러한 화물을 지정하고 포획하는 복잡한 시스템이 존재할 수 있음을 알고 있습니다.

선택적 오토파지라고 하는 이 과정은

주로 영양 스트레스 동안의 항상성 메커니즘인 “대량 오토파지”보다

대부분의 포유류 질병을 예방하는 데 더 중요할 수 있습니다.

세포의 여러 부분이

자가포식(autophagy)에 의해 분해될 대상으로

“선택”될 수 있습니다 (그림 2 및 표 2).

수많은 연구에서

미토콘드리아, ER, 퍼옥시좀, 지질 방울, 리보솜, 미드바디 링 및 핵을 비롯한

다양한 세포 기관의 선택적 자가포식이 보고되었습니다.

자가포식은

특정 신경 퇴행성 질환, 골격 및 심장 근육, 간 질환의 발병에 관여하는 단백질과 같이

응집되기 쉬운 잘못 접힌 단백질을 선택적으로 분해합니다.

또한,

신생 파고포어와 화물을 연결하는 어댑터 역할을 하는

개별 단백질과 특정 염증 및 면역 신호 전달 분자를 분해합니다.

또한,

선택적 자가포식은

액포 내 또는 세포질 내부에 존재하는 병원균을 리소좀 분해의 표적으로 삼을 수 있습니다.

화물이 포획되면,

화물 분해는 대량 자가포식과 동일한 분자 기전을 통해 진행됩니다.

다양한 형태의 선택적 자가포식은 화물에서 유래한 접두사(예: mito-, ER-, ribo-, nucleo-, pexo-, lipo-)와 접미사 “phagy”로 구성된 용어로 명명되는 경우가 많습니다. 미생물 침입자의 선택적 자가포식에는 xenophagy라는 용어가 일반적으로 사용됩니다.

Figure 2. Conceptual overview of selective autophagy.

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Shown are the diverse cargoes that are degraded by autophagy and the major known mechanisms by which cargo are attached to LC3 or GABARAP family members on the phagophore membrane. Also listed are currently known organelle-specific LC3/GABARAP-binding proteins, tags that label cargo destined for selective autophagic degradation, LC3/GABRAP-binding adaptor proteins, and factors that regulate the recognition of cargo by adaptors or LC3/GABARAP. Organelle-specific LC3/GABARAP-binding proteins and LC3/GABARAP-binding adaptor proteins interact with LC3/GABARAP via conserved W/F/YxxL/I/V motifs. See Table 2 for information about different types of selective autophagy and their possible roles in physiology and disease.

자가포식에 의해 분해되는 다양한 화물과, 화물이 식세포막에 있는 LC3 또는 GABARAP 가족에 부착되는 주요한 알려진 메커니즘이 표시되어 있습니다. 또한 현재 알려진 세포 소기관 특이적 LC3/GABARAP 결합 단백질, 선택적 자식작용 분해 대상 화물을 표시하는 태그, LC3/GABARAP 결합 적응 단백질, 및 적응 단백질이나 LC3/GABARAP에 의한 화물 인식 조절 인자가 목록으로 제시되어 있습니다. 세포 기관 특이적 LC3/GABARAP 결합 단백질과 LC3/GABARAP 결합 어댑터 단백질은 보존된 W/F/YxxL/I/V 모티프를 통해 LC3/GABARAP와 상호 작용합니다. 다양한 유형의 선택적 자가포식 및 생리학 및 질병에서 가능한 역할에 대한 정보는 표 2를 참조하십시오.

Table 2.

Types of Selective Autophagy and Possible Roles in Physiology and Disease

Process (Cargo)Physiological FunctionPossible Pathological Consequences of DefectsReferences

Proteins	Proteostasis	Aberrant signaling/cellular functions related to effects of increased protein (e.g. p62/SQSTMl inflammatory and pro-tumorigenic signaling; autoinflammatory disorders with defects in TRIM-mediated autophagy of inflammasome components; abnormal iron accumulation and ferroptosis in tissues with defects in ferritin degradation)	(Moscat et al., 2016) (Kimura et al., 2015) (Liu et al., 2016) (Latunde-Dada, 2017)
Aggrephagy (Protein aggregates)	Removal of misfolded aggregate-prone proteins labeled by ubiquitin	Enhanced accumulation and detrimental consequences of pathogenic proteins targeted by this mechanism (e.g. β-amyloid, mutant huntingtin, a-synuclein, mutant al-antitrypsin)	(Gatica et al., 2018) (Ueno and Komatsu, 2017)
Mitophagy (Mitochondria)	Mitochondrial quality control and homeostasis. Removal of damaged mitochondria, paternal mitochondria during embryogenesis, and mitochondria during erythrocyte differentiation Piecemeal degradation for respiratory chain turnover	Defective mitophagy may contribute to neurodegenerative diseases, aging, cancer, increased ROS-dependent inflammasome activation and genotoxic stress	(Rojansky et al., 2016) (Drake et al., 2017)
ER-phagy (Endoplasmic reticulum)	Control of ER morphology, turnover, ER luminal proteostasis and recovery from stress	Pathological consequences of defects not defined, but hypothetical role in pathologies associated with abnormal UPR and ER intraluminal proteostasis, including pancreatitis and certain metabolic disorders and aggregopathies. Mutation in ER LC3/GABARAP-binding protein, FAM134B leads to hereditary neuropathy in patients; loss of ER LC3-binding protein CCPG1 leads to injury of exocrine pancreas in mice. Defective ER-phagy in mice (due to AT-1 overexpression‎) leads to segmental progeria with multiple metabolic and inflammatory phenotypes	(Khaminets et al., 2015) (Grumati et al., 2017) (Smith et al., 2018) (Peng et al., 2018)
Ribophagy (Ribosomes)	Required for survival during nutrient starvation, providing source of nucleosides to cell	Not yet known if defects in pathway occur in vivo; if so, would be predicted to disrupt adaptive responses to starvation	(Wyant et al., 2018)
Lysophagy (Lysosomes)	Prevents cell destruction and inflammation due to leakage of lyso-somal contents when lysosomal membranes are damaged or ruptured	Defects predicted to be associated with increased cytosol invasion of pathogens, increased lysosomal cell death and inflammation, as well as disruption of lysosomal homeostasis (latter postulated to participate in neurodegeneration); lysosomal damage in autophagy-deficient mice results in acute kidney injury	(Maejima et al., 2013) (Yoshida et al., 2017) (Chauhan et al., 2016a)
Nucleophagy (Entire Nucleus)	Nuclear destruction necessary for terminal differentiation of keratinocytes; unknown if required for nuclear removal in red blood cells and lens fiber cells	Perturbations may occur in psoriasis, causing parakeratosis (retention of nuclei in stratum comeum of epidermis)	(Akinduro et al., 2016)
Nuclear lamina	Promotes Ras-oncogene-induced senescence	Autophagic degradation of lamin B proposed to be a mechanism of tumor suppression; defects may promote oncogenesis and phenotypes associated with decreased cellular senescence.	(Dou et al., 2015)
Micronuclei	Removal of micronuclei generated by mitotic aberration (and cytosolic DNA aggregates that resemble micronuclei)	Defects may contribute to genomic instability associated with autophagy deficiency and pro-inflammatory signaling via cGAS activation; neuroinflammatory autoimmune disorder Aicardi-Goutieres syndrome caused by mutation of DNA repair enzyme RNase H2 resulting in accumulation of micronuclei-like cytosolic DNA aggregates	(Rello-Varona et al., 2012) (Bartsch et al., 2017)
Retrotransposon RNA	Degradation of RNA granules containing retrotransposons may favor genomic stability	Deficiency results in increased retrotransposon insertions into the genome	(Guo et al., 2014)
Midbody Rings	Degradation of the midbody, an organelle that contains the remnants of cell division machinery, may regulate cellular fate	Deficiency predicted to alter cellular fate; several mutations affecting midbody proteins cause primary encephalopathies, which is also observed with genetically inherited syndromes associated with defects in autophagic flux	(Kuo et al., 2011) (Mandell et al., 2016)
Pexophagy (Peroxisomes)	Perixosomal quality control	Defects may contribute to neurodevelopmental disorders associated with mutations in genes involved in pexophagy, inflammation, aging and age-related diseases, diabetes, cancer and neurodegenerative disorders	(Cipolla and Lodhi, 2017)
Lipophagy (Lipid Droplets)	Facilitates transport of lipid droplets to lysosomes for catabolism by lysosomal acid lipase; contributes to lipid homeostasis	Defects are postulated to contribute to pathogenesis of metabolic syndrome, non-alcoholic fatty liver disease and alcoholic fatty liver disease; however, role of defects in lipophagy versus general autophagy pathway remain to be elucidated	(Zechner et al., 2017) (Zhang et al., 2018b)
Xenophagy (Intracellular pathogens)	Removal of cytoplasmic bacteria or viruses functions in cell-intrinsic immunity	Mutations in genes required for selective autophagy of pathogens result in enhanced microbial virulence in mouse models of tuberculosis and viral infections.	(Mitchell and Isberg, 2017) (Sumpter et al., 2016) (Franco et al., 2017)

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Major advances have been made in understanding certain aspects of selective autophagy, particularly how cargo binds to the forming phagophore (Figure 2). In most known instances, the cargo either contains an identifiable LC3-interacting region or LIR motif (W/F/Y1×2×3L/I/V4) that directly binds LC3, or it must be labeled with a tag such as ubiquitin, which then binds adaptor proteins that contain both a ubiquitin-binding domain and a LIR motif, thus serving as a bridge between the cargo and the LC3 (or GABARAPs) conjugated to the phagophore membrane. Alternatively, specific proteins (particularly those involved in the inflammasome or IFN signaling) can bind to TRIM (tripartite motif) family members, which serve as adaptors that interact with GABARAPs to target such proteins for autophagic degradation (Kimura et al., 2017). Of note, the proteins we refer to as “adaptors” are often called autophagy “receptors”; however, as these are bridging molecules that are not integral parts of cellular membranes that undergo ligand-dependent activation, the designation as “receptors” can be confusing.

선택적 자가포식의 특정 측면,

특히 화물이 형성되는 파고포어에 어떻게 결합하는지에 대한 이해가 크게 진전되었습니다 (그림 2).

대부분의 알려진 사례에서,

화물은 식별 가능한 LC3 상호작용 영역 또는 LIR 모티프 (W/F/Y1×2×3L/I/V4)를 포함하고 있어

LC3에 직접 결합하거나, 유비퀴틴과 같은 태그로 표시되어야 합니다.

이 태그는 유비퀴틴 결합 도메인과 LIR 모티프를 모두 포함하는 적응 단백질에 결합하여

화물과 파고포어 막에 결합된 LC3(또는 GABARAPs) 사이의 다리 역할을 합니다.

대안으로,

특정 단백질(특히 염증체나 IFN 신호전달에 관여하는 단백질)은

TRIM(삼중 모티프) 가족 구성원과 결합하여

GABARAPs와 상호작용하여

이러한 단백질을 자포작용 분해 대상으로 표적화하는 적응체 역할을 합니다(Kimura et al., 2017).

주목할 점은, 우리가 “어댑터”라고 부르는 단백질은 종종 자가포식 “수용체”라고 불리지만,

이들은 리간드 의존적 활성화를 거치는 세포막의 필수적인 부분이 아닌 연결 분자이기 때문에

“수용체”로 지칭하는 것은 혼동을 야기할 수 있습니다.

Several layers of control are needed to properly dictate the targeting of cargo for autophagy. In theory, cargo should be disposed of when it is constitutively harmful (e.g. intracellular pathogens), potentially dangerous to the cell (e.g. mitochondrial damage) or obsolete as a result of cellular differentiation (e.g. organelles during erythrocyte maturation). In the scenario where LC3 directly binds to a protein on an organelle containing a LIR motif, there must exist ways to hide or expose the LIR motif in a regulated-fashion. Two mechanisms identified thus far include (1) stimulatory and inhibitory phosphorylations of residues near or in the LIR motif (such as occurs for the mitochondrial outer membrane protein, FUNDC1, that mediates hypoxia-stimulated mitophagy (Lv et al., 2017)) and (2) the exposure of a normally hidden LIR motif (such as occurs when proteasomal-dependent rupture of the outer mitochondrial membrane exposes the inner mitochondrial membrane LC3-binding protein, prohibitin 2 [Wei et al., 2016]). Under circumstances where LC3 binds to an adaptor protein, there must exist ways to recruit the adaptor to the cargo destined for degradation. This process generally involves the concerted action of E3 ligases that ubiquitinate targets (e.g. Parkin, SMURF1), kinases that recruit E3 ligases (e.g. PINK1) or that phosphorylate LIR domains of adaptors (e.g. TBK1), deubiquitinating enzymes (DUBs) that counter E3 ligase activity (e.g. USP30, USP15) (Gatica et al., 2018), and acetylation/deacetylation of mitochondrial and ER target proteins (Peng et al., 2018; Webster et al., 2013).

자가포식 대상 물질을 적절하게 지정하기 위해서는

여러 단계의 제어가 필요합니다.

이론적으로 화물은 지속적으로 유해한 경우(예: 세포 내 병원체),

세포에 위험을 초래할 수 있는 경우(예: 미토콘드리아 손상),

는 세포 분화 과정에서 더 이상 필요하지 않은 경우(예: 적혈구 성숙 시 소기관)에 제거되어야 합니다.

LC3가 LIR 모티프를 포함하는 소기관에 있는 단백질과 직접 결합하는 경우,

LIR 모티프를 조절된 방식으로 숨기거나 노출시키는 메커니즘이 존재해야 합니다.

현재까지 확인된 두 가지 메커니즘은

(1) LIR 모티프 근처 또는 내부의 잔기들에 대한 자극적 및 억제적 인산화

(예: 저산소 자극성 미토파지를 매개하는 미토콘드리아 외막 단백질 FUNDC1에서 발생하는 경우 [Lv et al., 2017])

(2) 일반적으로 숨겨진 LIR 모티프의 노출

(예: 프로테아좀 의존적 미토콘드리아 외막 파열로 인해 내막에 위치한 LC3 결합 단백질인 프로히비틴 2가 노출되는 경우 [Wei et al., 2016]).

LC3가 적응 단백질에 결합된 상태에서,

적응 단백질을 분해 대상 화물에 모집하는 메커니즘이 존재해야 합니다.

이 과정은 일반적으로

E3 리가제가 표적을 유비퀴틴화하는 과정(예: Parkin, SMURF1),

E3 리가제를 모집하는 키나제(예: PINK1) 또는

적응 단백질의 LIR 도메인을 인산화하는 키나제(예: TBK1),

E3 리가제의 활성을 억제하는 디유비퀴틴화 효소(DUBs, 예: USP30, USP15) (Gatica et al., 2018),

그리고 미토콘드리아 및 ER 표적 단백질의 아세틸화/데아세틸화 (Peng et al., 2018; Webster et al., 2013).

All mechanisms for earmarking cargo must be tightly coordinated with the formation of autophagosomes to ensure final cargo disposal. Some potential nodes of coordination have recently been described. Certain autophagy adaptors, most notably the TRIM family proteins, bind not only substrate proteins and LC3/GABARAP family members but also assemble the ULK1 and Class III PI3K complexes to initiate autophagosome formation (Kimura et al., 2017). ULK1, a “master kinase” that phosphorylates multiple sites on downstream core autophagy proteins, is recruited to and phosphorylates proteins involved in selective autophagy, such as the LIR domain of the mitochondrial membrane protein, FUNDC1 (Wu et al., 2014). An organelle-specific LC3/GABARAP-binding protein, the ER membrane protein, CCPG1, interacts with a key component of the autophagy-initiating ULK1 complex, FIP200 (Smith et al., 2018). Thus, the machinery involved in selective autophagy substrate recognition may play an active role in autophagy initiation.

For selective autophagy targeting events that involve substrate ubiquitination, the precise mechanisms that dictate the choice between autophagic versus proteasomal degradation are uncertain. In yeast, substrate aggregation and oligmerization of the ubiquitin-binding proteins may favor autophagic degradation (Lu et al., 2017). The lysine residues used for linkage and the length and nature of the ubiquitin chains have also been proposed to contribute to pathway selection (Gatica et al., 2018), but definitive evidence is lacking. Moreover, despite elegant studies characterizing the ubiquitylome of selective autophagy cargo (such as mitochondria and intracellular pathogens) (Grumati and Dikic, 2018), the ubiquitin substrates required for autophagic targeting remain largely undefined.

화물을 지정하는 모든 메커니즘은

최종 화물 처리를 보장하기 위해

오토파고솜의 형성과 긴밀하게 협응되어야 합니다.

최근 몇 가지 잠재적인 협응력이 설명되었습니다.

특정 오토파지 어댑터,

특히 TRIM 가족 단백질은 기질 단백질 및 LC3/GABARAP 가족 구성원뿐만 아니라

ULK1 및 Class III PI3K 복합체를 조립하여 오토파고솜 형성을 시작합니다 (Kimura et al., 2017).

하류 핵심 자가포식 단백질의 여러 부위를 인산화하는 “마스터 키나아제”인 ULK1은 미토콘드리아 막 단백질 FUNDC1의 LIR 도메인과 같은 선택적 자가포식에 관여하는 단백질에 모집되어 인산화합니다 (Wu et al., 2014). 기관 특이적인 LC3/GABARAP 결합 단백질인 ER 막 단백질 CCPG1은 오토파지 개시 ULK1 복합체의 핵심 구성 요소인 FIP200과 상호 작용합니다 (Smith et al., 2018). 따라서 선택적 오토파지 기질 인식에 관여하는 기전은 오토파지 개시에 적극적인 역할을 할 수 있습니다.

기질 유비퀴티네이션과 관련된 선택적 자가포식 표적화 사건의 경우, 자가포식 분해와 프로테아좀 분해 중 어느 것을 선택할지 결정하는 정확한 메커니즘은 아직 확실하지 않습니다. 효모에서 기질 응집과 유비퀴틴 결합 단백질의 올리고머화는 자가포식 분해를 촉진할 수 있습니다 (Lu et al., 2017). 유비퀴틴 사슬의 연결에 사용되는 라이신 잔기, 사슬의 길이 및 성질도 경로 선택에 기여할 수 있다는 제안이 있습니다(Gatica et al., 2018), 하지만 결정적인 증거는 부족합니다.

또한, 선택적 자식 세포 사멸의 화물(미토콘드리아 및 세포 내 병원체 등)의 유비퀴티로ーム을 특성화한 정교한 연구(Grumati 및 Dikic, 2018)에도 불구하고, 자식 세포 사멸을 표적으로 하는 유비퀴틴 기질은 대부분 아직 밝혀지지 않았습니다.

Selective autophagy seems to involve multiple concurrent targeting mechanisms that act in a cooperative, potentially hierarchical and/or partially redundant manner to ensure proper removal of cargo. This “combinatorial design” may allow specific cell types to more precisely regulate when and how selective autophagy occurs for a given cargo. The partial redundancy also renders it more feasible to study loss-of-function phenotypes of genes required for selective autophagy but dispensable for bulk autophagy (as compared to those required for bulk autophagy), as they are less likely to be lethal to the cell or organism. As selective autophagy genes are partially redundant, this may explain why their loss-of-function mutation seems to be better tolerated in the human population than loss-of-function mutation of core ATG genes.

Indeed, mutations in many of the known molecules involved in selective autophagy are associated with susceptibility to a variety of human diseases (Table 1). Mutations in the genes encoding the adaptor proteins p62/SQSTM and optineurin, the E3 ligase Parkin, and the kinases PINK1 and TBK1, are among the most common causes of familial and early onset neurodegenerative diseases, including Parkinson’s disease, frontotemporal dementia and amyotrophic lateral sclerosis. Hereditary sensory and autonomic neuropathy type II is caused by mutations in an ER-specific LC3 binding protein, FAM134B, required for ER-phagy. Mutations in TRIM20 (also known as pyrin) that impair its ability to target inflammasome components for autophagic degradation result in an inherited autoinflammatory disorder, familial Mediterranean fever. Inflammatory bowel disease-associated genes encode proteins that function in multiple steps of autophagy, including the selective targeting of bacteria by the adaptor, CALCOCO2/NDP52, and the E3 ligase, SMURF1.

선택적 자식 세포 사멸은 화물의 적절한 제거를 보장하기 위해 협력적이고, 잠재적으로 계층적 및/또는 부분적으로 중복된 방식으로 작용하는 여러 가지 동시 표적화 메커니즘을 포함하는 것으로 보입니다.

이러한 “조합 설계”를 통해 특정 세포 유형은 주어진 화물에 대해 선택적 자가포식이 언제, 어떻게 일어나는지 더 정확하게 조절할 수 있습니다. 부분적 중복성은 또한 선택적 자가포식에 필요하지만 대량 자가포식에는 필요하지 않은 유전자의 기능 상실 표현형을 연구하기 더 용이하게 합니다 (대량 자가포식에 필요한 유전자와 비교).

선택적 자가포식 유전자는 부분적으로 중복되어 있기 때문에,

선택적 자가포식 유전자의 기능 상실 돌연변이가

핵심 ATG 유전자의 기능 상실 돌연변이보다 인간 집단에서 더 잘 견디는 것으로 보이는 이유를

설명할 수 있습니다.

실제로, 선택적 자가포식에 관여하는 많은 알려진 분자의 돌연변이는 다양한 인간 질병에 대한 감수성과 관련이 있습니다 (표 1). 어댑터 단백질 p62/SQSTM 및 옵티네우린, E3 리가제 파킨, 키나아제 PINK1 및 TBK1을 암호화하는 유전자의 돌연변이는 파킨슨병, 전두측엽 치매 및 근위축성 측삭 경화증 등 가족성 및 조기 발병 신경 퇴행성 질환의 가장 흔한 원인 중 하나입니다. 유전성 감각 및 자율 신경병증 유형 II는 ER 특이적 LC3 결합 단백질인 FAM134B의 돌연변이로 인해 발생하며, 이는 ER-phagy에 필수적입니다. TRIM20(피린으로도 알려져 있음)의 돌연변이는 염증체 구성 요소를 자식작용 분해 대상으로 표적화하는 능력을 손상시켜 유전성 자가염증 질환인 가족성 지중해 열을 유발합니다. 염증성 장 질환 관련 유전자는 어댑터인 CALCOCO2/NDP52 및 E3 리가제인 SMURF1에 의한 박테리아의 선택적 표적화 등 자가포식의 여러 단계에서 기능하는 단백질을 암호화합니다.

The numerous links between mutations in selective autophagy genes and human diseases underscore the likely physiological importance of different forms of selective autophagy (Table 2). However, the precise mechanisms that connect genotype to phenotype remain largely undefined. For example, it is not known why mutations in Parkin and PINK1 are associated with Parkinson’s disease, whereas mutations in optineurin, TBK1, and p62/SQSTM1 are associated with amyotrophic lateral sclerosis and frontotemporal dementia (Table 1). In addition to potential cell non-autonomous effects of mutations in these genes in tissues outside of the brain, cell type-specific differences in various populations of neurons and glia may exist with respect to (1) dependency on subsets of selective autophagy genes; (2) expression‎ and activity of DUBs and other negative feedback mechanisms that regulate selective autophagy; and/or (3) levels and types of stress that mandate different types of selective autophagy responses to maintain homeostasis (e.g. mitophagy or other forms of selective autophagy such as aggrephagy that are relevant to neurodegenerative diseases). Parkin knockout mice (unlike flies lacking Parkin) do not develop spontaneous neurodegeneration, but they do develop dopaminergic neuronal degeneration (resembling that observed in human Parkinson’s disease) when crossed with “mutator” mice with a proof-reading-defective mitochondrial DNA polymerase (PolG) that accumulate mitochondrial mutations (Pickrell et al., 2015). The localization of disease in dopaminergic neurons may be related to increased mitochondrial stress in these cells as compared to other neuronal populations in the brain. Intriguingly, the motor defect and neurodegeneration in Parkin-null/mutator mice can be rescued by deletion of STING, a regulator of Type I IFN responses to cytosolic DNA (Sliter et al. 2018). Thus, aberrant inflammatory signaling as a result of defects in mitophagy may contribute to the pathogenesis of neurodegenerative disease in patients with Parkin or PINK1 mutations.

선택적 자가포식 유전자의 돌연변이와 인간 질병 사이의 수많은 연관성은

다양한 형태의 선택적 자가포식의 생리적 중요성을 강조합니다 (표 2).

그러나 유전자형과 표현형 사이의 정확한 메커니즘은 여전히 대부분 미해결 상태입니다. 예를 들어, Parkin과 PINK1의 변이가 파킨슨병과 연관되는 이유, 반면 optineurin, TBK1, 및 p62/SQSTM1의 변이가 근위축성 측삭경화증과 전두측두엽 치매와 연관되는 이유는 알려져 있지 않습니다(표 1). 뇌 외 조직에서 이러한 유전자의 돌연변이가 세포에 미치는 잠재적인 비자립적 효과 외에도, 다양한 신경 세포 및 신경교 세포 집단에 따라 다음 사항에 대해 세포 유형별 차이가 존재할 수 있습니다. (1) 선택적 자가포식 유전자의 하위 집합에 대한 의존성 (2) 선택적 자가포식을 조절하는 DUB 및 기타 부정적 피드백 메커니즘의 발현 및 활동, 그리고/또는 (3) 항상성을 유지하기 위해 다양한 유형의 선택적 자가포식 반응을 요구하는 스트레스의 수준 및 유형(예: 미토파지 또는 신경 퇴행성 질환과 관련된 아그레파지 등 기타 형태의 선택적 자가포식)과 관련이 있을 수 있습니다. Parkin 결손 마우스(파킨이 결손된 파리와 달리)는 자발적인 신경퇴행을 보이지 않지만, 미토콘드리아 DNA 폴리메라제(PolG)의 교정 결함이 있는 “mutator” 마우스와 교배될 경우 인간 파킨슨병에서 관찰되는 것과 유사한 도파민 신경세포 퇴행을 나타냅니다(Pickrell et al., 2015). 도파민 신경세포에서의 질병 국소화는 뇌 내 다른 신경세포 집단에 비해 이 세포에서 증가된 미토콘드리아 스트레스와 관련될 수 있습니다. 흥미롭게도, 파킨 결핍/뮤테이터 마우스의 운동 장애와 신경퇴화는 세포질 DNA에 대한 Type I IFN 반응의 조절자인 STING을 삭제함으로써 회복됩니다(Sliter et al. 2018). 따라서 미토파지 결함으로 인한 비정상적인 염증 신호 전달이 파킨 또는 PINK1 돌연변이 환자의 신경 퇴행성 질환의 발병에 기여할 수 있습니다.

While most, if not all, forms of selective autophagy are likely to contribute to normal physiology and protection against disease, mitophagy has been the most extensively studied. Mitophagy is an essential component of mammalian developmental and differentiation processes, including elimination of paternal mitochondria from the fertilized egg (Rojansky et al., 2016), removal of mitochondria during red blood cell maturation (Sandoval et al., 2008) and beige-to-white adipocyte differentiation (Lu et al., 2018). In addition to Parkinson’s and other neurodegenerative diseases, defective mitophagy is thought to contribute to organ-specific and systemic inflammatory diseases (Zhao et al., 2018), cancer development and/or progression (Drake et al., 2017), and potentially aging (Lopez-Otin et al., 2016). The removal of damaged mitochondria by mitophagy maintains normal cellular metabolism, reduces mitochondrial generation of ROS that trigger inflammation and genotoxic stress, and prevents mitochondrial release of pro-apoptotic factors. Thus, maintenance of proper mitochondrial function by mitophagy is crucial for cellular and organismal health. Other forms of selective autophagy (including xenophagy) likely operate in a manner analogous manner to mitophagy, in that the mechanisms by which they regulate physiology and disease are a function of the normal “duties” of their substrate and the ensuing pathological consequences of abnormal substrate accumulation (see Table 2).

모든 형태의 선택적 자가포식이

정상적인 생리 기능과 질병 예방에 기여할 가능성이 높지만,

미토파지가 가장 광범위하게 연구되어 왔습니다.

미토파지는

포유류의 발달 및 분화 과정의 필수 구성 요소로,

수정된 난자에서 부계 미토콘드리아 제거(Rojansky et al., 2016),

적혈구 성숙 시 미토콘드리아 제거(Sandoval et al., 2008),

베이지-백색 지방세포 분화(Lu et al., 2018) 등에 관여합니다.

파킨슨병 및 기타 신경퇴행성 질환 외에도,

미토파지의 결함은 장기 특이적 및 전신성 염증성 질환(Zhao et al., 2018),

암의 발생 및/또는 진행(Drake et al., 2017), 그리고

잠재적으로 노화(Lopez-Otin et al., 2016)에 기여할 것으로 추정됩니다.

미토파지가

손상된 미토콘드리아를 제거함으로써

정상적인 세포 대사 유지, 염증과 유전독성 스트레스를 유발하는

미토콘드리아 유래 활성산소(ROS) 생성 감소,

세포 사멸을 촉진하는 인자 방출 방지 등이 이루어집니다.

따라서

미토파지를 통한 적절한 미토콘드리아 기능 유지가

세포 및 유기체 건강에 필수적입니다.

다른 형태의 선택적 자가포식 (이종포식 포함)은

미토파지와 유사한 방식으로 작동할 가능성이 높습니다.

즉, 생리학 및 질병을 조절하는 메커니즘이 기질의 정상적인 “역할”과

비정상적인 기질 축적에 따른 병리학적 결과의 기능이라는 점에서 유사합니다 (표 2 참조).

Our expanding knowledge of the mechanisms and physiological functions of selective autophagy may open up new — albeit unchartered — pathways for drug discovery. Knockdown of the mitochondrial deubiquitinase, USP30, rescues mitophagy defects and disease in flies with pathogenic mutations in Parkin (Bingol et al., 2014), suggesting a potential role for the inhibition of DUBs that target selective autophagy E3 ligases in the treatment of Parkinson’s and other diseases. Indeed, novel highly selective inhibitors of USP30 that accelerate mitophagy have recently been reported (Kluge et al., 2018). As phosphorylation of substrates is also a common mechanism involved in selective autophagic targeting, it may be possible to activate specific kinases to enhance selective autophagy. Potentially, it may also be possible to develop novel strategies to attach high-affinity LIR domains selectively to harmful cargo so that they can be more efficiently captured by an LC3-decorated nascent autophagosome.

선택적 자가포식의 메커니즘과 생리학적 기능에 대한 지식이 확대되면,

아직 알려지지 않은 새로운 약물 발견의 길이 열릴 수도 있습니다.

미토콘드리아 디유비퀴티나아제 USP30을 노크다운하면 파킨에 병원성 돌연변이가 있는 파리에서 미토파지 결함과 질병이 회복됩니다 (Bingol et al., 2014).

이는 선택적 자가포식 E3 리가제를 표적으로 하는 DUB의 억제가 파킨슨병 및 기타 질병의 치료에 잠재적인 역할을 할 수 있음을 시사합니다.

실제로,

미토파기를 촉진하는 USP30의 새로운 고선택성 억제제가

최근 보고되었습니다 (Kluge et al., 2018).

기질의 인산화는 선택적 자가포식 표적화에 관여하는 일반적인 메커니즘이기도 하기 때문에,

특정 키나아제를 활성화하여 선택적 자가포식을 강화할 수도 있습니다.

잠재적으로,

유해한 화물에 선택적으로 고친화성 LIR 도메인을 부착하여

LC3로 장식된 초기 자식 세포에 의해 더 효율적으로 포획될 수 있는

새로운 전략을 개발할 수도 있을 것입니다.

Autophagy Regulation: A Nexus for Therapeutics?

Autophagy was originally studied in yeast and mammalian cells as a nutrient stress response pathway. During the past decade, we have dramatically expanded our knowledge of autophagy regulation, particularly the spectrum of physiological and pathophysiological stimuli that control autophagy, the mechanisms that regulate the activity of the core autophagy proteins (Grumati and Dikic, 2018), and the interconnectivity of autophagy with other cellular stress response pathways (Kroemer et al., 2010). These concepts have been reviewed elsewhere; here, we highlight selected aspects relevant to physiology and disease.

Post-translational protein modifications such as phosphorylation, ubiquitination, and acetylation play a central role in coordinating the activity of ATG proteins. In most cases, the upstream kinases/phosphatases, ubiquitin ligases/DUBs, and acetyltransferases simultaneously modify both ATG proteins and proteins involved in other cellular stress-response pathways that are co-regulated with autophagy. As a result, pharmacological targeting of these enzymes will elicit broad-based modulation of multiple intertwined stress-response pathways. Depending on the enzyme and its substrates, such nonspecific targeting may be harmful in some instances, and useful in others.

One important example is the stimulation of AMPK, a low energy-sensing kinase activated by ATP depletion, which phosphorylates multiple proteins to both stimulate catabolic pathways (including autophagy) and restrain anabolic pathways (including mTORC1 signaling), thereby ensuring limitation of ATP consumption and generation of new ATP via breakdown of metabolic products (Herzig and Shaw, 2018). In recent years, AMPK has been shown to not only activate autophagy through inhibition of mTORC1, but also directly phosphorylate several ATG proteins, including ULK1, ATG9A, Beclin 1, and VPS34 (Egan et al., 2011; Kim et al., 2013a). In addition, AMPK promotes mitophagy through effects on ULK1 and stimulates TFEB-dependent activation of the CLEAR (Coordinated Lysosomal Expression‎ and Regulation) network of genes required for autophagy (Herzig and Shaw, 2018). This pro-autophagic activity of AMPK occurs concurrently with its effects on mitochondrial homeostasis and on lipid and glucose metabolism.

AMPK activation may underlie the beneficial effects of metformin, a drug widely prescribed for the treatment of diabetes (Herzig and Shaw, 2018). Metformin activates AMPK indirectly through mitochondrial depletion of ATP, and direct AMPK activators that yield effects similar to metformin are in pre-clinical development. The extent to which autophagy stimulation contributes to beneficial effects of AMPK activation in mice or patients is not known, but it seems likely that autophagy represents a critical part of an AMPK-activated hub that protects against various metabolic diseases, including diabetes, obesity, and non-alcoholic fatty liver disorders, as well as certain cancers and aging-related phenotypes. In Drosophila, deficiency of the Beclin 1 orthologue (ATG6) impairs the ability of metformin to prevent intestinal stem cell aging (Na et al., 2018), and lifespan extension by neuronal AMPK expression‎ requires the fly ULK1 orthologue, ATG1 (Ulgherait et al., 2014). In mice, AMPK upregulation of autophagy is correlated with improved function of aging muscle stem cells (White et al., 2018); additionally, muscle-specific AMPK deficiency results in defective autophagy, fasting-induced hypoglycemia, and aging-associated myopathy (Bujak et al., 2015). In yeast, core ATG genes are required for AMPK-mediated lipid droplet degradation and survival during acute glucose deprivation (Seo et al., 2017).

The lysine acetylation/deacetylation of ATG proteins has emerged as a central node of autophagic control regulated by metabolic sensors involved in lipid, glucose and protein metabolism. Moreover, this control center may function independently from, but intertwined with, AMPK and mTORC1 (Marino et al., 2014b; Su et al., 2017). During acute nutrient depletion, cells undergo a rapid decrease in levels of cytosolic acetyl coenzyme A (AcCoA), which leads to the deacetylation of cellular proteins (Marino et al., 2014b). Sirtuin 1 (which is downstream of AMPK) deacetylates multiple ATGs (e.g. ATG5, ATG7, ATG12, Beclin 1, VPS34, LC3) and thereby promotes autophagy, as does reduced activity of the acetyl transferase EP300 (Madeo et al., 2014; Su et al., 2017). Hence, endogenous activators of sirtuin-1 (e.g. nicotine adenine dinucleotide (NAD+)), endogenous inhibitors of EP300 (e.g. spermidine, a dietary polyamine), and reduced availability of AcCoA (a rate-limiting step for EP300 function) all stimulate autophagy (Madeo et al., 2014).

Compounds that act on these pathways, thereby mimicking the effects of caloric restriction (so-called “caloric restriction mimetics”), are an active area of investigation, and genetic evidence suggests that autophagy is essential for their beneficial effects in vivo. Reservatrol, an indirect sirtuin activator, requires the autophagy machinery for its favorable effects on longevity in nematodes (Morselli et al., 2010). Spermidine-induced autophagy is required for several of its beneficial health effects in model organisms, including lifespan extension in flies, worms, and mice; prevention of cardiac aging in mice; improvement in neuronal function in aging flies; and preservation of myocyte stemness in mice (reviewed in (Madeo et al., 2018)). Moreover, caloric restriction mimetics improve anti-tumor immune surveillance and enhance chemotherapy responses in autophagy-competent, but not autophagy-incompetent, mouse tumor allografts (Pietrocola et al., 2016).

Over the past decade, the lysosome — an organelle traditionally viewed as the downstream “workhorse” for autophagosomal cargo degradation — has been shown to also play a crucial role in the upstream regulation of autophagy (Napolitano and Ballabio, 2016; Shen and Mizushima, 2014). The nutrient-sensing kinase complex, mTORC1, detects both cytosolic and intra-lysosomal amino acids through distinct mechanisms to inhibit autophagy (Saxton and Sabatini, 2017). Amino acids (such as arginine) inside the lysosomal lumen are sensed by the amino acid transporter SLC38A9, which interacts with the lysosomal v-ATPase/Rag/Ragulator complex to activate mTORC1. This both restrains autophagy during baseline conditions and provides feedback inhibition to terminate autophagic responses to acute nutrient depletion. mTORC1 activation in the fed state and/or hyperactivation (as a result of mutations in regulatory signals) switches the cell to a state of anabolic growth and energy storage. Although essential for cell growth and proper metabolic regulation, sustained mTORC1 activation at the organismal level is associated with a variety of pathophysiological consequences, including impaired neonatal gluceoneogenesis and survival (Efeyan et al., 2013); accelerated age-related decline in pancreatic β-cell function (Shigeyama et al., 2008); late-onset muscle atrophy (Castets et al., 2013); altered lipogenesis and adipogenesis (Lee et al., 2016); immune suppression; epileptic seizures and autistic traits; tumorigenesis; and aging (Saxton and Sabatini, 2017). While in some cases, impaired induction of autophagy has been documented in mice with hyperactive mTORC1 signaling and is postulated to contribute to pathological phenotypes (e.g. impaired neonatal gluconeogenesis, late-onset muscle atrophy), the precise role of autophagy inhibition in most diseases associated with mTORC1 signaling remains unknown. There has been some interest in using FDA-approved mTOR inhibitors for the treatment of neurodegenerative disorders that may benefit from autophagy induction (Sarkar, 2013). However, the safety and efficacy of using mTOR inhibitors to induce therapeutic autophagy is uncertain, given the broad range of essential catabolic functions regulated by mTORC1 along with the lack of full specificity of existing agents to target mTORC1 rather than mTORC2 (which functions primarily as an effector of insulin/PI3K signaling).

Both AMPK and mTORC1 participate (in opposite directions) in a signaling axis that links autophagy, the lysosome, and the transcription factor EB (TFEB) and related family members. During the acute response to autophagic stimuli, transcriptional activation is not required, as evidenced by the observation that enucleated cells (cytoplasts) undergo autophagy (Morselli et al., 2011). However, sustained autophagy requires TFEB, a transcription factor that (when inactive) binds to Ragulator at the lysosomal membrane, is phosphorylated by mTORC1, and retained in the cytoplasm by 14–3–3 proteins. Following mTORC1 inhibition, TFEB dephosphorylation releases it from the cytoplasm, allowing its nuclear translocation and subsequent activation of the CLEAR gene network, which includes genes encoding lysosomal hydrolases, lysosomal v-ATPase pumps, lysosomal regulators and autophagy regulators (Puertollano et al., 2018). As noted above, AMPK also activates TFEB-dependent gene expression‎; this occurs through multiple different mechanisms. In addition, a recent study showed that phosphorylation of acetyl-CoA synthetase 2 (ACSS2) promotes its transport into the nucleus, where it binds to TFEB and favors the acetylation of histone H3 residues within the promoters of TFEB target genes (Li et al., 2017b).

In addition to TFEB, other transcription factors from the same family (such as micropthalmia-associated transcription factor [MITF] and TFE3) (Perera et al., 2015) or from other families (e.g. FOXO3A, HSF1 or TP53) stimulate autophagy (Cai et al., 2018; Kenzelmann Broz et al., 2013). Bromodomain 4 (BRD4), a transcription factor that represses autophagy and lysosomal genes, is displaced from chromatin in response to starvation by a signaling cascade involving an AMPK-SIRT1 axis (Sakamaki et al., 2017). Thus, multiple known (and probably yet-to-be-identified) transcription factors regulate the synthesis of genes required for autophagy (including both the formation of the autophagosome and degradation of its contents by lysosomes). Not surprisingly, the activity of these transcription factors is tightly regulated by numerous signaling factors that also regulate core ATG protein function by post-translational modifications.

Modulation of the activity of TFEB, a master regulator of both lysosomal biogenesis and autophagy, has emerged as a potential therapeutic strategy. Conceptually, this approach is attractive, since limitations in lysosomal numbers and function either occur intrinsically as part of many rare, but devastating, difficult-to-treat, primary diseases (such as lysosomal storage disorders [LSDs]) or are acquired during the progression of diseases associated with the clearance of toxic aggregates progress (such as Huntington’s, Parkinson’s, Alzheimer’s disease and tauopathies). In mice, TFEB overexpression‎ ameliorates several LSDs, neurodegenerative diseases, and α1-antitrypsin deficiency, and it also promotes lipophagy, thereby reducing obesity and associated metabolic syndrome (Napolitano and Ballabio, 2016). The mechanisms by which TFEB overexpression‎ partially corrects lysosomal malfunction in LSDs are not fully understood, but may involve induction of lysosomal exocytosis for the secretion of undigested material.

One potential obstacle to strategies for enhancing TFEB family activity is the risk of tumorigenesis associated with constitutive activation (e.g. renal clear cell carcinoma with TFEB and pancreatic cancer with MITF, TFE3, and TFEB) (Napolitano and Ballabio, 2016). While MITF/TFE3/TFEB-dependent autophagy-lysosomal activation is thought to sustain metabolic reprogramming in pancreatic cancer cells by maintaining intracellular amino acid pools (Perera et al., 2015), further genetic investigations are warranted to confirm‎ that ATG genes are involved in these effects. Moreover, enhanced activity of BRD4, a transcriptional repressor of autophagy, drives another type of cancer, NUT midline carcinoma (Sakamaki et al., 2017), suggesting the effects of transcriptional regulators of autophagy on tumorigenesis may be cell-type specific. It is unclear whether specific subsets of the TFEB-regulated gene network can be induced to avoid genes that contribute to tumorigenesis without losing beneficial effects on the autophagy-lysosomal pathway. An alternative strategy is to activate TFEB on an intermittent basis and/or for limited periods to avoid potential oncogenic effects.

Intriguingly, one of the most widely used medications in the world – aspirin – has been reported to upregulate TFEB in brain cells (via activation of PPARα), induce lysosomal biogenesis, and decrease amyloid plaque pathology in a mouse model of Alzheimer’s-like disease (Chandra et al., 2018). Aspirin (and its active metabolite salicylate) also induces autophagy via inhibition of the acetyltransferase EP300 (Pietrocola et al., 2018) and via AMPK activation/mTORC1 inactivation (Din et al., 2012). However, there is as-of-yet no direct genetic evidence that autophagy contributes to the health benefits of aspirin.

Highly specific activation of autophagy may be possible through strategies that enhance the activity of the upstream components in the core autophagy pathway, i.e. the ULK1 serine/threonine kinase complex and/or Beclin 1/VPS34 lipid kinase complexes. As a key allosteric regulator of VPS34 lipid kinase activity, Beclin 1 activity is tightly regulated by multiple post-translational modifications (ubiquitination, acetylation, phosphorylation) which govern its stability, heterodimeric binding to ATG14 or UVRAG, homodimerization in an inactive form, and/or binding to negative regulators, such as Bcl-2/Bcl-xL (Grumati and Dikic, 2018; Levine et al., 2015). Diverse stress kinases, including AMPK and MAPKAPK2/3, as well as the upstream ATG protein, ULK1, mediate stimulatory phosphorylations of Beclin 1 (Kim et al., 2013a; Park et al., 2018; Wei et al., 2015). The oncogenic kinases, Akt and EGFR, and the EP300 acetylase inhibit the autophagic activity of Beclin 1; mutation of their target post-translational sites in Beclin 1 demonstrates that suppression of Beclin 1-dependent autophagy promotes tumor growth in mouse xenograft models (Sun et al., 2015; Wang et al., 2012; Wei et al., 2013). Enhanced proteasome-mediated degradation of Beclin 1 due to decreased binding of the deubiquitinase, ataxin 3, may contribute to dysregulated autophagy in cells of patients with polyglutamine expansion protein-related diseases, such as Huntington’s and spinocerebellar ataxia type 3 (Ashkenazi et al., 2017).

Disruption of Bcl-2 binding to Beclin 1 represents a central mechanism by which autophagy is activated in response to stress stimuli (such as starvation, exercise, and immune signaling) (He et al., 2012; Wei et al., 2008). This disruption can be triggered by phosphorylation of the BH3 domain of Beclin 1 by DAPK, ubiquitination of the Beclin 1 BH3 domain by the E3 ligase TRAF6, phosphorylation of Bcl-2 by JNK1, or competition by BH3-only proteins (reviewed in [Levine et al., 2015]). Mice containing knock-in non-phosphorylatable mutations in Bcl-2 that prevent disruption of its binding to Beclin 1 are deficient in starvation and exercise-induced autophagy, have decreased exercise endurance, and fail to manifest the beneficial effects of exercise on glucose metabolism (He et al., 2012). Conversely, mice with a knock-in mutation in Beclin 1 that decreases Bcl-2 binding exhibit increased autophagy and extended lifespan and healthspan, including protection against Alzheimer’s-like disease and HER2-mediated breast cancer (Fernandez et al., 2018; Rocchi et al., 2017; Vega-Rubin-de-Celis et al., 2018). Thus, disruption of Beclin 1/Bcl-2 binding may be a safe and effective approach to induce autophagy in vivo; preclinical studies are in progress to develop agents that act through this mechanism (Chiang et al., 2018).

Cell-penetrating peptides (Tat-Beclin 1) derived from a flexible hinge region of Beclin 1 important for VPS34 membrane association and lipid kinase activity (Rostislavleva et al., 2015) are sufficient to induce autophagy in vitro and in vivo (Shoji-Kawata et al., 2013). In mice, Tat-Beclin 1 protects against West Nile virus, chikungunya virus and E. coli bacterial infections; lipopolysaccharide-induced cardiac dysfunction; pressure overload-induced heart failure; hyperammonemia in liver failure and urea cycle disorders, and bone loss in LSDs and in FGF-deficiency (Bartolomeo et al., 2017; Cinque et al., 2015; Shoji-Kawata et al., 2013; Soria et al., 2018; Sun et al., 2018). It also enhances chemotherapeutic effects of murine cancers in immune competent mice (Pietrocola et al., 2016), reduces the growth of human HER2-positive breast cancer xenografts in immune-deficient mice (Vega-Rubin-de-Celis et al., 2018), and acts synergistically with erastin to increase animal survival in an orthotopic pancreatic cancer model (Song et al., 2018). In rats, intrahippocampal injection of Tat-Beclin 1 improves long-term spatial memory (Hylin et al., 2018). In a zebrafish model of human polycystic kidney disease, Tat-Beclin 1 ameliorates renal cyst formation (Zhu et al., 2017). Further studies are needed to examine whether Tat-Beclin 1 induces these effects through autophagy, autophagy-independent effects of Beclin 1, or alternative mechanisms. Moreover, precise definition of its mechanism of action may lead to the development of novel small drug-like molecules that mimic its activity. Recent structural advances elucidating the atomic details of the Beclin 1/VPS34 complexes (reviewed in [Hurley and Young, 2017]) may provide a basis for rational drug design to selectively activate autophagy-specific Beclin 1-associated VP34 lipid kinase activity.

Autophagy in Tissue and Whole-Body Homeostasis

The health of multicellular organisms requires the coordinated regulation of cellular life and death decisions, cell fate determinations, preservation of genomic integrity, immune responses, and metabolic circuitries. The autophagy machinery, via its diverse functions described above (and yet-to-be-discovered mechanisms) plays a crucial role in these processes. Herein, we highlight some recent advances related to the role of autophagy in cell death, preservation of stem cells, tumor suppression, longevity and defense against metabolic diseases.

Autophagy as a Homeostat

During both routine “housekeeping” and responses to acute stress, cells must find ways to maintain adaptive cytoprotective levels of autophagy while simultaneously avoiding potentially maladaptive levels and/or detrimental effects of autophagy. This balance involves self-control of the levels of autophagy, mechanisms of preventing degradation products from becoming toxic to cells, avoidance of degrading essential cargo, and suppression of unwarranted cell death – which likely is a combined function of the aforementioned processes. Cellular self-titration of levels of autophagy involves multiple different inhibitory feedback loops, including feedback regulation of nutrient sensing signals by the generation of amino acids, acetyl-CoA and respiratory substrates (Galluzzi et al., 2014); cytosolic retention of pro-autophagic transcription factors by ATG7 (Simon et al., 2017); and TFEB-mediated activation of mTORC1 (Simon et al., 2017). Cellular toxicity by degradation products may be avoided during autophagy, as evidenced by the observation that the generation of lipid droplets generated by autophagy-dependent dismantling of lipid membranes during starvation-induced autophagy sequesters fatty acids, thereby protecting mitochondria against lipotoxicity and preserving cellular viability (Nguyen et al., 2017). It is not known whether starvation-induced autophagy preferentially induces removal of certain, perhaps aged, structures (and if so, by what mechanisms) or whether it is non-specific. Mitochondria elongate during starvation, which spares them from the autophagic capture that generally occurs after fission (Gomes et al., 2011). Numerous yet-to-be-discovered mechanisms likely protect mitochondria and other organelles from excessive autophagic capture during stress-induced autophagy.

The aforementioned negative feedback loops restrain autophagy to adaptive (rather than maladaptive) levels, allowing this homeostatic pathway to exert cytoprotective effects during stress, and thereby, prevent apoptotic and necroptotic cell death (Marino et al., 2014a). In addition, ATG gene-dependent processes, such as increased plasma membrane localization of the GLUT1 glucose transporter (Roy et al., 2017), may increase the threshold of damage required to kill cells and thereby promote successful organismal adaptation to stress. Promotion of cell survival in vivo during stress-induced autophagy may depend on concurrent antagonism of the Na+,K+-ATPase pump, which normally consumes a large fraction of the ATP available to the cell (Kheloufi et al., 2015). Interestingly, during acute bouts of exercise or nutrient limitation (potent physiological stimuli of autophagy that generally do not result in cell death), endogenous cardiac glycosides that target Na+,K+-ATPase are upregulated 50–500 fold, resulting in decreased cellular ATP consumption (Schoner, 2002).

However, when organisms are pushed beyond physiological limits of energy deprivation, adaptive mechanisms are insufficient to keep cells alive and to prevent tissue damage. In the liver of patients with anorexia nervosa or in neonatal rodents subjected to severe cerebral ischemic injury, a morphologically and genetically distinct form of cell death occurs called autosis, which requires both ATG genes and Na+,K+-ATPase activity (Kheloufi et al., 2015; Liu et al., 2013b). It is not clear how the cell’s major consumer of ATP (i.e. the Na+,K+-ATPase pump) and the cell’s major mobilizer of ATP-generating substrates during stress conditions (i.e. autophagy) interact to regulate life and death decisions of the cell. However, this interaction may represent a fundamental energy homeostatic mechanism that becomes pathologic during different types of ischemic conditions.

Another recently identified bona fide form of autophagic cell death (i.e. demonstrating a genetic requirement for ATG genes) involves GBA1 — the gene encoding the lysosomal enzyme, glucocerebrosidase (GCase) — which metabolizes glucosylceramide (GlcCer) to ceramide and glucose. GBA1 knockdown blocks autophagic cell death in resveratrol-treated lung cancer cells in vitro and developmental midgut death in Drosphila in vivo (Dasari et al., 2017). In humans, homozygous GBA1 mutations lead to Gaucher’s disease, a lysosomal storage disorder, while heterozygous mutations are the most important risk factor for Parkinson’s disease (Schapira, 2015). Interestingly, GBA1 deficiency in the brains of flies and mice leads to an accumulation of GlcCer, impaired autophagic-lysosomal flux, α-synuclein aggregate accumulation, and neurodegeneration; these features are similar to those observed in patients with Parkinson’s disease (Aflaki et al., 2017).

Thus, levels of cellular GCase are crucial for homeostasis; insufficient GCase activity results in a defect in autophagic-lysosomal function and neurodegeneration, whereas excessive GCase activity results in autophagic cell death. It is not yet known how GCase levels are physiologically titrated or how an excess of GCase activity converts adaptive autophagy into lethal autophagy. One possibility is that death occurs as a result of the generation of a metabolic intermediate, sphingosine, which affects lysosomal membrane permeabilization, as lysosome-membrane permeabilization by lysosomal-targeted Bax facilitates autophagic cell death in Bax/Bax knockout murine embryonic fibroblasts (Karch et al., 2017). Future studies are needed to determine the precise mechanisms that convert autophagy from a pro-survival to a cell death pathway, to specifically delineate the role of autophagic cell death pathways in pathophysiology, and to devise therapeutic strategies to block such death in vivo.

Autophagy in Stemness

Accumulating evidence indicates that autophagy is required for stem cell quality control (especially via mitophagy-mediated reduction in ROS levels), energy homeostasis, metabolic reprogramming, and the preservation of fitness. This requirement has multiple disease-related implications, depending on the type of stem cell (e.g. embryonic, adult, cancer) (see [Boya et al., 2018] for detailed recent review). In general, autophagy functions in adult stem cells, including muscle stem cells (satellite cells), hematopoietic stem cells (HSCs), and neural stem cells (NSCs) as a mechanism to prevent exhaustion and aging and to promote quiescence, allowing either self-renewal or differentiation (as needed). Conditional Atg7 deletion leads to a reduction of the muscle satellite cell pool in young mice and hallmarks of premature muscle aging (senescence and DNA damage), and autophagy activation reverses senescence and restores regenerative functions in satellite cells in aged animals (Garcia-Prat et al., 2016). Loss of Atg12 in HSCs impairs maintenance of HSC quiescence and stemness (Ho et al., 2017). Loss of FIP200 results in a progressive loss of neural stem cells (NSCs) and reduced neurogenesis in the adult brain (Wang et al., 2013). Thus, upregulation of autophagy may help stimulate muscle regeneration in sarcopenia, prevent late-onset diseases associated with immune cell senescence, and/or promote adult neurogenesis.

Several studies suggest a role for autophagy in the reprogramming of somatic cells to generate induced pluripotent stem cells (iPSCs) (Boya et al., 2018). The precise mechanisms by which autophagy functions in pluripotency reprogramming are debated, but may involve degradation of transcription factors, ability to sustain glycolytic metabolism (which favors stemness), degradation of mitochondria and other organelles, and mitophagy-mediated limitation of cellular ROS production. While the deletion of core ATG genes or loss of PINK1-dependent mitophagy impairs the reprogramming process (Boya et al., 2018), it is not known if autophagy upregulation improves the efficiency of pluripotent reprogramming for the optimization of stem-cell based therapies.

Additionally, autophagy is important in the origin, differentiation, and survival of cancer stem cells (CSCs), a unique niche which (similar to other stem cell populations) has the potential for self-renewal, but also has the potential for malignancy, initiation of tumor metastasis, and enhanced chemotherapy resistance (Pattabiraman and Weinberg, 2014). CSC formation is enhanced in regions of tumors that are hypoxic, nutrient-depleted and acidic — conditions that favor enhanced autophagy, and CSCs usually have higher rates of basal autophagy than non-cancer stem cells (Boya et al., 2018). Knockdown of core ATG genes in breast CSCs impairs their self-renewal in vitro and impairs their growth when xenografted into mice (Boya et al., 2018). This observation has raised interest in the therapeutic potential of autophagy inhibition of CSCs. However, it is not clear how such cells could be selectively targeted in vivo, and systemic inhibition of autophagy, even in adult mice, is associated with multiple severe toxicities, including multi-organ degeneration and fatal hypoglycemia during fasting (Karsli-Uzunbas et al., 2014). Moreover, this concept is further complicated by the plasticity of CSCs, i.e. the ability to convert to non-cancer stem cells and vice versa. In addition, the depletion of autophagy in HSCs favors the expansion of acute myeloid progenitor cells and development of frank hematological malignancies (Auberger and Puissant, 2017). Given these complexities, it may be premature to consider targeting autophagy in CSCs as an anti-cancer strategy.

Autophagy in Genomic Stability and Tumor Suppression

Besides eliminating ROS-producing dysfunctional mitochondria that are potentially mutagenic, autophagy may promote genomic stability through several additional mechanisms. In the setting of impaired autophagy, the accumulation of the autophagy adaptor and substrate, p62/SQSTM1 (1) inhibits the E3 ligase RNA168 that is essential for histone and chromatin ubiquitination and DNA damage responses (Wang et al., 2016) and (2) activates NRF2 transcription factor of MDM2, which acts through p53-dependent and –independent mechanisms to abrogate normal cell cycle checkpoints (Todoric et al., 2017). Genetic inhibition of autophagosome formation or lysosomal function results in a failure to degrade the small GTPase, RHOA, which leads to cytokinesis failure, multinucleation, and aneuploidy (Belaid et al., 2013). Selective autophagic removal of micronuclei and endogenous retrotransposons may also promote genomic stability (Table 2). Intriguingly, DNA damage repair pathway genes are involved in the selective autophagy of ROS-generating organelles, including pexophagy (e.g. ATM kinase (Zhang et al., 2015a)) and mitophagy (e.g. Fanconi anemia proteins (Sumpter et al., 2016)). Thus, there may be selective pressure for nuclear DNA damage pathways and autophagy proteins to function at multiple levels to protect the genome – both in the cytoplasmic removal of dysfunctional organelles that threaten genomic integrity and, more directly, in the regulation of nuclear events that maintain genomic stability.

The role of autophagy in promoting genomic stability is consistent with its role in tumor suppression. One of the most frequent genetic alterations in sporadic human breast and ovarian cancer is the allelic loss of beclin 1, which is associated with more aggressive cancers and worse patient survival (independently of allelic loss of the nearby tumor suppressor BRCA1) (Liang et al., 1999; Tang et al., 2015; Valente et al., 2014). Mice lacking a copy of beclin 1 develop spontaneous malignancies, demonstrating that it is a haploinsufficient tumor suppressor gene (Cicchini et al., 2014; Qu et al., 2003; Yue et al., 2003), and allelic loss of beclin 1 in immortalized mouse mammary epithelial cells promotes mammary tumorigenesis, DNA damage, and genomic instability in vivo (Karantza-Wadsworth et al., 2007). Similarly, partial autophagy defects in other mouse models (Ambra1+/−, Atg4c−/−, Sh3glb1−/− and mosaic Atg5−/−) are associated with an increased incidence of spontaneous or chemically-induced tumors (Rybstein et al., 2018). In mice, loss of the mitophagy receptor, BNIP3, accelerates progression to metastatic breast cancer (Chourasia et al., 2015), and loss of Parkin results in spontaneous liver tumors, increased radiation-induced lymphoma, enhanced colorectal adenoma development in Apc mutant mice and accelerated KRas-driven pancreatic tumorigenesis (Drake et al., 2017; Li et al., 2018a; Poulogiannis et al., 2010). In patients, PARKIN inactivating mutations are observed in glioblastomas, colorectal carcinoma and other malignancies (Drake et al.; 2017). Numerous oncogenic mutations suppress autophagy through mTORC1 activation (e.g. activating mutations in Akt/class I PI3K, PTEN loss, LKB1 loss), ULK1 and Parkin inhibition (e.g. cytoplasmic accumulation of TP53 mutant proteins) and/or inhibition of the activity of Beclin 1/Class III PI3K complex (e.g. Akt, EGFR, HER2, Bcl-2 amplification or activation) (Rybstein et al., 2018; Vega-Rubin-de-Celis et al., 2018; Levine et al., 2015).

Autophagy has additional cell-autonomous and non-cell-autonomous functions in tumor suppression. At the cell-autonomous level, together with its promotion of genomic instability, autophagy degrades the nuclear lamina (through an interaction with LC3 and lamin B) to promote oncogene-induced senescence (Dou et al., 2015), and it negatively regulates inflammatory signaling which is a strong oncogenic driver (Zhong et al., 2016). It has been proposed that deregulated inflammatory signaling due to defective autophagy may represent a common pro-oncogenic pathway for several cancer risk factors, including obesity, aging, alcohol abuse, chronic infections, and ATG16L1 deficiency/Crohn’ disease (Zhong et al., 2016).

At the cell non-autonomous level, autophagy acts to suppress tumor-promoting inflammatory signaling and to enhance anti-cancer immunity in myeloid cells in the tumor microenvironment. Autophagy in cancer cells is important for cross-presentation as well as facilitating the release of tumor antigens from dying cells and increasing their extracellular availability (Ma et al., 2013). The phenomenon of autophagy-dependent “immunogenic cell death” also leads to the release of ATP and other danger-associated molecular patterns (Michaud et al., 2011), which enhances anti-tumor CTL responses and contributes to the anti-cancer efficacy of chemotherapy and radiation therapy (Galluzzi et al., 2017b). Interestingly, the incidence of KRAS-induced non-small lung cancer is increased by genetic inhibition of autophagy and reduced by autophagy induction through a mechanism that requires T-cell-dependent antitumor immunity (Pietrocola et al., 2016). Taken together, autophagy acts both in cancer cells and myeloid cells to dampen pro-tumorigenic inflammation and to augment adaptive immunity that curtails cancer growth and progression. It is not yet known whether autophagy induction will act synergistically with immune checkpoint inhibitors to boost anti-cancer therapeutic responses.

In parallel with delineation of mechanisms of autophagy in tumor suppression and the promotion of anti-tumor immunity, numerous reports have demonstrated pro-tumorigenic roles of autophagy, primarily in cancers driven by KRAS that require high cellular metabolic activity to sustain survival (Kimmelman and White, 2017). The pro-tumorigenic effects are generally believed to result from the ability of autophagy to sustain tumor cell survival during metabolic stress in a cell-intrinsic fashion and/or in a cell-extrinsic fashion via the provision of nutrients to malignant cells by autophagy in stromal cells in the tumor microenvironment (Katheder et al., 2017; Kimmelman and White, 2017; Yang et al., 2018). These observations have piqued interest in developing autophagy inhibitors for treating certain cancers, a concept that has been explored in mice using two principal approaches: (1) tissue-specific or whole-body-inducible deletion of ATG genes, and (2) lysosomotropic agents such as a chloroquine and hyxdroxychloroquine that inhibit autophagic flux (along with other pharmacological effects). While both approaches reduce KRAS-driven tumor growth in mice (Kimmelman and White, 2017), ATG gene deletion results in inflammation and/or destruction of the organ (i.e. in pancreatic-specific or lung-specific knockouts) or in multi-system degeneration (in whole-body knockouts) (Guo et al., 2013; Karsli-Uzunbas et al., 2014; Rosenfeldt et al., 2013). Furthermore, an extensive examination of the effects of chloroquine on a panel of KRAS mutant tumors failed to show any ATG gene-dependent growth inhibition (Eng et al., 2016). Thus, despite the crucial role of autophagy in malignant cells and stromal cells in promoting tumor growth, conclusive data are lacking to support autophagy inhibition as a viable therapeutic approach, although a recent study of KRAS-driven pancreatic cancer using only mosaic genetic inhibition of autophagy may suggest efficacy with tolerable toxicity (Yang et al., 2018). This issue is further complicated by the aforementioned multiple roles of autophagy in tumor suppression and anti-tumor immunity, along with emerging evidence that autophagy inhibition may promote tumor metastasis (reviewed in [Dower et al., 2018]).

Autophagy in Metabolic Diseases

Growing evidence implicates functional defects in autophagy in various metabolic disorders, including obesity, diabetes, atherosclerosis, and non-alcoholic fatty liver disease (reviewed in [Ueno and Komatsu, 2017; Zhang et al., 2018a]). While these disorders involve genetic and epigenetic factors, excess caloric intake and decreased physical activity are principal driving forces, both of which suppress autophagy. Although there are divergent reports of whether autophagy is enhanced or suppressed in obesity, the preponderance of mouse genetic data indicate that decreased autophagy facilitates the transition from obesity to diabetes and increases the risk of atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Specifically, mice with whole-body partial mutation or tissue-specific (liver or pancreas) deletion of ATG genes or TFEB gain more weight when fed a high-fat diet and have an increased propensity to develop systemic inflammation, diabetes, and hepatic steatosis (Fernandez et al., 2017; Jung et al., 2008; Lim et al., 2014; Settembre et al., 2013a; Singh et al., 2009). In addition, mice with macrophage-specific deletion of Atg5 are more prone to the development of atherosclerotic plaques (Liao et al., 2012; Razani et al., 2012). In humans, genetic variants of IRGM1, a gene required for assembly and activation of the autophagy machinery, are associated with increased risk of NAFLD (Lin et al., 2016). Moreover, patients with NAFLD have elevated hepatic levels of Rubicon, an inhibitor of Beclin 1/VPS34 PI3KC3 activity, and hepatocyte-specific knockout of Rubicon protects mice against high-fat diet-induced impaired autophagy and steatosis (Tanaka et al., 2016). Interestingly, ethanol exposure also inhibits hepatocyte lipophagy by inactivating Rab7 (Schulze et al., 2017), raising the question of whether defective autophagy (lipophagy) may also contribute to alcoholic fatty liver disease.

The precise mechanisms by which deficient autophagy (and ATG gene functions) promote obesity and its metabolic complications are complex and may involve a variety of cell intrinsic effects (e.g. nutrient metabolism; mitochondria, peroxisome, ER and lipid droplet homeostasis), cell extrinsic effects (e.g. release of pro-inflammatory cytokines by aberrant inflammasome activation), and potentially, lack of feedback inhibition of insulin and mTORC1 signaling pathways. The mechanisms by which autophagy is inhibited during obesity are not well understood, but may involve a combination of abnormal lysosomal function in cells with lipid accumulation (Koga et al., 2010) as well as dysregulated endocrine signaling (Zhang et al., 2018a). Autophagy is normally tightly regulated by neuroendocrine signals that are decreased (e.g. insulin and insulin-like growth factors) or increased (e.g. glucagon, fibroblast growth factor 21 [FGF21]) during fasting, and the initiation of and cellular response to these signals are commonly dysregulated in obesity. In mice, deficiency of the fasting hormone FGF21 impairs TFEB activation in hepatocytes, resulting in defective autophagic-lysosomal function and increased lipid accumulation, demonstrating a nutrient-sensing hormonal link between the FGF21-TFEB signaling axis, lysosomal function, and lipid metabolism (Chen et al., 2017). Interestingly, not only do neuroendocrine signals regulate autophagy, but ATG genes may act within subpopulations of neurons to regulate metabolism and feeding behavior. Genetic ablation of certain ATG genes in hypothalamic proopiomelanocortin neurons results in adiposity, glucose intolerance and hyperphagia (Coupe et al., 2012; Malhotra et al., 2015; Quan et al., 2012). Postulated (and potentially non-autophagic) mechanisms involve leptin resistance as well as defects in the unconventional secretion of α-melanocyte-stimulating hormone.

Further studies are needed to determine whether general inducers of autophagy, specific activators of lipophagy, and/or regulators of anti-obesogenic CNS functions of ATG genes may be useful in reducing the morbidity and mortality due to obesity and its associated metabolic disorders. At least in mice, nutritional interventions and physical exercise exert favorable metabolic effects through ATG gene-dependent mechanisms. The benefits of intermittent fasting on high-fat diet-induced loss of pancreatic cells are blocked in autophagy-deficient Lamp2a−/− and Becn1+/− mice (Liu et al., 2017). Moreover, the benefits of prolongation of the intermeal time interval (i.e. feeding mice an isocaloric diet twice-a-day) on adiposity, lipid levels, gluconeogenesis, and age/obesity-associated metabolic defects are impaired in animals with knockout of Atg7 in different organs (proopiomelanocortin neurons, hepatocytes, white adipose tissue, skeletal muscle) (Martinez-Lopez et al., 2017). The ability of chronic exercise to protect against HFD-induced glucose intolerance is compromised in mice that cannot increase autophagy by virtue of a knock-in mutation in Bcl-2 that prevents its disruption from Beclin 1 (He et al., 2012). Physical exercise induces TFEB translocation into muscle fiber nuclei, allowing muscle to adapt by changes in the expression‎ of glucose transporters, glycolytic enzymes and other metabolism-relevant genes (Mansueto et al., 2017). Whether TFEB activation is causally involved in exercise-induced autophagy induction remains to be explored, but its dual role in coordinating insulin sensitivity and glucose homeostasis during exercise and as a master regulator of autophagic-lysosomal function is intriguing. Thus, lifestyle interventions (e.g. dietary and exercise) that are important interventions in preventing metabolic disease may exert such effects, at least in part, through autophagy.

Autophagy in Longevity

Genetic studies using yeasts, worms, flies and mice demonstrate the ATG genes are required for lifespan extension requirement in caloric restriction, loss-of-function insulin signaling and other conserved longevity paradigms (reviewed in (Hansen et al., 2018)). Systemic autophagy induction exerts anti-aging effects in worms, flies and mice (Lopez-Otin et al., 2016), and genetically engineered mice with constitutively increased autophagy have extended lifespan and improved healthspan (e.g. leanness, increased insulin sensitivity, improved muscle function, reduced cardiac and renal fibrosis, and decreased age-related spontaneous tumorigenesis) (Fernandez et al., 2018; Pyo et al., 2013). Interestingly, the offspring of people with exceptional longevity have enhanced activation-induced T cell autophagy and immune function compared to age-matched controls (Raz et al., 2017). Autophagy may prevent aging through improved organellar quality control and homeostasis (e.g. via selective autophagy pathways such as mitophagy, lipophagy, lysophagy, aggrephagy), enhanced insulin sensitivity, maintenance of stemness and promotion of genomic stability. Interestingly, tissue-specific autophagy in certain tissues (e.g. in the muscle, intestine and brain) may also exert favorable effects on longevity, potentially by modulating a range of inter-tissue interactions (Hansen et al., 2018). It is possible that ATG genes may have autophagy-independent effects that promote longevity; for example, their roles in secretion and exocytosis might contribute to inter-tissue effects.

Autophagy gene expression‎ and lysosomal function decline with aging in a range of tissues in worms, flies and mammals (including in human brains), resulting in an age-related decline in autophagic capacity (Hansen et al., 2018). This age-related decline likely contributes both to the aging process itself as well as the development of age-related diseases such as neurodegenerative diseases and cancer. Aside from autophagy inhibition produced by obesity, it is not known what factors contribute to this age-related decline. The molecular mechanisms underlying age-related declines in distinct steps in the autophagic-lysosomal pathway is a fascinating area of future autophagy research.

Concluding Remarks

Autophagy genes function in diverse cell biological pathways, not only in autophagy but also in other processes, to exert widespread physiological functions that protect mammals against aging and a broad range of medically important diseases (Figure 3). Accordingly, a large spectrum of mutations in genes required for autophagy-related pathways have now been implicated in the pathogenesis of human diseases (Table 1).

Figure 3. Diverse biological functions of autophagy genes contribute to their roles in the regulation of mammalian disease.

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Shown are the major known biological functions of ATG genes and the broad categories of diseases that they regulate as predicted based on mouse experimental data and human genetic associations. Below major disease categories, some representative specific diseases are noted. Many other examples exist but are not shown due to space limitations.

Some general mechanisms of disease pathogenesis emerge from these links: (1) mutations in genes that regulate mitophagy, such as PARKIN and PINK1, are causally linked to hereditary forms of Parkinson’s disease (and also observed in some cancers) suggesting a crucial role for the consequences of impaired mitophagy (e.g. cellular ROS accumulation, mitochondrial DNA accumulation provoking cGAS/STING dependent inflammatory signaling); (2) mutations in genes encoding autophagy adaptor proteins or their activating kinases (e.g. p62/SQSTM1, optineurin, TBK) contribute to familial forms of amyotrophic lateral sclerosis, frontotemproal dementia and primary open glaucoma, implying a role for deficient selective autophagy in their pathogenesis; (3) mutations in genes that disrupt lysosomal function perturb autophagy and contribute to lysosomal storage disorders (including those with CNS and bone manifestations), Alzheimer’s disease, and Parkinson’s disease; (4) mutations in genes that disrupt autophagolysosomal fusion are often associated with congenital neurodevelopmental disorders; (5) mutations in genes that result in the accumulation of excess protein cargo (e.g polyglutamine expansion proteins, presenilin 1, amyloid precursor protein, αB-crystallin, α1-anti-trypsin) exceed the turnover capacity of autophagy, leading to end-organ proteotoxicity and degeneration; (6) hypomorphic mutations in core autophagy or selective autophagy machinery increase the risk of cancer; (7) dysregulated inflammatory signaling (as a result of defective mitophagy and enhanced inflammasome activation and/or cGAS/STING-mediated interferon signaling) may represent a common downstream event that contributes to diseases that are associated with mutations in the autophagy pathway (e.g. Crohn’s disease, Parkinson’s disease, SLE); (8) specific disorders, such as Crohn’s disease, are associated with several different mutations/polymorphisms in genes (e.g. ATG16L1, CALCOCO2/NDP62, GPR65, IRGM, LRRK2, NOD2) that simultaneously compromise intertwined pathways (e.g. bacterial autophagy, inflammasome regulation, secretion of antimicrobial peptides by Paneth cells) that govern their pathological and clinical manifestations. These genetic links illustrate the wide-ranging impact of autophagy-related pathways on distinct cell types and homeostatic processes.

Therapeutic interventions may aim to restore the wild-type function of the mutated protein, to reverse the specific defects or downstream pathogenetic consequences caused by the gene mutation, and/or to broadly upregulate autophagic activity and lysosomal function. Preclinical studies in animal models with mutations in genes that impair autophagy and lysosomal function will be necessary to determine the optimal therapeutic approaches for diseases due to defects in autophagy-related pathways. Moreover, the intriguing possibility emerges that selectively enhancing or blocking autophagy-related sub-routines might indirectly influence other sub-routines, thus affecting a therapeutically relevant ecosystem of intersecting pathways involving ATG proteins.

ACKOWLEDGMENTS

The authors’ thanks H. Smith for assistance with manuscript preparation and A. Diehl for expert medical illustration. The work in the authors’ laboratories was supported by NIH grants RO1 CA109618 and U19 AI109725 (B.L.), Cancer Prevention Research Institute of Texas (CPRIT) grant RP120718 (B.L), and Fondation Leducq grant 15CBD04 (B.L., G.K.). G.K. was supported by Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR) – Projets blancs; ANR under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Chancelerie des universités de Paris (Legs Poix), Fondation pour la Recherche Médicale (FRM); European Research Area Network on Cardiovascular Diseases (ERA-CVD, MINOTAUR); the European Research Council (ERC); Fondation Carrefour; Institut National du Cancer (INCa); Inserm (HTE); Institut Universitaire de France; the LabEx Immuno-Oncology; the RHU Torino Lumière; the Seerave Foundation; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and the SIRIC Cancer Research and Personalized Medicine (CARPEM). We apologize to all those authors’ whose work could not be cited due to space limitations.

Footnotes

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CONFLICT OF INTEREST STATEMENT

B.L. is a scientific co-founder of Casma Therapeutics, Inc. G.K. is a scientific co-founder of Samsara Therapeutics, Ltd.

References:

1. Abdulrahman BA, Abdelaziz DH, and Schatzl HM (2018). Autophagy regulates exosomal release of prions in neuronal cells. J Biol Chem 293, 8956–8968. [DOI] [PMC free article] [PubMed] [Google Scholar]
2. Aflaki E, Westbroek W, and Sidransky E (2017). The complicated relationship between Gaucher disease and Parkinsonism: Insights from a rare disease. Neuron 93, 737–746. [DOI] [PMC free article] [PubMed] [Google Scholar]
3. Akinduro O, Sully K, Patel A, Robinson DJ, Chikh A, McPhail G, Braun KM, Philpott MP, Harwood CA, Byrne C, et al. (2016). Constitutive autophagy and nucleophagy during epidermal differentiation. J Invest Dermatol 136, 1460–1470. [DOI] [PubMed] [Google Scholar]
4. Akizu N, Cantagrel V, Zaki MS, Al-Gazali L, Wang X, Rosti RO, Dikoglu E, Gelot AB, Rosti B, Vaux KK, et al. (2015). Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction. Nat Genet 47, 528–534. [DOI] [PMC free article] [PubMed] [Google Scholar]
5. Amaravadi R, Kimmelman AC, and White E (2016). Recent insights into the function of autophagy in cancer. Genes Dev 30, 1913–1930. [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Ashkenazi A, Bento CF, Ricketts T, Vicinanza M, Siddiqi F, Pavel M, Squitieri F, Hardenberg MC, Imarisio S, Menzies FM, et al. (2017). Polyglutamine tracts regulate beclin 1-dependent autophagy. Nature 545, 108–111. [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Auberger P, and Puissant A (2017). Autophagy, a key mechanism of oncogenesis and resistance in leukemia. Blood 129, 547–552. [DOI] [PubMed] [Google Scholar]
8. Baixauli F, Lopez-Otin C, and Mittelbrunn M (2014). Exosomes and autophagy: coordinated mechanisms for the maintenance of cellular fitness. Front Immunol 5, 403. [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, Rioux JD, Brant SR, Silverberg MS, Taylor KD, Barmada MM, et al. (2008). Genome-wide association defines more than 30 distinct susceptibility loci for Crohn’s disease. Nat Genet 40, 955–962. [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Bartolomeo R, Cinque L, De Leonibus C, Forrester A, Salzano AC, Monfregola J, De Gennaro E, Nusco E, Azario I, Lanzara C, et al. (2017). mTORC1 hyperactivation arrests bone growth in lysosomal storage disorders by suppressing autophagy. J Clin Invest 127, 3717–3729. [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Bartsch K, Knittler K, Borowski C, Rudnik S, Damme M, Aden K, Spehlmann ME, Frey N, Saftig P, Chalaris A, et al. (2017). Absence of RNase H2 triggers generation of immunogenic micronuclei removed by autophagy. Hum Mol Genet 26, 3960–3972. [DOI] [PubMed] [Google Scholar]
12. Behrends C, Sowa ME, Gygi SP, a