PROTEIN MYRISTOYLATION IN S CEREVISIAE AND C NEOFORMANS

n -肉豆蔻酰基转移酶（Nmt）将14碳脂肪酸肉豆蔻酸酯共价连接到新生真核生物和病毒蛋白的n端甘氨酸上。这项资助支持了我们对酿酒葡萄球菌和新生隐球菌中蛋白n -肉豆蔻酰化的酶学和生物学意义的研究。遗传研究证实，NMT对新生芽孢杆菌的生存能力至关重要。我们发现纯化的真菌和人类Nmts具有不同的肽底物特异性，这些差异可用于开发一类具有杀真菌作用的拟肽抑制剂。同源Nmts之间肽底物特异性差异的结构基础需要被定义，以指导设计其他类别更有效的生物活性抑制剂。我们使用x射线晶体学，在2.9埃单位分辨率下，确定了S. cerevisiae Nmt1p与不可水解肉豆蔻酰基辅酶a类似物和拟肽抑制剂的三元配合物的结构。我们的具体目标是比较酿酒葡萄球菌、新生葡萄球菌和人类Nmts与结合肽底物的三元配合物的结构，并通过定点诱变测试结构/活性关系。使真菌病原体在静止期存活的因素尚不清楚，可能对发病机制有重要影响。以酿酒酵母为模型，我们发现n -肉豆蔻酰化蛋白的缺陷损害了静止期的存活，并加速了衰老。在一个野生型菌株中缺失了48个编码已知或推测的Nmt1p底物的基因，揭示了饥饿敏感性和快速衰老可以通过去除Sip2p来重现。Sip2p是一种与激酶（Snf1p）相关的n -肉豆荚酰基蛋白，参与调节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 -肉豆蔻酰基蛋白调节对营养剥夺和衰老的抵抗力的机制。sipp2通路将在酿酒酵母中进行遗传剖析。一种表达克隆策略将用于鉴定能够补充酿酒葡萄球菌中sip2delta产生的固定期（和其他）表型的新形态葡萄球菌的cdna。新形态C. SIP2的同源基因将被恢复，并产生一个空等位基因。基因缺失对新生生物抵御营养剥夺能力的影响将在培养和体内进行研究。这些研究可能为限制真菌病原体在营养匮乏的寄主隔间中生存的能力提供治疗靶点。它们还应该提供适用于其他生物体的抗营养剥夺和衰老之间关系的分子见解。

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.