Structure and Function of the CaaX Protease Ste24p

CaaX 蛋白酶 Ste24p 的结构和功能

基本信息

批准号：
8610715
负责人：
Michael Wiener
金额：
$ 45.11万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8610715
关键词：
Active Sites Address Adverse drug effect Antineoplastic Agents Antiviral Agents Architecture Aspartic Endopeptidases Binding Biological Biological Assay C-terminal Carrier Proteins Catalysis Cell Nucleus Cell membrane Chimeric Proteins Cleaved cell Complement Complex Conserved Sequence Cysteine Dependence Disease Disulfides Drug Design Electrostatics Elements Enzymes Excision Fatty acid glycerol esters Fluorescence Polarization Glycine Goals HIV Health High Pressure Liquid Chromatography Human In Vitro Intermediate Filament Proteins Ionic Strengths Lamin Type A Lipids Malignant Neoplasms Measurement Membrane Membrane Proteins Metalloproteases Molecular Structure Monomeric GTP-Binding Proteins Mutate Mutation N-terminal Nuclear Orthologous Gene Pathway interactions Peptide Hydrolases Peptides Pharmaceutical Preparations Phase Pheromone Premature aging syndrome Process Progeria Proteins Proteolytic Processing Reporting Research Research Proposals Role Site Specificity Structure Surface Testing Therapeutic Transmembrane Domain Yeasts Zinc a-mating factor base carbonyl group carboxymethylation design drug discovery in vivo isoprenoid isoprenylation mutant novel prelamin A protein complex protein function research study rho

项目摘要

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.

描述（由应用程序提供）：翻译后脂质提供了对某些蛋白质功能的关键调节。异普尼（即Farnesyl或黄烷基基团）附着在含有C末端CAAX序列基序的蛋白质中的半胱氨酸保留。异丙肾上腺化通常伴随着两个随后的加工步骤，AAX保留的蛋白水解切割和修饰的半胱氨酸住宅的新暴露的羰基的羧甲基化。一些异on基化蛋白还会进行其他蛋白水解加工，包括最初去除AAX保留的相同蛋白酶的额外裂解。由于CAAX加工的底物包括RAS和RHO，因此在癌症中经常突变的小GTP酶，CAAX途径的酶是癌症药物发现的验证靶标。由于它在A因子交配信息素的成熟中的作用，第一个识别和最佳特征的CAAX蛋白是酵母锌金属蛋白酶Ste24p。 Ste24p的人类直系同源物ZMPSTE24可以补充酵母菌Ste24p的完整功能。人类蛋白质的唯一已知底物是预胺A，这是核中间丝蛋白层状A的前体A。同样，ZMPSTE24受到旨在靶向HIV的天冬氨酸蛋白酶的抗病毒药物的抑制，这种脱靶效应可能会导致这些药物的某些严重副作用。我们确定了酵母Ste24p的晶体结构。它的核心结构是一个七个跨膜螺旋的环，该环包含一个大型（14,000Å3）内部体积（空腔），其中包含活动位置和底物结合区域（凹槽）。外部环境可以通过间隙（门户）（部分由非跨膜域遮住）进入腔体，这些腔位于非跨膜结构域，这些域之间位于成对的跨膜螺旋螺旋之间。人类ZMPSTE24由另一个群体解决了当代，具有相同的建筑。酵母Ste24p和人ZMPSTE24的结构非常相似，成对RMSD约为1.2Å。我们建议裂解通过底物插入，易位和射血的处理处理机制（PPM）进行。大型空腔的作用，具有不同序列的裂解位点的特定识别的机制，异常的双重裂解过程以及Farnesyl群体在识别中的作用是我们寻求通过这项研究提案来解决的一些问题，这些问题具有酵母和人类酶的结构功能研究。