Mechanism of protein retro-translocation from the endoplasmic reticulum

内质网蛋白质逆转位机制

• 批准号: 8148157
• 负责人: Yihong Ye
• 金额: $ 48.37万
• 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/8148157
• 关键词: Endoplasmic Reticulum; Proteins

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 dissected the role of intramembrane charged residues in ER quality control of T- cell receptor. We found that a TCR mutant lacking the intramembrane charged residues has a tendency to form homo-oligomer via interchain disulfide bond that involves a specific pair of cysteine residues. Covalent oligomerization of TCR appears to stabilize it at the ER membrane. The presence of a single lysine residue at specific positions within the TCR TM domain abolishes its oligomerization and causes its rapid degradation. Conversely, when TCR oligomerization is induced by a bivalent compound, the degradation of TCR is inhibited. Together, these results suggest that the intramembrane charged residues in TCR do not function as a signal for substrate recognition in ERAD. Instead, their primary role is to reduce TCR oligomerization to maintain it in a retrotranslocation competent state. Our results also suggest that the ERAD machinery is inefficient when coping with oligomerized substrates, indicating a requirement for chaperone-mediated protein disassembly in the ER lumen prior to retrotranslocation. We also studied the mechanism by which the human cytomegalovirus (HCMV) protein US2 hijacks the ER-associated degradation (ERAD) machinery to dispose of MHC class I heavy chain (HC) at the endoplasmic reticulum (ER). We established an in vitro permeabilized cell assay that recapitulates the retrotranslocation of MHC HC in US2-expressing cells. Using this assay, we demonstrate that the dislocation process requires ATP and ubiquitin, as expected. The retrotranslocation also involves the p97 ATPase. However, the mechanism by which p97 dislocates MHC class I HC in US2 cells is distinct from that in US11 cells: the dislocation reaction in US2 cells is independent of the p97 cofactor Ufd1-Npl4. Our results suggest that different retrotranslocation mechanisms can employ distinct p97 ATPase complexes to dislocate substrates.

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的多肽有时可能会采用异常构象，从而导致易折叠的蛋白质。错误折叠蛋白的积累代表了一种ER应激的形式，这与许多人类疾病的发病机理有关。为了保留ER稳态，真核生物已进化出一种称为恢复转移或脱位的保守质量控制途径，该途径通过将其导出到细胞质中，有效地消除了ER中有害蛋白质。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。复古转移的分子机制在很大程度上是未知的。例如，尚不清楚细胞如何将错误折叠的多肽与折叠过程中的多肽区分开。错误折叠的底物如何选择性地靶向ER膜上的易位位点，然后在整个膜上转移的底物是完全未知的。蛋白质传导通道的重新转换通道的身份仍在争论中。另外，病毒在入侵宿主细胞期间如何利用该细胞途径的方式尚不清楚。 我们先前已经鉴定出一种称为p97的胞质酶，该酶在恢复转移过程中提供了将底物移至胞质溶胶的主要驱动力。还需要两个p97，UFD1和NPL4的共同因素。 ATPase复合物以其ATP结合状态与从ER膜出现的底物相互作用，并且两个ATPase结构域在ATP水解中似乎在ATP水解中交替以一旦通过多泛素化而从ER膜中释放多肽。有趣的是，我们发现ATPase复合物包含几个特异性识别泛素链的泛素结合结构域。这部分解释了为什么ATPase复合物优先作用于多泛素化的底物。泛素链和p97之间的相互作用可能会触发ATP酶的ATP水解，从而使其可以将底物从ER膜中拉出。为了了解p97在ER膜上的功能，我们使用了一种亲和力纯化方法来识别两种新型的ER膜蛋白Derlin-1和VIMP，它们与P97相关。 VIMP充当将P97募集到ER膜的受体。保守的多跨度膜蛋白DERLIN-1在逆转转移中起着核心作用，也许是蛋白质传导通道的组成部分。它从ER腔内接收底物，并在ER膜的胞质侧接收粘膜，并与泛素化机械和“拉动” ATPase P97接收底物。因此，它在ER管腔中的底物识别与细胞质中的多肽脱位之间提供了联系。 我们还证明，有效消除错误折叠的ER蛋白还涉及与P97相关的去泛素化酶Ataxin-3。 ataxin-3中的突变已与3型脊椎动物共济失调有关，这是多谷氨酰胺诱导的神经退行性脱发酶家族的成员，但是ataxin-3的生理功能尚不清楚。我们表明，在去泛素化中有缺陷的ataxin-3突变体的过表达抑制了错误折叠的ER蛋白和触发ER应激的降解。通过突变体共生3稳定的错误折叠的多肽部分以多泛素化形式积累，这表明其去泛素化活性参与ERAD调节。我们证明，共生蛋白3通过p97和最近确定的Derlin-Vimp复合物瞬时与ER膜相关联，并且其从膜上释放似乎受P97 ATPase循环及其自身的去泛素化活性的控制。我们提供了证据，表明共生蛋白3可以促进与P97相关的去泛素化，以促进多肽从p97转移到蛋白酶体。 在过去的一年中，我们阐述了膜内电荷残基在T细胞受体的ER质量控制中的作用。我们发现，缺乏膜内电荷残基的TCR突变体倾向于通过涉及特定的半胱氨酸残基的链间二硫键形成同性恋者。 TCR的共价寡聚化似乎可以在ER膜上稳定。在TCR TM结构域中的特定位置存在单个赖氨酸残基会废除其寡聚化并导致其快速降解。 相反，当TCR寡聚通过二动化合物诱导时，抑制TCR的降解。总之，这些结果表明，TCR中膜内电荷残基不能充当ERAD中底物识别的信号。相反，他们的主要作用是减少TCR寡聚化，以将其保持在逆转录统计状态。我们的结果还表明，在应对寡聚底物时，ERAD机械效率低下，这表明在逆转倾斜之前，需要在ER管腔中脱离伴侣介导的蛋白质。 我们还研究了人类巨细胞病毒（HCMV）蛋白US2劫持与ER相关的降解（ERAD）机械以在内质网（ER）处置MHC I类重链（HC）的机制。我们建立了一种体外通透性细胞测定法，该测定法概括了表达US2的细胞中MHC HC的逆转录。使用此测定，我们证明了脱位过程需要如预期的那样，需要ATP和泛素。逆转录分配还涉及p97 ATPase。但是，p97脱位MHC I类HC中的机制与US11细胞中的机制不同：US2细胞中的脱位反应与p97辅助因子UFD1-NPL4无关。我们的结果表明，不同的逆转录机制可以采用不同的p97 ATPase复合物来摆脱底物。