DBP-D: UBIQUITYLATION AND POLYUBIQUITIN DYNAMICS AND NETWORKS

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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).