Molecular Mechanisms of Cigarette Smoke-Induced Down-Regulation of Sirtuin1 Deacetylase

Abstract

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Environmental Medicine, 2010.Sirtuin1 (SIRT1) is a NAD+-dependent histone deacetylase that regulates inflammation, aging, senescence, and apoptosis through the deacetylation of transcription factors and histones. SIRT1 has been shown to inhibit pro-inflammatory cytokine release and NF-B activation in chronic obstructive pulmonary disease (COPD), but cigarette smoke (CS), the main etiological factor in developing COPD, decreases SIRT1 protein, mRNA, and activity levels. COPD is a leading cause of mortality globally, and is characterized by a progressive destruction of the lower airways of the lung and irreversible airflow limitation, but unfortunately, there are no effective treatments for this disease. SIRT1 has been shown to modulate many of the processes involved in COPD pathology, such as inflammation, apoptosis, and senescence, making SIRT1 a potential target for pharmacologic intervention. CS is a complex mixture of oxidants, free radicals, and reactive aldehydes, and has been shown to induce irreversible covalent oxidative modifications on proteins, including SIRT1. As the mechanism by which CS causes loss of SIRT1 is unknown, it is hypothesized that CS down-regulates SIRT1 protein level and activity through oxidative modifications and cofactor depletion, leading to nucleocytoplasmic shuttling and proteasomal degradation. Studies using the human bronchial epithelial cell line BEAS-2B revealed that SIRT1 is carbonylated on cysteine residues by CS, oxidants, and reactive aldehydes, which was reversed by increasing the intracellular thiol pool. Modification of cysteine residues decreased SIRT1 activity and protein level. SIRT1 was shown to be a target of proteasomal degradation by CS through the use of proteasome inhibitors. This protein degradation was shown to occur in the cytoplasm, although inhibition of nucleocytoplasmic shuttling did not prevent loss of SIRT1 in response to CS. SIRT1’s NAD+ cofactor levels were also decreased in response to CS, however increasing NAD+ levels through pharmacologic inhibitors of NAD+-utilizing enzyme PARP-1 or NAD+ precursors could not restore SIRT1 activity in response to CS. Carbonylation was observed even in the presence of increased NAD+, and SIRT1 activity could not be reversed by a specific SIRT1 activator. Taken together, these data implicate carbonylation and proteasomal degradation in CS-induced decreased SIRT1 protein level and activity