Structure, function and regulation of the H2B ubiquitin-conjugating complex

H2B 泛素结合复合物的结构、功能和调控

• 批准号: 10165311
• 负责人: Mahesh B Chandrasekharan
• 金额: $ 5.66万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10165311
• 关键词: Active Sites; Address; Antibodies; Aspartate; Binding; Binding Proteins; Biochemical; Biochemistry; Biological Process; Catalytic Domain; Cell Wall; Cell physiology; Chromatin; Chromatin Structure; Complex; Crystallization; DNA; Data; Diabetes Mellitus; Disease; Enzymes; Epigenetic Process; Event; Gene Expression; Genetic Transcription; Genomics; Histone H2B; Histones; Human; Inherited; Link; Lysine; Malignant Neoplasms; Measures; Mediating; Mitogen-Activated Protein Kinases; Modeling; Modification; Molecular; Molecular Conformation; Molecular Structure; Monoubiquitination; Mutation Analysis; N-terminal; Nucleosomes; Pathogenesis; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Polyubiquitination; Process; Protein Dephosphorylation; Proteins; Proteomics; RNA; RNA Polymerase II; Regulation; Reporting; Saccharomycetales; Sclerosis; Serine; Signal Pathway; Signal Transduction; Site; Stroke; Structure; Testing; Transact; Transcription Elongation; Transcription Initiation; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; X-Ray Crystallography; Yeasts; base; cell growth regulation; dimer; epigenetic regulation; functional genomics; gamma-Glutamyl Hydrolase; genome-wide; histone methylation; in vivo; innovation; inorganic phosphate; insight; molecular recognition; multidisciplinary; mutant; nervous system disorder; novel; recruit; structured data; theories; ubiquitin-protein ligase

PROJECT SUMMARY: Protein ubiquitination functions in many biological processes. Histone ubiquitination, an epigenetic mark, governs DNA-related processes though chromatin structure and transactions. Misregulation in ubiquitination and epigenetic mechanisms are linked to numerous inherited and acquired diseases including diabetes, cancers and neurological disorders. Histone H2B monoubiquitination (H2Bub1) regulates gene transcription from yeast to humans. This proposal focuses on the evolutionarily conserved budding yeast H2B ubiquitin-conjugating complex (HUC) comprised of Rad6 (E2 conjugase), a homodimer of Bre1 (E3 ligase) and Lge1 (an accessory protein). Assembly, recruitment, and regulation of HUC complex are not fully understood. Using extensive functional, biochemical and structural studies including a preliminary crystal structure for Rad6-Bre1 sub-complex, we have identified multiple subunit interfaces that are distinct to the HUC complex compared to other ubiquitin-conjugating complexes including a novel interface between a non-catalytic region of Rad6 and a non-RING domain region of Bre1, an asymmetric conformational state of the Bre1 dimer, and Lge1 binding near the Bre1 RING domain. We will use X-ray crystallography to visualize these distinctive subunit contacts in Aim 1. Lge1, an essential but uncharacterized accessory protein, binds chromatin factors and Bre1 and is expendable for Rad6-Bre1's K63-linked polyubiquitination activity, prompting the intriguing hypotheses that Lge1 recruits the HUC complex to relevant sites on chromatin and restricts its activity to monoubiquitination, which will also be tested in Aim 1 using genomics to determine the genome-wide occupancy of the HUC complex and a biochemistry-proteomics combination to measure mono versus poly ubiquitination. Aim 2 builds on our confirmation of in vivo phosphorylation of serine-120 near Rad6's catalytic cleft. Our preliminary structure for Rad6 with the phosphomimetic aspartate substitution suggests that S120 phosphorylation is a molecular switch that `closes' down the active site to inhibit monoubiquitination but allow polyubiquitination. This will be tested by determining the structure of S120-phosphorylated Rad6. Our preliminary data also involves the discovery that the cell wall integrity pathway kinase Mpk1 phosphorylates Rad6. This motivates the model that this modification activates Rad6 to polyubiquitinate substrates during transcription initiation and in upstream signaling events, and that subsequent dephosphorylation makes Rad6 a H2B monoubiquitinase during transcription elongation promoting gene expression. This innovative regulatory switch theory will be tested in Aim 2 using a multipronged approach to determine genome-wide occupancies, transciptomes and ubiquitomes regulated by Rad6 and Rad6S120phos. In summary, our multidisciplinary studies will advance the ubiquitination and epigenetics fields by addressing fundamentally important functional and mechanistic questions regarding Rad6 and the HUC complex. Given the evolutionary conservation, our studies are applicable to the epigenetic and ubiquitination mechanisms in metazoans including humans.

项目概要：蛋白质泛素化在许多生物过程中发挥作用。组蛋白泛素化， 作为一种表观遗传标记，通过染色质结构和处理来控制DNA相关过程。 泛素化和表观遗传机制的失调与许多遗传和获得性疾病有关。 包括糖尿病、癌症和神经系统疾病在内的疾病。组蛋白H2 B单泛素化（H2 Bub 1） 调节从酵母到人类的基因转录。这个建议的重点是进化上保守的 芽殖酵母H2 B泛素缀合复合物（HUC），其由Rad 6（E2缀合酶）组成，Rad 6是 Bre 1（E3连接酶）和Lge 1（辅助蛋白）。大会，招聘，和HUC复杂的监管是 没有完全理解。利用广泛的功能，生物化学和结构研究，包括初步的 Rad 6-Bre 1亚复合物的晶体结构，我们已经确定了多个亚基界面， HUC复合物与其他泛素缀合复合物相比，包括一个新的界面， Rad 6的非催化区和Bre 1的非RING结构域区， Bre 1二聚体和Bre 1 RING结构域附近的Lge 1结合。我们将用X射线晶体学来观察 这些独特的亚基联系在目标1中。lge 1是一种必需的但未被表征的辅助蛋白， 染色质因子和Bre 1，并且对于Rad 6-Bre 1的K63连接的多聚泛素化活性是消耗性的， 有趣的假设是，Lge 1将HUC复合物募集到染色质上的相关位点，并限制其在染色质上的表达。 单泛素化的活性，这也将在目标1中使用基因组学进行测试，以确定全基因组 HUC复合物的占有率和生物化学-蛋白质组学组合，以测量单聚体与多聚体 泛素化目的2建立在我们证实的在体内丝氨酸-120磷酸化附近的Rad 6的催化， 裂缝我们对具有磷酸模拟天冬氨酸取代的Rad 6的初步结构表明，S120 磷酸化是一种分子开关，其“关闭"活性位点以抑制单泛素化， 聚泛素化这将通过确定S120-磷酸化Rad 6的结构进行测试。我们 初步数据还包括发现细胞壁完整性途径激酶Mpk 1磷酸化 Rad6.这激发了这样的模型，即这种修饰激活Rad 6以聚泛素化底物， 转录起始和上游信号事件，以及随后的去磷酸化使Rad 6成为一个 H2 B单泛素酶在转录延伸过程中促进基因表达。这种创新的监管 转换理论将在目标2中使用多管齐下的方法来确定全基因组范围的职业， 受Rad 6和Rad 6S 120 phos调节的转录组和遍在蛋白组。总之，我们的多学科 研究将推进泛素化和表观遗传学领域的解决根本重要的功能 以及关于Rad 6和HUC复合体的机械问题。考虑到进化保守性， 研究适用于后生动物包括人类的表观遗传和泛素化机制。