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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 鞘磷脂(SLS)介导包括细胞凋亡在内的细胞活动。SLS从头合成的第一步也是限速步骤是通过丝氨酸棕榈酰基转移酶(SPT)，它将丝氨酸与棕榈酰辅酶A缩合，产生狮身人面像碱基，这是所有SLS的主干。重要的是，许多刺激诱导SLS从头合成，包括化疗，但机制尚不清楚。我们有初步数据表明，在酵母中，热应激通过刺激培养液中丝氨酸的摄取来刺激SL的从头合成。此外，从头合成的脂肪酸成分来自于通过脂肪酸合成酶(FAS)进行的内源合成。基于这些发现，我们假设底物供应的调节有助于调节SLS的从头合成，以响应包括化疗药物在内的刺激。因此，这项建议的目的是：1)确定热应激刺激酵母外源丝氨酸吸收所需的机制；2)确定Fas和酰基辅酶A合成酶在热应激诱导的SPT活性中的作用。这2个目标将使用多方面的方法来实现，包括酵母基因片段、体外酶活性测定和脂质分析；3)确定底物供应在癌细胞对化疗药物反应中的作用。在这个目标中，我们将重点放在吉西他滨，它激活A549细胞中的从头通路，导致促凋亡因子的选择性剪接。我们假设底物的可用性可能调节吉西他滨诱导的从头合成SL，这将使新的化疗方法成为可能。

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.