STRUCTURAL STUDIES OF PROTEINS ASSOCIATED WITH HUMAN DISEASES

与人类疾病相关的蛋白质的结构研究

• 批准号: 8363521
• 负责人: ROBERT MCKENNA
• 金额: $ 5.36万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363521
• 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; Development; Enzyme Inhibition; Exhibits; Extracellular Matrix; Freezing; Funding; Gagging; Genetic Polymorphism; Glutamine; Grant; Growth; HIV; HIV-1; Human; Hydrogen Peroxide; Hydrolase; In Vitro; Infection; Isoenzymes; Kinetics; Label; Lead; Light; Malignant Neoplasms; Manganese Superoxide Dismutase; Modeling; Multi-Drug Resistance; National Center for Research Resources; Peptide Hydrolases; Predisposition; Principal Investigator; Property; Proteins; Research; Research Infrastructure; 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; cost; cytotoxicity; design; dimer; human disease; inhibitor/antagonist; metalloenzyme; mutant; novel therapeutics; pol Gene Products; pressure; protease C; tumor

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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 标本1：碳酸酐酶 碳酸酐酶（Ca）是锌 - 金糖酶，可催化CO2和HCO3-的可逆反转。最近，积累了令人信服的证据，这表明CA同工酶IX（CA IX）的过表达有助于细胞外基质的酸化，这被认为可以促进某些肿瘤的生长。鉴于这些观察结果，并基于结构比对同源性，使用晶体结构Ca II和Ca IX的序列，我们构建了一个Ca II的双重突变体，ALA 65被SER替换为SER，ASN 67替换为GLN，类似于Ca IX的活性位点。将使用X射线晶体学单独研究和与几种临床使用的CAS抑制剂进行复杂的X射线晶体学研究，并与野生型Ca II进行研究。此外，将在抑制和体外细胞毒性测定中评估该结构信息，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，这可能导致某些癌症的新治疗方法的发展。 我们还将尝试与CO2（与Sol和Chaeun（Chess）合作使用高压冻结，以捕获野生型和/或APO HCA II中的结合底物。 标本2：人类MN超氧化物歧化酶 将检查锰超氧化物歧化酶（MNSOD）中亚基之间界面的结构，稳定性和催化功能。 MNSOD催化了超氧化物产生O2和H2O2的比例。人类MNSOD是22 kDa亚基的同质二聚体，一个二聚体的二聚体，具有两个结构唯一的接口，一个二聚体和一个四聚体界面。这些界面的催化，稳定性和结构的功能不确定，并且将研究各种突变体MNSOD结构并与动力学和稳定性。由于与野生型相比，由于制备了MNSOD突变形式的MNSOD，例如表现出较少产物抑制作用的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少催化和增强产品抑制。 样品3：HIV-1亚型C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，在每个亚基中用99个氨基酸（标记为1-99和1'-99'）起强制性同型二聚体的作用。它的作用是将GAG和GAG/POL多蛋白分裂为结构和酶促蛋白，并诱导成熟的感染性病毒体的形成。该酶的抑制作用会产生未成熟的HIV病毒体，无法传播感染。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标。将研究未结合/结合的C PR的结构和C PR的各种抗抑制/结合的多药物抗性变体，以识别由于天然发生的多态性而导致的结构变化，并描述了它们对抗逆转录病毒耐药性/易感性的影响。