Structure and Function of the CaaX Protease Ste24p

描述(由申请人提供)：翻译后脂肪作用对某些蛋白质的功能提供关键的调节。在含有C端CAAX序列基序的蛋白质中，异戊二烯(即法尼基或香叶基)连接到半胱氨酸残基上。异戊二烯基化通常伴随着两个后续的加工步骤，即AAX残基的蛋白水解性裂解和修饰半胱氨酸残基新暴露的羰基的羧甲基化。一些异戊二烯基化的蛋白质还经历了额外的蛋白分解过程，包括最初去除AAX残基的同一种酶的额外切割。由于CAAX加工的底物包括在癌症中经常突变的小GTP酶ras和rho，CAAX途径的酶是癌症药物发现的有效靶点。由于其在a因子交配信息素成熟中的作用，首次发现的和 最具特性的CAAX蛋白酶是酵母锌金属蛋白酶Ste24p。人类Ste24p的同源基因ZMPSTE24可以补充酵母Ste24p的全部功能。人类蛋白质唯一已知的底物是前层蛋白A，它是核中间细丝蛋白Lam in A的前体。ZMPSTE24或Prelamin A的加工位点的突变与一系列被称为早衰症的早衰疾病有关。此外，ZMPSTE24被针对HIV天冬氨酸蛋白酶的抗病毒药物抑制，这种非靶点效应可能会导致这些药物的一些严重副作用。测定了酵母Ste24p的晶体结构。它的核心结构是一个由七个跨膜螺旋组成的环，包围着一个大的(14,000ä3)内部体积(腔)，其中包含活性部位和底物结合区域(槽)。空腔可以通过间隙(入口)进入外部环境，这些间隙(入口)部分被非跨膜结构域遮挡，这些非跨膜结构域位于一对展开的跨膜螺旋之间。人类ZMPSTE24，由另一组同时解出，具有相同的结构。酵母Ste24p和人ZMPSTE24的结构非常相似，成对的RMSD为~1.2？我们认为，卵裂是通过底物插入、移位和喷射的过程处理机制(PPM)进行的。大空腔的作用，具有不同序列的切割位点的特异性识别机制，不寻常的双重切割过程，以及法尼基在识别中的作用，这些都是我们试图通过这项研究提案解决的一些问题，该研究提案以酵母和人类酶的结构功能研究为特色。

DESCRIPTION (provided by applicant): Post-translational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CaaX sequence motifs. Isoprenylation is generally accompanied by two subsequent processing steps, proteolytic cleavage of the aaX residues and carboxymethylation of the newly exposed carbonyl group of the modified cysteine residue. Some isoprenylated proteins also undergo additional proteolytic processing, including an additional cleavage by the same protease that initially removes the aaX residues. As substrates for CaaX processing include ras and rho, small GTPases frequently mutated in cancers, the enzymes of the CaaX pathway are validated targets for cancer drug discovery. Due to its role in maturation of the a-factor mating pheromone, the first-identified and best-characterized CaaX protease is the yeast zinc metalloprotease Ste24p. A human ortholog of Ste24p, ZMPSTE24, can complement the full function of yeast Ste24p. The only known substrate for the human protein is prelamin A, the precursor to the nuclear intermediate filament protein lamin A. Mutations in either ZMPSTE24 or the processing site of prelamin A are associated with a spectrum of premature-aging diseases referred to as progeria. Also, ZMPSTE24 is inhibited by antiviral drugs designed to target the HIV aspartyl protease, and this off-target effect may give rise to some of the severe side-effects of these drugs. We determined the crystal structure of yeast Ste24p. Its core structure is a ring of seven transmembrane helices enclosing a large (14,000 Å3) interior volume (CAVITY) that contains the active-site and substrate binding region (GROOVE). The cavity is accessible to the external milieu via gaps (PORTALS), partially occluded by non-transmembrane domains, that are between pairs of splayed transmembrane helices. Human ZMPSTE24, solved contemporaneously by another group, possesses the same architecture. The structures of yeast Ste24p and human ZMPSTE24 are quite similar, with a pairwise RMSD of ~1.2 Å. We propose that cleavage proceeds via a processive processing mechanism (PPM) of substrate insertion, translocation and ejection. The role of the large cavity, the mechanism of specific recognition of cleavage sites with divergent sequences, the unusual dual cleavage process, and the role of the farnesyl group in recognition are some of the questions we seek to address with this research proposal that features structure-function studies of the yeast and human enzymes.