Laboratory set-up and personnel:? ? Since joining the NIEHS in late December 2006, I have set up my laboratory with all the essential equipments, obtained approval of all necessary safety protocols and animal study protocols, and recruited two postdoctoral fellows and a biologist.? ? Projects:? ? 1. Investigate the molecular mechanisms by which SIRT1 regulates the LXR signaling? We previously demonstrated that although the transcriptional activity of LXR is decreased in SIRT1 null animals and cells, the steady level of LXRalpha protein is increased. We found this is because SIRT1 is able to deacetylate LXRalpha and promote the ubiquitination and subsequent degradation. We further identified K432 as the single acetylation site of LXRalpha with tandem mass spectrometry, and showed that mutation of this lysine residue abolished SIRT1-mediated transactivation of LXRalpha. We propose that SIRT1 is required to reset (clear) LXRalpha targets promoter by promoting the ubiquitination of used LXRalpha. ? After arriving in NIEHS, by utilizing primary hepatocytes isolated from SIRT1 knockout animals, we were able to directly show that overexpressed wild type LXRalpha is much more acetylated in SIRT1 null cells than in SIRT1+/+ cells, and K432R mutant totally lost the acetylation signal. With these primary hepatocytes, we further showed that only wild type LXRalpha can be ubiquinated and destabilized by LXR ligand in SIRT1+/+ cells. Neither K432 mutants nor wild type LXRalpha are ubiquitinated or unstable in SIRT-/- cells. All these data demonstrated that K432 is the primary ubiquitination site in LXRalpha, and deacetylation of K432 by SIRT1 is necessary to trigger ubiquitination and instability. ? Since we lack good antibodies for LXRalpha protein, we are raising peptide antibodies to different regions of this protein in rabbits. We hope with a good antibody in hands, we will be able to confirm the acetylation and ubiquitination of endogenous LXRalpha protein. We also plan to further test our model by introducing K432 mutants into LXR null cells (hepatocytes and macrophages) and asking whether they are defective in inducing LXRalpha targets upon ligand treatment, and are less able to promote cholesterol efflux. Once these are confirmed, we plan to introduce these mutants in mice and directly analyze their activity in vivo. We believe that these studies will shed light on the possible protective effects of SIRT1 on cholesterol disorders at mechanistic levels, which may ultimately help to make SIRT1 an attractive drug target for these diseases.? ? ? 2. Study the role of SIRT1 in LXR-associated metabolic diseases? Our previous studies predict that high levels/activities of SIRT1 may decrease the risk of cholesterol diseases and other metabolic syndromes associated with diet and aging. Inhibition of SIRT1, on the other hand, could enhance the malignancy of these diseases. We will directly test this prediction by analyzing the onset and progression of age-associated metabolic diseases, particularly atherosclerosis, in tissue specific conditional SIRT1 knockout under various conditions.? Since our rederived founders arrived in NIEHS animal facility at the end of January, we have established three mouse strains: SIRT1 whole body knockout, SIRT1 conditional knockout parental strains, and liver-specific SIRT1 knockout. My first postdoc, Aparna Purushotham, joined my lab in the middle of April. Together we have expended our SIRT1 whole body knockout and liver-specific knokout and have obtained enough animals for the high fat diet feeding experiment. We are also in the middle of breeding SIRT1 whole body knockout into ApoE null mouse, a mouse model for atherosclerosis.
