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) 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。底物蛋白可以在一个（单泛素化）或多个位点（多泛素化）上用单个泛素进行修饰。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 et al., 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，因为泛素可以通过去泛素化酶从底物上去除。细胞中的多种 Ub 信号被多种受体识别，这些受体携带独特的泛素结合基序，可识别单泛素或多泛素化底物 (Hicke et al., 2005)。 调节细胞中去泛素酶的机制尚不清楚。在这里，我们表征了 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，许多这些酶在体外和细胞内被活性氧 (ROS) 氧化时会可逆地失活。 氧化优先发生在催化半胱氨酸上，消除异肽切割活性，而不影响这些酶与泛素的亲和力。 对氧化抑制的敏感性与 DUB 的激活有关，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调节对于 PCNA 在响应 DNA 氧化损伤时发生单泛素化至关重要，从而启动 DNA 损伤耐受程序。 这些发现建立了一种新的 DUB 调节机制，该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，氧化应激是与衰老和癌症密切相关的过程。 我们最近描述了 USP19 的功能和调节。我们将 Hsp90 确定为结合 USP19 催化结构域以促进底物结合的特定伙伴。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 主要存在于细胞质中，与 Hsp90 结合。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 耗尽对 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胞质溶胶中部分稳定，导致其去泛素化活性的自动抑制。这些结果阐明了 USP19 在 ERAD 中的作用，并提出了一种涉及分子伴侣关联和膜整合的新型 DUB 调节。