Mechanism of protein retro-translocation from the endoplasmic reticulum

内质网蛋白质逆转位机制

• 批准号: 9356081
• 负责人: Yihong Ye
• 金额: $ 93.78万
• 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/9356081
• 关键词: ATP phosphohydrolase; 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; Research; Role; Route; SGTA gene; Site; Solubility; Substrate Specificity; System; Ubiquitin; Ubiquitination; Virus; Virus Diseases; base; cofactor; endoplasmic reticulum stress; human disease; improved; inhibitor/antagonist; knock-down; 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 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. 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. We have also generated a highly potent inhibitor that blocks the ATPase activity of p97. We are using this tool to dissect the molecular mechanism by which p97 dislocates misfolded proteins out of the ER membrane.

内质网（ER）是真核生物中蛋白质生物合成的主要部位。进入ER的多肽可能经常采用异常构象，从而导致易折叠的蛋白质易发。错误折叠蛋白的积累会诱导ER应激，这与许多人类疾病的发病机理有关。为了保留ER蛋白稳态，真核生物进化了一种称为恢复转移/脱位或与ER相关的降解（ERAD）的保守质量控制途径，从而消除了ER中的错误折叠的蛋白质，通过将它们导出到细胞质中。胞质蛋白酶体处理了进行恢复重新定位的多肽。某些病毒劫持了复古转移途径，以破坏免疫反应所需的折叠细胞蛋白，从而使病毒逃避宿主的免疫监测。例如，人类免疫缺陷病毒使用名为VPU的蛋白质靶向新合成的CD4共受体降解，从而促进病毒感染。 我们先前鉴定出一种称为p97的胞质酶，该酶与两个辅助因子UFD1和NPL4作用，以将逆转录底物移动到细胞质中以降解。我们还使用了一种亲和力纯化方法来识别与p97相关的两种新型ER膜蛋白，即Derlin-1和VIMP。 VIMP充当将P97募集到ER膜的受体。保守的多跨膜膜蛋白Derlin-1在复古转移中起着核心作用。它似乎从ER管腔中接收底物，以通过尚未定义的膜孔来促进其易位。 我们进一步确定了泛素连接酶相关的多蛋白复合蛋白复合物，其中包括BAG6，UBL4A和TRC35，该复合物在途中转化为蛋白酶体以提高ERAD效率。 在体外，BAG6是该复合物的中心成分，它包含一种类似伴侣的活性，能够在展开但可溶的状态下维持易于聚集的底物。 这种持有酶活性的生理重要性强调了观察到的观察结果是，ERAD底物积聚在洗涤剂不溶性骨料中，在耗尽的BAG6或TRC35的细胞中，或TRC35（一种将BAG6都放在核外面的辅助因子，以使Bag6在核外面互动。 我们的结果揭示了一种泛素连接酶相关的持有酶，该酶保持多肽溶解度以增强哺乳动物细胞的蛋白质质量控​​制。 BAG6复合物还参与了其他几个蛋白质质量控​​制过程，但是BAG6在复杂的细胞环境中如何有效捕获错误折叠的多肽尚不清楚。我们最近发现了一个名为SGTA的新型ERAD介质，该介质与BAG6形成了伴侣级联，以帮助通道脱位的Erad底物，否则很容易聚集。 我们表明，SGTA包含一个异常的泛素样（UBL）结合基序，该基序通过静电与UBL4A中的非传统UBL结构域进行了专门相互作用。 这种相互作用增强了对BAG6的底​​物加载，以防止形成不可降解的蛋白质聚集体，从而提高了ERAD效率。 Bag6-ubl4a-Trc35复合物是一种多功能伴侣，可调节各种细胞过程。 由于BAG6的多种功能得到了其在细胞中的细胞质，细胞核和膜内质网（ER）中的无处不在的定位，因此我们最近研究了BAG6与ER膜的相关性。 我们发现，在与ER相关的降解（ERAD）途径中，BAG6可以通过其泛素样（UBL）域中的膜相关的泛素相关性泛素连接酶GP78与CUE域相互作用，但是这种交互的相对低亲密度但是这种相互作用的相对低亲度与Bag6的效果无关，而不是与Bag6的效果紧密界定。 在这里，我们证明了BAG6的UBL域与ER膜相互作用是必需的，尽管对GP78的亲和力较低。 除了GP78之外，我们还会发现BAG6 UBL域还结合了UBXD8中的UBL结合基序，UBXD8是GP78泛素化机械的重要组成部分。 重要的是，BAG6形成了一个大型的同源物体，使UBL结构域与含GP78的逆转录旋转复合物形成了多价相互作用。 GP78和UBXD8都包含p97识别的图案，从而将BAG6与膜中的此核心逆转录机械联系起来。 我们建议，与多种ERAD因子同时关联有助于将BAG6低聚物的一小部分固定在逆转录位置上，以提高ERAD效率。 我们的研究还解决了最近的研究中出现的一个令人惊讶的悖论，即泛素连接酶（E3S）和去泛素酶（DUB），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，而且还可以泛素化机械蛋白UBL4A，这是BAG6伴侣复合体的关键组成部分。值得注意的是，多泛素化不是靶向UBL4A降解，而是与Bag6的不可逆蛋白水解处理和失活有关。重要的是，我们将USP13识别为与GP78相关的DUB，它消除了UBL4A的泛素结合物以维持BAG6的功能。我们的研究揭示了一个意外的范式，其中配音防止了不需要的泛素化，从而增强了相关的泛素连接酶伴侣的底物特异性并促进ER质量控制。 去年，我们表征了HRD1和GP78之间的功能相互作用，这是两个在ERAD中具有重叠底物特异性重叠的泛素连接酶。 我们表征了含GP78的泛素连接酶复合物，并使用生化和最近开发的基于CRISPR的遗传工具来定义其与HRD1的功能相互作用。我们的数据表明，GP78复合物仅提供一个辅助函数，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介导的基因沉默对GP78复合物的瞬时灭活确实会导致腔内和膜ERAD底物的显着稳定。 但是，与HRD1在腔内和膜底物的逆转转换和泛素化中起着至关重要的作用不同，GP78的敲低不会影响这些过程。取而代之的是，GP78似乎在HRD1的下游作用，以通过与BAG6 Chaperone Complex的合作来促进ERAD底物的降解。我们得出的结论是，HRD1复合物形成了一个必不可少的逆转录模块，该模块在进化上是保守的，但是哺乳动物ERAD系统采用其他泛素连接酶在逆转录过程中帮助HRD1。 我们还产生了一种高度有效的抑制剂，该抑制剂阻断了p97的ATPase活性。 我们正在使用此工具来剖析p97将错误折叠蛋白从ER膜中脱位的分子机制。