단백질 오접힘과 응집을 방지하는 샤페론 감시 .. protostasis 탐구!!

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대부분의 단백질은 기능적 활성을 얻기 위해 정해진 3차원 구조로 접혀야 합니다. 그러나 세포 환경에서 새로 합성된 단백질은 비정상적인 접힘과 응집으로 인해 독성 종을 형성할 위험이 큽니다. 이러한 위험을 피하기 위해 세포는 복잡한 분자 샤프론 네트워크에 투자하며, 이 네트워크는 독창적인 메커니즘을 사용하여 응집을 방지하고 효율적인 폴딩을 촉진합니다. 단백질 분자는 매우 역동적이기 때문에 단백질 항상성(프로테오스테시스)을 보장하기 위해서는 지속적인 샤프론 감시가 필요합니다. 최근의 연구 결과에 따르면 노화와 관련된 단백질 항상성 능력의 감소는 알츠하이머병과 파킨슨병을 비롯한 다양한 단백질 응집 질환을 유발할 수 있다고 합니다. 이러한 질환 및 기타 여러 병리 상태에 대한 개입은 단백질체 유지의 기본이 되는 경로에 대한 상세한 이해에서 비롯될 수 있습니다.

Adv Exp Med Biol

. Author manuscript; available in PMC: 2021 Apr 2.

Published in final edited form as: Adv Exp Med Biol. 2020;1243:53–68. doi: 10.1007/978-3-030-40204-4_4

Challenging Proteostasis:

Role of the Chaperone Network to Control Aggregation-Prone Proteins in Human Disease

Tessa Sinnige 1, Anan Yu 1, Richard I Morimoto 1

• Author information
• Copyright and License information

PMCID: PMC8018701  NIHMSID: NIHMS1659903  PMID: 32297211

The publisher's version of this article is available at Adv Exp Med Biol

Abstract

Protein homeostasis (Proteostasis) is essential for correct and efficient protein function within the living cell. Among the critical components of the Proteostasis Network (PN) are molecular chaperones that serve widely in protein biogenesis under physiological conditions, and prevent protein misfolding and aggregation enhanced by conditions of cellular stress. For Alzheimer’s, Parkinson’s, Huntington’s diseases and ALS, multiple classes of molecular chaperones interact with the highly aggregation-prone proteins amyloid-β, tau, α-synuclein, huntingtin and SOD1 to influence the course of proteotoxicity associated with these neurodegenerative diseases. Accordingly, overexpression‎ of molecular chaperones and induction of the heat shock response have been shown to be protective in a wide range of animal models of these diseases. In contrast, for cancer cells the upregulation of chaperones has the undesirable effect of promoting cellular survival and tumor growth by stabilizing mutant oncoproteins. In both situations, physiological levels of molecular chaperones eventually become functionally compromised by the persistence of misfolded substrates, leading to a decline in global protein homeostasis and the dysregulation of diverse cellular pathways. The phenomenon of chaperone competition may underlie the broad pathology observed in aging and neurodegenerative diseases, and restoration of physiological protein homeostasis may be a suitable therapeutic avenue for neurodegeneration as well as for cancer.

요약

단백질 항상성(프로테오스타시스)은 

살아있는 세포 내에서 정확하고 효율적인 단백질 기능에 필수적입니다. 

단백질 항상성 네트워크(PN)의 중요한 구성 요소 중에는 생리적 조건에서 

단백질 생성에 광범위하게 작용하고 

세포 스트레스 조건에 의해 강화되는 

단백질 오접힘과 응집을 방지하는 분자 샤페론이 있습니다. 

알츠하이머, 파킨슨병, 헌팅턴병, 루게릭병의 경우 

여러 종류의 분자 샤프론이 응집성이 

높은 단백질인 아밀로이드-β, 타우, α-시누클린, 헌팅틴 및 SOD1과 상호작용하여 

이러한 신경 퇴행성 질환과 관련된 단백질 독성의 진행에 영향을 미칩니다. 

따라서 

분자 샤프론의 과발현과 열충격 반응의 유도는 

이러한 질환의 광범위한 동물 모델에서 보호 효과가 있는 것으로 나타났습니다. 

반대로 

암세포의 경우 샤프론의 상향 조절은 

돌연변이 종양 단백질을 안정화하여 

세포 생존과 종양 성장을 촉진하는 바람직하지 않은 효과를 가져옵니다. 

두 상황 모두에서 

분자 샤프론의 생리학적 수준은 

결국 잘못 접힌 기질의 지속성으로 인해 기능적으로 손상되어 

전체 단백질 항상성이 저하되고 

다양한 세포 경로의 조절 장애로 이어집니다. 

샤프론 경쟁 현상은 

노화 및 신경 퇴행성 질환에서 관찰되는 광범위한 병리의 기저에 있을 수 있으며, 

생리적 단백질 항상성의 회복은 암뿐만 아니라 

신경 퇴행에 적합한 치료 방법이 될 수 있습니다.

Keywords: Protein misfolding, Molecular chaperones, Neurodegenerative diseases, Proteostasis

4.1. Introduction

Protein homeostasis is regulated by the proteostasis network (PN) to control protein synthesis, folding and macromolecular assembly, localization, and degradation, processes that are essential for all living cells and organisms. An unbalance in the PN enhances the properties of destabilized mutant proteins that take advantage of the capacity of molecular chaperones to escape unfolding and degradation, leading to malignant phenotypes in cancer (see other chapters in this collection). The opposite scenario of failure of protein homeostasis is associated with aging and a plethora of protein misfolding diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS). In this chapter, we discuss how interactions between molecular chaperones and neurodegenerative disease-associated substrates amyloid-β (Aβ), tau, α-synuclein, polyglutamine-expansion proteins and SOD1 are exploited in vivo to counteract toxic protein aggregation, whereas these same interactions can lead to the sequestration of molecular chaperones and the collapse of proteostasis.

The main classes of molecular chaperones are also known as heat shock proteins (Hsps) after the discovery in Drosophila that the expression‎ of Hsps is induced by heat shock, a sudden increase in temperature. However, many of the genes encoding these chaperones are also constitutively expressed to ensure the proper balance of protein synthesis, folding, trafficking and translocation of a wide variety of proteins under physiological conditions. Under conditions of cellular stress such as heat shock, molecular chaperones such as Hsp70 and the J-domain protein Hdj-1 are titrated by misfolded client proteins from association with heat shock transcription factor 1 (HSF1) with which they interact under normal growth conditions (Zheng et al. 2016; Abravaya et al. 1992; Shi et al. 1998). Upon release from chaperones, HSF1 forms functional trimers that bind to heat shock elements in the promoters of genes encoding molecular chaperones and other components of the PN. This results in the release of the paused RNA polymerase II, posttranslational modifications of HSF1 and rapid inducible transcription of the heat shock genes. The heat shock transcriptional response can attenuate either during prolonged exposure to heat shock stress as translation is arrested at the heat shock temperature, or upon return to ambient conditions, in both scenarios reducing the requirement for chaperones to prevent further misfolding (Abravaya et al. 1992; Shi et al. 1998; Krakowiak et al. 2018).

Among the most ubiquitous chaperones are the Hsp70 family that is essential for protein synthesis and folding of a wide range of client proteins, via a mechanism that involves cycles of substrate binding and release driven by ATP hydrolysis. Hsp70 and its constitutively expressed counterpart Hsc70 function in a molecular machine that involves co-chaperones including Hsp40 proteins, which recruit substrates and regulate ATP hydrolysis of Hsp70, and Hsp110 and BAG proteins, which serve as nucleotide exchange factors (NEFs) (Kampinga and Craig 2010; Kim et al. 2013). Depending on the nucleotide binding and co-chaperone interactions, Hsp70 can function to hold non-native clients in a folding competent state or direct folding to a functional state (Freeman et al. 1995; Freeman and Morimoto 1996). Furthermore, specific combinations of Hsp70, Hsp40 and Hsp110 proteins form disaggregase machineries that can resolve luciferase aggregates (Nillegoda et al. 2015) and α-synuclein fibrils (Gao et al. 2015). The Hsp70 chaperone system also interacts with Hsp90, which is another ATP-dependent chaperone. Hsp90 interacts with specific co-chaperones downstream of Hsp70 to aid the folding of a wide array of clients including kinases, phosphatases, transcription factors and other signalling molecules (Morán Luengo et al. 2019). The chaperonins of the Hsp60 family, corresponding to GroEL in bacteria and TRiC/CCT in eukaryotes, exist as multimeric assemblies that form a cage in which substrates are allowed to obtain their native fold dependent upon ATP hydrolysis. This machinery is critical to fold large filamentous proteins such as actin and tubulin (Hayer-Hartl et al. 2016; Gestaut et al. 2019). The small Hsps (sHsps) are ATP-independent chaperones and can function in holding denatured or non-native protein conformations to prevent their misfolding and aggregation (Treweek et al. 2015).

In this chapter we will discuss the detailed modes of interaction between these classes of molecular chaperones and aggregation-prone proteins and peptides as characterised in vitro, and summarize evidence that chaperone activity is beneficial to combat protein aggregation in in vivo models of neurodegenerative diseases. Under these disease conditions, molecular chaperones may eventually become overwhelmed by these misfolding-prone proteins, leading to impaired protein homeostasis of physiological processes in the cell. The concept of chaperone competition not only serves as an explanation for the collapse of protein homeostasis during aging and neurodegenerative diseases, but also provides opportunities for therapeutic intervention. These approaches are also relevant to cancer in which it has been shown that the downregulation of certain molecular chaperones can be exploited to induce chaperone competition, promoting aggregation or degradation of oncoproteins and halting further proliferation of tumor cells (see other chapters in this collection).

4.1. 소개

단백질 항상성은 모든 살아있는 세포와 유기체에 필수적인 과정인 단백질 합성, 접힘 및 거대 분자 조립, 국소화 및 분해를 제어하기 위해 단백질 항상성 네트워크(PN)에 의해 조절됩니다.

PN의 불균형은 분자 샤프론의 능력을 이용하여 폴딩과 분해를 피하는

불안정한 돌연변이 단백질의 특성을 강화하여

암의 악성 표현형을 유발합니다(이 컬렉션의 다른 챕터 참조).

단백질 항상성 실패의 반대 시나리오는

노화와 알츠하이머병(AD), 파킨슨병(PD), 헌팅턴병(HD), 근위축성 측색 경화증(ALS)을 비롯한

수많은 단백질 오접힘 질환과 관련이 있습니다.

이 장에서는

분자 샤프론과 신경 퇴행성 질환 관련 기질인

아밀로이드-β(Aβ), 타우, α-시누클레인, 폴리글루타민 확장 단백질 및 SOD1 간의 상호작용이

생체 내에서 어떻게 독성 단백질 응집에 대응하기 위해 이용되는지,

그리고 이러한 동일한 상호작용이

분자 샤프론의 격리와 단백질 정체성의 붕괴로 이어질 수 있는지에 대해 설명합니다.

분자 샤프론의 주요 클래스는

열충격 단백질(Hsps)로도 알려져 있는데,

이는 초파리에서 열충격 단백질의 발현이

급격한 온도 상승인 열충격에 의해 유도된다는 사실을 발견한 이후부터입니다.

그러나

이러한 샤프론을 코딩하는 많은 유전자는

생리적 조건에서 다양한 단백질의 단백질 합성, 접힘, 이동 및 전위의 적절한 균형을 보장하기 위해

구성적으로 발현됩니다.

열충격과 같은 세포 스트레스 조건에서

Hsp70 및 J 도메인 단백질 Hdj-1과 같은 분자 샤프론은

정상적인 성장 조건에서 상호작용하는 열충격 전사인자 1(HSF1)과의 결합으로부터

잘못 접힌 클라이언트 단백질에 의해 적정됩니다(Zheng et al. 2016; Abravaya et al. 1992; Shi et al. 1998).

샤페론에서 방출되면

HSF1은 분자 샤페론과 PN의 다른 구성 요소를 코딩하는 유전자의 프로모터에서

열 충격 요소에 결합하는 기능성 트리머를 형성합니다.

그 결과 일시 정지된 RNA 중합효소 II가 방출되고,

HSF1의 번역 후 변형과 열 충격 유전자의 빠른 유도성 전사가 이루어집니다.

열충격 전사 반응은 열충격 온도에서 번역이 중단되어

열충격 스트레스에 장기간 노출되는 동안 또는 주변 조건으로 돌아오면 약화될 수 있으며,

두 시나리오 모두에서 추가적인 오접힘을 방지하기 위한

샤프론의 필요성이 감소합니다(Abravaya 외. 1992; Shi 외. 1998; Krakowiak 외. 2018).

가장 보편적인 샤페론 중에는

ATP 가수분해에 의해 구동되는 기질 결합 및 방출 주기를 포함하는 메커니즘을 통해

광범위한 클라이언트 단백질의 단백질 합성 및 폴딩에 필수적인 Hsp70 계열이 있습니다.

Hsp70과 구성적으로 발현되는 대응 단백질인 Hsc70은

기질을 모집하고 Hsp70의 ATP 가수분해를 조절하는 Hsp40 단백질과

뉴클레오티드 교환 인자(NEF) 역할을 하는 Hsp110 및 BAG 단백질 등의

공동 샤페론과 관련된 분자 기계에서 기능합니다(Kampinga and Craig 2010; Kim et al. 2013).

뉴클레오타이드 결합 및 공동 샤페론 상호 작용에 따라

Hsp70은 비네이티브 클라이언트를 폴딩 유능 상태로 유지하거나

폴딩을 기능 상태로 유도하는 기능을 할 수 있습니다(Freeman et al. 1995; Freeman and Morimoto 1996).

또한,

Hsp70, Hsp40 및 Hsp110 단백질의 특정 조합은

루시퍼라제 응집체(Nillegoda 등. 2015)와 α-시뉴클린 피브릴(Gao 등. 2015)을 분해할 수 있는

분해효소 메커니즘을 형성합니다.

Hsp70 샤프론 시스템은 또 다른 ATP 의존 샤프론인 Hsp90과도 상호 작용합니다.

Hsp90은 Hsp70의 하류에 있는 특정 공동 샤페론과 상호작용하여 키나아제, 포스파타제, 전사인자 및 기타 신호 분자를 포함한 광범위한 클라이언트의 폴딩을 지원합니다(Morán Luengo 외. 2019).

박테리아의 GroEL과 진핵생물의 TRiC/CCT에 해당하는 Hsp60 계열의 샤페로니닌은 ATP 가수분해에 따라 기질이 고유한 폴드를 얻을 수 있는 케이지를 형성하는 다합체 어셈블리로 존재합니다. 이 기계는 액틴과 튜불린과 같은 큰 필라멘트 단백질을 접는 데 매우 중요합니다(Hayer-Hartl 외. 2016; Gestaut 외. 2019). 작은 Hsp(sHsp)는 ATP 독립적인 샤프론으로 변성 또는 비본래 단백질 형태를 유지하여 단백질의 잘못된 접힘과 응집을 방지하는 기능을 합니다(Treweek et al. 2015).

이 장에서는

이러한 종류의 분자 샤프론과 응집에 취약한 단백질 및 펩타이드 간의

상세한 상호작용 방식을 시험관 내에서 특성화하고,

생체 내 신경 퇴행성 질환 모델에서 샤프론 활동이

단백질 응집을 막는 데 유익하다는 증거를 요약하여 설명합니다.

이러한 질병 조건에서 분자 샤프론은

결국 이러한 잘못 접히기 쉬운 단백질에 압도되어

세포의 생리학적 과정의 단백질 항상성을 손상시킬 수 있습니다.

샤프론 경쟁의 개념은

노화와 신경 퇴행성 질환 중 단백질 항상성 붕괴를 설명할 뿐만 아니라

치료적 개입의 기회도 제공합니다.

이러한 접근법은

특정 분자 샤프론의 하향 조절이 샤프론 경쟁을 유도하여

종양 단백질의 응집 또는 분해를 촉진하고

종양 세포의 추가 증식을 중단하는 데 이용될 수 있다는 사실이 밝혀진

암과도 관련이 있습니다(이 컬렉션의 다른 챕터 참조).

4.2. Molecular Chaperones and Protein Aggregation in Neurodegenerative Diseases4.2.1. Amyloid-β

Aβ is the main component of extracellular plaques that accumulate in the brains of AD patients. Processing of the amyloid precursor protein (APP) generates multiple isoforms of the Aβ peptide of which the most abundant species, the 42-residue Aβ peptide (Aβ42) is more aggregation-prone than the 40-residue form. In vitro, the aggregation of Aβ42 is delayed by the intracellular chaperones Hsp70 and Hsp90 at sub-stoichiometric ratios of ~1:50 (Evans et al. 2006). This effect is dependent on ATP, and the potency of Hsp70 is increased by the co-chaperone Hsp40 (family member DNAJB1), which stimulates ATPase activity, suggesting that to prevent aggregation the chaperones employ the same catalytic cycle that aids protein folding (Evans et al. 2006). Suppression of Aβ42 aggregation can also be achieved by sub-stoichiometric levels of DNAJB6, a member of the Hsp40 family, and quantitative analysis has revealed that DNAJB6 blocks both primary and secondary nucleation (Fig. 4.1) by preferentially binding to oligomeric species (Månsson et al. 2014a). The protective effects of Hsp70 against Aβ-associated toxicity have also been observed in vivo in the fruit fly Drosophila melanogaster. Overexpression‎ of Hsp70 effectively suppresses neurotoxicity associated with the extracellular deposition of Aβ42 in the fly disease model, irrespective of whether the chaperone is expressed intracellularly or targeted to the extracellular space (Fernandez-Funez et al. 2016; Martín-Peña et al. 2018). The beneficial effect of intracellular Hsp70 may occur by a general enhancement of global proteostasis, whereas in the extracellular space it likely depends on its ‘holding’ activity in the absence of ATP.

4.2. 신경 퇴행성 질환의 분자 샤페론 및 단백질 응집4.2.1. 아밀로이드-β

Aβ는 알츠하이머병 환자의 뇌에 축적되는 세포 외 플라크의 주성분입니다.

아밀로이드 전구체 단백질(APP)을 처리하면 Aβ 펩타이드의 여러 이소형이 생성되며,

이 중 가장 풍부한 42-잔기 Aβ 펩타이드(Aβ42)는 40-잔기 형태보다 응집 경향이 더 강합니다.

시험관 내에서 Aβ42의 응집은 ~1:50의 화학량론적 비율로

세포 내 샤프론인 Hsp70과 Hsp90에 의해 지연됩니다(Evans et al. 2006).

이 효과는 ATP에 의존하며,

Hsp70의 효능은 공동 샤프론인 Hsp40(가족 구성원인 DNAJB1)에 의해 증가하는데,

이는 ATPase 활동을 자극하여 응집을 방지하기 위해

샤프론이 단백질 폴딩을 돕는 동일한 촉매 주기를 사용함을 시사합니다(Evans et al. 2006).

Aβ42 응집의 억제는

또한 Hsp40 계열의 구성원인 DNAJB6의 화학량론적 수준 이하에 의해 달성될 수 있으며,

정량적 분석에 따르면

DNAJB6는 올리고머 종에 우선적으로 결합하여

1차 및 2차 핵 생성을 모두 차단합니다(그림 4.1)입니다(Månsson et al. 2014a).

Aβ 관련 독성에 대한 Hsp70의 보호 효과는 초파리 초파리 멜라노가스터에서 생체 내에서도 관찰되었습니다. Hsp70의 과발현은 샤프론이 세포 내에서 발현되는지 또는 세포 외 공간으로 표적화되는지에 관계없이 파리 질병 모델에서 Aβ42의 세포 외 침착과 관련된 신경 독성을 효과적으로 억제합니다(Fernandez-Funez 등. 2016; Martín-Peña 등. 2018).

세포 내 Hsp70의 유익한 효과는

전반적인 단백질 안정성의 일반적인 향상에 의해 발생할 수 있는 반면,

세포 외 공간에서는 ATP가 없을 때 '유지' 활동에 의존할 가능성이 높습니다.

Fig. 4.1.

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Schematic reaction of amyloid formation indicating where chaperones act to prevent protein misfolding in the case of amyloid-β, tau, α-synuclein, polyglutamine expansion proteins and/or SOD1. Inhibition of primary nucleation is inferred from the binding of chaperones to the monomeric proteins. Hsp90 alone and with its co-chaperone Aha1 has also been reported to promote aggregation in the case of tau

아밀로이드-β, 타우, α-시누클레인, 폴리글루타민 확장 단백질 및/또는 SOD1의 경우 단백질 오접힘을 방지하기 위해 샤프론이 작용하는 위치를 나타내는 아밀로이드 형성의 개략적인 반응입니다. 1차 핵 생성의 억제는 샤페론이 단량체 단백질에 결합하는 것으로 추론할 수 있습니다. Hsp90 단독 또는 공동 샤프론인 Aha1과 함께 사용하면 타우의 경우 응집을 촉진하는 것으로 보고되었습니다.

Another class of chaperone activities is represented by the Brichos domains of ProSP-C and Bri2 that inhibit Aβ42 aggregation in vitro. Whereas the Brichos domain of ProSP-C blocks secondary nucleation of Aβ42 by binding to the fibrillar surface (Cohen et al. 2015), the Brichos domain derived from Bri2 prevents elongation of Aβ42 in addition to secondary nucleation by binding both to the fibril surface and to fibril ends (Arosio et al. 2016) (Fig. 4.1). Brichos domains physiologically function in the extracellular space, and expression‎ of Brichos domains from ProSP-C and from Bri2 has been shown to be protective in Drosophila Aβ42 models by improving motor function and lifespan (Hermansson et al. 2014; Poska et al. 2016). Overexpression‎ of the Brichos domain of ProSP-C increases the levels of soluble versus insoluble Aβ42 and delays its deposition during aging of the flies, suggesting that this Brichos domain could act by preventing the formation of toxic oligomeric species (Hermansson et al. 2014). In further support, ProSP-C Brichos rescued the toxicity of a mixture of Aβ42 monomers and fibrils on mouse brain slices using an electrophysiology assay (Cohen et al. 2015).

A role for the TRiC/CCT chaperonin on Aβ42 phenotypes was demonstrated in a genetic screen of the chaperome in C. elegans expressing Aβ42 in the body wall muscle cells. Individual knock-down of each of the eight subunits of TRiC/CCT decreased the motility of the worms, and the screen also identified Hsc70, Hsp40, Hsp90 and its co-chaperones Cdc37 and Sti1 (Brehme et al. 2014). Many of the same chaperones were shown to be protective against toxicity in a C. elegans model expressing expanded polyglutamine, suggesting that this subset of chaperones may have a general beneficial effect on proteostasis, rather than or in addition to making direct interactions with Aβ42. The relevance of TRiC/CCT was furthermore underlined by several of its subunits being downregulated in the aging human brain and in patients with AD (Brehme et al. 2014).

sHsps, a class of ATP-independent chaperones, have also been linked to Aβ aggregation, as it was shown that expression‎ of human Aβ in C. elegans body wall muscle cells induces Hsp16 (Link et al. 1999), the overexpression‎ of which completely restores the paralysis phenotype of the Aβ worms (Fonte et al. 2008). This effect appears to result from a direct interaction that modulates aggregation, not only because Hsp16 co-localises with the deposits, but also because its overexpression‎ reduces the amyloid plaque load in the worms, leaving total Aβ levels unaltered. In vitro, the sHsp αB-crystallin binds along the sides and at the ends of Aβ42 fibrils, suggesting it can inhibit secondary nucleation and elongation of the fibrils (Shammas et al. 2011).

An important aspect of chaperone biology is that not all chaperones are protective against protein aggregation. The extracellular chaperone clusterin, which is a risk factor for late-onset AD, was reported to have more complex effects in mouse models of Aβ aggregation, presumably because it shifts the clearance pathways of Aβ, thus complicating the interpretation of its potential anti-aggregation effect (DeMattos et al. 2002; Wojtas et al. 2017). Extracellular chaperones may also alter Aβ toxicity by modulating targets that interact with toxic Aβ species. Sti1, which is a co-chaperone of Hsp90, but observed to be secreted by astrocytes, was shown to bind to the PrPC receptor and thereby block its interaction with Aβ oligomers in vitro and in cell culture (Ostapchenko et al. 2013). Increased levels of Sti1 have been observed in aged AD mice, as well as in human AD patients compared to control brains, consistent with a protective mechanism (Ostapchenko et al. 2013).

또 다른 종류의 샤페론 활동은 시험관 내에서 Aβ42 응집을 억제하는 ProSP-C와 Bri2의 브리코스 도메인으로 대표됩니다. ProSP-C의 브리코스 도메인은 피브릴 표면에 결합하여 Aβ42의 2차 핵 형성을 차단하는 반면(Cohen et al. 2015), Bri2에서 파생된 브리코스 도메인은 피브릴 표면과 피브릴 말단에 모두 결합하여 2차 핵 형성뿐만 아니라 Aβ42의 신장을 방지합니다(Arosio et al. 2016)(그림 4.1). 브리코스 도메인은 세포 외 공간에서 생리적으로 기능하며, ProSP-C와 Bri2에서 브리코스 도메인을 발현하면 운동 기능과 수명을 개선하여 초파리 Aβ42 모델을 보호하는 것으로 나타났습니다(Hermansson et al. 2014; Poska et al. 2016). ProSP-C의 브리코스 도메인을 과발현하면 수용성 및 불용성 Aβ42의 수준이 증가하고 파리의 노화 동안 침착이 지연되어 이 브리코스 도메인이 독성 올리고머 종의 형성을 방지하여 작용할 수 있음을 시사합니다(Hermansson et al. 2014). 이를 뒷받침하기 위해 전기생리학 분석법을 사용하여 마우스 뇌 절편에 대한 Aβ42 단량체와 피브릴의 혼합물의 독성을 ProSP-C Brichos가 완화했습니다(Cohen et al. 2015).

체벽 근육 세포에서 Aβ42를 발현하는 C. elegans의 샤페롬 유전자 스크린에서 Aβ42 표현형에 대한 TRiC/CCT 샤페로닌의 역할이 입증되었습니다. TRiC/CCT의 8개 하위 유닛 각각을 개별적으로 녹다운하면 웜의 운동성이 감소했으며, 이 스크린은 또한 Hsc70, Hsp40, Hsp90 및 공동 샤페론인 Cdc37 및 Sti1을 확인했습니다(Brehme et al. 2014). 동일한 샤프론 중 다수가 확장된 폴리글루타민을 발현하는 C. elegans 모델에서 독성으로부터 보호하는 것으로 나타났는데, 이는 이 샤프론의 하위 집합이 Aβ42와 직접적인 상호작용을 하는 것 외에도 단백질 안정성에 일반적으로 유익한 영향을 미칠 수 있음을 시사합니다. 또한 노화된 인간의 뇌와 알츠하이머병 환자에서 TRiC/CCT의 일부 하위 유닛이 하향 조절되는 것으로 밝혀져 그 관련성이 더욱 강조되었습니다(브레흐메 등. 2014).

ATP 독립적 샤프론의 일종인 sHsps도 Aβ 응집과 관련이 있는데, C. elegans 체벽 근육 세포에서 인간 Aβ의 발현이 Hsp16을 유도하는 것으로 나타났으며(Link et al. 1999), 이 과발현은 Aβ 벌레의 마비 표현형을 완전히 회복시킵니다(Fonte et al. 2008). 이 효과는 응집을 조절하는 직접적인 상호 작용에서 비롯된 것으로 보이는데, 이는 Hsp16이 침착물과 공동 위치할 뿐만 아니라 과발현이 웜의 아밀로이드 플라크 부하를 감소시켜 총 Aβ 수치는 변하지 않기 때문입니다. 시험관 내에서 sHsp αB-크리스탈린은 Aβ42 피브릴의 측면과 끝에 결합하여 피브릴의 이차 핵 생성 및 신장을 억제할 수 있음을 시사합니다(Shammas 등. 2011).

샤프론 생물학의 중요한 측면은 모든 샤프론이 단백질 응집으로부터 보호하는 것은 아니라는 점입니다. 후기 발병 AD의 위험 인자인 세포 외 샤페론 클러스터린은 Aβ 응집 마우스 모델에서 더 복잡한 영향을 미치는 것으로 보고되었는데, 이는 아마도 Aβ의 제거 경로를 이동시켜 잠재적인 항응집 효과의 해석을 복잡하게 만들기 때문일 것입니다(DeMattos 등. 2002; Wojtas 등. 2017). 세포 외 샤페론은 또한 독성 Aβ 종과 상호 작용하는 표적을 조절하여 Aβ 독성을 변화시킬 수 있습니다. Hsp90의 공동 샤프론이지만 성상세포에서 분비되는 것으로 관찰된 Sti1은 PrPC 수용체와 결합하여 시험관 및 세포 배양에서 Aβ 올리고머와의 상호작용을 차단하는 것으로 나타났습니다(Ostapchenko 등. 2013). 보호 메커니즘에 따라 대조군 뇌에 비해 노화된 AD 마우스와 인간 AD 환자에서 Sti1의 증가된 수준이 관찰되었습니다(Ostapchenko et al. 2013).

4.2.2. Tau

Tau is a microtubule-associated protein that forms intracellular aggregates in AD brains, and in patients suffering from various types of frontotemporal dementia (FTD) and ALS, collectively known as Tauopathies. Both Hsp70 and Hsp90 chaperones have been shown to directly interact with tau in vitro, which may seem surprising given the disordered and highly hydrophilic nature of tau. However, the sequence motifs that have β-strand propensity, and are involved in the formation of cross-β fibrils, contain hydrophobic residues that mediate binding to the constitutively expressed Hsc70 chaperone (Mitul et al. 2008). Hsp70 also interacts with tau monomers and oligomers to inhibit its nucleation as well as elongation in in vitro aggregation assays (Kundel et al. 2018). In C. elegans models expressing human tau in mechanosensory neurons, co-expression‎ of human Hsp70 has modest beneficial effects on restoration of the touch response (Miyasaka et al. 2005). In mice, overexpression‎ of Hsp70 reduces endogenous tau levels in aged animals, and especially reduces the insoluble high-molecular weight species (Petrucelli et al. 2004). Similar results have been observed using small molecule inhibitors of the ATPase activity of Hsp70 that lead to reduced levels of total and phosphorylated tau in tau transgenic mice. This suggests that both overexpression‎ and inhibition of the folding cycle of Hsp70 may converge to promote tau degradation by the ubiquitin-proteasome system (Jinwal et al. 2009). Both Hsc70/Hsp70 and Hsp90 are co-localized with tau tangles in a transgenic mouse model and in human AD brains; moreover upregulation of these chaperones suppresses the formation of tau aggregation in cellular models by partitioning tau into a productive folding pathway that restores tau binding to microtubules (Luo et al. 2007).

Studies on Hsp90 have shown that the site of Hsp90 binding on tau includes a broad region encompassing the hydrophobic motifs, generating an extended interaction surface held together by a combination of weak hydrophobic and elecrostatic interactions (Karagöz et al. 2014). The levels of phosphorylated tau but not total tau levels in a transgenic mouse model are reduced when the ATPase activity of Hsp90 is inhibited by small molecules (Dickey et al. 2007). Hsp90 inhibition also leads to activation of the heat shock response and the subsequent increase in expression‎ of chaperones including Hsp70, but drug treatment may have a stronger effect than only inducing the heat shock response. Reduction in phospho-tau levels upon the inhibition of Hsp90 may depend on increased activity of its co-chaperone CHIP, which mediates ubiquitination and proteasomal degradation (Dickey et al. 2007). Another small molecule inhibitor of Hsp90 also reduces levels of phosphorylated tau as well as total tau, while increasing Hsp70 levels in mouse models expressing wild-type tau or the FTD-associated tau mutant P301L (Luo et al. 2007). In these studies, Hsp90 interacts directly with the mutant but not wild-type tau, suggesting that the mechanisms promoting tau clearance may differ between the two models, and thus potentially between AD and other types of (familial) dementia (Luo et al. 2007).

Modulation of Hsp90 activity involves co-chaperones such as Aha1, and together these can promote tau aggregation both in vitro and in a tau transgenic mouse model. Moreover, a small molecule that blocks the Hsp90-Aha1 interaction reduces this effect, demonstrating the power of combining mechanistic insights from in vitro experiments with in vivo models in developing therapeutic avenues (Shelton et al. 2017). Co-chaperones may also affect protein aggregation independently, as noted above for Aβ. For tau, it has been shown that the Hsp40 protein DnaJA2 is a potent inhibitor of its aggregation in vitro (Mok et al. 2018). DnaJA2 binds to monomeric tau, and also reduces seeded aggregation in cells, suggesting that DnaJA2 may have an effect on multiple steps of the aggregation process. In AD patient neurons with tau pathology, DnaJA2 is highly abundant and is localised with aggregated tau, perhaps by being upregulated as a protective, but insufficient cellular response (Mok et al. 2018). In contrast, FKBP51, another co-chaperone of Hsp90 that co-localises with tau pathology in AD brains may have a role in promoting tau misfolding (Blair et al. 2013). Overexpression‎ of FKBP51 in tau transgenic mice results in increased overall tau levels and neuronal loss, whereas the numbers of tau tangles are decreased. Consistent with these results, in vitro experiments have suggested that FKBP51 in complex with Hsp90 may especially increase the formation of oligomeric tau species (Blair et al. 2013).

A role for the sHsp Hsp27 in modulating tau was shown in transgenic mice overexpressing Hsp27 that reduced tau levels and ameliorated the defects in long-term potentiation. This effect was shown to depend on the oligomerisation of Hsp27, since a phosphorylation mutant of Hsp27 that cannot undergo this cycle did not affect tau pathology or other phenotypes of the mouse (Abisambra et al. 2010).

4.2.3. α-Synuclein

The 140-residue, largely disordered protein α-synuclein is found in intracellular inclusions termed Lewy Bodies, which form primarily in dopaminergic neurons of the substantia nigra in PD patients. Several Hsps have been identified as components of Lewy Bodies (Auluck et al. 2002; McLean et al. 2002; Outeiro et al. 2006), and sub-stoichiometric concentrations of Hsp70 are sufficient in vitro to suppress the formation of α-synuclein fibrils in the absence of ATP (Dedmon et al. 2005; Luk et al. 2008; Roodveldt et al. 2009; Aprile et al. 2017). This effect is dependent on the interaction of Hsp70 with the hydrophobic NAC region of α-synuclein, which is essential for fibril formation (Luk et al. 2008). Upon addition of ATP, Hsp70 delays fibril formation primarily by binding to the fibril ends, thus inhibiting elongation (Aprile et al. 2017). Furthermore, a complex of the constitutively expressed Hsc70 together with specific Hsp40 co-chaperones and a Hsp110 NEF can dissociate preformed α-synuclein fibrils (Gao et al. 2015). The effect of Hsp70 on α-synuclein aggregation in vivo is thus likely to depend on the relative levels of specific co-chaperones and ATP.

Similarly, in yeast expressing human α-synuclein, induction of Hsp70 expression‎ by a brief heat shock is protective against α-synuclein-induced apoptosis and the generation of reactive oxygen species. Similar effects have been obtained by direct overexpression‎ of the yeast Hsp70 orthologue Ssa3 or by inhibiting Hsp90 using geldanamycin, which also induces the heat shock response (Flower et al. 2005). On the other hand, in C. elegans, knock-down of Hsp70 does not affect α-synuclein inclusion formation in muscle cells, suggesting that this chaperone does not have a beneficial effect in this model system (Van Ham et al. 2008). However, knock-down of Hip, an Hsp70 co-chaperone, significantly increases the number of inclusions in this C. elegans model, suggesting that the Hsp70-Hip complex acts against inclusion formation (Roodveldt et al. 2009).

In a Drosophila model of α-synuclein, co-expression‎ of human Hsp70 with wild-type α-synuclein or the familial mutants A53T or A30P in dopaminergic neurons restores locomotion and lifespan without affecting the number or size of Lewy Body-like inclusions (Auluck et al. 2002). This is further supported by geldanamycin treatment which induces the heat shock response and similarly protects against neurodegeneration (Auluck and Bonini 2002; Auluck et al. 2005), whereas LB-like pathology is not affected and the levels of insoluble α-synuclein are even increased (Auluck et al. 2005), suggesting that Hsp70 may reduce the presence of toxic oligomeric species.

Rodent models for expression‎ of human α-synuclein have yielded inconsistent results. Overexpression‎ of rat Hsp70 in a mouse model resulted in a strong decrease in both high molecular weight α-synuclein species, and Triton X-100 insoluble protein (Klucken et al. 2004), which contrasts with another study in which human Hsp70 and α-synuclein A53T were co-overexpressed and the levels of high molecular weight and insoluble α-synuclein were unaffected (Shimshek et al. 2010). Another study found beneficial effects from co-expressing Hsp70 with α-synuclein in the rat brain, showing a reduction in the number of dystrophic neurites which typically precede neurodegeneration (Moloney et al. 2014). The protective effects of Hsp70 may depend on the ratio of Hsp70, its co-chaperones and α-synuclein in these models as mentioned above, and potential differences in the binding affinities between Hsp70 from different species and wild-type and A53T α-synuclein could furthermore affect the outcome.

In addition to Hsp70 and Hsp40, the expression‎ of Hsp27 is increased upon viral expression‎ of α-synuclein in mouse brains (St Martin et al. 2007). Likewise, mice expressing α-synuclein A53T had increased levels of Hsp25 and αB-crystallin, Hsp25 being primarily increased in astrocytes rather than neurons. αB-crystallin inhibits in vitro aggregation of α-synuclein isolated from the mouse brain (Wang et al. 2008), which is consistent with another in vitro result that αB-crystallin interacts directly with α-synuclein fibrils to prevent fibril elongation from pre-formed seeds (Waudby et al. 2010). It has also been reported to interact with early intermediates in in vitro aggregation reactions (Rekas et al. 2007). In line with these findings, expression‎ of αB-crystallin in the fly eye ameliorates the rough eye phenotype induced by α-synuclein expression‎ (Tue et al. 2012).

Hsp90 can inhibit α-synuclein aggregation in vitro in seeded aggregation assays of α-synuclein A53T. This activity is ATP-independent, and relies on the interaction of Hsp90 with oligomeric α-synuclein species (Daturpalli et al. 2013). The yeast disaggregase Hsp104 can inhibit α-synuclein aggregation and remodel pre-formed α-synuclein fibrils in vitro, and overexpressing it together with the A30P α-synuclein mutant in rats reduces inclusion formation and neuronal loss (Lo Bianco et al. 2008).

4.2.4. Polyglutamine Expansions

Nine human neurodegenerative diseases are associated with genetic expansions leading to the production of different proteins with expanded polyglutamine (polyQ) tracts. Irrespective of the protein, disease symptoms occur beyond a pathogenic threshold of ~35–40 glutamine residues, accompanied by the formation of cytoplasmic and nuclear inclusions in neuronal tissue (Lieberman et al. 2019). The onset of pathology and disease is correlated with the length of the polyQ tract, with longer polyQ lengths having increasing aggregation propensity in vitro and in cellular models. HD is the most preval‎ent of these polyQ disorders, and is related to an expansion within the huntingtin gene HTT. In particular, a fragment of the huntingtin protein corresponding to the first exon of the gene in which the polyQ expansion is located is found to accumulate in disease, and this fragment is sufficient to drive neurodegeneration and inclusion formation in mouse models (Mangiarini et al. 1996; Scherzinger et al. 1997).

In the nematode worm C. elegans, expression‎ of a construct comprising 40 glutamine residues (Q40) with a YFP-tag for visualisation in the body wall muscle cells is sufficient for protein aggregation and formation of toxic immobile inclusions (Morley et al. 2002). Expression‎ of 35 residues (Q35) also leads to aggregation and toxicity, but later in adulthood, whereas shorter polyQ lengths of 19 or 24 glutamine residues remain diffuse. These polyQ lines were used for a genome-wide genetic screen to identify genetic modifiers of protein aggregation which identified components of the proteostasis network for transcription and splicing, translation, folding, transport and degradation including the chaperones Hsp70, Hsp40 and subunits of TRiC/CCT (Nollen et al. 2004). The mechanism by which TRiC/CCT inhibits polyQ aggregation has been further explored in vitro, and it was shown to interact with the tips of mutant huntingtin fibrils and to encapsulate smaller oligomers (Shahmoradian et al. 2013).

Other genetic screens in yeast and Drosophila have additionally identified multiple Hsps, including Hsp70, Hsp40, Hsp90 and sHsps, as well as Hsp104, the yeast disaggregase (Krobitsch and Lindquist 2000; Willingham et al. 2003; Kazemi-Esfarjani and Benzer 2000; Giorgini et al. 2005; Zhang et al. 2010; Jimenez-Sanchez et al. 2015). Overexpression‎ of Hsp70 and Hsp40 ameliorates multiple phenotypes in Drosophila and mouse polyQ disease models, typically without altering the numbers of mature protein aggregates (Warrick et al. 1999; Chan et al. 2000; Cummings et al. 2001; Hay et al. 2004; Labbadia et al. 2012). Consistent with these findings, Hsp70 has been shown to associate with huntingtin oligomers in vitro, but not with monomers or detergent-insoluble inclusions, and it is able to prevent further aggregation together with Hsp40 and in the presence of ATP (Lotz et al. 2010).

More mechanistic studies on the activities of Hsp70, Hsp40 and Hsp110 against huntingtin exon 1 aggregation performed in cell culture have revealed that DNAJB6 and DNAJB8 members of the Hsp40 family are highly effective (Hageman et al. 2010). Subsequently, DNAJB6 was shown to inhibit protein aggregation in an HD mouse model, delaying the onset of symptoms and extending lifespan (Kakkar et al. 2016). In vitro, DNAJB6 inhibits the primary nucleation of polyQ peptides which depends on a serine-threonine rich region on its surface (Kakkar et al. 2016). This activity does not, however, depend on the presence of Hsp70 or ATP (Månsson et al. 2014b), providing an interesting example of independent chaperone activity of Hsp40 proteins.

4.2.4. 폴리글루타민 확장

9가지 인간 신경 퇴행성 질환은 폴리글루타민(polyQ) 관이 확장된 다양한 단백질의 생산으로 이어지는 유전적 확장과 관련이 있습니다. 단백질에 관계없이 질병 증상은 ~35-40 글루타민 잔류물의 병원성 역치 이상으로 발생하며, 신경 조직에 세포질 및 핵 내포물이 형성됩니다(Lieberman et al. 2019). 병리 및 질병의 발병은 polyQ 관의 길이와 상관관계가 있으며, polyQ 길이가 길수록 시험관 및 세포 모델에서 응집 성향이 증가합니다. HD는 이러한 폴리큐 장애 중 가장 흔한 질환으로, 헌팅틴 유전자 HTT의 확장과 관련이 있습니다. 특히, polyQ 확장이 위치한 유전자의 첫 번째 엑손에 해당하는 헌팅틴 단백질 조각이 질병에 축적되는 것으로 밝혀졌으며, 이 조각은 마우스 모델에서 신경 퇴행 및 내포 형성을 유도하기에 충분합니다(Mangiarini 등. 1996; Scherzinger 등. 1997).

선충 선충 C. elegans에서는 체벽 근육 세포에서 시각화를 위한 YFP 태그가 있는 40개의 글루타민 잔기(Q40)로 구성된 구조의 발현이 단백질 응집과 독성 부동 내포물의 형성에 충분합니다(Morley et al. 2002). 35개의 잔기(Q35)의 발현도 응집과 독성을 유발하지만, 19개 또는 24개의 글루타민 잔기의 짧은 polyQ 길이는 성인이 된 후에도 확산 상태를 유지합니다. 이러한 polyQ 라인은 게놈 전체 유전자 스크린에 사용되어 단백질 응집의 유전적 조절자를 식별하는 데 사용되었으며, 이를 통해 전사 및 스플라이싱, 번역, 접힘, 수송 및 분해를 위한 단백질 정체 네트워크의 구성 요소와 샤프론 Hsp70, Hsp40 및 TRiC/CCT의 하위 단위(Nollen et al. 2004)를 확인했습니다. TRiC/CCT가 polyQ 응집을 억제하는 메커니즘은 시험관 내에서 추가로 연구되었으며, 돌연변이 헌팅틴 피브릴의 끝단과 상호 작용하고 더 작은 올리고머를 캡슐화하는 것으로 나타났습니다(Shahmoradian 등. 2013).

효모와 초파리의 다른 유전자 스크린에서는 효모 분해 효소인 Hsp104뿐만 아니라 Hsp70, Hsp40, Hsp90 및 sHsp를 포함한 여러 Hsp를 추가로 확인했습니다(Krobitsch and Lindquist 2000; Willingham 외 2003; Kazemi-Esfarjani and Benzer 2000; Giorgini 외 2005; Zhang 외 2010; Jimenez-Sanchez 외 2015). Hsp70과 Hsp40의 과발현은 일반적으로 성숙한 단백질 응집체의 수를 변경하지 않고 초파리와 마우스 polyQ 질병 모델에서 여러 표현형을 개선합니다(Warrick 외. 1999; Chan 외. 2000; Cummings 외. 2001; Hay 외. 2004; Labbadia 외. 2012). 이러한 연구 결과와 일관되게, Hsp70은 시험관 내에서 헌팅틴 올리고머와 결합하지만 단량체나 세제 불용성 내포물과는 결합하지 않는 것으로 나타났으며, Hsp40과 함께 그리고 ATP가 있을 때 추가 응집을 방지할 수 있습니다(Lotz et al. 2010).

세포 배양에서 수행된 헌팅틴 엑손 1 응집에 대한 Hsp70, Hsp40 및 Hsp110의 활성에 대한 보다 기계적인 연구에 따르면 Hsp40 계열의 DNAJB6 및 DNAJB8 구성원이 매우 효과적이라는 것이 밝혀졌습니다(Hageman et al. 2010). 그 후, DNAJB6는 HD 마우스 모델에서 단백질 응집을 억제하여 증상의 발병을 지연시키고 수명을 연장하는 것으로 나타났습니다(Kakkar et al. 2016). 시험관 내에서 DNAJB6는 표면의 세린-트레오닌이 풍부한 영역에 의존하는 폴리큐 펩타이드의 1차 핵 생성을 억제합니다(Kakkar 외. 2016). 그러나 이 활성은 Hsp70 또는 ATP의 존재에 의존하지 않으며(Månsson 외. 2014b), 이는 Hsp40 단백질의 독립적인 샤프론 활성에 대한 흥미로운 예를 제공합니다.

4.2.5. SOD1

Point mutations in superoxide dismutase 1 (SOD1) are one of the causes of familial forms and sporadic cases of ALS (Cook and Petrucelli 2019). In contrast to the proteins discussed above that are largely disordered, SOD1 is a well folded soluble globular protein that binds copper and zinc ions and is stabilized by a disulfide bond. Disease-associated mutations are thought to destabilize the native state of SOD1, rendering it more prone to aggregation (Lindberg et al. 2005; Prudencio et al. 2009).

In a genome-wide RNAi screen on a C. elegans strain expressing SOD1 with G85R mutation throughout the neurons, the majority of hits belonged to the category of protein quality control, including the regulator of the heat shock response HSF1, several chaperones and components of the degradation machinery (Wang et al. 2009). Induction of the heat shock response is protective against SOD1 G93A aggregation and toxicity in mice, as demonstrated by the overexpression‎ of SIRT1, which has HSF1 amongst its substrates. The beneficial effect in this case was found to be limited by the expression‎ levels of inducible Hsp70, which were not sufficient to restore the phenotype of mice with higher levels of SOD1 (Watanabe et al. 2014).

A role for the Hsp70/Hsp40/Hsp110 machinery in aggregate disassembly as described above for α-synuclein has also been suggested for SOD1. Overexpression‎ of Hsp110 in mice expressing YFP-tagged SOD1 G85R in motor-neurons extends their lifespan, and a reduction of aggregates has been observed in a subset of animals (Nagy et al. 2016). Addition of YFP-SOD1 G85R to isolated squid axoplasm inhibits axonal transport, and supplementing Hsc70, but more so Hsp110, was found to suppress these defects (Song et al. 2013). Likewise, overexpression‎ of the Hsp40 family member DNAJB2 reduces aggregation of SOD1 G93A in late stages of disease progression in mice, and improves motor-neuron survival and muscle strength. DNAJB2 has been found to associate with the SOD1 aggregates, providing evidence for a direct interaction which was suggested to reduce aggregate formation by promoting ubiquitination (Novoselov et al. 2013).

sHsps have also been proposed to modulate SOD1 aggregation and toxicity. Hsp25 and αB-crystallin co-elute with insoluble SOD1 mutant protein from mice (Wang et al. 2003), and in in vitro studies using a brain homogenate, αB-crystallin inhibits aggregation (Wang et al. 2005a). This is further supported by experiments with recombinant Hsp27 and αB-crystallin that inhibit SOD1 G93A aggregation by interfering with aggregate growth, rather than the primary nucleation step (Yerbury et al. 2013). Mutations in Hsp27 have been identified in sporadic cases of ALS, which may be related to the inability to prevent SOD1 aggregation (Capponi et al. 2016).

4.3. Chaperone Competition as a Basis for Proteostasis Collapse in Protein Misfolding Diseases4.3.1. The Chaperone

Competition Hypothesis

The interaction of molecular chaperones with intermediate states of highly aggregation-prone disease-related proteins is a finely tuned process, in which chaperones can either direct and maintain functional client interactions for cellular health or result in protein aggregation and proteome mismanagement in aging and stress. This imbalance can be enhanced by increased protein expression‎, coding mutations, posttranslational modifications or alterations in the composition and functional properties of the proteostasis network to shift the balance towards aggregation and proteotoxicity. While misfolded and aggregated proteins have been directly linked to cellular toxicity (Bucciantini et al. 2002; Baglioni et al. 2006; Marsh et al. 2000; Fath et al. 2002), the diverse protein misfolding diseases have very complex pathologies likely resulting from multiple molecular defects. The chaperone competition hypothesis provides an explanation why aggregation of a disease-associated protein can interfere with multiple cellular pathways (Yu et al. 2014; Yu et al. 2019). The multifaceted roles of chaperone networks raise the possibility that competition between misfolding proteins and endogenous clients for limited chaperone resources will have consequences on protein functionality. The higher localized concentration of misfolded proteins in aggregates results in a spatial redistribution of chaperones and other components of the proteostasis network. Kinetically, this will have negative effects on many chaperone-regulated processes resulting in multiple pathological symptoms, exacerbating disease progression. Chaperone sequestration initiated by intracellular accumulation of misfolded and aggregated proteins is common to all protein misfolding diseases, supporting the hypothesis that the loss of chaperone function upon protein aggregation can accelerate cellular toxicity.

4.3.2. Chaperone Sequestration During Protein Misfolding

Multiple families of chaperones and co-chaperones form extensive protein-protein interaction networks to assist in the folding of diverse clients. Chaperone-client interactions are transient in nature to allow reversible engagement with multiple substrates including nascent polypeptides, unfolded and misfolded proteins and folding intermediates (Kim et al. 2013; Hiller 2019; Koldewey et al. 2017). Compared with on-pathway substrates for Hsc70, misfolded species are more likely to expose hydrophobic regions to allow Hsc70 to bind with higher apparent avidity (Kundel et al. 2018; Pemberton et al. 2011). Consequently, Hsc70 is preferentially occupied by aberrantly folded protein substrates in stressed cells or upon expression‎ of metastable proteins. When protein aggregates form after stable interaction of misfolded proteins, Hsc70 as well as other interacting partners become sequestered into the aggregates (Fig. 4.2).

Fig. 4.2.

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Aggregate-driven chaperone competition explains the pathological complexity associated with disease-associated aggregation-prone proteins. Shown is a model depicting chaperone competition between protein aggregates and the protein folding and vesicular trafficking arms of the proteostasis network. Under normal conditions (left), Hsc70 is at sufficiently high levels to mediate CME as well as basal protein client folding. Under disease conditions where protein aggregates have accumulated and the Hsc70 relocalizes to aggregates, both protein folding and CME are inhibited (right). This can be reversed by increasing the levels of Hsc70 by small molecule activation of HSF1 to restore chaperone function

Histochemical and biochemical studies have revealed that Hsc70 and other chaperones colocalize with a variety of protein inclusions in multiple cell and animal models of disease-associated protein aggregation and in patient-obtained brain tissues. Proteomic analysis of laser dissected amyloid plaques (Liao et al. 2004) and tau tangles (Wang et al. 2005b) from AD patient brains show that these inclusions sequester molecular chaperones, and other proteins that may be conformationally challenged. Hsc70 and the proteasome also co-localize with intracellular Aβ aggregates in a cellular model (Bückig et al. 2002). A human cell system for seeding-dependent tau aggregation has shown that the chaperones Hsc70/Hsp70, Hsp90 and J-domain co-chaperones are sequestered by tau aggregates (Yu et al. 2019). Likewise in PD, immunohistochemistry and proteomics have identified major chaperones including Hsc70, Hsp90, Hsp40 and Hsp27 as constituents of filamentous Lewy bodies, co-localizing with α-synuclein (Uryu et al. 2006; Leverenz et al. 2007).

In the context of HD, Hsc70 binds specifically to the N-terminal flanking region of huntingtin exon 1. Using a conditional human cell system and immunofluorescence, the chaperones BiP/GRP78, Hsp70, Hsp40, proteasome subunits and other aggregation-prone proteins were shown to colocalize with the perinuclear inclusions of huntingtin exon 1 with an expanded polyQ (Waelter et al. 2001). Proteomic profiling of HD inclusions revealed widespread sequestration of proteins into the mutant huntingtin inclusion bodies (Hosp et al. 2017). Similarly, chaperones colocalize with ataxin 1 and ataxin 3 polyQ protein inclusions (Cummings et al. 1998; Chai et al. 1999). For ALS, mutant SOD1 is a substrate of interactions with Hsc70/Hsp70, and mice expressing mutant SOD1 form inclusions containing ubiquitin, the proteasome and Hsc70 (Zetterström et al. 2011). Hsc70-positive inclusions have also been detected in sporadic ALS cases (Watanabe et al. 2001). Chaperone association has also been detected in cells expressing an artificial aggregation-prone β-sheet protein (Olzscha et al. 2011). Collectively, these observations provide strong evidence for sequestration of key components of the proteostasis network such as molecular chaperones and constituents of the protein degradation machinery as a unifying feature of protein misfolding diseases. The delicate balance between the levels of available molecular chaperones and client proteins is further under-scored by the fact that many types of cancer cells depend on elevated levels of chaperones for their survival, accommodating for the increased demand by destabilized and misfolding-prone oncoproteins (see other chapters in this collection).

4.3.3. Functional Consequence of Chaperone Sequestration

As described above, many cellular processes are affected by the sequestration of chaperones by protein aggregation. The functional consequences of chaperone competition were determined by measuring multiple Hsc70-mediated cellular processes (Yu et al. 2014, 2019). Clathrin-mediated endocytosis (CME), the main entry route of biological molecules into the cell (Kirchhausen et al. 2014) involves the self-assembly of trimeric clathrin units on the plasma membrane to cause membrane curvature and the formation of coated pits. The released vesicles rapidly lose their clathrin coats in a process catalyzed by Hsc70 together with the co-chaperone auxilin in order to fuse with intracellular endosomes (Massol et al. 2006). Reducing cellular levels of Hsc70 by siRNA inhibits CME, indicating the requirement of Hsc70 for the assembly and disassembly of clathrin-coated vesicles (Yu et al. 2014).

The kinetic correlation between cytosolic Hsc70 concentration and CME therefore provides a highly sensitive functional readout of chaperone competition in the presence of protein aggregation. By monitoring CME in prostate cancer PC-3 cells expressing different aggregation-prone proteins including polyglutamine, huntingtin, ataxin1 and SOD1, a reduction of CME due to the sequestration of Hsc70 by aggregates was observed (Yu et al. 2014). The sensitivity of CME to Hsc70 depletion suggests that chaperone abundance is rate-limiting in cells expressing aggregation-prone proteins. Moreover, suppression of CME by protein aggregation could be reversed by conditionally increasing Hsc70. These effects were also observed in neuronal cells showing that protein aggregation causes dysregulated internalization of the AMPA receptor, a neuron-specific CME cargo.

The observations of chaperone competition extend beyond Hsc70, as other chaperones, including Hsp90, Hsp40 and Hsp27, are also sequestered in tau inclusions. Single-cell analysis of protein folding and CME in a cell model of tau aggregation reveals that both chaperone-dependent activities are impaired by tau aggregation (Yu et al. 2019). These observations are further supported by the finding in yeast that sequestration of Sis1p, a low-abundant Hsp40 homolog chaperone, by polyQ aggregates interferes with nuclear degradation of misfolded proteins and leads to the formation of cytosolic inclusions (Park et al. 2013). Consequently, the decline in chaperone-dependent arms of the proteostasis network will have profound negative effects on the protein quality control capacity of the cell. Besides cytosolic chaperones, various species of misfolded proteins and aggregates interact with and sequester components of the protein degradation machinery. This sequestration will further compromise the protein quality control capacity (Thibaudeau et al. 2018; Guo et al. 2018; Yang et al. 2015; Holmberg et al. 2004).

4.3.4. Restoration of Chaperone Homeostasis as a Therapeutic Strategy

The functional dissection of chaperone competition will lead to a better understanding of the early events of protein aggregation, and may uncover novel strategies to intervene at early stages of protein misfolding diseases. All Hsc70-regulated processes will be negatively affected by a subcellular redistribution of Hsc70 among its clients, resulting in a decline in multiple chaperone-dependent processes leading to multiple pathological symptoms that exacerbate disease progression. Consequently, restoring Hsc70 homeostasis could be an effective strategy to battle age-related protein conformational diseases. The appearance of a misfolded conformational state of tau associated with CME failure can be detected before the appearance of mature tau inclusions and the stable sequestration of Hsc70 (Yu et al. 2019). The timing of these cellular events therefore suggests that the inhibition of CME is an early cellular event of tauopathy and precedes failure of other cellular processes such as chaperone-dependent protein folding. Moreover, both CME and protein folding can be restored by small molecule regulators of HSF1 resulting in expression‎ of cytosolic chaperones (Yu et al. 2019), suggesting that enhancing chaperone expression‎ could have beneficial consequences on chaperone function and cellular health without reversing tau aggregation. Similarly, overexpressing protein sequestrated by polyQ aggregates has been shown to rescue loss of function phenotypes and relieve polyQ dependent toxicity (Park et al. 2013).

4.4. Outlook

Further studies will be required to bridge the gap between the kinetic studies of protein aggregation and their modulation by molecular chaperones in vitro and in vivo in healthy and disease states. Cellular probes capable of detecting aggregating protein species, especially in the early and oligomeric states, may allow real-time monitoring of chaperone engagement of disease-associated substrates. Furthermore, molecular chaperones do not operate in isolation and the coordination of chaperone networks to maintain all arms of the proteostasis network needs to be further characterized for each of the aggregation-prone proteins in disease-relevant tissues and cell types. The systems approach for proteostasis in neurodegenerative diseases likely will differ from cancer, although both classes of diseases are related by the consequence of aging on the robustness of cell stress responses and the functional capacity of the proteostasis network. The use of primary patient derived cells and live cell-based approaches to measure the cellular state of proteostasis during disease progression and in the context of aging will provide a basis to screen for small molecules that restore cellular proteostasis. Targeting the restoration of proteostasis, in particular chaperone homeostasis, can potentially serve as a major therapeutic avenue to treat many forms of protein misfolding disorders ranging from neurodegenerative diseases to cancer.

Acknowledgements

We thank Christopher M. Dobson for critical reading of part of the manuscript. This work was supported by National Institutes of Health (National Institute on Aging), the Daniel F. and Ada L. Rice Foundation to RIM and research grants from Eli Lilly & Co Ltd, Lilly Research Centre and the Tau Consortium of the Rainwater Foundation.

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