Regulation and function of deubiquitinating enzyme USP19

去泛素化酶USP19的调控和功能

• 批准号: 9356202
• 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/9356202
• 关键词: Active Sites; Affect; Affinity; Aging; Binding; Catalytic Domain; Cell physiology; Cells; Cellular biology; Cleaved cell; Cysteine; Cytosol; DNA Damage; Deubiquitinating Enzyme; Deubiquitination; Encapsulated; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Enzymes; Exposure to; Goals; Homeostasis; Human; In Vitro; Link; Lysine; Malignant Neoplasms; Membrane; Molecular Chaperones; Mono-S; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Polyubiquitin; Post-Translational Protein Processing; Process; Proteasome Inhibitor; Protein Secretion; Proteins; Quality Control; Reactive Oxygen Species; Regulation; Reporting; Role; Signal Transduction; Site; Surface; Transmembrane Domain; Ubiquitin; Ubiquitination; base; cell type; late endosome; member; novel; overexpression; oxidation; oxidative DNA damage; programs; protein misfolding; receptor; response; secretion process

Protein modification by ubiquitin (Ub) is a critical regulatory process for virtually all aspects of cell biology. Substrate proteins can be modified with single ubiquitin on one (monoubiquitylation) or multiple sites (multi-ubiquitylation). Alternatively, several rounds of ubiquitination can occur on ubiquitin itself, leading to the formation of a polyubiquitin chain. Any of the seven lysines, or the amino terminus, of ubiquitin can be used to polymerize ubiquitin (Peng et al., 2003), so there are a huge number of differently linked polyubiquitin signals that can be formed. Ub signals are reversible as ubiquitin can be removed from substrates by deubiquitinating enzymes. The diverse Ub signals are recognized in cells by a myriad of receptors that carry distinct ubiquitin binding motifs recognizing mono- or polyubiquitinated substrates (Hicke et al., 2005). The mechanisms that regulate deubiquitinases in the cells are unclear. Here we characterize 34 human DUBs including 25 USP, 4 OTU, 1 Josephin and 4 UCHL subfamily members. We show that many of these enzymes are reversibly inactivated when oxidized by reactive oxygen species (ROS) in vitro and in the cell. Oxidation occurs preferentially on the catalytic cysteine, abrogating the isopeptide-cleaving activity without affecting these enzymes affinity to ubiquitin. Sensitivity to oxidative inhibition is associated with the activation of the DUBs wherein the active site cysteine is converted to a deprotonated state prone to oxidation. We further demonstrate that this redox-dependent regulation is essential for mono-ubiquitination of PCNA to occur in response to oxidative DNA damage, which initiates a DNA damage tolerance program. These findings establish a novel mechanism of DUB regulation that may be integrated with other redox-dependent signaling circuits to govern cellular adaptation to oxidative stress, a process intimately linked to aging and cancer. We recently characterized the function and regulation of USP19. We identify Hsp90 as a specific partner that binds the catalytic domain of USP19 to promote substrate association. Intriguingly, although overexpressed USP19 interacts with Derlin-1 and other ERAD machinery factors in the membrane, endogenous USP19 is mostly in the cytosol where it binds Hsp90. Accordingly, we detect neither interaction of endogenous USP19 with Derlin-1 nor significant effect on ERAD by USP19 depletion. The USP19 transmembrane domain appears to be partially stabilized in the cytosol by an interaction with its own catalytic domain, resulting in auto-inhibition of its deubiquitinating activity. These results clarify the role of USP19 in ERAD and suggest a novel DUB regulation that involves chaperone association and membrane integration. We also discover a new cellular process that is regulated by USP19. Specifically, we report a pathway termed Misfolding-Associated Protein Secretion (MAPS), which uses the endoplasmic reticulum (ER)-associated deubiquitinase USP19 to preferentially export aberrant cytosolic proteins. Intriguingly, the catalytic domain of USP19 possesses an unprecedented chaperone activity, allowing recruitment of misfolded proteins to the ER surface for deubiquitination. Deubiquitinated cargos are encapsulated into ER-associated late endosomes and secreted to cell exterior. USP19 deficient cells cannot efficiently secrete unwanted proteins and grow more slowly than wild-type cells upon exposure to a proteasome inhibitor. Together, our findings delineate a protein quality control (PQC) pathway, which unlike degradation-based PQC mechanisms, promotes protein homeostasis by exporting misfolded proteins through an unconventional protein secretion process.

泛素（ubiquitin，Ub）对蛋白质的修饰是细胞生物学各个方面的重要调控过程。底物蛋白可以在一个位点（单泛素化）或多个位点（多泛素化）上被单个泛素修饰。或者，几轮泛素化可以发生在泛素本身上，导致多聚泛素链的形成。泛素的七个赖氨酸或氨基末端中的任一个都可用于泛素化（Peng et al.，2003），因此可以形成大量不同连接的多聚泛素信号。泛素信号是可逆的，因为泛素可以通过去泛素化酶从底物中去除。在细胞中，多种多样的Ub信号被携带识别单或多泛素化底物的不同泛素结合基序的无数受体识别（Hicke等人，2005年）。 调节细胞中去泛素化酶的机制尚不清楚。在这里，我们表征了34个人DUB，包括25个USP，4个OTU，1个Josephin和4个UCHL亚家族成员。 我们发现，许多这些酶是可逆失活时，在体外和细胞中的活性氧（ROS）氧化。 氧化优先发生在催化半胱氨酸上，废除了异肽裂解活性，而不影响这些酶对泛素的亲和力。 对氧化抑制的敏感性与DUB的活化有关，其中活性位点半胱氨酸转化为易于氧化的去质子化状态。 我们进一步证明，这种氧化还原依赖性的调节是必不可少的单泛素化的PCNA发生在氧化DNA损伤，启动DNA损伤耐受程序。 这些发现建立了一种新的DUB调节机制，可以与其他氧化还原依赖性信号通路整合，以控制细胞对氧化应激的适应，这一过程与衰老和癌症密切相关。 我们最近描述了USP 19的功能和调节。我们确定热休克蛋白90作为一个特定的合作伙伴，结合催化结构域的USP 19，以促进底物协会。有趣的是，尽管过表达的USP 19与膜中的Derlin-1和其他ERAD机制因子相互作用，但内源性USP 19主要存在于细胞质中，在那里它结合Hsp 90。因此，我们既未检测到内源性USP 19与Derlin-1的相互作用，也未检测到USP 19消耗对ERAD的显著影响。USP 19跨膜结构域似乎通过与其自身催化结构域的相互作用在胞质溶胶中部分稳定，导致其去泛素化活性的自抑制。这些结果阐明了USP 19在ERAD中的作用，并提出了一种新的DUB调节，涉及伴侣缔合和膜整合。 我们还发现了一个受USP 19调控的新细胞过程。 具体来说，我们报告了一个途径称为错误折叠相关蛋白分泌（MAPS），它使用内质网（ER）相关的去泛素化酶USP 19优先出口异常的胞质蛋白。有趣的是，USP 19的催化结构域具有前所未有的伴侣活性，允许错误折叠的蛋白质募集到ER表面进行去泛素化。去泛素化的货物被封装到ER相关的晚期内体中并分泌到细胞外部。 USP 19缺陷型细胞不能有效分泌不需要的蛋白质，并且在暴露于蛋白酶体抑制剂时比野生型细胞生长更慢。总之，我们的研究结果描绘了一个蛋白质质量控制（PQC）途径，它不同于基于降解的PQC机制，通过非常规的蛋白质分泌过程输出错误折叠的蛋白质来促进蛋白质稳态。

项目成果

Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells.

• DOI: 10.1038/ncb3372
• 发表时间: 2016-07
• 期刊: NATURE CELL BIOLOGY
• 影响因子: 21.3
• 作者: Lee, Jin-Gu;Takahama, Shokichi;Zhang, Guofeng;Tomarev, Stanislav I.;Ye, Yihong
• 通讯作者: Ye, Yihong