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: 7380818
• 负责人: Cecile M. Pickart
• 金额: $ 20.95万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7380818
• 关键词: DBP; UBIQUITYLATION; POLYUBIQUITIN; DYNAMICS; NETWORKS

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).

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。该DBP的目标是A）发现和表征新的多聚泛素信号（目的1，2）和B）以评估乙酰化和泛素化之间的串扰的发生（目的3）。如果没有技术核心项目（TCP）1至4中正在开发的技术，这些研究就无法进行。岩心3的建模专业知识将有助于数据解释。具体目标（DBP-D）1.应激过程中多聚泛素信号的动态调节。通过K48和K29（蛋白酶体蛋白水解）和K63（DNA耐受性）连接的多聚泛素链已定义了特定的信号传导功能。其他四种类型的多聚泛素链的功能尚不清楚，但有几条证据表明某些类型的非典型链与应激反应有关。我们将使用定量质谱（TCP-3，4）来确定当细胞受到已知会改变细胞泛素化的应激时，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，和C）DNA损伤。我们特别感兴趣的是泛素本身的泛素化，即个体多聚泛素连接的稳态含量的变化。后续的生物化学和遗传学研究（TCP-2）将阐明新型多聚泛素链的信号传导功能以及所选非泛素靶点的泛素化所起的作用。我们也希望获得新的见解所发挥的作用，泛素化在应激反应。2.新的多聚泛素信号的发现。26S蛋白酶体是K48和（可能）K29 - 1连接的多聚泛素链的主要受体。其他链仍然不确定其潜在的结合伙伴。我们将筛选酵母蛋白质组芯片（TCP-1），其具有通过至少六个泛素的赖氨酸残基连接的均聚多聚泛素链。选择新的结合蛋白将通过生物化学和分子遗传学（TCP-2）的方法，以深入了解如何聚泛素链的形成是用来赋予多样性的泛素信号。3.泛素化与其他赖氨酸修饰的相互作用。A）DNA聚合酶持续合成因子PCNA（增殖细胞核抗原）可以在相同的赖氨酸残基上用单泛素、SUMO或K63 - 1连接的多聚泛素链修饰。这些修改的动力学描述得很差。在概念验证实验中，我们将使用工程酵母菌株，结合质谱法（TCP-3，4），以定量这些位点特异性修饰在正常和DNA损伤的酵母细胞在稳定状态下，并作为一个功能的时间后，施加DNA损伤。B）在最近的几项研究中已经观察到乙酰化和泛素化之间的功能性拮抗作用。我们假设，这种监管的相互作用发生在一个更显着的规模比目前的赞赏。为了检验这一假设，我们将使用质谱（TCP-4），以确定如何基板（和网站）的泛素化的变化，在芽殖酵母删除选定的赖氨酸乙酰化酶和脱乙酰酶。新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。