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）修饰的蛋白质修饰是细胞生物学几乎所有方面的关键调节过程。可以在一个（单素化）或多个位点（多泛素化）上用单个泛素修饰底物蛋白。另外，在泛素本身上可能会发生几轮泛素化，从而导致多泛素链的形成。泛素的七种赖氨酸或氨基末端中的任何一种均可用来聚合泛素（Peng等，2003），因此可以形成大量不同的链接的多泛素信号。 UB信号是可逆的，因为可以通过去泛素化酶从底物中去除泛素。多种UB信号在细胞中被多种受体识别，这些受体具有识别单或多泛素化底物的不同泛素结合基序（Hicke等，2005）。 调节细胞中的去泛素酶的机制尚不清楚。在这里，我们表征了34个人配音，包括25个USP，4个OTU，1个Josephin和4个UCHL亚家族成员。 我们表明，当用活性氧（ROS）在体外和细胞中用活性氧（ROS）氧化时，这些酶中的许多酶被可逆地灭活。 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将活性位点半胱氨酸转化为易于氧化的去质子化状态的配音的激活有关。 我们进一步证明，这种依赖氧化还原的调节对于氧化DNA损伤响应PCNA的单素化是必不可少的，该损伤启动了DNA损伤耐受性程序。 这些发现建立了一种新型的配音调节机制，该机制可能与其他氧化还原依赖性信号传导电路集成，以控制细胞对氧化应激的适应，这是与衰老和癌症密切相关的过程。 我们最近表征了USP19的功能和调节。我们将HSP90识别为结合USP19催化域以促进底物关联的特定合作伙伴。有趣的是，尽管过表达的USP19与膜中的Derlin-1和其他ERAD机械因子相互作用，但内源性USP19主要在结合HSP90的细胞质中。因此，我们既未检测到内源性USP19与DERLIN-1的相互作用，也没有对USP19耗竭对ERAD的显着影响。 USP19跨膜结构域似乎通过与其自身催化结构域的相互作用在细胞质中部分稳定，从而导致自动抑制其去泛素活性。这些结果阐明了USP19在ERAD中的作用，并提出了一种涉及伴侣关联和膜整合的新型配音调节。