Mechanism of protein quality control at the endoplasmic reticulum

内质网蛋白质质量控制机制

基本信息

批准号：
10919405
负责人：
Yihong Ye
金额：
$ 77.97万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10919405
关键词：
Address Adopted Affinity Affinity Chromatography Anemia Animals Basement membrane Binding CRISPR screen Cell Nucleus Cell physiology Cells Client Collaborations Collagen Complex Cytoplasm Cytosol Data Degradation Pathway Deposition Detergents Development Dislocations Drosophila genus Electrostatics Endoplasmic Reticulum Ensure Environment Enzymes Eukaryota Goals HIV Hemoglobin Homeostasis Homo Human Immune response Immunologic Surveillance Impairment In Vitro Link Lysine Lysosomes Mammalian Cell Mediator Membrane Membrane Proteins Modeling Molecular Molecular Chaperones Molecular Conformation Multiprotein Complexes Mus Names Pathogenesis Pathway interactions Physiological Play Polyubiquitination Process Production Protein Biosynthesis Protein Secretion Protein translocation Proteins Proteolytic Processing Quality Control Research Ribosomes Role Route SGTA gene Site Solubility Substrate Specificity TRAPP transport protein particle Ubiquitin Ubiquitination Virus Virus Diseases cofactor coping endoplasmic reticulum stress erythroid differentiation genome-wide human disease improved misfolded protein multicatalytic endopeptidase complex neuron development novel polypeptide preservation prevent protein aggregation proteostasis receptor recruit sensor stress reduction stress tolerance ubiquitin isopeptidase ubiquitin ligase

项目摘要

The endoplasmic reticulum (ER) is the major site of protein biosynthesis in eukaryotes. Polypeptides entering the ER may frequently adopt aberrant conformations, resulting in aggregation-prone, misfolded proteins. Accumulation of misfolded proteins induces ER stress, which has been implicated in the pathogenesis of many human diseases. To preserve ER protein homeostasis, eukaryotes have evolved a conserved quality control pathway termed retro-translocation/dislocation or ER-associated degradation (ERAD), which eliminates misfolded proteins from the ER by exporting them into the cytosol. Polypeptides undergoing retro-translocation are disposed of by the cytosolic proteasome. The retro-translocation pathway is hijacked by certain viruses to destroy folded cellular proteins required for immune response, allowing the virus to evade host immune surveillance. For example, the Human Immunodeficiency Virus uses a protein named Vpu to target newly synthesized CD4 co-receptor for degradation, which promote viral infection. We previously identified a cytosolic enzyme called p97, which acts with two co-factors Ufd1 and Npl4 to move retrotranslocating substrates into the cytosol for degradation. We also used an affinity purification approach to identify two novel ER membrane proteins, Derlin-1 and VIMP, which associate with p97. VIMP functions as a receptor to recruit p97 to the ER membrane. The conserved multi-spanning membrane protein Derlin-1 plays a central role in retro-translocation. It appears to receive substrates from the ER lumen to promote their translocation via a yet-to-be defined membrane pore. We further identified an ubiquitin ligase-associated multiprotein complex comprising Bag6, Ubl4A, and Trc35, which chaperones retrotranslocated polypeptides en route to the proteasome to improve ERAD efficiency. In vitro, Bag6, the central component of the complex, contains a chaperone-like activity capable of maintaining an aggregation-prone substrate in an unfolded yet soluble state. The physiological importance of this holdase activity is underscored by observations that ERAD substrates accumulate in detergent insoluble aggregates in cells depleted of Bag6, or of Trc35, a cofactor that keeps Bag6 outside the nucleus for engagement in ERAD. Our results reveal an ubiquitin ligase-associated holdase that maintains polypeptide solubility to enhance protein quality control in mammalian cells. The Bag6 complex also participates in several other protein quality control processes, but how Bag6 effectively captures misfolded polypeptides in the complex cellular environment is unclear. We recently found a novel ERAD mediator named SGTA, which forms a chaperone cascade with Bag6 to help channel dislocated ERAD substrates that are otherwise prone to aggregation. We show that SGTA contains an unusual ubiquitin-like (UBL) binding motif that interacts specifically with a non-canonical UBL domain in Ubl4A via electrostatics. This interaction enhances substrate loading to Bag6 to prevent the formation of non-degradable protein aggregates, and thus improve the ERAD efficiency. The Bag6-Ubl4A-Trc35 complex is a multifunctional chaperone that regulates various cellular processes. Because the diverse functions of Bag6 are supported by its ubiquitous localization to the cytoplasm, the nucleus, and membranes of the endoplasmic reticulum (ER) in cells, we recently investigated how Bag6 is associated with the ER membrane. We found that in the ER-associated degradation (ERAD) pathways, Bag6 can interact with the CUE domain in the membrane-associated ubiquitin ligase gp78 via its ubiquitin-like (UBL) domain, but the relative low affinity of this interaction does not reconcile with the fact that a fraction of Bag6 is tightly bound to the membrane. Here, we demonstrate that the UBL domain of Bag6 is required for its interaction with the ER membrane despite the low affinity to gp78. We findthat in addition to gp78, the Bag6 UBL domain also binds a UBL-binding motif in UbxD8, an essential component of the gp78 ubiquitinating machinery. Importantly, Bag6 forms a large homo-oligomer, allowing the UBL domain to form multivalent interactions with the gp78-containing retrotranslocation complex. Both gp78 and UbxD8 contain motifs for recognition by p97, thus linking Bag6 to this core retrotranslocation machinery in the membrane. We propose that simultaneous association with multiple ERAD factors helps to anchor a fraction of Bag6 oligomer to the site of retrotranslocation to enhance ERAD efficiency. Our research also addressed a surprising paradox emerging from recent studies that ubiquitin ligases (E3s) and deubiquitinases (DUBs), enzymes with opposing activities, can both promote ERAD. We demonstrate that the ERAD E3 gp78 can ubiquitinate not only ERAD substrates, but also the machinery protein Ubl4A, a key component of the Bag6 chaperone complex. Remarkably, instead of targeting Ubl4A for degradation, polyubiquitination is associated with irreversible proteolytic processing and inactivation of Bag6. Importantly, we identify USP13 as a gp78-associated DUB that eliminates ubiquitin conjugates from Ubl4A to maintain the functionality of Bag6. Our study reveals an unexpected paradigm in which a DUB prevents undesired ubiquitination to sharpen substrate specificity for an associated ubiquitin ligase partner and to promote ER quality control. We recently show that ribosome stalling during protein translocation induces the attachment of UFM1, a ubiquitin-like modifier, to two conserved lysine residues near the COOH-terminus of the 60S ribosomal subunit RPL26 (uL24) at the ER. Strikingly, RPL26 UFMylation enables the degradation of stalled nascent chains, but unlike ERAD or previously established cytosolic ribosome-associated quality control (RQC), which uses proteasome to degrade their client proteins, ribosome UFMylation promotes the targeting of a translocation-arrested ER protein to lysosomes for degradation. RPL26 UFMylation is upregulated during erythroid differentiation to cope with increased secretory flow, and compromising UFMylation impairs protein secretion, and ultimately hemoglobin production. We propose that in metazoan, co-translational protein translocation (TAQC) into the ER is safeguarded by a UFMylation-dependent, translocation-associated protein quality control mechanism, which when impaired causes anemia in mice and abnormal neuronal development in humans. More recently, we used a genome-wide CRISPR/Cas9 screen to identify an uncharacterized membrane protein named SAYSD1 that facilitates TAQC. SAYSD1 associates with the Sec61 translocon and engages a stalled nascent chain-ribosome complex by directly recognizing both ribosome and UFM1, ensuring the export of stalled substrates via the TRAPP complex for lysosomal degradation. Like UFM1 deficiency, SAYSD1 depletion causes the build-up of translocation-stalled proteins at the ER, which triggers ER stress. Importantly, disrupting UFM1- and SAYSD1-dependent quality control in Drosophila leads to the accumulation of translocation-stalled collagen, defective collagen deposition, abnormal basement membranes, and reduced stress tolerance. Together, our data support a model that SAYSD1 acts as an ER-associated UFM1 sensor to collaborate with ribosome UFMylation at the site of translocon-jamming, safeguarding ER homeostasis during animal development.

内质网（ER）是真核生物蛋白质生物合成的主要场所。进入内质网的多肽可能经常采用异常构象，导致易于聚集、错误折叠的蛋白质。错误折叠蛋白的积累会引起内质网应激，这与许多人类疾病的发病机制有关。为了保持内质网蛋白稳态，真核生物进化出了一种保守的质量控制途径，称为逆向易位/错位或内质网相关降解（ERAD），通过将错误折叠的蛋白输出到细胞质中来消除内质网中的错误折叠蛋白。经历逆转位的多肽被胞质蛋白酶体处理。逆转录易位途径被某些病毒劫持，以破坏免疫反应所需的折叠细胞蛋白，从而使病毒能够逃避宿主免疫监视。例如，人类免疫缺陷病毒使用一种名为Vpu的蛋白质来靶向新合成的CD4辅助受体进行降解，从而促进病毒感染。我们之前发现了一种称为 p97 的胞质酶，它与两个辅因子 Ufd1 和 Npl4 一起作用，将逆转位底物移动到胞质溶胶中进行降解。我们还使用亲和纯化方法鉴定了两种新型 ER 膜蛋白：Derlin-1 和 VIMP，它们与 p97 相关。 VIMP 作为受体将 p97 招募到 ER 膜上。保守的多跨膜蛋白 Derlin-1 在逆转录易位中发挥核心作用。它似乎从内质网腔接收底物，通过尚未定义的膜孔促进它们的易位。我们进一步鉴定了包含 Bag6、Ubl4A 和 Trc35 的泛素连接酶相关多蛋白复合物，该复合物在通往蛋白酶体的途中陪伴逆向转位多肽以提高 ERAD 效率。在体外，Bag6（该复合物的核心成分）具有类分子伴侣的活性，能够将易于聚集的底物维持在未折叠但可溶的状态。这种保持酶活性的生理重要性通过以下观察得到强调：ERAD 底物在缺乏 Bag6 或 Trc35 的细胞中积累在去污剂不溶性聚集物中，Trc35 是一种将 Bag6 保持在细胞核外以参与 ERAD 的辅助因子。我们的结果揭示了一种泛素连接酶相关的保持酶，它可以维持多肽的溶解度，从而增强哺乳动物细胞中的蛋白质质量控制。 Bag6复合物还参与其他几个蛋白质质量控制过程，但Bag6如何在复杂的细胞环境中有效捕获错误折叠的多肽尚不清楚。我们最近发现了一种名为 SGTA 的新型 ERAD 介体，它与 Bag6 形成分子伴侣级联，以帮助通道移位的 ERAD 底物，否则这些底物容易聚集。我们发现 SGTA 包含一个不寻常的类泛素 (UBL) 结合基序，它通过静电与 Ubl4A 中的非典型 UBL 结构域特异性相互作用。这种相互作用增强了 Bag6 的底物负载，以防止形成不可降解的蛋白质聚集体，从而提高 ERAD 效率。 Bag6-Ubl4A-Trc35 复合物是调节各种细胞过程的多功能伴侣。由于 Bag6 的多种功能是由其普遍定位于细胞内质网 (ER) 的细胞质、细胞核和膜所支持的，因此我们最近研究了 Bag6 如何与 ER 膜相关。我们发现，在ER相关降解（ERAD）途径中，Bag6可以通过其泛素样（UBL）结构域与膜相关泛素连接酶gp78中的CUE结构域相互作用，但这种相互作用的相对低亲和力并不协调事实上，Bag6 的一部分与膜紧密结合。在这里，我们证明了 Bag6 的 UBL 结构域是其与 ER 膜相互作用所必需的，尽管与 gp78 的亲和力较低。我们发现除了 gp78 之外，Bag6 UBL 结构域还结合 UbxD8 中的 UBL 结合基序，UbxD8 是 gp78 泛素化机制的重要组成部分。重要的是，Bag6 形成一个大的同源寡聚体，允许 UBL 结构域与含有 gp78 的逆转录转位复合物形成多价相互作用。 gp78 和 UbxD8 都包含 p97 识别的基序，从而将 Bag6 与膜中的核心逆转位机制连接起来。我们建议同时与多个 ERAD 因子关联有助于将 Bag6 寡聚体的一部分锚定到逆转录位点，以提高 ERAD 效率。我们的研究还解决了最近研究中出现的一个令人惊讶的悖论，即泛素连接酶 (E3) 和去泛素酶 (DUB) 这两种具有相反活性的酶都可以促进 ERAD。我们证明 ERAD E3 gp78 不仅可以泛素化 ERAD 底物，还可以泛素化机械蛋白 Ubl4A（Bag6 分子伴侣复合物的关键组成部分）。值得注意的是，多泛素化不是针对 Ubl4A 进行降解，而是与不可逆蛋白水解加工和 Bag6 失活相关。重要的是，我们将 USP13 确定为 gp78 相关的 DUB，它消除了 Ubl4A 中的泛素缀合物以维持 Bag6 的功能。我们的研究揭示了一种意想不到的范例，其中 DUB 可以防止不需要的泛素化，从而提高相关泛素连接酶伴侣的底物特异性并促进 ER 质量控制。我们最近表明，蛋白质易位过程中核糖体停滞会诱导 UFM1（一种泛素样修饰剂）附着到内质网 60S 核糖体亚基 RPL26 (uL24) COOH 末端附近的两个保守赖氨酸残基上。引人注目的是，RPL26 UFMylation 能够降解停滞的新生链，但与 ERAD 或之前建立的胞质核糖体相关质量控制 (RQC)（使用蛋白酶体降解其客户蛋白）不同，核糖体 UFMylation 促进易位阻滞的 ER 蛋白的靶向溶酶体进行降解。 RPL26 UFMylation 在红细胞分化过程中上调，以应对分泌流量的增加，而 UFMylation 的受损会损害蛋白质分泌，并最终损害血红蛋白的产生。我们提出，在后生动物中，共翻译蛋白易位 (TAQC) 进入内质网受到 UFMylation 依赖性、易位相关的蛋白质质量控制机制的保护，当该机制受损时，会导致小鼠贫血和人类神经元发育异常。最近，我们使用全基因组 CRISPR/Cas9 筛选来鉴定一种名为 SAYSD1 的未表征膜蛋白，该蛋白有助于 TAQC。 SAYSD1 与 Sec61 易位子结合，通过直接识别核糖体和 UFM1 来接合停滞的新生链核糖体复合物，确保通过 TRAPP 复合物输出停滞的底物以进行溶酶体降解。与 UFM1 缺陷一样，SAYSD1 缺失会导致内质网易位停滞蛋白的积聚，从而引发内质网应激。重要的是，破坏果蝇中依赖于 UFM1 和 SAYSD1 的质量控制会导致易位停滞胶原蛋白的积累、胶原蛋白沉积缺陷、基底膜异常和应激耐受性降低。总之，我们的数据支持这样一个模型：SAYSD1 充当 ER 相关 UFM1 传感器，与易位子干扰位点的核糖体 UFMylation 协作，从而在动物发育过程中维护 ER 稳态。