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）是真核生物蛋白质合成的主要部位。进入内质网的多肽可能经常采用异常构象，导致容易聚集、错误折叠的蛋白质。错误折叠蛋白的积累诱导内质网应激，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，称为逆转录易位/错位或内质网相关降解（ERAD），它通过将内质网中的错误折叠蛋白输出到细胞质中来消除它们。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。例如，人类免疫缺陷病毒使用一种名为Vpu的蛋白质靶向新合成的CD4共受体进行降解，从而促进病毒感染。