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: 8171501
• 负责人: ROBERT MCKENNA
• 金额: $ 9.52万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171501
• 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

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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 样品1：碳酸酐酶碳酸酐酶（CA）是锌 - 金糖酶，可催化CO2和HCO3-的可逆反转化。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。该CAIX模拟物将使用X射线晶体学单独使用，并与几种临床使用的CAS抑制剂进行复杂化，并与野生型Ca II进行比较。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。 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 catalyst of the interfaces between subunits in manganese superoxide dismutase (MnSOD) will be examined. MnSOD catalyzes the超氧化物对人类的o2和H2O2的比例是22 kDa亚基的同质体，这是两个二聚体的二聚体，一个结构上独特的接口，一个二聚体和一个四聚体界面在催化剂，稳定性和稳定性中的结构MNSOD的突变形式是作为可能的抗增殖剂制备的，例如与野生型相比表现出更少的产品抑制作用的H30N MNSOD，应该预期，二聚体界面处其他残留物的改变可能会减少催化和增强产物抑制。样品3：HIV-1子型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，在每个亚基中具有99个氨基酸的强制性同型二聚体（标记为1-99和1。它的作用是将GAG和GAG/POL多蛋白清除到结构和酶促蛋白中，并诱导成熟的，传染病的形成。这种酶的抑制会产生未成熟的HIV病毒，无法传播感染。由于其在获得病毒感染中的重要作用，HIV-1 PR被认为是发现新的和潜在的抗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。