Autophagy genes in biology and disease

[문형철]

• Review Article
• Published: 12 January 2023

Autophagy genes in biology and disease

• Hayashi Yamamoto, 
• Sidi Zhang & 
• Noboru Mizushima 

Nature Reviews Genetics volume 24, pages382–400 (2023)Cite this article

Abstract

Macroautophagy and microautophagy are highly conserved eukaryotic cellular processes that degrade cytoplasmic material in lysosomes. Both pathways involve characteristic membrane dynamics regulated by autophagy-related proteins and other molecules, some of which are shared between the two pathways. Over the past few years, the application of new technologies, such as cryo-electron microscopy, coevolution-based structural prediction and in vitro reconstitution, has revealed the functions of individual autophagy gene products, especially in autophagy induction, membrane reorganization and cargo recognition. Concomitantly, mutations in autophagy genes have been linked to human disorders, particularly neurodegenerative diseases, emphasizing the potential pathogenic implications of autophagy defects. Accumulating genome data have also illuminated the evolution of autophagy genes within eukaryotes as well as their transition from possible ancestral elements in prokaryotes.

거대 오토파지와 미세 오토파지는 리소좀에서 세포질 물질을 분해하는 고도로 보존된 진핵 세포 과정입니다. 

두 경로 모두 오토파지 관련 단백질 및 기타 분자에 의해 조절되는 

특징적인 막 역학을 수반하며, 

그 중 일부는 두 경로 간에 공유됩니다. 

지난 몇 년 동안 극저온 전자 현미경, 공진화 기반 구조 예측 및 시험관 내 재구성과 같은 새로운 기술의 적용을 통해 특히 오토파지 유도, 막 재구성 및 화물 인식에서 개별 오토파지 유전자 산물의 기능이 밝혀졌습니다. 

이와 함께 오토파지 유전자의 돌연변이는 

인간 질환, 특히 신경 퇴행성 질환과 연관되어 

오토파지 결함의 잠재적 병원성 영향을 강조하고 있습니다. 

또한, 축적된 게놈 데이터는 

진핵생물 내 자가포식 유전자의 진화와 

원핵생물의 조상 요소로부터의 전환에 대해서도 밝혀냈습니다.

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Introduction

Autophagy (‘self-eating’) is a collection of processes by which cellular components such as proteins and organelles are delivered to the lysosome or vacuole for degradation. Two autophagy pathways are well conserved in eukaryotes: macroautophagy and microautophagy (Box 1). In macroautophagy, a cup-shaped membrane structure (known as the phagophore or isolation membrane) emerges near the endoplasmic reticulum (ER), elongates, bends and finally closes via membrane fission to form a double-membraned structure called the autophagosome, capturing part of the cytoplasm inside itself (Fig. 1). The autophagosome then fuses with lysosomes (or the vacuole in yeasts and plants), where its contents are degraded. In microautophagy, the endosomal or lysosomal membrane invaginates inwards to capture part of the cytoplasm directly (Fig. 2).

자가포식('스스로 먹는 것')은 

단백질과 

세포 소기관과 같은 세포 구성 요소를 

리소좀이나 액포에 전달하여 분해하는 일련의 과정입니다. 

진핵생물에는 거대 오토파지와 미세 오토파지라는 두 가지 오토파지 경로가 잘 보존되어 있습니다(상자 1). 

거대 자가포식에서는

컵 모양의 막 구조(식세포 또는 분리막으로 알려진)가 소포체(ER) 근처에서 출현하여

길어지고 구부러진 다음

막 분열을 통해 닫혀 오토파지좀이라는 이중 막 구조를 형성하여

세포질의 일부를 내부에 포획합니다(그림 1).

그런 다음 오토파지솜은

리소좀(또는 효모와 식물의 액포)과 융합하여

내용물이 분해됩니다.

Fig. 1: Membrane dynamics of macroautophagy.

a, (1) At initiation of macroautophagy, the ULK complex assembles near the ER membrane upon starvation and recruits ATG9 vesicles via its interaction with the ATG13–ATG101 subcomplex. (2) Alternatively, cargo adaptors such as p62, NDP52 and TAX1BP1 induce assembly of the ULK complex via interaction with FIP200, whereas ATG9 vesicles are recruited by OPTN. At the membrane-elongation step, the ULK complex recruits the class III phosphatidylinositol 3–kinase complex I (PI3KC3–C1) that produces PI(3)P, which further recruits its effector proteins, DFCP1 to omegasomes and WIPI2 and WIPI4 to phagophores. WIPI4 directs ATG2 to the phagophore membrane, which transfers phospholipids from the ER in concert with ATG9, VMP1 and TMEM41B (see b). WIPI2 recruits the ATG12–ATG5–ATG16L1 complex to promote LC3 lipidation on the phagophore membrane. Autophagosomes are closed by the action of the ESCRT machinery. Subsequently, PLEKHM1, EPG5 and RAB7 tether autophagosomes with lysosomes, and the two SNARE complexes, STX17–SNAP29–VAMP7/8 and YKT6–SNAP29–STX7, trigger fusion. After prolonged starvation, lysosomal membrane proteins on autolysosomes are recycled via autophagic lysosome reformation (ALR), whereas autophagosomal membrane proteins are recycled via autophagosomal components recycling (ACR). b, The lipid transfer protein ATG2 tethers the ER and phagophore membranes and transfers phospholipids from the ER to the phagophore. ATG9 on the phagophore membrane and VMP1 and TMEM41B on the ER membrane scramble phospholipids. c, In selective macroautophagy, cargos are recognized in a ubiquitin (Ub)-dependent manner (through Ub-binding adaptors) or a Ub-independent manner. Cargo adaptors/receptors bind to ATG8 on the autophagic membrane. d, In macro-fluidophagy, the phagophore membrane adheres to fluid-like condensates via membrane wetting.

1) 거대 오토파지가 시작될 때, ULK 복합체는 

굶주림에 의해 ER 막 근처에 조립되고 

ATG13-ATG101 하위 복합체와의 상호 작용을 통해 

ATG9 소포체를 모집합니다. 

(2) 또는 p62, NDP52 및 TAX1BP1과 같은 화물 어댑터는 

FIP200과의 상호 작용을 통해 ULK 복합체의 조립을 유도하는 반면, 

ATG9 소포는 OPTN에 의해 모집됩니다. 

막 연장 단계에서 ULK 복합체는 

PI(3)P를 생성하는 클래스 III 포스파티딜이노시톨 3-키나아제 복합체 I(PI3KC3-C1)을 모집하여 

이펙터 단백질인 DFCP1을 오메가좀으로, WIPI2와 WIPI4를 식세포로 추가 모집합니다.

 WIPI4는 ATG2를 식세포막으로 안내하고, 이 식세포막은 ATG9, VMP1 및 TMEM41B와 함께 인지질을 ER로부터 이동시킵니다(그림 b 참조). WIPI2는 ATG12-ATG5-ATG16L1 복합체를 모집하여 식세포막에서 LC3 지질화를 촉진합니다. 

오토파지는 ESCRT 기계의 작용에 의해 닫힙니다. 그 후 PLEKHM1, EPG5, RAB7이 오토파지와 리소좀을 연결하고, 두 개의 SNARE 복합체, 즉 STX17-SNAP29-VAMP7/8 및 YKT6-SNAP29-STX7이 융합을 촉발합니다. 

장기간의 기아 후, 

자가 리소좀의 리소좀 막 단백질은 

자가 리소좀 변형(ALR)을 통해 재활용되는 반면, 

자가 포식체 막 단백질은 

자가 포식체 성분 재활용(ACR)을 통해 재활용됩니다. 

b, 지질 전달 단백질 ATG2는 

ER과 식세포막을 묶고 

인지질을 ER에서 식세포로 전달합니다. 

식세포막의 ATG9와 ER 막의 VMP1 및 TMEM41B는 인지질을 스크램블합니다. 

c, 선택적 거대 오토파지에서 화물은 

유비퀴틴(Ub)에 의존하는 방식(Ub 결합 어댑터를 통해) 또는

 Ub와 독립적인 방식으로 인식됩니다. 

화물 어댑터/수용체는 자가포식 막의 ATG8에 결합합니다. 

d, 거대 유체포식에서 

식세포 막은 막 습윤을 통해 유체와 같은 응축물에 부착합니다.

마이크로 오토파지에서는

엔도솜 또는 리소좀 막이 안쪽으로 침입하여

세포질의 일부를 직접 포획합니다(그림 2).

Fig. 2: Membrane dynamics of microautophagy.

a, Microautophagy involves the invagination of endosomal membranes (left) or lysosomal membranes (right) to incorporate cytoplasmic material. The resulting intraluminal vesicles are degraded inside lysosomes or the vacuole. b, Cargos are recognized by (1) ATG8, (2) Nbr1, (3) HSC70 or (4) other proteins. Microautophagy can also uptake cytoplasmic materials non-selectively. c, Several types of microautophagy in mammals, yeasts, flies and plants are summarized. The numbers in the ‘Recognition’ column correspond to those in panel b. The ‘ATGs’ column indicates dependency on autophagy-related (ATG) proteins. Asterisks indicate dependence on only ATG8. Sp, Schizosaccharomyces pombe. Ub, ubiquitin.

The macroautophagy pathway was first observed in mammalian cells in the 1960s, and its discovery in Saccharomyces cerevisiae in the early 1990s fuelled rapid progress in the field, including the isolation of autophagy-deficient mutants, the identification of autophagy-related (ATG) genes and the characterization of related functional protein complexes1. Similarly, much of the progress in microautophagy research occurred in S. cerevisiae and other yeast species2.

거시적 오토파지 경로는 

1960년대에 포유류 세포에서 처음 관찰되었으며, 

1990년대 초에 사카로마이세스 세레비지애에서 발견되면서 

오토파지 결핍 돌연변이체 분리, 

오토파지 관련(ATG) 유전자 확인, 

관련 기능성 단백질 복합체 특성 규명 등 이 분야의 급속한 발전을 촉진했습니다1. 

마찬가지로, 마이크로 오토파지 연구의 많은 진전은 S. 세레비지애와 다른 효모 종에서 이루어졌습니다2.

The most fundamental physiological function common to both macro- and microautophagy is thought to be the supply of nutrients in times of need, such as during starvation or development2,3,4,5,6. In addition, autophagy maintains cellular homeostasis by selectively degrading proteins, organelles and foreign entities2,3,4; this process is particularly important for long-living cells such as neurons. Occasionally, autophagy also delivers vacuolar hydrolases to fulfill biosynthetic roles in yeasts7,8. Multiple ATG genes have also been implicated in the function of non-autophagic pathways, including endocytosis, secretion and extracellular vesicle biogenesis9. Owing to these versatile functions, ATG genes have attracted attention in various research fields, and the scope of research continues to grow.

거시적 오토파지와 미시적 오토파지 모두에서 공통적으로 나타나는 

가장 기본적인 생리적 기능은 

굶주림이나 

발달 과정과 같이 필요한 시기에 영양분을 공급하는 것으로 생각됩니다2,3,4,5,6. 

또한 자가포식은 

단백질, 

세포 소기관 및 이물질2,3,4을 선택적으로 분해하여 

세포의 항상성을 유지하며, 

이 과정은 신경세포와 같이 수명이 긴 세포에 특히 중요합니다. 

때때로 오토파지는 효모에서 생합성 역할을 수행하기 위해 액포 가수분해효소를 전달하기도 합니다7,8. 

여러 ATG 유전자는 

세포 내 분해, 

분비 및 세포 외 소포 생성을 포함한 

비자가포식 경로의 기능에도 관여합니다9. 

이러한 다양한 기능으로 인해 ATG 유전자는 다양한 연구 분야에서 주목을 받고 있으며, 그 연구 범위는 계속 확대되고 있습니다.

In recent years, much progress has been made in understanding the functions of individual ATG genes as well as their roles in disease and physiology. The molecular functions of key players have been revealed, and the importance of cargo-driven mechanisms and phase separation have been highlighted. These findings were made possible by the application of new technologies such as cryo-electron microscopy, coevolution-based structural prediction and in vitro reconstitution. In addition to the molecular machinery, another focus of autophagy research is its role in pathophysiology. Genetic analyses of human diseases (for example, neurodegenerative disorders and autoimmune diseases) have identified several ATG genes as causative genes or risk factors in the past decade4,10. Last, recent genome data have elucidated the evolution of autophagy genes within eukaryotes and possibly from prokaryotes.

최근 몇 년 동안 개별 ATG 유전자의 기능과 질병 및 생리학에서의 역할에 대한 이해에 많은 진전이 있었습니다.

핵심 플레이어의 분자 기능이 밝혀지고

화물 구동 메커니즘과 상 분리의 중요성이 강조되고 있습니다.

이러한 발견은 극저온 전자 현미경, 공진화 기반 구조 예측 및 시험관 내 재구성과 같은 새로운 기술의 적용을 통해 가능했습니다. 분자 메커니즘 외에도 오토파지 연구의 또 다른 초점은 병리 생리학에서의 역할입니다.

인간 질병(예: 신경 퇴행성 질환 및 자가 면역 질환)에 대한 유전자 분석을 통해

지난 10년 동안 여러 ATG 유전자가 원인 유전자 또는

위험 요인으로 밝혀졌습니다4,10.

마지막으로,

최근의 게놈 데이터는

진핵생물과 원핵생물에서

오토파지 유전자의 진화를 밝혀냈습니다.

In this Review, we summarize recent progress in the molecular and cellular biology of genes involved in macro- and microautophagy, the pathological relevance of these genes and key evolutionary aspects. We do not cover the third type of autophagy, chaperone-mediated autophagy, which does not involve membrane dynamics and is regulated by an entirely different set of proteins11.

이 리뷰에서는 

거시적 및 미시적 오토파지에 관여하는 유전자의 분자 및 세포 생물학, 

이러한 유전자의 병리학적인 관련성 및 

주요 진화적 측면에 대한 최근의 진전을 요약합니다. 

세 번째 유형의 오토파지인 샤프론 매개 오토파지는 

막 역학에 관여하지 않고 

완전히 다른 단백질 집합에 의해 조절되는 오토파지에 대해서는 다루지 않습니다11.

Box 1 Evolution of autophagy genes

Bacteria, archaea (collectively the prokaryotes) and eukaryotes constitute the three domains of life (their common ancestor is LUCA, the last universal common ancestor). Thaumarchaeota,Aigarchaeota, Crenarchaeota, and Korarchaeota (TACK), Euryarchaeota and Asgard are major branches in archaea. The autophagy pathway, which requires intracellular membranous compartments, is present in eukaryotes, but absent from prokaryotes. Most, if not all, of the core autophagy-related (ATG) proteins were probably already present in the last eukaryotic common ancestor (LECA; see the figure), with subsequent extensive duplications and losses among eukaryotic lineages contributing to the observed functional diversity180.

While many of the duplication events occurred in vertebrates and plants, two ATG protein families (the Atg1/ULK and β-propellers that bind polyphosphoinositides family proteins, PROPPINs) had possibly already diversified into different subgroups in or shortly after the LECA, although not all subgroups are involved in autophagy.

In ATG conjugation systems, canonically, ATG12 is covalently conjugated to ATG5, but Toxoplasma, Plasmodium (both Alveolata, which belongs to the Stramenopiles, Alveolata and Rhizaria (SAR) supergroup) and Komagataella (a yeast genus) lost the necessary proteins and/or residues (that is, the E2-like enzyme ATG10 and/or the C-terminal glycine of ATG12) for conjugation and thus rely on noncovalent interactions between ATG12 and ATG5 instead181 (see the figure). The noncovalent form is considered adaptive because it does not require ATP or enzymes. Such covalent-to-noncovalent transitions may have occurred as many as 16 times in eukaryotes180.

Many breakthroughs in autophagy research, including the first identification of the ATG genes, were made in the budding yeast Saccharomyces cerevisiae, which is often regarded as the standard model. However, studies in other species have revealed that the autophagy system in S. cerevisiae has many unconventional features (see the figure). The Atg1 complex in S. cerevisiae lacks ATG101, which forms a complex with and stabilizes ATG13 in other species and contains Atg29 and Atg31 in a complex with Atg17 (ref. 182). Whether the acquisition of Atg29 and Atg31 and the loss of ATG101 are functionally linked remains unknown. Additionally, S. cerevisiae lacks VMP1, the endoplasmic reticulum (ER)-resident downstream (of hisT) Escherichia coli DNA gene A (DedA) superfamily protein that is required for autophagy in many other species, including metazoa, Dictyostelium and possibly green algae. Finally, S. cerevisiae has a unique pathway known as the cytoplasm-to-vacuole targeting (Cvt) pathway, which is a biosynthetic pathway that delivers vacuolar hydrolases to the vacuole. Schizosaccharomyces pombe has another biosynthetic pathway, termed the Nbr1-mediated vacuolar targeting (NVT) pathway (see details in the main text).

Expansions of gene families are also observed among proteins associated with selective autophagy. NBR1 (Atg19 in S. cerevisiae) is broadly distributed in eukaryotes183, and SQSTM1 could be the result of NBR1 duplication followed by NBR1 domain loss. The OPTN and CALCOCO families are conserved in most metazoan species, and expansions of these families probably occurred in vertebrate lineages.

Even though many ATG proteins are eukaryote-specific, some may have originated in prokaryotes. Indeed, most of the functional complexes in the autophagy pathway contain at least one protein with remote homologues in prokaryotes (for example, the DedA superfamily proteins, including TMEM41B and VMP1, the Hop1, Rev7 and Mad2 (HORMA)-domain-containing proteins, including ATG13 and ATG101, the transmembrane portion of ATG9, the chorein-N domain at the N termini of lipid transfer proteins, including ATG2, and the ubiquitin-like ATG conjugation systems) (see the figure), suggesting that the recruitment of pre-existing genes was important for the evolution of autophagy180,184.

The endosomal sorting complex required for transport (ESCRT) proteins, which are required for both macro- and microautophagy, also originated in prokaryotes. Although ESCRT-I, -II and -III function sequentially at the site of membrane fission in eukaryotes, the ESCRT-III proteins evolved first, in the form of PspA/Vipp1 and CdvB proteins widely distributed among bacteria and archaea, respectively185. By contrast, the ESCRT-I and -II proteins represent later additions and probably originated in the Asgard archaea group186. Therefore, the Asgard group, from which eukaryotes have been hypothesized to have emerged, already had a complete ESCRT system (though without ESCRT-0, which only occurs in the Opisthokonta).

Genes regulating macroautophagy

Initiation of macroautophagy

The ULK complex — which is composed of the scaffold protein FIP200 (also known as RB1CC1), ATG13, ATG101 and the serine/threonine kinases ULK1 or ULK2 — is the central regulator in the initiation of macroautophagy (Table 1, Fig. 1a). Its activity is suppressed primarily by mechanistic target of rapamycin complex 1 (mTORC1). Upon starvation, mTORC1 is inactivated, leading to the activation and assembly of the ULK complex in the vicinity of the ER membrane, which recruits downstream ATG proteins to initiate autophagosome formation (Fig. 1a, Initiation). In yeasts, the homologous Atg1 complex forms similar assembled structures known as pre-autophagosomal structures (PASs). The PAS is a higher-order assembly of the Atg1 complexes, in which Atg13 tethers Atg1 (homologous to ULK1/2) and the Atg17–Atg29–Atg31 subcomplex (Atg17 is homologous to part of FIP200)12,13. The PAS is a fluid-like condensate resulting from liquid–liquid phase separation, which is driven by the multivalent interactions between Atg1, Atg13 and Atg17 (ref. 13), and provides an environment that intermolecularly auto-activates the Atg1 kinase14. Thus, ULK/Atg1 complex assembly is a key step in initiating macroautophagy.

스캐폴드 단백질 FIP200(RB1CC1이라고도 함), ATG13, ATG101, 세린/트레오닌 키나아제 ULK1 또는 ULK2로 구성된 ULK 복합체는 매크로오토파지의 시작에 있어 핵심적인 조절 인자입니다(표 1, 그림 1a). 이 효소의 활성은 주로 라파마이신 복합체 1(mTORC1)의 기계적인 표적에 의해 억제됩니다. 

굶주림이 발생하면 

mTORC1이 비활성화되어 

ER 막 근처에서 ULK 복합체가 활성화되고 조립되며, 

이 복합체는 하류 ATG 단백질을 모집하여 

오토파지솜 형성을 시작합니다(그림 1a, 개시). 

효모에서 상동성 Atg1 복합체는 오토파지 전 구조(PAS)로 알려진 유사한 조립 구조를 형성합니다. PAS는 Atg1 복합체의 고차 조립체로, Atg13이 Atg1(ULK1/2와 상동성)과 Atg17-Atg29-Atg31 하위 복합체(Atg17은 FIP200의 일부와 상동성)12,13를 묶는 구조입니다. PAS는 액체-액체 상 분리로 인해 생성되는 유체와 같은 응축물로, Atg1, Atg13 및 Atg17 간의 다원적 상호 작용에 의해 구동되며(참조 13), 분자 간 자동 활성화 환경을 제공합니다14. 따라서 ULK/Atg1 복합체 조립은 매크로 오토파지를 시작하는 핵심 단계입니다.

Table 1 The autophagy-related (ATG) proteins required for autophagosome formation

In addition to starvation-induced assembly, ULK/Atg1 complex assembly is also driven by autophagy cargos (Fig. 1a, Initiation). The soluble autophagy cargo adaptor SQSTM1 (also called p62) forms fluid-like condensates with ubiquitinated proteins and interacts with FIP200 to recruit the ULK complex15. (In this Review, ‘adaptor’ refers to a soluble protein that mediates binding between a cargo and the autophagy machinery, whereas ‘receptor’ refers to a cargo-resident protein that binds to the autophagy machinery.) During Parkin-dependent mitophagy (see below) and selective degradation of Salmonella, NDP52 (also called CALCOCO2) — another soluble autophagy adaptor — localizes to ubiquitinated mitochondria and the cytosol-invading bacteria and recruits the ULK complex via interaction with FIP200 (refs. 16,17). TAX1BP1 also recruits FIP200 to SQSTM1 condensates18. Ubiquitin-independent selective macroautophagy also involves the cargo-driven assembly of the ULK complex: the ER-phagy receptor CCPG1 interacts with the Claw domain of FIP200 to recruit the ULK complex19,20.

기아에 의한 조립 외에도, 

ULK/Atg1 복합체 조립은 오토파지 화물에 의해 주도됩니다(그림 1a, 개시). 

수용성 오토파지 화물 어댑터인 SQSTM1(p62라고도 함)은 유비퀴틴화된 단백질과 유체와 같은 응축물을 형성하고 FIP200과 상호작용하여 ULK 복합체를 모집합니다15. (본 리뷰에서 '어댑터'는 화물과 오토파지 기계 사이의 결합을 매개하는 수용성 단백질을 말하며, '수용체'는 오토파지 기계에 결합하는 화물 상주 단백질을 말합니다.). 파킨 의존성 미토파지(아래 참조)와 살모넬라균의 선택적 분해 과정에서 또 다른 수용성 오토파지 어댑터인 NDP52(칼코코2라고도 함)는 유비퀴틴화된 미토콘드리아와 세포질 침입 박테리아에 국한되어 FIP200과의 상호작용을 통해 ULK 복합체를 모집합니다(참고자료 16,17). TAX1BP1은 또한 FIP200을 SQSTM1 응축물로 모집합니다18. 유비퀴틴 독립적인 선택적 거대 오토파지는 또한 화물 주도의 ULK 복합체 조립을 포함합니다: ER-파지 수용체 CCPG1은 FIP200의 클로 도메인과 상호 작용하여 ULK 복합체를 모집합니다19,20.

Upon assembly, the ULK complex recruits ATG9 vesicles (Fig. 1a, ‘Initiation’), which are thought to be the seeds for autophagosome formation21. ATG9 vesicle recruitment is achieved via interactions between the HORMA domains of the ATG13–ATG101 subcomplex and the most C-terminal region of ATG9A22 (Table 1). During Parkin-dependent mitophagy, ATG9 vesicles are also recruited through binding with the autophagy adaptor OPTN, which is present on ubiquitinated mitochondria23. Similarly, in yeast, Atg9 vesicles are recruited via two pathways: one through interaction with the HORMA domain of Atg13 during starvation-induced macroautophagy, and the other through the cargo adaptor Atg11 during selective macroautophagy24.

조립이 완료되면 ULK 복합체는 

자가포식소체 형성의 씨앗으로 여겨지는 ATG9 소포(그림 1a, '개시')를 모집합니다21. 

ATG9 소포 모집은 

ATG13-ATG101 하위 복합체의 HORMA 도메인과 ATG9A22의 가장 C-말단 영역 간의 상호 작용을 통해 이루어집니다(표 1). 파킨 의존성 미토파지 과정에서 ATG9 소포는 유비퀴틴화된 미토콘드리아에 존재하는 오토파지 어댑터 OPTN과의 결합을 통해서도 모집됩니다23. 마찬가지로 효모에서 Atg9 소포는 두 가지 경로를 통해 모집되는데, 하나는 굶주림으로 유도된 거대 오토파지 동안 Atg13의 HORMA 도메인과의 상호작용을 통해, 다른 하나는 선택적 거대 오토파지 동안 화물 어댑터 Atg11을 통해 모집됩니다24.

Membrane elongation by lipid transfer

The ULK complex also recruits the class III phosphatidylinositol 3-kinase complex I (PI3KC3–C1), which produces PI(3)P in autophagic membranes (Fig. 1a, ‘Membrane elongation’). The PI3KC3–C1 is composed of five subunits: VPS34, VPS15, BECN1 (Vps30 in yeast), ATG14 and NRBF2 (Atg38 in yeast) (Table 1). PI3KC3–C1 binds to membranes via ATG14, BECN1 and VPS34, after which VPS34 generates PI(3)P25,26.

ULK 복합체는 

또한 클래스 III 포스파티딜이노시톨 3-키나아제 복합체 I(PI3KC3-C1)을 모집하여 

자가포식막에서 PI(3)P를 생성합니다(그림 1a, '막 신장'). 

PI3KC3-C1은 5개의 서브유닛으로 구성되어 있습니다: VPS34, VPS15, BECN1(효모의 Vps30), ATG14 및 NRBF2(효모의 Atg38)(표 1). PI3KC3-C1은 ATG14, BECN1, VPS34를 통해 막에 결합하고, 그 후 VPS34가 PI(3)P25,26을 생성합니다.

The PI(3)P effectors include the β-propellers that bind polyphosphoinositides (PROPPIN) family proteins (WIPI proteins in mammals, and Atg18, Atg21 and Hsv2 in yeast), which further recruit ATG2. ATG2 forms a rod-shaped structure that attaches to the ER membrane with its N-terminal tip and the autophagic membrane with its C-terminal tip27,28 (Fig. 1b). In vitro studies have shown that phospholipids are transferred through a hydrophobic cavity in ATG2, suggesting that this process occurs between the ER and phagophore in vivo29,30,31. However, the mechanism by which the transfer activity is regulated and what drives it remain to be elucidated.

PI(3)P 이펙터에는 폴리포스포노시타이드(PROPPIN) 계열 단백질(포유류의 경우 WIPI 단백질, 효모의 경우 Atg18, Atg21, Hsv2)과 결합하는 β-프로펠러가 포함되며, 이 단백질은 ATG2를 추가로 모집합니다. ATG2는 막대 모양의 구조를 형성하여 N-말단 끝으로 ER 막에 부착하고 C-말단 끝으로 자가포식 막에 부착합니다27,28(그림 1b). 시험관 내 연구에 따르면 인지질은 ATG2의 소수성 공동을 통해 전달되며, 이는 이 과정이 생체 내에서 ER과 식세포 사이에서 발생한다는 것을 시사합니다29,30,31. 그러나 전달 활동이 조절되는 메커니즘과 그 원동력은 아직 밝혀지지 않았습니다.

There are four PROPPIN family proteins (WIPI1–4) in mammals. ATG2 is directed to PI(3)P-rich autophagic membranes, probably through the interaction with WIPI3 or WIPI4 (refs. 27,32). Of the four homologues, WIPI2 functions dominantly, as depletion of only WIPI2 profoundly suppresses autophagy33, upstream of WIPI3 and WIPI4 (ref. 34). WIPI2 binds to ATG16L1, a component of the ATG12–ATG5–ATG16L1 complex (Table 1), which has an E3-like activity to promote lipidation of ATG8 proteins (LC3 and GABARAP family proteins in mammals, which are collectively called ATG8 hereafter). ATG8 can further recruit ATG2, as ATG2 has a LC3-interacting region (LIR) (see below)35. Thus, WIPI2 also contributes to ATG2 recruitment indirectly.

포유류에는 4개의 PROPPIN 계열 단백질(WIPI1-4)이 있습니다. ATG2는 아마도 WIPI3 또는 WIPI4와의 상호작용을 통해 PI(3)P가 풍부한 자가포식막으로 이동합니다(참고 문헌 27,32). 네 개의 상동 단백질 중 WIPI2가 지배적으로 기능하는데, 이는 WIPI2만 결핍되면 WIPI3와 WIPI4의 상류에 있는 자가포식33이 크게 억제되기 때문입니다(참고 문헌 34). WIPI2는 ATG12-ATG5-ATG16L1 복합체(표 1)의 구성 요소인 ATG16L1에 결합하는데, 이 복합체는 E3와 유사한 활성을 가지고 있어 ATG8 단백질(포유류의 LC3 및 가바랩 계열 단백질, 이하 통칭하여 ATG8이라고 함)의 지질화를 촉진합니다. ATG2는 LC3 상호 작용 영역(LIR)을 가지고 있기 때문에(아래 참조)35) ATG8은 ATG2를 추가로 모집할 수 있습니다. 따라서 WIPI2는 간접적으로 ATG2 모집에도 기여합니다.

After ATG2 transfers lipids to the outer leaflet of the autophagic membrane, ATG9 scrambles phospholipids in the membrane (Fig. 1b). ATG9 forms a trimer and translocates phospholipids between the outer and inner leaflets36,37,38. There are also two phospholipid scramblases required for autophagosome formation in the ER membrane: VMP1 and TMEM41B38,39,40 (Fig. 1b). Both proteins are multi-spanning membrane proteins with a conserved DedA domain predicted to have two characteristic re-entrant loops41,42 (Box 1). Because ATG2A interacts with ATG9 as well as VMP1 and TMEM41B38, VMP1/TMEM41B–ATG2–ATG9 may be considered a lipid transfer unit. These scramblases can equilibrate the imbalance of phospholipid density on each leaflet caused by lipid transfer (Fig. 1b). However, if local lipid synthesis in the outer leaflet of the ER membrane produces pressure for directional lipid transfer, lipid scrambling may weaken that activity. Moreover, VMP1 and TMEM41B are important for the formation of not only autophagosomes but also lipid droplets, lipoproteins and the double-membrane structures required for the replication of several RNA viruses (including SARS-CoV-2)43. Therefore, their scrambling activity may be crucial for a fundamental function of the ER, not only in providing lipids to ATG2. Yeast has a TMEM41B-like protein, Tvp38, but it is not required for autophagosome formation41. The mechanism by which yeast can form autophagosomes without DedA family proteins has yet to be elucidated.

Two ubiquitin-like conjugation systems, the ATG8 and ATG12 systems, also have key roles in autophagosome formation. In concert with the E1-like enzyme ATG7 and the E2-like enzymes ATG3 and ATG10, ATG8 and ATG12 are conjugated to phosphatidylethanolamine (PE) and ATG5, respectively. The ATG12–ATG5–ATG16(L1) complex has an E3-like activity for ATG8–PE conjugation. Although ATG conjugation systems are essential for autophagosome formation in yeasts, seemingly normal autophagosomes can be generated in mammalian cells even when these systems are disrupted44,45,46. They seem to be more important for fusion with lysosomes or the degradation of the autophagosomal inner membrane47,48 and, therefore, are still crucial for autophagic flux.

Cargo recognition

Because autophagosomes engulf part of the cytoplasm (approximately 0.5–1 μm in diameter), the majority of soluble cargos are believed to be incorporated non-selectively. However, autophagosomes can also selectively recognize various cargos, including damaged organelles, intracellular bacteria and certain proteins49. Besides its crucial role in membrane dynamics, ATG8 has a central function in cargo recognition in selective macroautophagy (Fig. 1c). ATG8 directly interacts with the LIR motifs (or the Atg8-interacting motif in yeast) in cargos or autophagy adaptors, which are classified into ubiquitin-dependent and -independent types (Table 2). Ubiquitin-recognizing soluble cargo adaptors include SQSTM1, NBR1, NDP52, OPTN, TAX1BP1 and TOLLIP49. The ubiquitin-independent category includes organelle-bound receptors for ER-phagy (for example, CCPG1, TEX264, FAM134B, SEC62, RTN3L and ATL3) and mitophagy (for example, BNIP3, BNIP3L/NIX, FUNDC1, FKBP8 and BCL2L13) as well as LIR-containing soluble proteins such as CALCOCO1 (ref. 49). Besides cargo recognition, LIRs are used for the recruitment of some core autophagy factors, including FIP200, ULK1 and ATG13 (ref. 50), as well as the tethering factors PLEKHM1 and EPG5 (refs. 51,52). As discussed above, selective cargos can also recruit the ULK complex, constituting another layer of cargo recognition (Fig. 1a, ‘Initiation’).

Table 2 Non-ATG molecules in macroautophagy and microautophagy

Full size table

In addition to organelles and individual proteins, fluid-like condensates formed by liquid–liquid phase separation are degraded by macroautophagy entirely or in a piecemeal manner (termed fluidophagy)53 (Fig. 1d). As shown in the degradation of SQSTM1 condensates, the adherence of fluid-like condensates to ATG8-positive autophagic membranes is promoted by a membrane-wetting effect53.

Closure of autophagosomes

Autophagosome closure is mediated by membrane scission of the phagophore membrane into the outer and inner autophagosomal membranes (Fig. 1a). This is topologically identical to membrane scission of the intraluminal vesicle formation in multivesicular bodies and virus budding at the plasma membrane. Indeed, like these two processes, the endosomal sorting complex required for transport (ESCRT) complex has a pivotal role in autophagosome closure54,55,56 (Table 2). How the ESCRT complex localizes to the open rim of the phagophore remains unknown in mammals. However, in yeast, Rab5-dependent interactions between Atg17 and Snf7, an ESCRT-III component, may account for the ESCRT recruitment to phagophores55.

Autolysosome formation and recycling

After closure, autophagosomes fuse with lysosomes to become autolysosomes. In mammals, fusion is mediated by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins STX17 and YKT6, which are recruited to autophagosomes when they are closed57,58 (Table 2), thereby preventing premature fusion between unclosed autophagosomes and lysosomes. STX17 and YKT6 form SNARE bundles with cytosolic SNAP29 and lysosomal VAMP7/VAMP8 and STX7, respectively (Fig. 1a, ‘Lysosome fusion’). The relationship and functional differences between these two autophagosomal SNAREs (STX17 and YKT6) are not well understood; thus far, they seem to function redundantly. The role of YKT6 in autophagosome fusion is conserved in yeast59,60, but STX17 is absent in yeast. Tethering between autophagosomes and lysosomes is mediated by multiple tethering factors, including HOPS, PLEKHM1 and EPG5 (ref. 61). PLEKHM1 (ref. 51) and EPG5 (ref. 52) have a LIR motif and therefore interact with autophagosomal ATG8.

After fusing with lysosomes, the inner autophagosomal membrane is degraded. In yeast, autophagosomes fuse with the large vacuole, releasing autophagic bodies surrounded by the autophagosomal inner membranes. The vacuolar phospholipase Atg15 is responsible for degrading the membranes of autophagic bodies (derived from the autophagosomal inner membrane)62. Most organisms, including mammals, do not have Atg15 homologues but instead possess multiple lysosomal phospholipases, which might be redundantly involved in inner membrane degradation. One major remaining question is how degradation is limited to only the inner membrane when both the outer and inner membranes, which are derived from the same phagophore membrane, are exposed to lysosomal enzymes.

After prolonged starvation, autolysosomes deform to generate protolysosomes that then mature into functional lysosomes, in a process called autophagic lysosome reformation (ALR)63 (Fig. 1a, ‘Recycling’). This process recycles lysosomal membrane proteins and is triggered by the reactivation of mTORC1 in response to increased amino acid levels owing to prolonged macroautophagy. Upon induction of ALR, the PI(4)P 5-kinase PIP5K1B produces PI(4,5)P2 on the membrane of autolysosomes, generating PI(4,5)P2-rich microdomains that are further organized by clathrin and adaptor protein 2 (AP2)64 (Table 2). Lysosomal membrane proteins are captured in these microdomains, which subsequently undergo tubulation driven by the kinesin heavy chain KIF5B65. The mechanism by which protolysosomes mature into lysosomes remains to be elucidated.

An additional autolysosome recycling mechanism called autophagosomal components recycling (ACR) was recently reported66. This mechanism recycles autophagosome-derived membrane-anchoring proteins such as ATG9 and STX17 via the budding of autolysosomal membranes depending on the SNX4–SNX5–SNX17 recycler complex and the dynein–dynactin complex (Table 2) (Fig. 1a, ‘Recycling’). Thus, autophagosomal and lysosomal membrane proteins are recycled by ACR and ALR, respectively. ACR occurs earlier than ALR.

Non-autophagic functions

Although ATG proteins were originally identified in yeast as factors required for autophagy, it is now apparent that most of them also participate in non-autophagic processes67 (Table 1). We discuss only a few of these functions here owing to space limitations. Many of the non-autophagic functions of ATG proteins are related to membrane dynamics. BECN1 is a component of not only PI3KC3–C1 but also PI3KC3–C2 (containing UVRAG instead of ATG14), which is involved in the endocytic pathway. The tumour-suppressing activity of BECN1 is at least partially attributable to its non-autophagic function in PI3KC3–C2 (ref. 68). Another well recognized non-autophagic process is the conjugation of ATG8 to single-membrane compartments in the endocytic pathway rather than double-membrane autophagosomes, which is known as conjugation of ATG8 to endolysosomal single membranes (CASM) or single-membrane ATG8 conjugation (SMAC), including LC3-associated phagocytosis (LAP) and LC3-associated endocytosis (LANDO)69,70,71. LAP is one of the phagocytotic pathways and degrades its contents by fusion with lysosomes. LAP does not require the ULK complex but does require the ATG conjugation systems. Notably, the WD40 repeat domain of ATG16L1, which is absent in yeast Atg16 (Table 1), is essential for LAP through binding to V-ATPase, but not for canonical autophagy72,73,74,75,76. The PI3KC3–C2 component UVRAG and RUBCN are also required. A recent study reported that during LAP (or CASM), ATG8 is conjugated not only to PE, but also to phosphatidylserine (PS)77. It is unknown whether ATG8–PS has a unique function distinct from that of ATG8–PE. Secretory autophagy (autophagy-based unconventional secretion) is another type of non-autophagic process. In secretory autophagy, closed autophagosomes do not fuse with lysosomes, but instead fuse with the plasma membrane, secreting cytosolic proteins lacking conventional leader sequences. ATG proteins are required to form autophagosomes in this pathway, and inhibition of autophagosome–lysosome fusion leads to upregulation of secretory autophagy78. In addition, autophagy-related genes are involved in viral replication and transmission. Many viruses exploit autophagosome-like vesicles for replication and exocytosis79. A genome-wide CRISPR screen identified TMEM41B and VMP1 as host factors for flaviviruses and coronaviruses, including SARS-CoV-2, where they are thought to participate in the formation of specialized replication organelles43. Other examples of non-autophagic membrane-related processes that require ATG genes include ER-to-Golgi trafficking (ULK1 and ULK2)80, protection against plasma membrane permeabilization (ATG9A)81, and TFEB activation during lysosomal damage or lysosomal transient receptor potential mucolipin channel 1 (TRPML1) activation (ATG conjugation systems)82,83,84. In apicomplexan parasites such as Plasmodium and Toxoplasma, ATG8 (and the ATG conjugation systems) are essential for the biogenesis of apicoplasts85,86, non-photosynthetic plastids specific to this lineage that support key metabolic functions.

ATG genes can also regulate various non-membrane-related processes, such as cell cycle progression and cell death. ATG7 directly interacts with p53, and ATG7-deficient cells show impaired cell cycle arrest and increased apoptosis (both are p53-mediated) upon nutrient starvation87. ATG12 promotes mitochondrial apoptosis by binding to and inactivating Bcl-2 family proteins88. GABARAPs are required for interferon-γ (IFNγ)-mediated antimicrobial responses through binding to ADP-ribosylation factor 1 (ref. 89).

Genes regulating microautophagy

Membrane dynamics of microautophagy

Microautophagy was first described in mammalian cells as a process involving the invagination of lysosomal membranes that incorporate cytosolic material into lysosomes, followed by membrane fission and degradation90,91 (Fig. 2a). Because observing microautophagy in small lysosomes is difficult by light microscopy, the underlying molecular mechanisms have been primarily revealed in yeasts and plants, where the vacuole is sufficiently large for optical observation. Like macroautophagy, microautophagy can be both non-selective and selective; the process non-selectively enwraps cytosolic material but also selectively recognizes organelles, such as peroxisomes (micropexophagy), mitochondria (micromitophagy), lipid droplets (microlipophagy), a subdomain of the ER (microER-phagy), a portion of the nucleus (micronucleophagy), and photodamaged chloroplasts (microchlorophagy)2,92,93. Microautophagy requires the ESCRT machinery at the membrane fission step2. However, its dependency on ATG proteins is complicated and may differ among cargo types and inducing conditions. While ATG proteins seem to be dispensable in general microautophagy and microER-phagy, at least some of the ATG proteins are required for microlipophagy94,95,96, micropexophagy97,98, micromitophagy99 and micronucleophagy100,101 in yeast as well as microchlorophagy102 in plants (Fig. 2b). In mammals, micromitophagy and microlipophagy seem to be independent of ATG proteins103,104, whereas micronucleophagy mediated by cGAS requires the ATG8 conjugation system for cargo recognition105. Thus, microautophagy can be roughly divided into two types: ATG-independent and ATG-dependent (Fig. 2c). In the latter type, ATG proteins can be involved in the formation of additional membrane structures, the remodelling of vacuolar morphology, and/or the recognition of selective cargos (see below)2,92,93.

In mammalian cells, multivesicular body formation of endosomes is considered to be a type of microautophagy referred to as endosomal microautophagy106. Endosomal microautophagy occurs constitutively and is also induced during early periods of amino acid starvation, leading to the degradation of cytosolic proteins, particularly selective macroautophagy adaptors, such as SQSTM1, NDP52, NBR1, TAX1BP1 and NCOA4 (an adaptor for ferritin)5 (Fig. 2b). The multivesicular body pathway in yeast is also known to be induced by starvation and contributes to early proteome remodelling during starvation6. Starvation-induced endosomal microautophagy in mammals requires ESCRT-III (CHMP4B) and VPS4 but not ESCRT-0, -I or -II (ref. 5). The necessity of ATG proteins in this pathway is also complicated. The ATG8 conjugation system is required for the degradation of SQSTM1 and NDP52 and partially required for NBR1 and TAX1BP1 but not for NCOA4, whereas FIP200 and VPS34 are not required for any of them5. Therefore, ATG proteins should be important for cargo recognition rather than membrane dynamics in this case (Fig. 2c).

Endosomal intraluminal vesicles formed by microautophagy are directed to lysosomes for degradation, but they are also secreted to the extracellular space in mammals. Several RNA-binding proteins, including HNRNPK and SAFB, are incorporated into endosomal intraluminal vesicles by the LC3-dependent endosomal microautophagy-like pathway, referred to as LC3-dependent extracellular vesicle loading and secretion (LDELS)107. This process differs from canonical endosomal microautophagy in that it is independent of ESCRTs; however, it is dependent on ceramide produced by neutral sphingomyelinase 2 (nSMase2, also known as SMPD3), which is an alternative pathway of endosomal membrane invagination (Table 2).

Cargo recognition

For cargo recognition, ATG-dependent microautophagy in yeast utilizes Atg8 and/or Atg11, which interact(s) with organelle-bound selective macroautophagy receptors, including Atg30 in micropexophagy108 and Atg39 in micronucleophagy101 (part (1) of Fig. 2b,c). Additionally, in mammals, the ER-phagy receptor SEC62 mediates microER-phagy in an ATG8 binding-dependent manner109.

By contrast, cargo recognition in ATG-independent microautophagy is not well understood. Specific subdomain formation may be important. For example, in microER-phagy in yeast, the ER membrane forms multilamellar whorls consisting of ribosome-free ER membrane to be subjected to microautophagy110. In Schizosaccharomyces pombe, microautophagy is used for a biosynthesis pathway for vacuolar enzymes, termed the Nbr1-mediated vacuolar targeting (NVT) pathway (part (2) of Fig. 2b,c), which is functionally similar to the cytoplasm-to-vacuole targeting (Cvt) pathway in S. cerevisiae but uses a different route8,111. This pathway is independent of ATG proteins but requires Nbr1 to recognize its cargos, such as aminopeptidases (Ape2, Ape4 and Lap2) and α-mannosidase (Ams1). In the NVT pathway, recruitment of Nbr1 to the endosomal membranes is mediated by ubiquitin8, in sharp contrast to the process of macroautophagy, in which mammalian NBR1 (or its yeast homologue Atg19) is recruited by ATG8.

In mammalian cells, fluid-like ferritin–NCOA4 condensates are subjected to macroautophagy and endosomal microautophagy, both of which require TAX1BP1 for incorporation112,113. Because TAX1BP1 interacts with NCOA4, TAX1BP1 can bridge autophagosomal ATG8 and ferritin–NCOA4 condensates in macroautophagy. However, the mechanism by which TAX1BP1 mediates the incorporation of ferritin–NCOA4 condensates to endosomes has yet to be elucidated, because it is largely independent of ATG8 (ref. 5).

During endosomal microautophagy in mammals, cargo recognition is achieved, in part, by the cytosolic chaperone HSC70/HSPA8 (part (3) of Fig. 2b,c)106. HSC70 recognizes the KFERQ-like motif contained in selective cargos and incorporates them into endosomes (the KFERQ-like motif was originally identified as a signal for chaperone-mediated autophagy11) (Fig. 2b). In this process, HSC70 binds to PS on the endosomal membrane via its cationic domain and induces inward membrane deformation106,114. This KFERQ-dependent endosomal microautophagy is also conserved in Drosophila, despite its lack of chaperone-mediated autophagy115. Notably, this pathway requires Atg1 and Atg13, but not Atg5, Atg7 or Atg12.

In LDELS, ATG8 is required for recognizing the LIR sequence of RNA-binding proteins such as HNRNPK and SAFB107. Membrane invagination occurs even without ATG8 lipidation, but resultant intraluminal vesicles do not contain selective cargos. How ATG8 translocates to endosomes is unknown. A mechanism similar to LAP may be used. Another issue warranting investigation is why only a subset of microautophagy cargos depend on ATG8 to be recognized in both ESCRT-dependent endosomal microautophagy and LDELS.

Autophagy gene mutations and polymorphisms in human diseases

Given the crucial roles of autophagy in various physiological processes, including stress responses and intracellular clearance, it has been postulated that autophagy is involved in the pathogenesis of human diseases. However, it is difficult to determine which diseases are associated with changes in autophagy owing to a lack of methods with which to measure autophagic activity in humans. Nevertheless, recent genetic studies have identified a number of mutations in autophagy-related genes associated with human diseases, suggesting that autophagy alteration contributes to the development of these diseases. Moreover, studies using acute systemic Atg7 knockout and Fip200 knockout mice116,117 and brain-rescued systemic Atg5 knockout mice118 suggest that organs highly susceptible to autophagy deficiency include the nervous system, immune system, liver and intestine. Consistent with these findings, these tissues are often affected in autophagy-gene-related diseases (Tables 3,4). In this section, mutations and polymorphisms of genes involved in general and selective autophagy are discussed. However, it is important to consider that, as emphasized above, most of these autophagy genes also have non-autophagic functions (Table 1). Therefore, the identification of mutations in autophagy genes does not directly implicate a defect in canonical autophagy in the disease phenotype. The involvement of non-autophagic function should always be considered in the interpretation of these mutations.

Table 3 Mendelian diseases associated with autophagy-related-gene mutations

Full size table

Table 4 Autophagy-related risk factor genes in human diseases

Full size table

Mendelian disorders caused by autophagy gene mutations

Autophagy-related diseases include Mendelian disorders caused by mutations in autophagy genes (Table 3). The most frequently affected tissue seems to be the nervous system. Homozygous mutations in ATG5 and ATG7 were found to be associated with human neurological diseases119,120. Autophagy is suppressed in these diseases, but only partially, because small amounts of either the ATG12–ATG5 conjugate or LC3-II (the lipidated form) can be detected. Patients with these diseases arising from mutations in ATG5 and ATG7 show some overlapping phenotypes, including ataxia and developmental delay. Patients with ATG7 mutations also show abnormal cerebellum and corpus callosum structure and facial dysmorphism (it is unknown whether patients with ATG5 mutations have these abnormalities). SQSTM1 accumulates inpatient-derived cells, confirm‎ing reduced autophagic flux119,120.

Mutations in the PROPPIN family of proteins also cause neurodegenerative diseases, but their phenotypes are somewhat different. A homozygous mutation in WIPI2 was found in patients with a complex developmental disorder known as intellectual developmental disorder with short stature and variable skeletal anomalies (IDDSSA)121,122,123,124. The detected Val249Met mutation reduces WIPI2–ATG16L1 binding and autophagic flux123. Homozygous nonsense mutations in WDR45B/WIPI3 cause neurodevelopmental disorder with spastic quadriplegia and brain abnormalities with or without seizures (also called El-Hattab–Alkuraya syndrome)125,126,127,128. The WDR45/WIPI4 gene is found on the X chromosome, and its heterozygous mutations in women and hemizygous mutations in men cause β-propeller protein-associated neurodegeneration (BPAN; originally called static encephalopathy of childhood with neurodegeneration in adulthood)129,130,131. This is a biphasic disease that demonstrates infant-onset, non-progressive psychomotor retardation, epilepsy and autism as well as adolescent-onset dystonia, Parkinsonism and dementia. Iron accumulation in the globus pallidus and substantia nigra is one of the hallmarks of this disease, but its relationship with ferritinophagy is unclear because iron accumulation has not been reported in other diseases related to autophagy gene mutations. Given that the deletion of either WIPI3 or WIPI4 suppresses autophagy only mildly compared with deletion of WIPI2, ATG5 or ATG7 (N.M., unpublished results), it is plausible that defects in yet-unknown non-autophagic functions of WIPI3 and WIPI4 may account for the severe phenotype observed in these diseases.

Mutations in genes related to selective autophagy also cause disease (Table 3). The mitophagy-related genes PARK2/PRKN (encoding Parkin) and PARK6/PINK1 are mutated in juvenile-onset familial Parkinson disease132. Parkin, a ubiquitin ligase, ubiquitinates various proteins in depolarized mitochondria in a PINK1-dependent manner, recruiting autophagy adaptors such as NDP52 (ref. 16) and OPTN (Fig. 1a, ‘Initiation’)23. Although Parkin- and PINK1-dependent mitophagy is clearly observed in cell culture, its physiological relevance was initially unclear because Prkn or Pink1 knockout mice show almost normal basal mitophagy levels without an obvious phenotype under normal conditions133,134. However, recent studies revealed that aged Prkn knockout mice develop locomotor impairments associated with dopaminergic neuronal loss135. Intestinal infection could also promote neurodegeneration in Prkn knockout mice136. Furthermore, Parkin- and PINK1-dependent mitophagy is physiologically important to suppress the release of mitochondrial DNA into the cytosol and subsequent inflammation under stress conditions in vivo137,138. By contrast, another report suggests that PINK1-dependent mitophagy in endothelial cells could be pro-inflammatory via the release of mitochondrial formyl peptides139. Thus, the pathophysiological role of Parkin- and PINK1-dependent mitophagy may depend on cell type or context. Mutations in autophagy adaptors such as SQSTM1 (ref. 140 and the ER-phagy receptor FAM134B141 are also found in childhood-onset neurodegeneration and hereditary sensory and autonomic neuropathy type II, respectively, suggesting that defects in selective autophagy may cause these diseases (Table 3).

Although the diseases listed above are recessive, some exhibit dominant inheritance, which includes amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) (these two are often associated) and Paget disease of bone. Autosomal dominant mutations in the selective autophagy-related genes SQSTM1, OPTN and TBK1 are found in association with these diseases (Table 4). TBK1 phosphorylates and regulates OPTN, but in addition to this canonical role, TBK1 can also directly recruit the PI3KC3–C1 complex in OPTN-dependent mitophagy142. In general, many ALS/FTD-related gene mutations, for example, those in SOD1 and TARDBP (encoding TDP-43), are thought to be gain-of-function mutations and show dominant inheritance143,144. However, the ALS/FTD-related mutations of OPTN and TBK1 are likely to be loss-of-function mutations144. Both autosomal recessive and dominant inheritance patterns have been reported in ALS with OPTN mutations145. Thus, haploinsufficiency of OPTN and TBK1 could contribute to the ALS/FTD spectrum. Although some of the ALS/FTD-associated mutations affect the adaptor function of SQSTM1, OPTN and TBK1 (refs. 146,147), it remains unknown whether this is the general mechanism. The pathogenic effects of these mutations might be mediated by their non-autophagic roles; for example, the TBK1–OPTN axis is also important for the innate immune and RIPK1-dependent cell death pathways144. However, a gain-of-function hypothesis cannot be entirely excluded. Of particular interest is that, like other ALS-related proteins148, autophagy adaptors such as SQSTM1 (refs. 149,150,151) and OPTN152 form liquid-like biomolecular condensates. Mutations of these genes may exhibit some gain of toxicity.

Although most Mendelian disorders associated with autophagy gene mutations are related to the nervous system, there are some diseases involving other tissues and organs (Table 3). An example is Paget disease of bone, which is characterized by one or multiple focal regions with increased bone remodelling. Of its causative genes, SQSTM1 is the major one; however, it remains elusive whether its autophagy adaptor function is involved in the pathogenesis of this disease153,154.

Genetic risk factors for human diseases

The second category includes diseases whose susceptibility is associated with polymorphism of autophagy-related genes (Table 4). The core autophagy gene first shown to be associated with human disease by a genome-wide association study (GWAS) is ATG16L1; the single nucleotide polymorphism resulting in the Thr300Ala (T300A) substitution is a risk factor for Crohn’s disease155,156; Thr300 is located immediately upstream of the WD40 repeat domain (Table 1). Atg16L1T300A knock-in mice exhibit abnormalities in Paneth cells in the intestine157 and gut microbiota158. The WD40 repeat domain in ATG16L1 is essential for LAP but not for canonical autophagy72; however, the effect of the T300A substitution on autophagy and LAP is relatively small or undetectable157,159,160. Nevertheless, it is possible that autophagy is associated with Crohn’s disease because the disorder has also been linked to other autophagy genes (ULK1, ATG9A, NDP52 and ATG4C)161,162,163,164. However, the fact that ATG16L2, which is considered to be unnecessary for autophagy165,166, is also associated with Crohn’s disease167 suggests that a yet unknown non-autophagic function shared by ATG16L1 and ATG16L2 may be involved.

Autophagy genes such as ATG5, ATG7 and MAP1LC3B have also been identified as susceptibility genes in autoimmune diseases, including systemic lupus erythematosus (SLE)168 (Table 4). This may reflect the role of autophagy in mitochondrial quality control to suppress the release of SLE-inducible damage-associated molecular patterns from mitochondria169,170. In addition to canonical autophagy, these genes are also required for LAP. Because LAP is important for interferon production in response to the incorporation of DNA-containing immune complexes171, the role of autophagy genes in LAP may be related to their genetic association with autoimmune diseases. Association of ATG16L2 with SLE was also identified, but the role of ATG16L2 in LAP is unclear.

Whole-exome sequencing and subsequent missense variant searches in patients with non-alcoholic fatty liver disease revealed an enrichment of the Phe426Leu and Val471Ala variants of ATG7 (ref. 172); both are loss-of-function mutations. However, these findings are inconsistent with results obtained in mice showing that a loss of autophagy instead suppresses liver steatosis173. Thus, partially reduced autophagic activity in humans may have an impact on the liver different from that caused by the complete loss of autophagy observed in autophagy gene knockout mice.

The relationship between autophagy and cancer has attracted much attention. However, although there are numerous reports suggesting the association of specific tumours with autophagy gene single nucleotide polymorphisms, recurrent or driver mutations of core autophagy genes in human cancers are rather rare174. Thus, autophagy may be still functional in most cancers and could even be important. In fact, mouse studies have suggested that, while the deletion of autophagy genes might promote tumorigenesis, it also affects tumour growth either through cell-autonomous or -nonautonomous mechanisms175. Nevertheless, mutations in core ATG genes might be associated with familial cancers. For example, a linkage study identified an association of a germline nonsense mutation of ATG7 (c.2000C>T p.Arg659*) with familial cholangiocarcinoma176. In cancer cells, somatic deletion of ATG7 occurs in the complementary allele, leading to complete inhibition of autophagy. This case suggests that autophagy suppression could also be tumorigenic in humans.

Conclusions and perspective

A quarter century has passed since the first autophagy gene ATG1, named APG1 initially, was cloned in yeast in 1997 (ref. 177). During this period, our understanding of the molecular biology underlying autophagy has grown exponentially. In particular, recent structural biological approaches have provided crucial evidence to explain the unique membrane dynamics of autophagy at the molecular level. However, despite the increasing clarity of the functions of individual autophagy gene products, several key cell biological questions remain unanswered. For example, what is the mechanism of unidirectional transport of lipids from the ER to autophagosomes? How is the size of autophagosomes regulated? How is the timing of autophagosome–lysosome fusion regulated? To address these questions, new approaches, including biophysics, theoretical modelling and molecular dynamics simulation, will be useful.

1997년 효모에서 최초의 자가포식 유전자 ATG1(초기에는 APG1로 명명)이 복제된 지 25년이 흘렀습니다(참고 177). 이 기간 동안 오토파지의 기초가 되는 분자 생물학에 대한 이해는 기하급수적으로 증가했습니다. 특히 최근의 구조 생물학적 접근법은 오토파지의 독특한 막 역학을 분자 수준에서 설명할 수 있는 중요한 증거를 제공했습니다. 그러나 개별 오토파지 유전자 산물의 기능이 점점 더 명확해지고 있음에도 불구하고 몇 가지 주요 세포 생물학적 질문은 여전히 해결되지 않은 채로 남아 있습니다. 

예를 들어, 

지질이 ER에서 오토파지솜으로 단방향으로 운반되는 메커니즘은 무엇일까요? 

오토파지솜의 크기는 어떻게 조절되나요? 

오토파지좀-리소좀 융합의 타이밍은 어떻게 조절될까요? 

이러한 질문을 해결하기 위해서는 생물물리학, 이론적 모델링, 분자 역학 시뮬레이션 등 새로운 접근 방식이 유용할 것입니다.

Although we have aimed to summarize the latest knowledge about microautophagy, our efforts may seem incomplete because its mechanisms are still less understood than those of macroautophagy. It is intriguing that some ATG proteins (for example, ATG8) and selective autophagy adaptors (for example, NBR1) are used by both macroautophagy and microautophagy. Whether these molecules exert similar functions in both pathways needs to be elucidated in future studies. In addition, some cargos (for example, ferritin) are selectively degraded by both pathways, but the regulation mechanisms of sorting remain unknown. Further research will reveal a more complete picture of autophagy.

우리는 마이크로 오토파지에 대한 최신 지식을 요약하고자 했지만, 마이크로 오토파지의 메커니즘이 거대 오토파지에 비해 아직 덜 이해되었기 때문에 우리의 노력이 불완전해 보일 수 있습니다. 일부 ATG 단백질(예: ATG8)과 선택적 오토파지 어댑터(예: NBR1)가 매크로 오토파지와 마이크로 오토파지 모두에서 사용된다는 점은 흥미롭습니다. 이러한 분자가 두 경로 모두에서 유사한 기능을 발휘하는지 여부는 향후 연구를 통해 밝혀져야 합니다. 또한 일부 화물(예: 페리틴)은 두 경로 모두에서 선택적으로 분해되지만, 분류의 조절 메커니즘은 아직 알려지지 않았습니다. 추가 연구를 통해 오토파지에 대한 보다 완전한 그림이 드러날 것입니다.

As we mentioned above, one of the apparent bottlenecks in autophagy research is the lack of methods with which to monitor autophagy in humans. It would be ideal if we could estimate autophagic activity by measuring some metabolites (that is, biomarkers) in the blood or urine that are secreted via autophagy-dependent pathways. Alternative techniques may be noninvasive imaging such as fluorescence molecular tomography178 and positron emission tomography179.

위에서 언급했듯이, 오토파지 연구의 명백한 장애물 중 하나는 인간의 오토파지를 모니터링할 수 있는 방법이 부족하다는 것입니다. 자가포식 의존 경로를 통해 분비되는 혈액이나 소변의 일부 대사물질(즉, 바이오마커)을 측정하여 자가포식 활동을 추정할 수 있다면 이상적일 것입니다. 형광 분자 단층 촬영178 및 양전자 방출 단층 촬영179과 같은 비침습적 영상 기법이 대안이 될 수 있습니다.

Finally, although many diseases have been found to be linked to autophagy gene mutations or associated with polymorphisms of autophagy genes, the phenotypes of these diseases are diverse. Thus, it is still difficult to offer a unified explanation of their pathogenesis. This may be due to complementation by homologues, differences in tissue expression‎ and involvement of non-autophagic functions. More investigations will be required to reveal the exact mechanisms by which autophagy gene defects cause a wide range of human diseases.

마지막으로, 많은 질병이 오토파지 유전자 돌연변이와 관련이 있거나 오토파지 유전자의 다형성과 연관된 것으로 밝혀졌지만, 이러한 질병의 표현형은 다양합니다. 따라서 발병 기전에 대한 통일된 설명을 제공하기는 여전히 어렵습니다. 이는 상동체에 의한 보완, 조직 발현의 차이 및 비자가포식 기능의 관여 때문일 수 있습니다. 오토파지 유전자 결함이 다양한 인간 질병을 유발하는 정확한 메커니즘을 밝히기 위해서는 더 많은 연구가 필요합니다.

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