Regulation of deubiquitinating enzymes

去泛素化酶的调节

• 批准号: 8939700
• 负责人: Yihong Ye
• 金额: $ 57.06万
• 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/8939700
• 关键词: Active Sites; Affect; Affinity; Aging; Binding; Catalytic Domain; Cells; Cellular biology; Cleaved cell; Cysteine; Cytosol; DNA Damage; Deubiquitinating Enzyme; Endoplasmic Reticulum Degradation Pathway; Enzymes; Goals; Human; In Vitro; Link; Lysine; Malignant Neoplasms; Membrane; Molecular Chaperones; Mono-S; Oxidation-Reduction; Oxidative Stress; Polyubiquitin; Post-Translational Protein Processing; Process; Proteins; Reactive Oxygen Species; Regulation; Role; Signal Transduction; Site; Transmembrane Domain; Ubiquitin; Ubiquitination; member; novel; overexpression; oxidation; oxidative DNA damage; programs; receptor; response

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.

泛素(Ub)修饰蛋白质是细胞生物学各个方面的重要调控过程。底物蛋白质可以用单一泛素在一个(单泛素化)或多个位点(多泛素化)修饰。或者，泛素本身可以发生几轮泛素化，导致多泛素链的形成。泛素的七个赖氨酸或氨基末端中的任何一个都可以用来聚合泛素(Peng等人，2003)，因此可以形成大量不同连接的多泛素信号。UB信号是可逆的，因为泛素可以通过脱泛素酶从底物中去除。不同的Ub信号在细胞中被无数的受体识别，这些受体携带识别单一或多泛素底物的不同泛素结合基序(Hicke等人，2005年)。 调节细胞中去泛素酶的机制尚不清楚。在这里，我们描述了34个人类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，在体外和细胞内，当被活性氧物种(ROS)氧化时，这些酶中的许多都是可逆的失活。氧化优先发生在催化半胱氨酸上，取消了异肽的裂解活性，而不影响这些酶与泛素的亲和力。对氧化抑制的敏感性与DUBS的激活有关，其中活性部位半胱氨酸被转化为易于氧化的去质子化状态。我们进一步证明，这种氧化还原依赖的调节对于在氧化DNA损伤时发生的增殖细胞核抗原的单一泛素化是必不可少的，这启动了DNA损伤耐受计划。这些发现建立了一种新的DUB调节机制，该机制可能与其他依赖氧化还原的信号通路整合，以管理细胞对氧化应激的适应，这一过程与衰老和癌症密切相关。 我们最近对USP19的功能和调控进行了研究。我们确定Hsp90是一个特定的合作伙伴，它结合USP19的催化结构域来促进底物结合。有趣的是，尽管过表达的USP19与膜上的Derlin-1和其他ERAD机械因子相互作用，但内源性USP19主要在与Hsp90结合的胞浆中。因此，我们既没有检测到内源性USP19与Derlin-1的相互作用，也没有检测到USP19缺失对ERAD的显著影响。USP19跨膜结构域似乎通过与其自身的催化结构域相互作用而部分稳定在细胞质中，导致其脱泛素化活性的自动抑制。这些结果阐明了USP19在ERAD中的作用，并提出了一种新的涉及伴侣结合和膜整合的DUB调控。