Roles of protein misfolding in neurodegenerative diseases

蛋白质错误折叠在神经退行性疾病中的作用

• 批准号: 10697852
• 负责人: Yihong Ye
• 金额: $ 89.25万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697852
• 关键词: Adult; Alzheimer' s Disease; Attenuated; Autophagocytosis; COS-7 Cell; Cell Differentiation process; Cell membrane; Cells; Chemicals; Collection; Coupled; Defect; Drosophila genus; Endoplasmic Reticulum; Endosomes; Eukaryotic Cell; Excision; Extracellular Space; Failure; Functional disorder; Goals; Heat shock proteins; Homeostasis; Inherited; Lead; Lipofuscin; Lysosomes; Mammalian Cell; Mediating; Membrane Fusion; Membrane Proteins; Methods; Modeling; Molecular Chaperones; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Ceroid-Lipofuscinosis; Neurons; Parkinson Disease; Pathway interactions; Patients; Physiological; Population; Process; Proliferating; Protein Family; Protein Isoforms; Protein Secretion; Protein translocation; Proteins; Proteomics; Quality Control; Reporting; Research Project Grants; Role; Route; SNAP23 gene; Serum; Stains; Starvation; Stress; Surface; Synapses; Triage; Ubiquitin; Variant; Vesicle; Western Blotting; exosome; extracellular vesicles; fly; human disease; inhibitor; knock-down; late endosome; member; misfolded protein; multicatalytic endopeptidase complex; mutant; neurotoxic; overexpression; polypeptide; protein degradation; protein folding; protein misfolding; proteostasis; proteotoxicity; receptor; secretion process; synuclein; tau Proteins; transmission process

Misfolded proteins pose a major threat to the protein homeostasis network of eukaryotic cells, particularly in terminally differentiated cells like neurons, which cannot proliferate to dilute aberrant polypeptides to a level below aggregation threshold. To safeguard the protein homeostasis network, cells have evolved several protein quality control (PQC) strategies, including chaperone-assisted protein folding, proteasomal degradation, and autophagy-mediated protein turnover. As anticipated, failures in PQC lead to accumulation of aggregationprone polypeptides, culminating in proteotoxic stress that can cause a variety of human diseases. Misfolding-associated protein secretion (MAPS) is a recently uncovered PQC mechanism, which targets misfolded cytosolic proteins to a unconventional protein secretion (UPS) pathway for export into the extracellular space. This process uses the endoplasmic reticulum (ER)-associated deubiquitinase USP19 as a receptor, which enriches misfolded proteins on the ER surface by an intrinsic chaperone activity. Subsequently, misfolded polypeptides are moved into the lumen of a population of ER-associated vesicles via an unknown protein translocation mechanism, and eventually secreted when partial or complete membrane fusion occurs between these vesicles and the plasma membrane (PM). Interestingly, many MAPS substrates are also ubiquitinated in the cell and targeted for degradation by the proteasome. The interaction of USP19 with these substrates causes the removal of ubiquitin chains by USP19, which is required for the subsequent exporting processing, at least for the substrates studied to date. Existing evidence suggests that MAPS is a supplementary PQC process, in parallel with the proteasome to alleviate proteotoxic stress caused by haploinsufficiency of the proteasome. Although USP19 is known to interact with Hsp90 and Hsp70, the chaperone requirement for MAPS has been unclear. In addition, the pathway has so far only been characterized with a few substrates, and therefore, it is unclear whether MAPS can export most or just a subset of misfolded cytosolic proteins. Moreover, among the polypeptides examined, -synuclein (-Syn), an intrinsically misfolded soluble protein implicated in Parkinsons disease is efficiently secreted, whereas the Alzheimers disease-associated Tau protein, when fused with a GFP tag was not subject to secretion by MAPS. Thus, it is unclear to what extent MAPS may contribute to the widely reported cell-to-cell transmission of misfolded proteins in neurodegenerative diseases. In addition to MAPS, several UPS routes have been reported previously. A few studies suggested exosome or extracellular vesicles as a carrier for misfolded -Syn and Tau, However, our results as well as other studies showed that misfolded -Syn and Tau released into the cell exterior are mostly not bound to vesicles. Thus, the relative contribution of different protein secretion routes to the intercellular propagation of misfolded neurotoxic proteins under physiological conditions needs to be further evaluated. Interestingly, a recent study also suggested a mechanism reminiscent of MAPS for disposal of Tau and -Syn, which involves the cytosolic chaperone HSC70, its co-chaperone DNAJC5 and a vesicle fusion regulator SNAP23. However, the functional relationship between this process and the MAPS pathway is unclear. In this study, we identify additional MAPS substrates, which now cover a collection of aberrant proteins associated with neurodegenerative diseases. Importantly, we characterize the role of two USP19-interacting chaperones; while Hsp90 is dispensable for MAPS, HSC70 and its co-chaperone DNAJC5 function together with USP19 to form a critical protein-triaging hub in MAPS. These findings unify two UPS routes that were previously deemed unrelated. Importantly, our study suggests that MAPS, as an exosome-independent secretion process, may contribute to the export of diverse neurotoxic misfolded proteins known to propagate between neurons. Because chaperone-mediated autophagy (CMA) is a known mechanism that imports cytosolic proteins bearing a specific CMA motif to lysosomes for degradation and because late endosomes and lysosomes overlap significantly in mammalian cells, we determined here whether CMA is involved in targeting protein cargoes to the lumen of late endosomes in MAPS. Using HEK293T and COS-7 cells and immunoblotting and -staining and coimmunoprecipitation methods, we show that, unlike CMA, the secretion of misfolded proteins in MAPS does not require cargo unfolding, is inhibited by serum starvation, and is not dependent on the CMA motif in cargo. Intriguingly, knockdown of lysosome-associated membrane protein 2 (LAMP2), which consists of three isoforms, including a variant proposed to form a protein channel on lysosomes for CMA, attenuated MAPS. However, this could not be attributed to the proposed channel function of the LAMP2a isoform because overexpression of a cytosolic MAPS stimulator, DnaJ heat shock protein family (Hsp40) member C5 (DNAJC5), fully rescued the secretion defect associated with LAMP2 deficiency. We conclude that, in MAPS, cargoes use a CMA-independent mechanism to enter a nondegradative prelysosomal compartment. Mutations in DNAJC5/CSP are associated with adult neuronal ceroid lipofuscinosis (ANCL), a dominant-inherited neurodegenerative disease featuring lysosome-derived autofluorescent storage materials (AFSMs) termed lipofuscin. Functionally, DNAJC5 has been implicated in chaperoning synaptic proteins and in misfolding-associated protein secretion (MAPS), but how DNAJC5 dysfunction causes lipofuscinosis and neurodegeneration is unclear. Here we report two functionally distinct but coupled chaperoning activities of DNAJC5, which jointly regulate lysosomal homeostasis: While endolysosome-associated DNAJC5 promotes ESCRT-dependent microautophagy, a fraction of perinuclear and non-lysosomal DNAJC5 mediates MAPS. Functional proteomics identifies a previously unknown DNAJC5 interactor SLC3A2/CD98hc that is essential for the perinuclear DNAJC5 localization and MAPS but dispensable for microautophagy. Importantly, uncoupling these two processes, as seen in cells lacking SLC3A2 or expressing ANCL-associated DNAJC5 mutants, generates DNAJC5-containing AFSMs resembling NCL patient-derived lipofuscin and induces neurodegeneration in a Drosophila ANCL model. These findings suggest that MAPS safeguards microautophagy to avoid DNAJC5-associated lipofuscinosis and neurodegeneration.

错误折叠的蛋白质对真核细胞的蛋白质稳态网络构成重大威胁，特别是在神经元等终末分化细胞中，这些细胞无法增殖以将异常多肽稀释至低于聚集阈值的水平。为了维护蛋白质稳态网络，细胞进化出了多种蛋白质质量控​​制（PQC）策略，包括分子伴侣辅助的蛋白质折叠、蛋白酶体降解和自噬介导的蛋白质周转。正如预期的那样，PQC 的失败会导致易于聚集的多肽积累，最终导致蛋白毒性应激，从而导致多种人类疾病。 错误折叠相关蛋白分泌 (MAPS) 是最近发现的一种 PQC 机制，它将错误折叠的胞浆蛋白靶向非常规蛋白分泌 (UPS) 途径，然后输出到细胞外空间。该过程使用内质网 (ER) 相关去泛素酶 USP19 作为受体，通过内在的伴侣活性富集 ER 表面错误折叠的蛋白质。 随后，错误折叠的多肽通过未知的蛋白质易位机制移动到内质网相关囊泡群体的管腔中，并最终在这些囊泡和质膜（PM）之间发生部分或完全膜融合时被分泌。有趣的是，许多 MAPS 底物在细胞中也被泛素化，并被蛋白酶体降解。 USP19 与这些底物的相互作用导致 USP19 去除泛素链，这是后续导出处理所必需的，至少对于迄今为止研究的底物而言是如此。现有证据表明，MAPS 是一个补充性 PQC 过程，与蛋白酶体并行，可减轻因蛋白酶体单倍体不足引起的蛋白毒性应激。 尽管已知 USP19 与 Hsp90 和 Hsp70 相互作用，但 MAPS 的伴侣要求尚不清楚。 此外，迄今为止，该途径仅用少数底物进行了表征，因此，尚不清楚 MAPS 是否可以输出大部分或仅部分错误折叠的胞浆蛋白。此外，在所检查的多肽中，-突触核蛋白（-Syn）是一种与帕金森病有关的本质上错误折叠的可溶性蛋白，可以有效分泌，而与阿尔茨海默病相关的 Tau 蛋白在与 GFP 标签融合时不会被 MAPS 分泌。因此，目前尚不清楚 MAPS 在多大程度上有助于广泛报道的神经退行性疾病中错误折叠蛋白的细胞间传播。 除了 MAPS 之外，此前还报道过几条 UPS 路线。一些研究表明外泌体或细胞外囊泡是错误折叠的-Syn和Tau的载体，然而，我们的结果以及其他研究表明，释放到细胞外部的错误折叠的-Syn和Tau大多不与囊泡结合。因此，需要进一步评估生理条件下不同蛋白质分泌途径对错误折叠神经毒性蛋白质细胞间传播的相对贡献。有趣的是，最近的一项研究还提出了一种类似于 MAPS 处理 Tau 和 -Syn 的机制，其中涉及胞质伴侣 HSC70、其辅助伴侣 DNAJC5 和囊泡融合调节因子 SNAP23。然而，该过程与 MAPS 途径之间的功能关系尚不清楚。 在这项研究中，我们确定了其他 MAPS 底物，这些底物现在涵盖了与神经退行性疾病相关的一系列异常蛋白质。重要的是，我们描述了两个 USP19 相互作用伴侣的作用；虽然 Hsp90 对于 MAPS 是不可或缺的，但 HSC70 及其辅助伴侣 DNAJC5 与 USP19 一起发挥作用，形成 MAPS 中关键的蛋白质分类中心。这些发现将之前被认为不相关的两条 UPS 路线统一起来。 重要的是，我们的研究表明，MAPS 作为一种不依赖于外泌体的分泌过程，可能有助于输出已知在神经元之间传播的多种神经毒性错误折叠蛋白。 由于伴侣介导的自噬 (CMA) 是一种已知的机制，它将带有特定 CMA 基序的胞浆蛋白导入溶酶体进行降解，并且由于晚期内体和溶酶体在哺乳动物细胞中显着重叠，因此我们在此确定 CMA 是否参与将蛋白质货物靶向 MAPS 中晚期内体的管腔。使用 HEK293T 和 COS-7 细胞以及免疫印迹和染色以及免疫共沉淀方法，我们表明，与 CMA 不同，MAPS 中错误折叠蛋白的分泌不需要货物展开，受到血清饥饿的抑制，并且不依赖于货物中的 CMA 基序。有趣的是，溶酶体相关膜蛋白 2 (LAMP2) 由三种亚型组成，其中包括一种在溶酶体上形成 CMA 蛋白质通道的变体，敲低该蛋白可减弱 MAPS。然而，这不能归因于所提出的 LAMP2a 亚型的通道功能，因为胞质 MAPS 刺激剂、DnaJ 热休克蛋白家族 (Hsp40) 成员 C5 (DNAJC5) 的过度表达，完全挽救了与 LAMP2 缺陷相关的分泌缺陷。我们的结论是，在 MAPS 中，货物使用独立于 CMA 的机制进入非降解性前溶酶体隔室。 DNAJC5/CSP 突变与成人神经元蜡样脂褐素沉着症 (ANCL) 相关，ANCL 是一种显性遗传性神经退行性疾病，其特征是溶酶体衍生的自发荧光储存材料 (AFSM)，称为脂褐质。从功能上讲，DNAJC5 与陪伴突触蛋白和错误折叠相关蛋白分泌 (MAPS) 有关，但 DNAJC5 功能障碍如何导致脂褐质沉着症和神经变性尚不清楚。在这里，我们报告了 DNAJC5 的两种功能不同但耦合的陪伴活动，它们共同调节溶酶体稳态：虽然内溶酶体相关的 DNAJC5 促进 ESCRT 依赖性微自噬，但核周和非溶酶体 DNAJC5 的一小部分介导 MAPS。功能蛋白质组学鉴定了一个以前未知的 DNAJC5 相互作用蛋白 SLC3A2/CD98hc，它对于核周 DNAJC5 定位和 MAPS 至关重要，但对于微自噬来说是可有可无的。重要的是，如在缺乏 SLC3A2 或表达 ANCL 相关 DNAJC5 突变体的细胞中所见，解开这两个过程会产生类似于 NCL 患者来源的脂褐素的含有 DNAJC5 的 AFSM，并在果蝇 ANCL 模型中诱导神经变性。这些发现表明 MAPS 可以保护微自噬，以避免 DNAJC5 相关的脂褐质沉着症和神经变性。