This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Driving biological Project BSIRT3 (a Silent Information Regulator family member) is a human NAD*-dependent mitochondrial deacetylase encoded by a nuclear gene located on chromosome 11p15.5, that we discovered in a search for candidate tumor suppressor genes in thisregion. It is of potential importance, as 1) it is the first silencing-related gene localized to mitochondria, 2) homologues in yeast and C. elegans control lifespan, and 3) a mammalian deacetylase targets p53 controlling its ability to induce apoptosis.Furthermore, our discovery uncovers a previously unknown biochemical mechanism in mitochondria, namely deacetylation, and by implication, prior acetylation. Despite its importance, we have been stymied in our efforts to identify the function of SIRT3, because nothing is known about mitochondrial protein acetylation. This Roadmap proposal offers an opportunity to uncover this function, as well as the nature of mitochondrial acetylation, since the only reasonably fruitful strategy is an integrated proteomic and micro analytic approach that is at the heart of this Center. The long-term objective of this proposal is to dissect the molecular pathways of SIRT3 and to foster a deeper understanding of the function of the gene and its substrates in mitochondrial biology. Four highly integrated and innovative proteomics technologies to be developed in the technology center for networks and pathways of lysine modification will be applied to this problem. Mitochondrial protein arrays (TCP-1); chromatographic/massspectrometry measurement of the dynamics and quantification of post-translationalmodifications (TCP-3 and 4); yeast genetic interaction technologies (TCP-2) and active site labeling for acetyltransferases (TCP-5) will form the foundation of two aims, each targeting a fundamental knowledge gap on the function of SIRT3 in mitochondria.
Specific Aim 1 : To identify SIRT3 mitochondrial substrates.
This aim will clarify the specific role(s) of SIRT3 in the mitochondria by identifying candidate substrates using mitochondrial protein chip arrays developed under Technology Core Project I(TCP-1), and mass spectrometry analysis of acetylated and deacetylated mitochondrial proteins (TCP-3 and TCP-4). To validate SIRT3 substrate authenticity we will utilize mass spectrometry approaches (TCP-3), in combination withRNA interference (RNAi) technology and studies on SIRT3 knockout mice. We will utilize yeast genetic interaction technologies (TCP-2) to verify SIRT3 substrates that have yeast homologues.
Specific Aim 2 : Elucidation of SIRT3 biochemical pathways in mitochondria.
This aim will elucidate SIRT3 biochemical acetylation and deacetylation pathways in the mitochondria, using an integrated approach of bioinformatic tools, mitochondrial protein chip arrays (TCP-1), yeast genetics interaction technologies (TCP-2), chromatographic/mass spectrometry analysis (TCP-3) and acetyltransferase active site labeling (TCP-
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