Studies on the cellular functions of lipid-modified proteins

许多细胞蛋白被脂质修饰，如酰基（棕榈酰或肉豆蔻酰）和/或戊基（香叶酰或香叶酰）残基。本课题研究了脂质修饰蛋白的生物化学功能，并探索了其形成的微生物抑制剂，作为抗癌或抗病毒药物的新途径。HIV Gag蛋白在n端甘氨酸残基上有myrisstoylate，在病毒颗粒结合中起重要作用。利用本课课组发现的特异性酰基辅酶a合成酶抑制剂triacsin控制豆芽糖酰化水平，研究其对颗粒出芽的影响。我们发现三聚乳酸对Gag肉豆蔻酰化的抑制遵循剂量依赖性动力学，但颗粒出芽表现出突然关闭动力学，这表明只有相对较小比例的Gag分子需要进行肉豆蔻酰化才能有效出芽，并表明有效的抗hiv治疗需要完全抑制肉豆蔻酰化。癌基因产物Ras蛋白在c端附近的半胱氨酸残基处发生翻译后法尼基化，其中涉及蛋白法尼基转移酶（PFTase）。PFTase被认为是一种新的抑制靶点，因为抑制会改变Ras蛋白的膜定位和阻断Ras蛋白的激活。利用酿酒酵母对PTFase进行了高效筛选，发现了两个新的PTFase抑制剂系列：青霉sp.FO-3929产生的andrastins和拟青霉sp.FO-3684产生的kurasoins。对其结构进行了立体化学、生物合成和全合成。对PFTase的抑制活性（IC_<50>）为10 ~ 60mum。这些化合物是否能抑制细胞中的PFTase并显示出体内抗癌活性还有待研究。

Many cellular proteins are modified with lipids such as acyl(palmitoyl or myristoly)and/or prenly(farnesly or geranylgeranyl)residues. In this researach project, the biochemical functions of lipid-modified proteins were studied and searaach for microbial inhibitors of their formation wes carried out as a new approach for anticancer or antiviral agents.HIV Gag protein is myrisstoylataed at the N-terminal glycine residue, which plays an important role in virus particlebdding. The myristoylation levels were controlled to investigate the effect on the particle budding by using the specifiv acyl-CoA synthetase inhibitor triacsin previousy discovered by our group. We showed that the inhibition of Gag myristoylation by triacsin follows dose-dependent kinetics but that the particle budding exhibits sudden shutoff kinetics, suggesting that only a relatively small proportion of total Gag molecules need to be myristoylated for efficient budding and indicating that total inhibition of myristoylation will be required for effective anti-HIV therapy.Oncogene product Ras proteins are posttranslationally farnesylated at the cysteine residue near the C-terminus, in which protein farnesyltransferase(PFTase)is involved. PFTase is expected as a novel target of inhibition since the inhibition causes altering membrane localization and blocking activation of Ras proteins. An efficient screening system was conducted by utilizing Saccharomyces cerevisiae, resulting in discovery of two series of new PTFase inhibitors, andrastins produced by Penicillium sp.FO-3929 and kurasoins by Paecilomyces sp.FO-3684. The structure elucidation including stereochemistries, biosynthesis of andrastin and total sylnthesis of kurasoin were studied. Their inhibitory activity against PFTase (IC_<50>) was 10-60muM.It still remains to be investigated whether or not these compounds inhibit PFTase in cells and show in fvivo anticancer activity.