STRUCTURE AND MECHANISM OF PROTEIN PRENYLTRANSFERASES

本研究涉及哺乳动物蛋白戊烯基转移酶蛋白：法尼基转移酶（FTase）和香叶基转移酶（GGTase-1）的结构和机制。这些蛋白质催化类异戊二烯脂质的添加到一些小的调节g蛋白。这种修饰（戊烯酰化）对于g蛋白的活性和靶向细胞膜是必不可少的。fase介导的Ras癌基因蛋白的法尼化对其致癌和细胞转化活性是绝对必需的。在组织培养细胞和动物模型中，已证明用FTase抑制剂处理Ras转化细胞可导致转化表型的逆转，且无明显毒性。抑制Ras法尼化目前被认为是最有希望的抗癌靶点之一。超过25%的人类癌症与Ras的致癌形式有关。因此，阐明FTase及其底物复合物的三维结构对于理解信号转导的基本过程以及开发具有药理活性的抗癌药物或改进现有药物的衍生物都很重要。近年来，GGTase-1作为药物开发的潜在靶点也备受关注。细胞中的大多数戊酰化蛋白都被香叶基类异戊二烯修饰。GGTase-1可以催化Ras超家族中参与细胞增殖的几个成员的戊烯化。抑制GGTase-1可能不仅对癌症化疗很重要，而且对心血管疾病的治疗和抗真菌化合物的开发也很重要。此外，GGTase-1的结构，特别是与相关FTase的结构相比较，可能会进一步加深我们对肽和类异戊二烯底物特异性机制的理解。此外，了解FTase和GGTase底物特异性差异的分子基础可能有助于开发更具选择性的FTase（和GGTase-1）抑制剂用于抗癌治疗。本课题的总体目标是利用x射线晶体学研究哺乳动物蛋白丙烯基转移酶蛋白的机制和底物特异性的分子基础，以1)确定哺乳动物FTase底物、产物和抑制剂复合物的高分辨率三维结构2)确定哺乳动物GGTase-1的三维结构3)确定GGTase-1与底物、产物和抑制剂的结构。

This proposal is concerned with the structure and mechanism of mammalian protein prenyltransferase proteins: farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase-1). These proteins catalyze the addition of an isoprenoid lipid to a number of small regulatory G-proteins. This modification (prenylation) is essential for the activity and targeting of G-proteins to cell membranes. The FTase-mediated farnesylation of Ras oncogene proteins is absolutely required for their oncogenic, cell transforming activity. Treatment of Ras transformed cells with inhibitors of FTase has been shown to result in reversion of the transformed phenotype in tissue culture cells and animal models without apparent toxicity. Inhibition of Ras farnesylation is currently considered one of the most promising anti-cancer targets. Over 25 percent of human carcinomas are associated with oncogenic forms of Ras. Elucidation of the three-dimensional structure of FTase and its substrate complexes is therefore important both for understanding fundamental processes in signal transduction and for the development of pharmocologically active anti-cancer drugs or improved derivatives of existing drugs. GGTase-1 has also received attention recently as a potential target of the development of pharmaceuticals. The majority of prenylated proteins in the cell are modified by the geranylgeranyl isoprenoid. GGTase-1 catalyzes the prenylation of several members of the Ras superfamily involved in cell proliferation. Inhibition of GGTase-1 may be important not only for cancer chemotherapy, but also in the treatment of cardiovascular disease and the development of anti-fungal compounds. Furthermore, the structure of GGTase-1, particularly when compared to the structure of the related FTase, may further our understanding of the mechanisms of both peptide and isoprenoid substrate specificity. In addition, understanding the molecular basis for the differences between FTase and GGTase substrate specificities may facilitate the development of more selective FTase (and GGTase-1) inhibitors for anti-cancer therapy. The overall goal of this proposal is to study the molecular basis for the mechanism and substrate specificity of mammalian protein prenyltransferase proteins using X-ray crystallography to 1) determine the high resolution, three-dimensional structures of mammalian FTase substrate, product, and inhibitor complexes 2) determine the three-dimensional structure of mammalian GGTase-1 3) determine the structures of GGTase-1 together with substrates, products, and inhibitors.