STRUCTURAL STUDIES OF PROTEINS ASSOCIATED WITH HUMAN DISEASES

与人类疾病相关的蛋白质的结构研究

基本信息

批准号：
7955564
负责人：
ROBERT MCKENNA
金额：
$ 5.76万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7955564
关键词：
Active Sites Amino Acids Anti-HIV Agents Antiretroviral drug resistance Bicarbonates Binding Biological Assay Carbon Dioxide Carbonic Anhydrase II Catalysis Cleaved cell Collaborations Complex Computer Retrieval of Information on Scientific Projects Database Development Enzyme Inhibition Exhibits Extracellular Matrix Freezing Funding Gagging Genetic Polymorphism Glutamine Grant Growth HIV HIV-1 Human Hydrogen Peroxide Hydrolase In Vitro Infection Institution Isoenzymes Kinetics Label Lead Light Malignant Neoplasms Manganese Superoxide Dismutase Modeling Multi-Drug Resistance Peptide Hydrolases Predisposition Property Proteins Research Research Personnel Resources Role Source Specimen Structure Structure-Activity Relationship Superoxides United States National Institutes of Health Variant Viral Virion X-Ray Crystallography Zinc antiproliferative agents base carbonate dehydratase cytotoxicity design dimer human disease inhibitor/antagonist metalloenzyme mutant novel therapeutics pol Gene Products pressure protease C tumor

项目摘要

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.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以出现在其他 CRISP 条目中列出的机构是。对于中心来说，它不一定是研究者的机构。样本 1：碳酸酐酶碳酸酐酶 (CA) 是催化 CO2 和 HCO3- 可逆相互转化的锌金属酶。最近，已经积累了大量证据，表明 CA 同工酶 IX ( CA) 的过度表达。 IX) 有助于细胞外基质的酸化，根据这些观察结果并基于结构排列，这被认为促进某些肿瘤的生长。同源性，利用晶体结构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。使用 X 射线晶体学研究，单独或与几种临床使用的 CA 抑制剂复合，并与野生型 CA II 进行比较，进一步评估该结构信息与抑制和体外细胞毒性测定以及相关结构活性关系的关系。这些研究将提供一个有用的模型来设计更多的同工酶特异性 CA IX 抑制剂，这可能会导致开发一些癌症的新治疗方法。我们还将尝试使用 CO2 高压冷冻（与 Sol 和Chaeun (CHESS) 捕获野生型和/或 apo HCA II 中的结合底物样本 2：人锰超氧化物歧化酶亚基之间界面的结构、稳定性和催化功能。将检查锰超氧化物歧化酶 (MnSOD) 催化超氧化物歧化产生 O2 和 H2O2。人类 MnSOD 是 22 kDa 亚基的同四聚体，是二聚体的二聚体，具有两个结构独特的界面，即二聚体和四聚体界面。这些界面的催化功能、稳定性和结构是不确定的，并且各种突变体 MnSOD 结构将由于 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产物抑制的 H30N MnSOD，因此应该预期二聚体界面处其他残基的改变。可能会减弱催化作用并增强产物抑制作用样本 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，作为必需同二聚体发挥作用，每个亚基包含 99 个氨基酸（标记为 1-99 和 1¿ ¿ “-99¿”) 的作用是将 gag 和 gag/pol 多蛋白裂解成结构蛋白和酶蛋白，并诱导形成成熟的感染性病毒颗粒。对该酶的抑制会产生不成熟的 HIV 病毒颗粒，无法传播病毒。由于其在获得病毒感染性方面的重要作用，HIV-1 PR 已被认为是发现新的有效抗 HIV 药物的有吸引力的靶标。未结合/结合的 C PR 和各种药物的结构。将研究 C PR 的未结合/结合多重耐药变体，以确定由于自然发生的多态性而引起的结构变化，并描述它们在抗逆转录病毒药物耐药性/敏感性中的影响。