Studies in posttranslational regulation of the transsulfuration pathway and truncation of SIRT6
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Abstract
The science of aging and longevity has recently become a growing area of study. As old age becomes an increasingly prevalent cause of mortality, it has become more obvious that the metabolic processes and genomics that relate to aging are largely unknown. A genetically conserved family of proteins known as sirtuins have been found to have a profound effect on metabolic regulation. SIRT6 is one of the seven proteins (SIRT1-SIRT7) in this family having recently been found to prevent metabolic effects of aging. An NAD+ dependent deacetylase specific for H3K9 (Histone 3, Lysine 9) and H3K56 (Histone 3, Lysine 56) and an ADP ribosyl transferase, SIRT6 regulates genome stability, DNA double strand break repair, telomere integrity as well as gene transcription through the use of the transcription factor Sp1. The overexpression of SIRT6 in B6 male and female mice both preserved glucose homeostasis and hepatic glucose output in their old age, repressed glycolysis, and activated pathways such as AMP kinase (Roichman, et al, 2021). All of these effects helped maintain homeostasis in the mice as they aged by promoting normoglycemia. In the liver specifically, SIRT6 was found to repress glycolysis, triglyceride synthesis, and increase beta oxidation. Structurally, SIRT6 is composed of a core domain of 276 amino acids. The C terminal is mainly responsible for nuclear localizations where the N terminal is the primary site of the catalytic activity. While the overall location of catalytic activity in SIRT6 is known, little research has gone into pinpointing the Sp1 binding site of SIRT6 (Tennen, et al, 2010). •Gaseous H2S has also been found to have a positive effect on health and aging by preventing hypertension and protecting against neurodegeneration associated with Huntington's disease, atherosclerosis, and type 1 diabetes (Hine, et al, 2014). A major product of the transsulfuration (TSS) pathway, H2S is generated by CBS and CGL, enzymes in the TSS pathway. This pathway is present in both humans and mice. It has been found in our lab that the mouse CBS protein is acetylated on lysine 386 (K386). Further research discovered that this acetylation regulates CBS’s H2S production activity. However, in the human CBS, this lysine residue is substituted with an arginine (R), which is traditionally used in molecular biology for the mimicking of constant deacetylation. Thus, this region in CBS in humans resembles a constant state of deacetylation, whereas in mice this region resembles a variable state of acetylation. Little is known about the effects of the differing residues in human and mice CBS, and consequently how the acetylation/deacetylation states of CBS affects metabolic activity. •Our first goal was to determine the effects of acetylation on CBS activity, H3K9 acetylation, and CGL concentration. Secondly, our goal was to successfully truncate SIRT6 in order to determine the location of the Sp1 binding site in the future.