The Chemistry of Gene Silencing

Johns Hopkins University, Baltimore MD

Investigators

Abstract

The Sir2 proteins, also known as sirtuins, comprise an ancient family of NAD+-dependent enzymes found in all organisms, where they play a central role in diverse biological processes including transcriptional silencing, suppression of rDNA recombination, regulation of the p53 tumor suppressor, fat mobilization, neuronal regeneration, and aging. Most sirtuins deacetylate lysine residues in an NAD+-dependent reaction that releases the deacetylated peptide, nicotinamide, and O-acetyl ADP ribose. The unusual chemistry catalyzed by sirtuins allows some of these enzymes to catalyze a second reaction, ADP ribosylation, in which NAD+ is cleaved and the ADP ribose product is attached to target side chains. In the past funding period, significant progress was made towards elucidating the structure-based mechanism of NAD+-dependent deacetylation. In addition, the first direct evidence for ADP ribosylation by a sirtuin was obtained and the modified side chains identified. The present proposal builds upon this work, applying approaches using x-ray crystallography, biochemistry, and computational biology to understand the underlying mechanism of the ADP ribosyltransferase activity of sirtuins. Structures of complexes with substrates and trapped reaction intermediates will be determined using x-ray crystallography in order to uncover the mechanism by which sirtuins ADP ribosylate acetylated and unacetylated substrates. Solution studies and quantum mechanical calculations on the new structures will complement these structural studies and will shed further light on the enzymatic mechanism. In a second area of inquiry, structural and biochemical studies will be carried out on the complex formed by the yeast Sir2 protein with Net1, which is responsible for rDNA silencing and suppression of rDNA recombination. These studies will shed light on how Net1 recruits Sir2 to the nucleolus and establishes a chromatin-bound complex that silences transcription and suppresses recombination. In addition to elucidating how Sir2 interacts with partner proteins, these studies will also contribute to an understanding of why the enzymatic activity of Sir2 is altered when it becomes part of a complex. BROADER IMPACT OF THE PROPOSED ACTIVITY: The proposed experiments will be carried out exclusively by graduate students and will provide excellent training in using an integrated approach that applies the tools of x-ray crystallography and solution biochemistry to important biological problems. The principal investigator's ongoing work in structural biology of sirtuins has provided an opportunity to reach out to and encourage women and underrepresented groups to pursue a career in science. The principal investigator participates in the "Women Serious About Science" club at a city high school with a largely minority student body. She has also devised and taught a curriculum for "Crystal Day" as part of a new summer program for inner-city 5th graders that is taught annually.