Biochemical Characterization of Cytochrome b5 Reductase in Candida albicans

描述(申请人提供)：白色念珠菌是人类微生物区系的共生成员，是最常见的真菌相关疾病的病原体，在免疫功能受损的艾滋病毒疾病/艾滋病患者中具有特别重要的意义，从轻微的临床表现，如口腔鹅口疮到更严重的播散性念珠菌病。新出现的耐药性是假丝酵母菌的一个主要问题，其原因是参与外源物质和药理学试剂解毒的酶。其中，细胞色素b5还原酶(Cb5r)具有很高的药理学意义，因为它在脂肪酸延长、麦角甾醇(或哺乳动物中的胆固醇)生物合成以及细胞色素P450介导的外源生物解毒中发挥作用。本项目的目标是鉴定念珠菌cb5r及其电子受体细胞色素b5(Cb5)，作为潜在的新药物靶点。因此，我们从白色念珠菌中克隆了cb5r和cb5，并对重组蛋白在大肠杆菌中的表达进行了优化。特异的AIM 1将表征假丝酵母菌cb5r的生化、动力学和药理特性，以检验真菌酶不同于哺乳动物酶的假设。特异性AIM 2将评估两种cb5R亚型CBR1和MCR1在白念珠菌和酵母模型系统酿酒酵母中对pH、热休克和氧化应激反应的细胞生长和活力的敲除表型。这将使我能够检验这一假设，即cb5r基因敲除会影响对各种环境压力的敏感性。特定的AIM 3将利用蛋白质下拉试验和蛋白质组学分析来鉴定与白念珠菌cb5r和cb5相互作用的细胞质和/或膜结合蛋白。因此，在最终的特定目标中，我将检验假丝酵母菌cb5r除了cb5之外还与其他细胞蛋白相互作用的假设。系统发育分析表明，念珠菌的cb5r和cb5与植物的同源性最高，而与人类cb5r和cb5的同源性较低。有趣的是，植物cb5r已被确定为一种新的除草靶标，这使得这些植物抑制剂有望成为一类新的抗真菌药物。这项提案将描述念珠菌的一种新的药物靶标，念珠菌是一种机会真菌，会影响免疫受损的人，如艾滋病患者。因此，识别新的药物靶点对于对抗机会性真菌病原体的多重耐药具有特别重要的意义。与公共健康相关：我的总体长期目标是从事分子微生物学、传染病和生物医学研究。我以前在这些领域有过经验，我知道留在研究领域是我的激情和渴望。我发现特别令人兴奋的是，这个项目将传染病与生物化学和蛋白质组学结合在一起，总体目标是为一种重要的人类病原体确定新的药物靶点。这项建议中的研究培训计划使我能够学习生物化学和蛋白质组学方面的新的尖端方法，同时也加强了我作为分子微生物学家的培训。为了实现这些目标，这个项目将增进我对实验设计的了解，并伴随着文献回顾。它将进一步提高我在分子方法方面的技能，例如定向基因破坏(基因敲除)和重组蛋白表达。我还将学习生物化学和蛋白质组学中使用的新技术，如质谱学、蛋白质下拉分析和其他涉及蛋白质表征的方法。

DESCRIPTION (provided by applicant): Candida albicans is a commensal member of the human microflora and is the most common causative agent of fungal-related disease with particular significance in immuno-compromised individuals with HIV disease/AIDS, ranging from mild clinical manifestations such as oral thrush to a more serious disseminated candidiasis. Emerging drug resistance is a major problem in Candida, contributed by enzymes involved in the detoxification of xenobiotics and pharmacological agents. One such enzyme, cytochrome b5 reductase (cb5r), has a high pharmacological significance owing to its role in fatty acid elongation, ergosterol (or cholesterol in mammals) biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics. The objective of this project is to characterize Candida cb5r and its electron acceptor, cytochrome b5 (cb5), as potential novel drug targets. Hence we have cloned cb5r and cb5 from C. albicans and have optimized recombinant protein expression in E. coli. SPECIFIC AIM 1 will characterize the biochemical, kinetic, and pharmacological properties of Candida cb5r in order to test the hypothesis that the fungal enzyme differs from its mammalian counterpart. SPECIFIC AIM 2 will assess the knockout phenotype of the two cb5r isoforms, CBR1 and MCR1, on cell growth and viability in response to pH, heat shock, and oxidative stress in Candida albicans and the yeast model system, Saccharomyces cerevisiae. This will allow me to test the hypothesis that the cb5r knockouts affect susceptibility to various environmental stresses. SPECIFIC AIM 3 will utilize protein pull-down assays and proteomic analysis to identify cytoplasmic and/or membrane-bound proteins that interact with C. albicans cb5r and cb5. Hence, in the final specific aim I will test the hypothesis that Candida cb5r interacts with other cellular proteins in addition to cb5. Phylogenetic analysis revealed that both cb5r and cb5 from Candida have highest homology to their plant counterparts, but lower homology to human cb5r and cb5. Interestingly, plant cb5r has been identified as a novel herbicidal target, making these plant inhibitors promising candidates as a new class of antifungals against Candida cb5r. This proposal will characterize a novel drug target in Candida, an opportunistic fungus that affects immuno-compromised individuals such as AIDS patients. Hence, identification of new drug targets has particular significance to combat multiple drug resistance in opportunistic fungal pathogens. PUBLIC HEALTH RELEVANCE: My overall long-term goal is to pursue a career in molecular microbiology, infectious disease, and biomedical research. I have had previous experience in these fields and know it is my passion and desire to remain in research. I find it particularly exciting that this project merges infectious disease with biochemistry and proteomics, with the overall goal of identifying novel drug targets for an important human pathogen. The research training plan in this proposal allows me to learn new cutting edge methods in biochemistry and proteomics, while also enhancing my training as a molecular microbiologist. To accomplish these goals, this project will advance my knowledge of experimental design and accompanying literature review. It will furthermore improve my skills in molecular methods, such as targeted gene disruption (gene knockout) and recombinant protein expression. I will also learn new techniques used in biochemistry and proteomics, such as mass spectrometry, protein pull-down assays, and other methods involved in the characterization of proteins.