Studies on the cellular functions of lipid-modified proteins

许多细胞蛋白质被脂类修饰，如酰基(棕榈酰基或月桂醇)和/或戊烯基(法尼基或香叶基)残基。在本研究项目中，研究了脂质修饰蛋白的生化功能，并寻找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-末端的甘氨酸残基上被肉豆蔻酰化，在病毒颗粒中起着重要的作用。用本课题组以前发现的特异性酰辅酶A合成酶抑制剂Triacsin控制肉豆蔻酰化水平，以观察对颗粒萌发的影响。我们发现Triacsin抑制GAG肉豆蔻酰化的作用遵循剂量依赖的动力学，但颗粒萌发呈现突然关闭动力学，这表明只需要相对较小比例的总GAG分子肉豆蔻化就能有效地萌发，这表明有效的抗HIV治疗将需要完全抑制肉豆蔻酰化。癌基因产物RAS蛋白在C末端附近的半胱氨酸残基被翻译后法尼化，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，因为这种抑制会改变膜的定位并阻断RAS蛋白的激活。利用酿酒酵母建立了一套高效的筛选系统，发现了青霉FO-3929产生的两个新的PTFase抑制剂系列和由拟青霉FO-3684产生的苦参素。对苦参素的立体化学、生物合成和总合成为苦参素进行了研究。它们对PFTase的抑制活性(IC_&lt；50&gt；)为10-60微米，这些化合物是否在细胞内对PFTase有抑制作用以及在体内是否具有抗癌活性仍有待进一步研究。

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.