Determining the mechanism of specificity for the SAGA deubiquitinating module

确定 SAGA 去泛素化模块的特异性机制

• 批准号: 8396860
• 负责人: Michael Thomas Morgan
• 金额: $ 4.92万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8396860
• 关键词: Acetyltransferase; Active Sites; Affect; Affinity; Arginine; Binding; Biochemical; Biochemistry; Biological Assay; Biological Process; Biophysics; C-terminal; Catalysis; Chemicals; Chemistry; Chromatin; Cleaved cell; Code; Complex; DNA; DNA-Directed RNA Polymerase; Deubiquitination; Disease; Elements; Enzyme Kinetics; Equilibrium; Eukaryota; Event; Excision; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Histone Acetylation; Histone Code; Histone H2B; Histones; In Vitro; Kinetics; Lead; Ligation; Link; Measures; Mediating; Methods; Methylation; Modification; Molecular Chaperones; Monoubiquitination; Mutagenesis; Mutate; Mutation; Nucleosome Core Particle; Nucleosomes; Pattern; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Chemistry; Proteins; RNA Polymerase II; Recruitment Activity; Roentgen Rays; Series; Solvents; Specificity; Structure; Surface; Techniques; Transcript; Transcriptional Activation; Ubiquitin; Ubiquitination; Work; Yeasts; Zinc Fingers; base; histone modification; improved; intein; interest; mutant; novel; physical process; preference; protein complex; success; tool; transcription factor; ubiquitin isopeptidase

DESCRIPTION (provided by applicant): Post-translational modifications of histone proteins create a "histone code" that regulates transcription. One such modification is the monoubiquitination of histone H2B (forming H2B-Ub), which is a marker for active transcription in chromatin and mediates several important processes in pre-initiation complex formation before it must be removed for transcript elongation. H2B-Ub also enhances transcription efficiency by recruiting FACT, a histone chaperone that disassembles the nucleosome ahead of RNA Polymerase II and reassembles it in its wake. The SAGA deubiquitinating module (DUBm) cleaves the isopeptide bond of H2B-Ub, removing ubiquitin, and thereby enhances transcription within a subset of SAGA-regulated genes. The dynamic state of the nucleosome during transcription suggests that H2B-Ub occurs in the context of the nucleosome and the H2A/H2B heterodimer, prompting us to wonder whether the DUBm may be acting preferentially against either complex. Our lab recently determined the crystal structure of the DUBm in the apo- and ubiquitin-bound states, allowing us to form hypotheses about how this complex specifically targets H2B-Ub. First, we will determine whether DUBm has a significant preference for H2B-Ub in the context of the nucleosome or H2A/H2B heterodimer using an approach that combines equilibrium binding assays with Michaelis-Menten kinetics. This goal is enabled by new techniques for the semisynthesis of natively linked H2B-Ub from intein-derivatized proteins using native chemical ligation (NCL). This work may reveal a novel functional linkage between SAGA and H2B-Ub, but will provide important information for the study of SAGA-DUBm regardless. In our second aim, we consider which parts of the DUBm complex governs specificity for H2B-Ub. Sgf11 is a DUBm component that is necessary for efficient cleavage of H2B-Ub and contains several conserved arginines on its zinc-finger (Sgf11-ZnF). We will mutate these residues and measure the effect on binding and catalysis to better understand their importance. The Sgf11-ZnF domain contacts Ubp8, the enzymatic subunit of DUBm, proximal to the active site residues. We also consider the possibility that upon recognition of substrates by Sgf11-ZnF residues, a small change at the interface between Ubp8 and Sgf11-ZnF drives a conformational change of a conserved loop that produces the reactive complex. We will explore this possibility with mutagenesis of the involved residues and crystallize mutants that show altered activity due to mutation. Finally, we will determine the structural basis of DUBm specificity for H2B-Ub by attempting to crystallize the DUBm bound to H2B-Ub in the context of the nucleosome, the H2A/H2B heterodimer, and free H2B-Ub. If crystals are not produced, we will use small angle X-ray scattering as an alternative method to determine how DUBm is oriented as it interacts with substrates. This work will answer important questions about DUBm catalysis and specificity, and will significantly improve our understanding of how SAGA interacts with chromatin. PUBLIC HEALTH RELEVANCE: The SAGA complex regulates gene activity with a sub-complex (DUBm) that allows it to cleave the bond linking ubiquitin to histone H2B. We will reveal how DUBm recognizes its target and performs its function using the tools of biochemistry and biophysics. Our findings will improve our understanding of gene regulation, which can lead to disease if disrupted.

描述（由申请人提供）：组蛋白的翻译后修饰创建了调节转录的“组蛋白代码”。一种这种修饰是组蛋白H2b（形成H2B-UB）的单样泛素化，它是染色质中主动转录的标记，并在必须将其去除前发射复合物形成中的几个重要过程以进行笔录伸长。 H2B-UB还通过募集事实来提高转录效率，这是一种组蛋白伴侣，它在RNA聚合酶II之前分解了核小体，并在其后重新组装它。传奇去泛素化模块（DUBM）裂解H2B-UB的异肽键，去除泛素，从而增强了SAGA调节基因子集中的转录。转录过程中核小体的动态状态表明H2B-UB发生在核小体和H2A/H2B异二聚体的背景下，促使我们怀疑DUBM是否可能优先针对任何一种复合物。我们的实验室最近确定了载脂蛋白和泛素结合状态中DUBM的晶体结构，从而使我们能够形成有关该复合物如何专门针对H2B-UB的假设。首先，我们将使用将平衡结合测定法与Michaelis-Menten动力学相结合的方法来确定DUBM是否在核小体或H2A/H2B异二聚体的背景下对H2B-UB具有显着偏爱。该目标是通过使用天然化学连接（NCL）的新技术来实现新技术的新技术来实现内部链接的H2B-UB的半合成。这项工作可能揭示了传奇和H2B-UB之间的新功能联系，但无论如何，都将为SAGA-DUBM的研究提供重要信息。在我们的第二个目标中，我们考虑DUBM复合体的哪些部分控制着H2B-UB的特异性。 SGF11是一种DUBM组件，是H2B-UB有效切割所必需的，并在其锌指（SGF11-ZNF）上包含几种保守的精氨酸。我们将突变这些残基并衡量对结合和催化的影响，以更好地理解它们的重要性。 SGF11-ZNF域接触UBP8，DUBM的酶亚基，靠近活性位点残基。我们还考虑了SGF11-ZNF残基识别底物的可能性，UBP8和SGF11-ZNF之间的界面发生了很小的变化，驱动了产生反应性复合物的保守环的构象变化。我们将通过涉及残基的诱变并结晶突变体探索这种可能性，这些突变体显示出突变引起的活性改变。最后，我们将通过试图在核小体，H2A/H2B异二聚体和游离H2B-UB的背景下结晶与H2B-UB结合的DUBM来确定H2B-UB的DUBM特异性。如果未产生晶体，我们将使用小角度X射线散射作为替代方法，以确定DUBM与底物相互作用时的定向。这项工作将回答有关DUBM催化和特异性的重要问题，并将大大提高我们对Saga如何与染色质相互作用的理解。 公共卫生相关性：传奇综合体通过亚复合物（DUBM）调节基因活性，使其可以裂解将泛素连接到组蛋白H2B的键。我们将透露DUBM如何使用生物化学和生物物理学工具来识别其目标并执行其功能。我们的发现将提高我们对基因调节的理解，如果破坏可能会导致疾病。