SUBSTRATE SUPPLY IN DE NOVO SPHINGOLIPID SYNTHESIS: REGULATION/IMPACT ON CHEMOTH

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sphingolipids (SLs) mediate cellular activities including apoptosis. The first and rate-limiting step in de novo synthesis of SLs is through serine palmitoyltransferase (SPT), which condenses serine with palmitoyl-CoA, producing a sphingoid base, the backbone of all SLs. Importantly, many stimuli induce de novo synthesis of SLs, including chemotherapy, but the mechanisms are unknown. We have preliminary data that in yeast, heat stress stimulates de novo SL synthesis by stimulating serine uptake from media. Moreover, the fatty acid component for de novo synthesis derives from endogenous synthesis via fatty acid synthase (FAS). Based on these findings, we hypothesize that regulation of substrate supply serves to modulate de novo synthesis of SLs in response to stimuli including chemotherapeutic agents. Therefore, the aims of this proposal are: 1) to determine the mechanisms required for heat stress stimulation of exogenous serine uptake in yeast; 2) to determine the role of FAS and acyl-CoA synthetase in heat-stres induced SPT activity. These 2 aims will be accomplished using a multifaceted approach with yeast genetic sreens, in vitro enzyme activity determinations, and lipid analysis; and 3)to determine the role of substrate supply in cancer cells in response to chemotherapeutic agents. In this aim, we will focus on gemcitabine, which activates the de novo pathway in A549 cells, leading to alternative splicing of pro-apoptotic factors. We hypothesize that substrate availability may regulate gemcitabine-induced de novo SL synthesis, which would allow novel approaches to chemotherapy.