PROTEIN MYRISTOYLATION IN S CEREVISIAE AND C NEOFORMANS

酿酒酵母和新生酵母中的蛋白质肉豆蔻酰化

• 批准号: 6510598
• 负责人: JEFFREY I GORDON
• 金额: $ 30.8万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6510598
• 关键词: Cryptococcus neoformans; Saccharomyces cerevisiae; X ray crystallography; acyltransferase; aging; enzyme complex; enzyme mechanism; enzyme structure; expression cloning; fatty acylation; fungal genetics; gene mutation; laboratory mouse; laboratory rabbit; myristates; nutrient bioavailability; site directed mutagenesis

N-myristoyltransferase (Nmt) covalently links the 14 carbon fatty acid, myristate, to the N-terminal glycine of nascent eukaryotic and viral proteins. This grant has supported our efforts to examine the enzymology and biological significance of protein N-myristoylation in S. cerevisiae and Cryptococcus neoformans. Genetic studies established that NMT is essential for the viability of C. neoformans. We found that purified fungal and human Nmts have divergent peptide substrate specificities and that these differences can be used to develop a class of peptidomimetic inhibitors that are fungicidal. The structural basis for the differences in peptide substrate specificities between orthologous Nmts needs to be defined to guide design of additional classes of more potent, biologically active inhibitors. We have used X-ray crystallography to determine, at 2.9 Angstrom units resolution, the structure of a ternary complex of S. cerevisiae Nmt1p with a nonhydrolyzable myristoylCoA analog and peptidomimetic inhibitor. Our specific aim 1 will be to compare the structures of ternary complexes of S. cerevisiae, C. neoformans and human Nmts with bound peptide substrates and to test structure/activity relationships by site-directed mutagenesis. The factors that allow fungal pathogens to survive during stationary phase are poorly understood and may have an important impact on pathogenesis. Using S. cerevisiae as a model, we found that defects in protein N-myristoylation impair survival during stationary phase and also accelerate aging. Deletion of 48 genes encoding known or putative Nmt1p substrates in a wild type strain disclosed that starvation sensitivity and rapid aging can be recapitulated by removing Sip2p, a N-myristoylprotein associated with a kinase (Snf1p) involved in regulating global cellular responses to glucose starvation. Our specific aim 2 will be to characterize the mechanisms by which N-myristoylproteins regulate resistance to nutrient deprivation and aging. The Sip2p pathway will be dissected genetically in S. cerevisiae. An expression cloning strategy will be used to identify C. neoformans cDNAs that can complement the stationary phase (and other) phenotypes produced by sip2delta in S. cerevisiae. The C. neoformans ortholog of SIP2 will be recovered and a null allele generated. The impact of the gene deletion on C. neoformans' ability to withstand periods of nutrient deprivation will be examined in culture and in vivo. These studies may yield therapeutic targets for limiting the ability of fungal pathogens to survive in host compartments where nutrients are scarce. They should also provide molecular insights about the relationship between resistance to nutrient deprivation and aging that are applicable to other organisms.

N-肉豆蔻酰转移酶（Nmt）将十四碳脂肪酸肉豆蔻酸酯共价连接至新生真核和病毒蛋白的N-末端甘氨酸。 该基金支持了我们的工作，研究蛋白质N-豆蔻酰化在S。酿酒酵母和新型隐球菌。遗传学研究表明，NMT对C.新人类 我们发现，纯化的真菌和人类NMT具有不同的肽底物特异性，这些差异可用于开发一类杀真菌的拟肽抑制剂。 需要定义邻位Nmts之间肽底物特异性差异的结构基础，以指导设计其他类别的更有效的生物活性抑制剂。 我们用X射线晶体学方法测定了S的三元配合物的结构，分辨率为2.9埃单位。酿酒酵母Nmt 1 p与不可水解的肉豆蔻酰CoA类似物和拟肽抑制剂。 我们的具体目标1将是比较S的三元配合物的结构。酿酒酵母C.新型人和人Nmt与结合的肽底物，并通过定点诱变测试结构/活性关系。 使真菌病原体在稳定期存活的因素知之甚少，可能对发病机制产生重要影响。 利用S.作为一个模型，我们发现蛋白N-豆蔻酰化的缺陷损害在稳定期的生存，也加速老化。在野生型菌株中缺失48个编码已知或推定的Nmt 1 p底物的基因，揭示了饥饿敏感性和快速衰老可以通过去除Sip 2 p来重现，Sip 2 p是一种与激酶（Snf 1 p）相关的N-肉豆蔻酰蛋白，参与调节对葡萄糖饥饿的整体细胞反应。 我们的具体目标2将是表征N-肉豆蔻酰蛋白调节对营养剥夺和衰老的抵抗力的机制。 Sip 2 p途径将在S.啤酒。 表达克隆策略将用于鉴定C。可以补充由S. neoformans中sip 2 δ产生的稳定期（和其它）表型的cDNA。啤酒。 梭将回收SIP 2的neoformans直系同源物并产生无效等位基因。 研究了该基因缺失对C.将在培养物和体内检查新生动物耐受营养缺乏时期的能力。 这些研究可能会产生限制真菌病原体在营养缺乏的宿主区室中生存的能力的治疗靶点。 他们还应该提供适用于其他生物体的关于营养剥夺抵抗力和衰老之间关系的分子见解。