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）是真核生物蛋白质合成的主要部位。进入内质网的多肽可能偶尔会采用异常构象，导致容易聚集、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，这与许多人类疾病的发病机制有关。为了保持内质网的稳态，真核生物进化出了一种保守的质量控制途径，称为逆转录或位错，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的蛋白质。进行逆转录的多肽被胞质蛋白酶体处理掉。逆转录易位途径被某些病毒劫持，破坏免疫应答所需的折叠细胞蛋白，使病毒逃避宿主免疫监视。逆转录易位的分子机制在很大程度上是未知的。例如，细胞是如何区分错误折叠的多肽和那些处于折叠过程中的多肽的，目前还不是很清楚。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，并随后在膜上转移是完全未知的。逆转录易位的蛋白质传导通道的身份仍在争论中。此外，病毒在入侵宿主细胞时如何利用这一细胞途径尚不清楚。