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: 10396526
• 负责人: Mahesh B Chandrasekharan
• 金额: $ 32.03万
• 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 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10396526
• 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; 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.

项目摘要：蛋白质泛素化在许多生物过程中发挥作用。组蛋白泛素化， 一种表观遗传标记，通过染色质结构和交易来管理DNA相关的过程。 泛素化和表观遗传机制中的错误调节与许多遗传和获得性有关 包括糖尿病、癌症和神经疾病在内的疾病。组蛋白H_2B单素化(H_2Bub1) 调节从酵母到人类的基因转录。这项建议关注的是进化上保守的 芽期酵母H_2B泛素偶联复合体(Huc)，由RAD6(E_2偶联酶)组成。 Bre1(E3连接酶)和Lge1(一种辅助蛋白)。HUC综合体的组装、招募和管理是 没有完全理解。使用广泛的功能、生化和结构研究，包括初步的 Rad6-Bre1亚络合物的晶体结构，我们已经确定了多个亚基界面，这些界面与 与其他泛素结合的复合体相比，Huc复合体包括一个新的界面， Rad6的非催化区域和Bre1的非环结构域区域，不对称构象状态 Bre1二聚体，以及Bre1环结构域附近的Lge1结合。我们将使用X射线结晶学来可视化 这些不同的亚基与Aim 1相联系。Lge1是一种必需的但尚未鉴定的辅助蛋白，它与 染色质因子和BRE1是RAD6-BRE1‘S K63连锁多泛素化活性的消耗品，提示 耐人寻味的假设是Lge1将Huc复合体招募到染色质上的相关位置并限制其 对单一泛素化的活性，这也将在Aim 1中进行测试，使用基因组学来确定全基因组 Huc复合体的占有率和生物化学-蛋白质组学组合来测量单核苷酸和多核苷酸 泛素化。目的2建立在我们确认在RAD6‘S催化附近丝氨酸-120的体内磷酸化的基础上 克里夫特。我们对模拟天冬氨酸取代的Rad6的初步结构表明，S120 磷酸化是一种分子开关，它关闭活性部位以抑制单泛素化，但允许 多泛素化。这将通过确定S120磷酸化的Rad6的结构来进行测试。我们的 初步数据还包括发现细胞壁完整性途径的激酶Mpk1被磷酸化 RAD6.这激发了这种修饰激活Rad6到多泛素化底物的模型 转录启动和上游信号事件中，随后的去磷酸化使Rad6 a 过氧化氢酶在转录延伸过程中促进基因表达。这一创新的监管 开关理论将在目标2中使用多管齐下的方法进行测试，以确定全基因组的占有率， 受Rad6和Rad6S120phos调控的转移体和泛素体。总而言之，我们的多学科 研究将通过解决基本的重要功能来推进泛素化和表观遗传学领域 以及关于Rad6和Huc建筑群的机械问题。考虑到进化守恒，我们的 研究适用于包括人类在内的后生动物的表观遗传和泛素化机制。

项目成果

Structure-function analysis of histone H2B and PCNA ubiquitination dynamics using deubiquitinase-deficient strains.

使用去泛素酶缺陷菌株对组蛋白H2B和PCNA泛素化动力学的结构 - 功能分析。

• DOI: 10.1038/s41598-023-43969-z
• 发表时间: 2023-10-04
• 期刊: SCIENTIFIC REPORTS
• 影响因子: 4.6
• 作者: Radmall, Kaitlin S.;Shukla, Prakash K.;Leng, Andrew M.;Chandrasekharan, Mahesh B.
• 通讯作者: Chandrasekharan, Mahesh B.

Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting.

• DOI: 10.3390/mps5050074
• 发表时间: 2022-09-24
• 期刊: METHODS AND PROTOCOLS
• 影响因子: 2.4
• 作者: Leng, Andrew M;Radmall, Kaitlin S;Shukla, Prakash K;Chandrasekharan, Mahesh B
• 通讯作者: Chandrasekharan, Mahesh B

Rapid purification of rabbit immunoglobulins using a single-step, negative-selection chromatography.

使用单步负选择色谱法快速纯化兔免疫球蛋白。

• DOI: 10.1016/j.pep.2023.106270
• 发表时间: 2023
• 期刊: Protein expression and purification
• 影响因子: 1.6
• 作者: Shukla,PrakashK;Radmall,KaitlinS;Chandrasekharan,MaheshB
• 通讯作者: Chandrasekharan,MaheshB