DBP-D: UBIQUITYLATION AND POLYUBIQUITIN DYNAMICS AND NETWORKS

DBP-D：泛素化和多泛素动力学和网络

• 批准号: 7622847
• 负责人: Cecile M. Pickart
• 金额: $ 22.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7622847
• 关键词: 26S proteasome; Acetylation; Acetylesterase; Automobile Driving; Binding; Binding Proteins; Biochemical Genetics; Biochemistry; Biological; Cells; Computer Retrieval of Information on Scientific Projects Database; DNA Damage; DNA polymerase A; Data; Engineering; Funding; Goals; Grant; Heat Stress Disorders; Individual; Institution; Light; Link; Lysine; Mass Spectrum Analysis; Methods; Modeling; Modification; Molecular Genetics; Polyubiquitin; Proliferating Cell Nuclear Antigen; Proteasome Inhibition; Proteins; Proteolysis; Proteome; Purpose; Regulation; Research; Research Personnel; Resources; Role playing therapy; Saccharomycetales; Signal Transduction; Site; Source; Stress; Technology; Testing; Time; Ubiquitin; United States National Institutes of Health; Yeasts; biological adaptation to stress; concept; follow-up; insight; interest; multicatalytic endopeptidase complex; novel; receptor; research study; yeast genetics

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Driving Biological Project-D The goal of this DBP is to A) discover and characterize novel polyubiquitin signals (Aims 1, 2) and B) to assess the occurrence of cross-talk between acetylation and ubiquitylation (Aim 3). These studies could not be performed without the technologies being developed in Technology Core Projects (TCPs) 1 through 4. Data interpretation will be facilitated by the modeling expertise of Core 3. SPECIFIC AIMS (DBP-D) 1. Dynamic regulation of polyubiquitin signals during stress. Specific signaling functions have been defined for polyubiquitin chains linked through K48 and K29 (proteasome proteolysis) and K63 (DNA tolerance). No function is yet known for the four other types of polyubiquitin chains, but several lines of evidence implicate some types of non-canonical chains in stress responses. We will use quantitative mass spectrometry (TCPs-3, 4) to determine how the abundance of specific ubiquitylated proteins changes when cells are subjected to stresses that are known to alter cellular ubiquitylation: A) proteasome inhibition, B) heat stress, and C) DNA damage. We are particularly interested in the ubiquitylation of ubiquitin itself, ie changes in the steady-state contents of individual polyubiquitin linkages. Follow-up biochemical and genetic studies (TCP-2) will shed light on the signaling functions of novel polyubiquitin chains and the purposes served by the ubiquitylation of selected non-ubiquitin targets. We also expect to gain new insights into the roles played by ubiquitylation in stress responses. 2. Discovery of novel polyubiquitin signals. The 26S proteasome is the primary receptor for K48 and (possibly) K29-1inked polyubiquitin chains. Other chains remain uncharacterized with respect to their potential binding partners. We will screen yeast proteome chips (TCP-1) with homopolymeric polyubiquitin chains linked through at least six of ubiquitin''s lysine residues. Selected novel binding proteins will be characterized through approaches of biochemistry and molecular genetics (TCP-2) in order to gain insight into how polyubiquitin chain formation is used to impart diversity in ubiquitin signaling. 3. Interplay of ubiquitylation with other lysine modifications. A) The DNA polymerase processivity factor PCNA (Proliferating Cell Nuclear Antigen) can be modified on the same lysine residue with monoubiquitin, SUMO, or a K63-1inked polyubiquitin chain. The dynamics of these modifications are poorly described. In proof of concept experiments, we will use engineered yeast strains, in conjunction with mass spectrometry (TCP-3, 4), to quantify these site-specific modifications in normal and DNA-damaged yeast cells in the steady state and as a function of time after imposition of DNA damage. B) Functional antagonism between acetylation and ubiquitylation has been observed in several recent studies. We hypothesize that this regulatory interplay occurs on a more significant scale than is currently appreciated. To test this hypothesis we will use mass spectrometry (TCP-4) to determine how substrates (and sites) of ubiquitylation change in budding yeast deleted for selected lysine acetylases and deacetylases. Novel modification sites will be further characterized through methods of biochemistry and yeast genetics (TCP-2).

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 驾驶生物项目-D 该DBP的目的是a）发现并表征新颖的多泛素信号（Aims 1， 2）和b）评估乙酰化和泛素化之间的串扰发生（AIM 3）。这些 没有技术核心项目中开发的技术，就无法进行研究 （TCP）1至4。数据解释将通过Core 3的建模专业知识来促进。 具体目标（DBP-D） 1。在应力过程中对多泛素信号的动态调节。特定的信号功能已经 针对通过K48和K29（蛋白酶体蛋白水解）和K63（DNA）连接的多泛素链定义 宽容）。其他四种类型的多泛素链尚未闻名，但是有几条证据暗示了压力反应中某些类型的非典型链。我们将使用定量质谱法（TCPS-3，4）来确定当细胞受到已知会改变细胞泛素化的应力时，特定泛素化蛋白的丰度如何变化：a）蛋白酶体抑制，b）热应激，c）热应激，c）DNA损伤。我们对泛素本身的泛素化特别感兴趣，即在单个多泛素链接的稳态内容中变化。随访的生化和遗传研究（TCP-2）将阐明新型多泛素链的信号传导功能以及所选非泛素靶标的泛素化所提供的目的。我们还期望对泛素化在压力反应中扮演的角色获得新的见解。 2。发​​现新型多泛素信号。 26S蛋白酶体是K48和（可能是）K29-1键多泛素链的主要受体。其他连锁店相对于其潜在的约束伙伴仍然没有表征。我们将筛选酵母蛋白质组碎片（TCP-1），并与至少六个泛素赖氨酸残基相连的均聚糖链链。选定的新型结合蛋白将通过生物化学和分子遗传学（TCP-2）的方法来表征，以便深入了解如何使用多泛素链形成来赋予泛素信号传导多样性。 3。泛素化与其他赖氨酸修饰的相互作用。 A）DNA聚合酶加工性因子PCNA（增殖细胞核抗原）可以在与单纤维蛋白，Sumo或K63-1关联的多泛素链的同一赖氨酸残基上修饰。这些修饰的动力学描述很差。在概念实验证明中，我们将使用工程酵母菌菌株与质谱法（TCP-3，4）一起使用在稳态状态下的正常和DNA受损酵母细胞中量化这些位点特异性修饰，并在施加DNA损伤后的时间函数。 b）功能 在最近的几项研究中，已经观察到乙酰化和泛素化之间的拮抗作用。我们假设这种调节性相互作用发生在比目前更为重要的规模上。 为了检验这一假设，我们将使用质谱（TCP-4）来确定在选定的赖氨酸乙酰基酶和脱乙酰基酶中缺失萌芽酵母中泛素化变化的底物（和位点）。新颖的修饰位点将通过生物化学和酵母遗传学方法（TCP-2）进一步表征。