Acetyltransfer in gene regulation: Structural and biochemical studies of human SIRT1 deacetylase, its FoxO1 substrate, and archaeal PAT acetyltransferase
Reversible protein acetylation is a mechanism used by the cell to regulate gene expression and other important activities such as stress response and apoptosis. Human SIRT1 is an NAD+ dependent deacetylase that regulates both chromatin, through its deacetylation of histone proteins H3 and H4, and non-histone proteins including p53 tumor suppressor and the FoxO transcription factors. Chapter 1 of this thesis describes the purification and biochemical characterization of SIRT1, and the pursuit of SIRT1 crystals for structure determination by X-ray diffraction to aid in drug design efforts. Chapter 2 reports the results of high-throughput compound screening to identify molecules that regulate SIRT1 activity and may serve as lead molecules for drug design. Chapter 3 reports the identification of potent in vitro inhibition of SIRT1 by HIV Tat protein and the implications for viral replication and disease progression are discussed.
The FoxO transcription factors are substrates of SIRT1 and regulate the transcription of genes that control metabolism, stress tolerance, apoptosis and possibly lifespan. FoxO is regulated by several post-translational modifications including acetylation and phosphorylation. Chapter 4 reports the crystal structures of FoxO1 bound to three different DNA elements. The structures reveal differences for FoxO binding to insulin response element (IRE) versus Daf-16 family binding element (DBE) DNA. DNA binding assays revealed significant changes in DNA affinity with acetylation and phosphorylation of FoxO1. These findings suggest that modulation of FoxO-DNA affinity by post-translational modification is an important component of its regulation in health and misregulation in disease.
The Sulfolobus solfataricus protein acetyltransferase (PAT) acetylates ALBA, an abundant nonspecific DNA binding protein, on K16 to reduce its DNA affinity, and archaeal Sir2 deacetylase reverses the modification to cause transcriptional repression. This “primitive” form of chromatin regulation is analogous to histone modification in eukaryotes. Chapter 5 reports the determination of a 1.84 Å crystal structure of PAT in complex with coenzyme A and biochemical characterization of PAT by site-directed mutagenesis. The structure reveals homology to both prokaryotic GNAT acetyltransferases and eukaryotic histone acetyltransferases (HATs), and it suggests that PAT could represent the ancestral scaffold from which the eukaryotic HATs have evolved.