Mechanism of protein retro-translocation from the endoplasmic reticulum

内质网蛋白质逆转位机制

• 批准号: 9148777
• 负责人: Yihong Ye
• 金额: $ 88.76万
• 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/9148777
• 关键词: Address; Adopted; Affect; Affinity; Affinity Chromatography; Binding; Biochemical; Cell Nucleus; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytoplasm; Cytosol; Data; Degradation Pathway; Detergents; Dislocations; Electrostatics; Endoplasmic Reticulum; Environment; Enzymes; Eukaryota; Gene Silencing; Genetic; Goals; HIV; Homeostasis; Homo; Immune response; Immunologic Surveillance; In Vitro; Link; Mammalian Cell; Mediating; Mediator of activation protein; Membrane; Membrane Proteins; Molecular; Molecular Chaperones; Molecular Conformation; Multiprotein Complexes; Names; Pathogenesis; Pathway interactions; Physiological; Play; Polyubiquitination; Process; Protein Biosynthesis; Proteins; Proteolytic Processing; Quality Control; Recruitment Activity; Relative (related person); Research; Role; Route; Site; Solubility; Substrate Specificity; System; Ubiquitin; Ubiquitination; Virus; Virus Diseases; base; cofactor; endoplasmic reticulum stress; human disease; improved; luminal membrane; multicatalytic endopeptidase complex; novel; polypeptide; prevent; protein aggregate; protein misfolding; receptor; small hairpin RNA; tissue/cell culture; tool; ubiquitin ligase

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 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. 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. In last year, we characterize the functional interplay between Hrd1 and gp78, two ubiquitin ligases that have overlapping substrate specificity in ERAD. we characterize the gp78-containing ubiquitin ligase complex and define its functional interplay with Hrd1 using biochemical and recently developed CRISPR-based genetic tools. Our data show that the gp78 complex only provides an accessory function that can be compensated when this complex is permanently inactivated in tissue culture cells. Intriguingly, transient inactivation of the gp78 complex by short hairpin RNA-mediated gene silencing does cause significant stabilization of both luminal and membrane ERAD substrates. However, unlike Hrd1, which plays an essential role in retrotranslocation and ubiquitination of both luminal and membrane substrates, knockdown of gp78 does not affect these processes. Instead, gp78 appears to act downstream of Hrd1 to promote the degradation of ERAD substrates via cooperation with the BAG6 chaperone complex. We conclude that the Hrd1 complex forms an essential retrotranslocation module that is evolutionarily conserved, but the mammalian ERAD system employs additional ubiquitin ligases to assist Hrd1 during retrotranslocation.

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，它与两个辅助因子 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，以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。 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。 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。 去年，我们描述了 Hrd1 和 gp78 之间的功能相互作用，这两种泛素连接酶在 ERAD 中具有重叠的底物特异性。 我们使用生化和最近开发的基于 CRISPR 的遗传工具表征了包含 gp78 的泛素连接酶复合物，并定义了其与 Hrd1 的功能相互作用。我们的数据表明，gp78 复合物仅提供辅助功能，当该复合物在组织培养细胞中永久失活时，该功能可以得到补偿。有趣的是，短发夹 RNA 介导的基因沉默使 gp78 复合物瞬时失活确实会导致管腔和膜 ERAD 底物显着稳定。 然而，与在管腔和膜底物的逆转位和泛素化中发挥重要作用的 Hrd1 不同，gp78 的敲低不会影响这些过程。相反，gp78 似乎在 Hrd1 下游发挥作用，通过与 BAG6 伴侣复合物合作促进 ERAD 底物的降解。我们得出的结论是，Hrd1 复合物形成了一个重要的逆转录易位模块，该模块在进化上是保守的，但哺乳动物 ERAD 系统在逆转录易位期间采用额外的泛素连接酶来协助 Hrd1。