How do cells eliminate unassembled cytocolic proteins?

细胞如何消除未组装的胞质蛋白？

• 批准号: 9549994
• 负责人: Yihong Ye
• 金额: $ 28.13万
• 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/9549994
• 关键词: Aneuploidy; Cell Death; Cell Nucleus; Cell Survival; Cells; Chemicals; Complex; Cytoplasm; Cytoplasmic Protein; Cytosol; Elements; Endoplasmic Reticulum; Enzymes; Eukaryota; Eukaryotic Cell; Genomic Instability; Goals; Hydrophobicity; Individual; Lead; Lectin; Malignant Neoplasms; Mammalian Cell; Mediating; Membrane; Membrane Proteins; Messenger RNA; Methionine; Modeling; Molecular Chaperones; Monitor; N-terminal; Names; Nuclear Protein; Pathway interactions; Play; Process; Proteins; Quality Control; Regulation; Research Project Grants; Ribosomes; Role; Saccharomycetales; System; Translations; Ubiquitination; Yeasts; base; cancer cell; cytotoxicity; endoplasmic reticulum stress; misfolded protein; multicatalytic endopeptidase complex; novel; polypeptide; protein aggregation; protein complex; protein degradation; protein function; proteostasis; stoichiometry; targeted cancer therapy; tumor; ubiquitin ligase

Many eukaryotic proteins function in multi-subunit complexes with a stoichiometry that needs to be strictly maintained. It is thought that the degradation of unassembled subunits might be an essential mechanism that controls complex stoichiometry. Elimination of unassembled proteins is also crucial for protein homeostasis because unassembled proteins often contain exposed hydrophobic segments that can lead to protein aggregation and cytotoxicity. In fact, a major effort in developing anti-cancer therapies that target proteostasis-addicted tumors is based on the assumption that unbalanced protein assembly due to aneuploidy or other genome instabilities in cancer cells render them more susceptible to chemicals that disturb the proteostasis network. In this regard, identification of cellular components mediating the degradation of unassembled proteins may reveal novel anti-cancer targets. Membrane and secreted protein complexes are usually assembled in the endoplasmic reticulum (ER) after individual subunits have been imported into the ER. The assembly process is subject to a strict checkpoint regulation enforced by an efficient protein quality control (PQC) mechanism. The ER PQC pathway employs chaperones, lectins and other enzymes to monitor the assembly process, identifying unassembled polypeptides for retrotranslocation, ubiquitination and proteasomal degradation in the cytosol. This conserved process is termed ER-associated protein degradation (ERAD), which is critical for cell viability because unassembled polypeptides can interfere with normal protein assembly when they become misfolded or form non-specific interactions. Unassembled ER proteins can also co-aggregate with essential cellular factors such as chaperones to cause ER stress, which if not rectified, can lead to cell death. Many proteins in the cytosol and nucleus also form multi-subunit assemblies, but the mechanism by which cells eliminate unassembled soluble proteins is not well understood. Several studies have investigated the mechanism of cytoplasmic and nuclear PQC, which remove misfolded or damaged proteins from the cytoplasm and nucleus, respectively. These studies identified several pathways that target misfolded proteins of different classes to the proteasome for degradation. For example, the ribosome-associated ubiquitin ligase Ltn1 in budding yeast recognizes and ubiquitinates defective translation products due to non-stop messenger RNAs. In mammalian cells, a chaperone-associated ubiquitin ligase named RNF126 targets mislocalized membrane proteins for degradation. However, these studies did not use substrates representing unassembled polypeptides. Therefore, it is unclear whether these cytosolic PQC pathways play a role in unassembled soluble protein degradation (USPD). To date, the best-characterized cytosolic quality control pathway is the N-end rule pathway, which mediates the degradation of substrates bearing an N-terminal destabilizing element termed degron. The N-end rule substrates have been classified into three major groups: those with an N-terminal destabilizing residue, those with an exposed acetylated N-terminal methionine residue and a group of proteins with an N-terminal initiator methionine followed by a hydrophobic residue. A major ubiquitin ligase responsible for degradation of non-acetylated N-end rule substrates is UBR1 and the related enzymes UBR2 and UBR3. In yeast, a protein named CNOT4 was recently identified as the ubiquitin ligase for an unassembled soluble protein carrying an exposed acetylated N-terminal methionine. It is conceivable that some USPD substrates may carry one of the above-mentioned degrons, but for those without a predicted N-end rule degron, how they are targeted for degradation is unclear. We have established model substrates to study N-end rule independent USPD in mammalian cells. Our study establishes HUWE1 as an enzyme that ubiquitinates substrates bearing exposed hydrophobic residues due to incomplete assembly to cause their degradation by the proteasome. We identify endogenous HUWE1 substrates, which reveal a new surveillance system that safeguards the proteostasis network of the eukaryotic cells.

许多真核蛋白质在多生育络合物中的功能具有严格维护的化学计量。人们认为，未组装亚基的降解可能是控制复杂化学计量法的基本机制。消除未组装的蛋白质对于蛋白稳态也至关重要，因为未组装的蛋白通常包含裸露的疏水片段，这可能导致蛋白质聚集和细胞毒性。实际上，针对靶向蛋白质的肿瘤的抗癌疗法的主要努力是基于这样的假设：癌细胞中由于非整倍性或其他基因组不稳定性而导致的蛋白质组装不平衡，使它们更容易受到扰乱蛋白质治疗网络的化学物质。在这方面，介导未组装蛋白质降解的细胞成分的鉴定可能揭示了新型的抗癌靶标。 膜和分泌的蛋白质复合物通常在将单个亚基进口到ER后的内质网中（ER）。组装过程受到有效蛋白质质量控​​制（PQC）机制强制执行的严格检查点调节。 ER PQC途径使用伴侣，凝集素和其他酶来监测装配过程，从而识别细胞质中的未组装多肽，用于逆转录，泛素化和蛋白酶体降解。该保守的过程称为ER相关的蛋白质降解（ERAD），这对于细胞活力至关重要，因为当未组装的多肽变成错误折叠或形成非特异性相互作用时，它们可能会干扰正常的蛋白质组装。未组装的ER蛋白还可以与必需的细胞因子（例如伴侣引起ER胁迫）共聚集，如果不矫正，则可能导致细胞死亡。 细胞质和细胞核中的许多蛋白质也形成了多生成组件，但是细胞消除未组装的可溶性蛋白质的机制尚不清楚。几项研究研究了细胞质和核PQC的机制，这些机制分别从细胞质和核中清除了错误折叠或受损的蛋白质。这些研究确定了几种将不同类别的蛋白质错误折叠蛋白靶向蛋白酶体降解的途径。例如，萌芽酵母中与核糖体相关的泛素连接酶LTN1识别和泛素识别由于不间断的Messenger RNA而导致的有缺陷的翻译产物。在哺乳动物细胞中，称为RNF126的伴侣相关的泛素连接酶靶向错误定位的膜蛋白降解。但是，这些研究并未使用代表未组装多肽的底物。因此，尚不清楚这些胞质PQC途径是否在未组装的可溶性蛋白降解（USPD）中起作用。 迄今为止，最佳特征的胞质质量控制途径是N端规则途径，它介导了带有N端不稳定元件的底物的降解，称为DEGRON。 N末端规则底物已分为三个主要组：具有N末端不稳定的残基的基质，那些患有乙酰化N末端蛋氨酸残基和一组具有N端启动蛋氨酸的蛋白质的蛋白质，然后是疏水性残基。负责非乙酰化N端规则底物降解的主要泛素连接酶是UBR1和相关酶UBR2和UBR3。在酵母中，最近将一种名为CNOT4的蛋白鉴定为携带暴露于乙酰化N末端蛋氨酸的未组装可溶性蛋白的泛素连接酶。可以想象，某些USPD底物可能会携带上述DEGRON之一，但是对于那些没有预测的n端规则DEGRON的人来说，如何将其靶向降解。 我们已经建立了模型底物来研究哺乳动物细胞中独立的USPD。我们的研究将HUWE1确定为一种酶，该酶泛素化，这是由于组装不完整而导致蛋白酶体降解的底物，该底物带有暴露的疏水残基。我们确定了内源性HUWE1底物，该底物揭示了一种新的监视系统，该系统可以保护真核细胞的蛋白质量网络。