STRUCTURAL STUDIES OF PROTEINS ASSOCIATED WITH HUMAN DISEASES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 样本1：碳酸酐酶 碳酸酐酶（Carbonic anhydrases，CA）是一类催化CO2和HCO 3-可逆相互转化的锌金属酶。近年来，大量的研究表明，CA同工酶IX（CA isozyme IX，CA IX）的过表达促进了细胞外基质的酸化，从而促进了某些肿瘤的生长。根据这些观察结果，并基于结构比对同源性，使用晶体结构CA II和CA IX的序列，我们构建了CA II的双突变体，其中Ala 65被Ser取代，Asn 67被Gln取代，以类似于CA IX的活性位点。将使用X射线晶体学单独和与几种临床使用的CA抑制剂复合研究这种CAIX模拟物，并与野生型CA II进行比较。此外，将评价与抑制和体外细胞毒性试验相关的结构信息，并将开发相关的结构-活性关系。这些研究将提供一个有用的模型，以设计更多的同工酶特异性CA IX抑制剂，可能会导致一些癌症的新的治疗方法的发展。 我们还将尝试使用CO2高压冷冻（与Sol和Chaeun（CHESS）合作）来捕获野生型和/或载脂蛋白HCA II中的结合底物。 样本2：人锰超氧化物歧化酶 锰超氧化物歧化酶（MnSOD）的亚基之间的界面的结构，稳定性和催化功能将被检查。MnSOD催化超氧化物歧化反应生成O2和H2 O2。人MnSOD是22 kDa亚基的同源四聚体，二聚体的二聚体，具有两个结构独特的界面，二聚体和四聚体界面。这些接口的催化，稳定性和结构的功能是不确定的，各种突变MnSOD结构将被研究和相关的动力学和稳定性。由于MnSOD的突变形式被制备为可能的抗增殖剂，例如与野生型相比表现出较少的产物抑制的H30 N MnSOD，因此应该预期二聚体界面处的其他残基的改变可能会减少催化并增强产物抑制。 标本3：HIV-1 C亚型蛋白酶 人类免疫缺陷病毒1型（HIV-1）蛋白酶（PR）是一种天冬氨酸水解酶，其功能为每个亚基（标记为1-99和1 '-99'）中具有99个氨基酸的专性同源二聚体。其作用是将gag和gag/pol多聚蛋白切割成结构蛋白和酶蛋白，并诱导成熟的感染性病毒体的形成。这种酶的抑制产生不成熟的HIV病毒粒子，不能传播感染。由于其在获得病毒感染性中的重要作用，HIV-1 PR被认为是发现新的和有效的抗HIV药物的有吸引力的靶点。将研究未结合/结合的C PR和各种未结合/结合的多药耐药C PR变体的结构，以确定由于天然存在的多态性引起的结构变化，并描述其在抗逆转录病毒药物耐药/敏感性中的意义。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Specimen 1: Carbonic anhydrase Carbonic anhydrases (CA)s are zinc-metalloenzyme that catalyzes the reversible inter-conversion of CO2 and HCO3-. Recently, a convincing body of evidence has been accumulated, that suggests the over-expression of CA isozyme IX (CA IX) contributes to the acidication of the extracellular matrix, which is thought to promote growth of certain tumors. In light of these observations, and based on structural alignment homology, using the crystal structure CA II and the sequence of CA IX, we have constructed a double mutant of CA II with Ala 65 replaced by Ser and Asn 67 replace by Gln to resemble the active site of CA IX. This CAIX mimic will be studied using X-ray crystallography, alone and in complex with several clinically used CAs inhibitors and compared to wild-type CA II. Further this structural information will be evaluated in relationship to inhibition and in vitro cytotoxicity assays and a correlated structure-activity relationship will be developed. These studies will provide a useful model to design more isozyme specific CA IX inhibitors that may lead to development of new therapeutic treatments of some cancers. We will also be trying to use high pressure freezing with CO2 (in collaboration with Sol and Chaeun (CHESS) to capture the bound substrate in wild-type and/or apo HCA II. Specimen 2: human Mn SuperOxide Dismutase The function in structure, stability, and catalysis of the interfaces between subunits in manganese superoxide dismutase (MnSOD) will be examined. MnSOD catalyzes the disproportionation of superoxide to produce O2 and H2O2. Human MnSOD is a homotetramer of 22 kDa subunits, a dimer of dimers, with two structurally unique interfaces, a dimeric and a tetrameric interface. The function in catalysis, stability, and structure of these interfaces is uncertain and various mutants MnSOD structures will be studied and correlated to kinetic and stability properties. As mutant forms of MnSOD are prepared as possible antiproliferative agents, such as H30N MnSOD that exhibits less product inhibition compared with wild-type, it should be expected that alteration of other residues at the dimeric interface are likely to diminish catalysis and enhance product inhibition. Specimen 3: HIV-1 subype C protease Human Immunodeficiency Virus type 1 (HIV-1) protease (PR) is an aspartic hydrolase that functions as an obligatory homodimer with 99 amino acids in each subunit (labeled 1-99 and 1'-99'). Its role is to cleave the gag and gag/pol polyproteins into structural and enzymatic proteins and to induce the formation of mature, infectious virions. The inhibition of this enzyme yields immature HIV virions, incapable of spreading the infection. Because of its essential role in gaining viral infectivity, HIV-1 PR has been considered an attractive target for discovering new and potent anti-HIV drugs. The structure of the unbound/bound C PR and various unbound/bound multi-drug resistant variant of C PR will be studied to identify structural changes due to the naturally occurring polymorphisms and delineate their implications in antiretroviral drug resistance/susceptibility.