Structure and Function of the CaaX Protease Ste24p

描述（由申请方提供）：翻译后脂化提供了某些蛋白质功能的关键调节。类异戊二烯（即，法呢基或香叶基香叶基）连接到含有C-末端CaaX序列基序的蛋白质中的半胱氨酸残基上。异戊二烯化通常伴随两个后续加工步骤，aaX残基的蛋白水解裂解和修饰的半胱氨酸残基的新暴露的羰基的羧甲基化。一些异戊二烯化的蛋白质还经历另外的蛋白水解加工，包括由最初去除aaX残基的相同蛋白酶进行的另外的切割。由于CaaX加工的底物包括ras和rho，即在癌症中经常突变的小GTP酶，因此CaaX途径的酶是癌症药物发现的经验证的靶标。由于其在a因子交配信息素成熟中的作用， 最佳表征的CaaX蛋白酶是酵母锌金属蛋白酶Ste 24 p。Ste 24 p的人类直系同源物ZMPSTE 24可以补充酵母Ste 24 p的全部功能。人类蛋白质的唯一已知底物是前核纤层蛋白A，核中间丝蛋白核纤层蛋白A的前体。ZMPSTE 24或前层蛋白A加工位点的突变与一系列称为早衰症的早衰性疾病相关。此外，ZMPSTE 24被设计为靶向HIV乙酰基蛋白酶的抗病毒药物抑制，这种脱靶效应可能导致这些药物的一些严重副作用。我们测定了酵母Ste 24 p的晶体结构。它的核心结构是一个由7个跨膜螺旋组成的环，包围着一个包含活性位点和底物结合区（GROOVE）的大（14，000 μ 3）内部体积（CAVITY）。腔可经由间隙（门户）进入外部环境，所述间隙部分地被非跨膜结构域封闭，所述非跨膜结构域在成对的张开的跨膜螺旋之间。人类ZMPSTE 24，由另一个小组同时解决，具有相同的结构。酵母Ste 24 p和人ZMPSTE 24的结构非常相似，成对RMSD为~1.2 μ g。我们建议，裂解收益通过基板插入，易位和弹出的过程处理机制（PPM）。的作用的大腔，特异性识别的切割位点与不同的序列，不寻常的双切割过程中，和法呢基基团的作用，在识别的机制是一些问题，我们试图解决这个研究建议，功能的酵母和人类酶的结构-功能研究。

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.