Mechanism of protein retro-translocation from the endoplasmic reticulum

内质网蛋白质逆转位机制

基本信息

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
项目类别：
财政年份：
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

项目摘要

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，我们确定了包含BAG6，UBL4A和TRC35的泛素连接酶相关的多蛋白络合物，该复合物在通往蛋白酶体的途径的伴侣转循环的多肽中均转化为质量，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。我们发现，除了GP78外，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。