Mechanism of protein retro-translocation from the endoplasmic reticulum

内质网蛋白质逆转位机制

• 批准号: 8741408
• 负责人: Yihong Ye
• 金额: $ 74.7万
• 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/8741408
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATPase Domain; Address; Adopted; Affinity; Affinity Chromatography; Binding; Cell Nucleus; Cell physiology; Cells; Complex; Cytoplasm; Cytosol; Degradation Pathway; Detergents; Deubiquitinating Enzyme; Deubiquitination; Dislocations; Electrostatics; Endoplasmic Reticulum; Environment; Enzymes; Eukaryota; Family; Goals; Homeostasis; Homo; Immune response; Immunologic Surveillance; In Vitro; Link; MJD1 protein; Machado-Joseph Disease; Mammalian Cell; Mediator of activation protein; Membrane; Membrane Proteins; Molecular; Molecular Chaperones; Molecular Conformation; Multiprotein Complexes; Mutation; Names; Nerve Degeneration; Pathogenesis; Pathway interactions; Physiological; Play; Process; Protein Biosynthesis; Proteins; Quality Control; Recruitment Activity; Regulation; Relative (related person); Role; Route; Side; Site; Solubility; Ubiquitin; Ubiquitination; Virus; cofactor; driving force; endoplasmic reticulum stress; human disease; improved; member; multicatalytic endopeptidase complex; mutant; novel; overexpression; p97 ATPase; polyglutamine; polypeptide; prevent; protein aggregate; protein misfolding; receptor; ubiquitin ligase

The endoplasmic reticulum (ER) is the major site of protein biosynthesis in eukaryotes. Polypeptides entering the ER may occasionally adopt aberrant conformations, resulting in aggregation-prone, misfolded proteins. The accumulation of misfolded proteins represents a form of ER stress, which has been implicated in the pathogenesis of many human diseases. To preserve ER homeostasis, eukaryotes have evolved a conserved quality control pathway termed retro-translocation or dislocation, which efficiently eliminates unwanted 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. The molecular mechanism of retro-translocation is largely unknown. For example, it is not well understood how cells can distinguish misfolded polypeptides from those that are in the folding process. How misfolded substrates are selectively targeted to the translocation site at the ER membrane, and subsequently transferred across the membrane are completely unknown. The identity of the protein-conducting channel for retro-translocation is still under debate. In addition, how viruses can exploit this cellular pathway during their invasion into the host cell is unclear. We have previously identified a cytosolic enzyme called p97, which provides the major driving force to move substrates into the cytosol during retro-translocation. Two co-factors of p97, Ufd1 and Npl4, are also required. The ATPase complex interacts in its ATP bound state with substrates emerging from the ER membrane, and the two ATPase domains appear to alternate in ATP hydrolysis to release polypeptides from the ER membrane once they are modified by poly-ubiquitination. Interestingly, we found that the ATPase complex contains several ubiquitin binding domains that specifically recognize ubiquitin chains. This partially explains why the ATPase complex preferentially acts on poly-ubiquitinated substrates. The interaction between the ubiquitin chains and p97 may trigger ATP hydrolysis by the ATPase, allowing it to pull substrates out of the ER membrane. To understand how p97 functions at the ER membrane, we 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, perhaps as a component of the protein-conducting channel. It receives substrates from the ER lumen, and also associates on the cytosolic side of the ER membrane with both the ubiquitination machinery and the "pulling" ATPase p97. Thus, it provides a link between substrate recognition in the ER lumen and polypeptide dislocation in the cytosol. We also demonstrated that efficient elimination of misfolded ER proteins also involves a p97-associated deubiquitinating enzyme, ataxin-3. Mutations in ataxin-3 have been linked to type-3 spinocerebellar ataxia, a member of the poly-glutamine induced neurodegenerative diesease family, but the physiological function of ataxin-3 is unclear. We show that overexpression of an ataxin-3 mutant defective in deubiquitination inhibits the degradation of misfolded ER proteins and triggers ER stress. Misfolded polypeptides stabilized by mutant ataxin-3 are accumulated in part as poly-ubiquitinated form, suggesting an involvement of its deubiquitinating activity in ERAD regulation. We demonstrate that ataxin-3 transiently associates with the ER membrane via p97 and the recently identified Derlin-VIMP complex, and its release from the membrane appears to be governed by both the p97 ATPase cycle and its own deubiquitinating activity. We present evidence that ataxin-3 may promote p97-associated deubiquitination to facilitate the transfer of polypeptides from p97 to the proteasome. In the past year, we identify 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 find that 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.

内质网（ER）是真核生物蛋白质生物合成的主要场所。进入内质网的多肽有时可能会采用异常构象，导致易于聚集、错误折叠的蛋白质。错误折叠蛋白的积累代表了内质网应激的一种形式，它与许多人类疾病的发病机制有关。为了保持内质网稳态，真核生物进化出了一种保守的质量控制途径，称为逆向易位或错位，通过将不需要的蛋白质输出到细胞质中，有效地消除内质网中不需要的蛋白质。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。逆向易位的分子机制很大程度上是未知的。例如，目前尚不清楚细胞如何区分错误折叠的多肽和处于折叠过程中的多肽。错误折叠的底物如何选择性地靶向内质网膜上的易位位点，并随后跨膜转移是完全未知的。逆转录转位的蛋白质传导通道的身份仍在争论中。此外，病毒在侵入宿主细胞期间如何利用这种细胞途径尚不清楚。 我们之前已经鉴定出一种称为 p97 的胞质酶，它提供了在逆向易位期间将底物移动到胞质溶胶中的主要驱动力。还需要 p97 的两个辅因子 Ufd1 和 Npl4。 ATP 酶复合物在其 ATP 结合状态下与 ER 膜上出现的底物相互作用，并且两个 ATP 酶结构域似乎在 ATP 水解中交替，一旦被多聚泛素化修饰，就会从 ER 膜上释放多肽。有趣的是，我们发现 ATP 酶复合物包含几个特异性识别泛素链的泛素结合域。这部分解释了为什么 ATP 酶复合物优先作用于多聚泛素化底物。泛素链和 p97 之间的相互作用可能会触发 ATP 酶水解 ATP，从而将底物从 ER 膜中拉出。为了了解 p97 如何在 ER 膜上发挥作用，我们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 在逆向易位中发挥着核心作用，可能是蛋白质传导通道的一个组成部分。它从 ER 腔接收底物，并且还在 ER 膜的胞质侧与泛素化机制和“拉动”ATP 酶 p97 结合。因此，它提供了内质网腔中的底物识别和胞质溶胶中的多肽错位之间的联系。 我们还证明，有效消除错误折叠的 ER 蛋白还涉及 p97 相关的去泛素化酶 ataxin-3。 ataxin-3 的突变与 3 型脊髓小脑共济失调有关，这是多聚谷氨酰胺诱导的神经退行性疾病家族的一员，但 ataxin-3 的生理功能尚不清楚。我们发现去泛素化缺陷的ataxin-3突变体的过度表达会抑制错误折叠的内质网蛋白的降解并引发内质网应激。由突变ataxin-3稳定的错误折叠多肽部分以多泛素化形式积累，表明其去泛素化活性参与了ERAD调节。我们证明，ataxin-3 通过 p97 和最近鉴定的 Derlin-VIMP 复合物与 ER 膜短暂结合，并且其从膜上的释放似乎受到 p97 ATP 酶循环及其自身的去泛素化活性的控制。我们提供的证据表明，ataxin-3 可能促进 p97 相关的去泛素化，以促进多肽从 p97 转移到蛋白酶体。 去年，我们鉴定了一种包含 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。 通过观察发现，ERAD 底物在缺乏 Bag6 或 Trc35（一种将 Bag6 保持在细胞核外参与 ERAD 的辅助因子）的细胞中积累在去污剂不溶性聚集物中，这一观察结果强调了这种保持酶活性的生理重要性。 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 复合物是一种调节多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与Bag6的一部分与膜紧密结合的事实不相符。 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。