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稳态中起着关键作用，从生物体中清除不需要的细胞，包括受损和病毒感染的细胞。细胞凋亡反应的中断与癌症（细胞死亡太少）和退行性疾病（细胞死亡太多）有关。半胱氨酸蛋白酶家族被称为caspase，与哺乳动物白细胞介素1b转换酶（ICE/caspase-1）和CED-3有关，CED-3是线虫自杀基因的产物，在细胞凋亡程序的启动和下游执行中发挥核心作用。来自加利福尼亚亲笔核多角体病毒（杆状病毒）的基因p35抑制广泛的半胱天蛋白酶，抑制细胞凋亡。需要通过结构研究来解决P35与caspase结合的抑制机制与P35切割之间的关系。最近我们确定了P35的晶体结构，这是第一个明确的目标。最近，我们在动物王国中发现了p35的第一个同源基因slp49，这是一个来自沿海夜蛾核多角体病毒（SINPV）的基因。slp49序列预测了一个49kDa多肽（446个氨基酸），与P35（299个氨基酸）的同源性为48.8%。P35的第一个同源物的突变和功能分析的可用性，以及P35和SLP49结构的测定能力，为本研究提供了框架，旨在确定P35样凋亡抑制因子（或调节剂）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样基因来发现无脊椎动物和脊椎动物中是否存在p35样基因家族铺平道路，并为合成具有凋亡抑制/调节活性的新型小肽或肽样分子建立骨架。

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.