STRUCTURAL STUDIES OF PROTEINS ASSOCIATED WITH HUMAN DISEASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 标本1：碳酸酐酶(CA)S是催化CO_2和HCO_3~-可逆相互转化的锌金属酶。最近，大量令人信服的证据表明，CA同工酶IX(CA IX)的过度表达有助于细胞外基质的酸化，这被认为促进了某些肿瘤的生长。根据这些观察结果，根据结构比对同源性，利用CA II的晶体结构和CA IX的序列，我们构建了一个CA II的双突变体，其中Ala 65用Ser替换，Asn 67用Gln替换，与CA IX的活性部位相似。该CAIX模拟物将用X射线结晶学进行研究，单独和与几种临床使用的CA抑制剂复合使用，并与野生型CA II进行比较。此外，还将评估这些结构信息与抑制和体外细胞毒性试验的关系，并建立相关的构效关系。这些研究将为设计更具同工酶特异性的CA IX抑制剂提供一个有用的模型，这些抑制剂可能会导致一些癌症的新治疗方法的开发。我们还将尝试使用二氧化碳高压冷冻(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将研究锰超氧化物歧化酶(MnSOD)亚基之间界面的结构、稳定性和催化功能。MnSOD催化超氧化物歧化生成O2和H2O2。人MnSOD是由22 kDa亚基组成的同源四聚体，是二聚体的二聚体，具有两个结构独特的界面，一个是二聚体界面，一个是四聚体界面。这些界面在催化、稳定性和结构上的作用是不确定的，将研究各种突变的MnSOD结构，并将其与动力学和稳定性性质相关联。由于突变形式的MnSOD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H30N MnSOD，应该可以预见，二聚体界面上其他残基的改变可能会降低催化作用，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，其功能是必需的同源二聚体，每个亚基含有99个氨基酸(标记为1-99和1？“-99？”)。它的作用是将Gag和Gag/Poll多蛋白切割成结构蛋白和酶蛋白，并诱导成熟的、有感染性的病毒粒子的形成。这种酶的抑制会产生未成熟的HIV病毒粒子，不能传播感染。由于其在获得病毒感染性方面的重要作用，HIV-1PR一直被认为是发现新的有效抗HIV药物的有吸引力的靶点。将研究非结合/结合C-PR和各种非结合/结合多药耐药变异体的结构，以确定由于自然发生的多态引起的结构变化，并阐明它们在抗逆转录病毒耐药性/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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.