BmNPV多角体蛋白“操纵”细胞自噬以利于多角体形成的分子机制

Mounting evidence suggests that selective autophagy is mediated by some autophagic adapter proteins. As an autophagic adapter, three features, a direct interaction between the adapter and the autophagosomal marker protein LC3, the inherent ability to polymerize or aggregate as well as to specifically recognize substrates, are required. Previously, the protein polyhedrin encoded by Bombyx mori nucleopolyhedrovirus (BmNPV), was found to be directly interacted and co-localized with LC3 to the envelope of autophagosome, implying the involvement of polyhedrin in cellular autophagy. Further, polyhedrin may function as an autophagic adapter which was testified by the results that polyhedrin bound to the sequestosome 1 (SQSTM1, namely p62), a well-known cellular autophagic adapter that targets ubiquitinated substrates to the autophagosomes by direct binding to LC3 and plays an important role in clearance of protein aggregates, damaged mitochondria, midbody rings, peroxisomes and invading microbes. Moreover, the most striking observations in the present work were that over-expression of p62 led to remarkable decrease in polyhedrin expression and polyhedra production in the nucleus of infected cells, suggesting anti-viral activity of p62 against infection of BmNPV. Besides, the amount of p62 decreased accompanying the progress of BmNPV infection. We inferred that the highly expressed polyhedrin and p62 competitively bound to LC3, thereby enabling p62 to be degraded by cellular pathway rapidly. The aim of this study is to investigate the post-translational modifications of polyhedrin and p62 to bind to LC3, thereby to elucidate the mechanism of polyhedrin to get involved in cellular autophagy to overcome host anti-viral defense systems and to optimize intracellular micro-environment for polyhedra formation.

选择性自噬由“货物”受体蛋白介导。该类蛋白具三个特征：与自噬体标记蛋白LC3互作；能聚集或多聚(aggregate/polymerize)；特异性地识别“货物”。BmNPV多角体蛋白(polyhedrin)具备上述特征，与家蚕LC3共定位于自噬体膜而参与细胞自噬。p62是细胞的一个“货物”受体蛋白。令人感兴趣的是：过表达p62，再感染BmNPV T3，将使多角体蛋白量降低，呈现多角体的细胞数量显著减少，表明p62具抗病毒活性；p62还与polyhedrin结合；随BmNPV继续感染，细胞内p62量降低。推测：持续、高水平表达的polyhedrin与p62竞争结合LC3，使p62通过自噬通路降解，从而优化适于多角体生成的细胞内微环境。本项目以polyhedrin和p62为对象，从蛋白翻译后修饰研究它们与LC3结合的分子基础，阐明polyhedrin“操纵”细胞自噬以利于多角体形成的分子机制。

p62，又名sequestosome-1，是细胞自噬通路上的一个重要受体蛋白之一，参与各种信号通路及细胞自噬的调控。p62的多个功能结构域使之成为细胞多个信号通路的中心环节，能够介导蛋白聚集体、中间体环、可溶性蛋白、过氧化物酶体、受损的线粒体、吞噬的膜残余物、病毒衣壳蛋白、入侵的细菌以及细菌前体等通过细胞自噬降解。本项目对家蚕自噬受体蛋白p62和ref(2)P在BmNPV感染中的生物学功能进行了研究。①家蚕基因组只含有一个p62基因。由于转录起始位点和翻译起始位点的不同，该基因的蛋白产物有两个：一个是p62，另一个被命名为ref(2)P。从氨基酸序列上看，ref(2)P比p62缺少PB1结构域，其余氨基酸序列，两蛋白完全一致，表明p62与ref(2)P的生物学特性有差异，也有共性。②在细胞中过表达家蚕p62后，再感染BmNPV T3，发现polyhedrin表达量降低，呈现多角体的细胞数也显著减少，表明家蚕p62和ref(2)P具有限制或约束BmNPV感染的生物学功能。③p62、ref(2)P限制或约束BmNPV感染归因于它们的3个生物学特性：核质穿梭特性、与polyhedrin结合的特性、在细胞质中形成聚集体的特性。推测p62、ref(2)P能将转运到细胞核内的polyhedrin滞留于细胞质中形成聚集体，一定程度上限制子代多角体的生成。④BmNPV感染细胞后，内源性p62、ref(2)P的表达谱带发生很大变化。除p62、ref(2)P本身大小的蛋白条带外，还观察到分子量增加和减小的条带。分子量增加的条带可能是p62、ref(2)P被修饰的产物，分子量减小的条带可能是p62、ref(2)P被某种蛋白酶切割后产生的多肽。进一步的实验证实ref(2)P在BmNPV感染后被切割，并确定了切割位点。ref(2)P被切割后产生的两个多肽不具有在细胞质内形成聚集体的特性。