DBP-D: UBIQUITYLATION AND POLYUBIQUITIN DYNAMICS AND NETWORKS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。推动生物学项目-D这个DBP的目标是A)发现和表征新的多泛素信号(目标1，2)和B)评估乙酰化和泛素化之间的串扰的发生(目标3)。这些研究离不开技术核心项目(TCP)1至4中正在开发的技术。核心3.特定目标(DBP-D)的建模专业知识将促进数据解释1.应激过程中多泛素信号的动态调节。通过K48和K29(蛋白酶体蛋白分解)和K63(DNA耐受性)连接的多泛素链的特定信号功能已经被定义。目前还不知道其他四种类型的多泛素链的功能，但有几条证据表明，某些类型的非正则链与应激反应有关。我们将使用定量质谱仪(TCPs-3，4)来确定当细胞受到已知会改变细胞泛素化的压力时，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，以及C)DNA损伤。我们特别感兴趣的是泛素本身的泛素化，即单个多泛素连接的稳态含量的变化。后续的生化和遗传学研究(TCP-2)将阐明新的多泛素链的信号功能以及选定的非泛素靶标的泛素化所达到的目的。我们还希望对泛素化在应激反应中所扮演的角色有新的见解。2.发现了新的多泛素信号。26S蛋白酶体是K48和(可能)K29-1连接的多泛素链的主要受体。其他链仍未确定其潜在的约束性伙伴的特征。我们将筛选酵母蛋白质组芯片(tcp-1)，其中均聚泛素链通过至少6个S赖氨酸残基连接。选择新的结合蛋白将通过生物化学和分子遗传学(TCP-2)的方法进行表征，以深入了解多泛素链的形成是如何在泛素信号中赋予多样性的。3.泛素化与其他赖氨酸修饰的相互作用。A)DNA聚合酶加工因子(增殖细胞核抗原)可以用单泛素、相扑或K63-1连接的多泛素链在相同的赖氨酸残基上进行修饰。这些修改的动态没有得到很好的描述。在概念验证实验中，我们将使用工程酵母菌株，结合质谱仪(TCP-3，4)，在稳定状态下以及施加DNA损伤后作为时间函数的正常和DNA损伤的酵母细胞中量化这些特定部位的修饰。B)在最近的几项研究中观察到乙酰化和泛素化之间的功能拮抗。我们假设，这种监管相互作用发生的规模比目前认识到的更大。为了验证这一假说，我们将使用质谱仪(TCP-4)来确定特定赖氨酸乙酰基酶和脱乙酰基酶缺失的芽酵母泛素化底物(和位置)是如何变化的。新的修饰位点将通过生物化学和酵母遗传学(TCP-2)的方法进一步表征。

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).