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How do cells eliminate unassembled cytocolic proteins?

细胞如何消除未组装的胞质蛋白？

基本信息

批准号：
10004462
负责人：
Yihong Ye
金额：
$ 75.12万
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/10004462
关键词：
Anemia Aneuploidy Biogenesis Cell Death Cell Nucleus Cell Survival Cells Chemicals Client Complex Cytoplasm Cytoplasmic Protein Cytosol Elements Endoplasmic Reticulum Enzymes Eukaryota Eukaryotic Cell Genomic Instability Goals Hemoglobin Human Hydrophobicity Impairment Individual Lead Lectin Lysine Lysosomes Mammalian Cell Mediating Membrane Membrane Proteins Messenger RNA Methionine Modeling Molecular Chaperones Monitor Mus N-terminal Names Nuclear Protein Pathway interactions Play Process Production Protein Secretion Protein translocation Proteins Quality Control Regulation Research Project Grants Ribosomes Role Saccharomycetales System Translations Ubiquitin Ubiquitination Yeasts anti-cancer base cancer cell cancer therapy cytotoxicity endoplasmic reticulum stress erythroid differentiation misfolded protein multicatalytic endopeptidase complex neuron development novel polypeptide protein aggregation protein complex protein degradation protein function proteostasis stoichiometry targeted cancer therapy tumor ubiquitin ligase

项目摘要

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. In addition, we have started to investigate ribosome-quality control. In eukaryotic cells, protein biogenesis at the endoplasmic reticulum (ER) is monitored by a protein quality control system named ER-associated protein degradation (ERAD). While there has been substantial progress in understanding how ERAD eliminates defective polypeptides generated from erroneous folding, how cells remove nascent chains stalled in the translocon during co-translational protein insertion into the ER is unclear. Here we show that ribosome stalling during protein translocation at the ER induces the attachment of UFM1, a ubiquitin-like modifier, to two conserved lysine residues near the COOH-terminus of the 60S ribosomal subunit RPL26 (uL24). Strikingly, RPL26 UFMylation enables the degradation of stalled nascent chains, but unlike ERAD or previously established cytosolic ribosome-associated quality control (RQC), which uses proteasome to degrade their client proteins, ribosome UFMylation promotes the targeting of a translocation-arrested ER protein to lysosomes for degradation. RPL26 UFMylation is upregulated during erythroid differentiation, which helps cells to cope with increased secretory flow, and compromising UFMylation impairs protein secretion, and ultimately hemoglobin production. We propose that in metazoan, co-translational protein translocation into the ER is safeguarded by a UFMylation-dependent protein quality control mechanism, which when impaired causes anemia in mice and abnormal neuronal development in humans.

许多真核蛋白质在多亚基复合体中发挥作用，其化学计量比需要严格保持。人们认为，未组装亚基的降解可能是控制复杂化学计量学的一个重要机制。消除未组装的蛋白质对蛋白质动态平衡也是至关重要的，因为未组装的蛋白质通常含有暴露的疏水片段，可导致蛋白质聚集和细胞毒性。事实上，针对蛋白平衡成瘾肿瘤开发抗癌疗法的主要努力是基于这样的假设，即癌细胞中非整倍体或其他基因组不稳定性导致的蛋白质组装不平衡使它们更容易受到干扰蛋白平衡网络的化学物质的影响。在这一点上，鉴定介导未组装蛋白降解的细胞成分可能揭示新的抗癌靶点。膜和分泌的蛋白质复合体通常在内质网(ER)中组装后，个别亚基被引入内质网。组装过程受到由有效的蛋白质质量控制(PQC)机制执行的严格检查点监管。内质网PQC途径利用伴侣、凝集素和其他酶来监控组装过程，识别胞浆中逆转录易位、泛素化和蛋白酶体降解的未组装多肽。这种保守的过程被称为内质网相关蛋白降解(ERAD)，这对细胞存活至关重要，因为未组装的多肽在错误折叠或形成非特异性相互作用时会干扰正常的蛋白质组装。未组装的ER蛋白也可以与基本的细胞因子(如伴侣)共同聚集，导致内质网应激，如果不加以纠正，可能导致细胞死亡。胞浆和胞核中的许多蛋白质也形成多亚基组合，但细胞清除未组装的可溶性蛋白的机制尚不清楚。一些研究已经探讨了细胞质和核PQC的机制，PQC分别从细胞质和细胞核中去除错误折叠或损坏的蛋白质。这些研究确定了几条将不同类别的错误折叠蛋白靶向蛋白酶体进行降解的途径。例如，萌芽酵母中的核糖体相关泛素连接酶Ltd n1识别并泛化由于持续信使RNA而产生的有缺陷的翻译产物。在哺乳动物细胞中，一种名为RNF126的伴侣相关泛素连接酶针对错误定位的膜蛋白进行降解。然而，这些研究没有使用代表未组装多肽的底物。因此，目前还不清楚这些胞质PQC通路是否在未组装的可溶性蛋白降解(USPD)中起作用。到目前为止，最具特征性的细胞质质量控制途径是N-末端规则途径，它介导含有N-末端不稳定元件的底物的降解，称为degron。N-末端规则底物被分为三大类：具有N-末端不稳定残基的底物，具有暴露的乙酰化N-末端蛋氨酸残基的底物，以及具有N-末端引发剂蛋氨酸和疏水残基的蛋白质组。负责降解非乙酰化N-末端规则底物的主要泛素连接酶是UBr1及其相关酶UBr2和UBR3。在酵母中，一种名为CNOT4的蛋白质最近被鉴定为泛素连接酶，它是一种携带暴露的乙酰化N-末端蛋氨酸的未组装的可溶性蛋白质。可以想象，一些USPD底物可能带有上述退化之一，但对于那些没有预测的N端规则退化的底物，它们如何被降解尚不清楚。我们已经建立了模型底物来研究哺乳动物细胞中N-端规则非依赖性USPD。我们的研究确定HUWE1是一种泛素化底物，由于不完全组装而泛素化暴露的疏水残基，导致它们被蛋白酶体降解。我们鉴定了内源性HUWE1底物，这揭示了一个新的监测系统，它保护了真核细胞的蛋白平衡网络。此外，我们已经开始研究核糖体的质量控制。在真核细胞中，内质网(ER)的蛋白质生物发生由一种名为ER相关蛋白降解(ERAD)的蛋白质质量控制系统来监控。虽然在理解ERAD如何消除错误折叠产生的缺陷多肽方面已经取得了实质性进展，但细胞如何在共翻译蛋白插入内质网的过程中移除停滞在转位蛋白中的新生链尚不清楚。在这里，我们证明了在内质网蛋白质转位过程中的核糖体停滞导致UFM1，一种泛素样修饰物，与60S核糖体亚基RPL26(UL24)的COOH末端附近的两个保守的赖氨酸残基结合。值得注意的是，RPL26 UFM化使停滞的新生链能够降解，但与ERAD或先前建立的细胞质核糖体相关质量控制(RQC)不同，核糖体UFM化促进易位受阻的ER蛋白靶向溶酶体降解。RPL26 UFM化在红系分化过程中上调，这有助于细胞应对增加的分泌流量，而受损的UFM化会损害蛋白质分泌，最终导致血红蛋白的产生。我们认为，在后生动物中，共翻译的蛋白质移位到内质网受到UFM化依赖的蛋白质质量控制机制的保护，当受到损害时，会导致小鼠贫血和人类神经元发育异常。