STRUCTURAL STUDIES OF PROTEINS THAT INHIBIT APOPTOSIS

细胞凋亡是一种正常的生理细胞自杀程序，在脊椎动物和无脊椎动物中高度保守。这种细胞死亡程序在正常发育和组织动态平衡过程中起着关键作用，从生物体中清除不需要的细胞，包括受损和受病毒感染的细胞。细胞凋亡反应的中断与癌症和退行性疾病有关，前者细胞死亡太少，后者细胞死亡太多。与哺乳动物白介素1b转换酶(ICE/caspase-1)和线虫自杀基因产物CED-3相关的半胱氨酸蛋白酶家族caspase在细胞凋亡程序的启动和下游执行中起着核心作用。来自加州链球线虫核型多角体病毒的基因p35，一种杆状病毒，抑制了广泛的半胱氨酸天冬氨酸酶，抑制了细胞凋亡。需要进行结构研究来解决P35与caspase结合的抑制机制与P35-裂解之间的关系。最近，我们已经确定了P35的晶体结构，这是父母资助的第一个具体目标。最近，我们在动物界发现了第一个p35的同源物，slp49，一个来自斜纹夜蛾核型多角体病毒(SINPV)的基因。SLP49序列预测了一个49 kDa的多肽(446个氨基酸)，与P35(299个氨基酸)有48.8%的同源性。P35的第一个同源物可用于突变和功能分析，加上确定P35和SLP49结构的能力，为这项研究提供了框架，旨在确定P35样凋亡抑制因子(或调节因子)中保守的重要基序。这将为通过从其他杆状病毒和生物体中分离出p35类基因来发现无脊椎动物和脊椎动物中是否存在p35类基因家族铺平道路，并为合成具有凋亡抑制/调节活性的新型小肽或类肽分子奠定基础。

Apoptosis is a normal physiological cell suicide program highly conserved among vertebrates and invertebrates. This cell death program plays a critical role during normal development and tissue homeostasis, eliminating unwanted cells from the organism, including damaged and virus-infected cells. Disruptions of the apoptotic response are associated with cancer, where there is too little cell death, and with degenerative diseases, where is too much cell death. A family of cysteine proteases called caspases, related to the mammalian interleukin-1b converting enzyme (ICE/caspase-1) and to CED-3, the product of a suicidal gene from the nematode C. elegans, play a central role in the initiation and downstream execution of the apoptotic program. The gene p35 from the Autographa californica nucleopolyhedrovirus, a baculovirus, inhibits a broad spectrum of caspases, suppressing apoptosis. Structural studies are required to resolve the relationship between the inhibitory mechanism of P35 that involves binding to the caspase and P35-cleavage. Recently we have determined the crystal structure of P35 the first specific aim in the parent grant. Most recently we discovered the first homologue of p35 in the animal kingdom, slp49, a gene from the Spodoptera littoralis nucleopolyhedrovirus (SINPV). The slp49 sequence predicted a 49kDa polypeptide (446 amino acids) with 48.8 percent identity to P35 (of 299 amino acids). The availability of the first homologue of P35 for mutagenesis and functional analysis, together with the capability of determination of the structures of P35 and SLP49 provides the framework for this study that aims to define important motifs conserved in P35-like apoptotic suppressors (or modulators). This will pave the way to find out if a p35-like gene family exists in invertebrates and vertebrates through the isolation of p35-like genes from other baculoviruses and organisms, and establish the backbone to synthesize novel small peptide or peptide-like molecules with apoptotic suppressing/modulating activity.