Role of Interactions between Autographa californica Multiple Nucleopolyhedrovirus Procathepsin and Chitinase Chitin-Binding or Active-Site Domains in Viral Cathepsin Processing
Role of Interactions between Autographa californica Multiple Nucleopolyhedrovirus Procathepsin and Chitinase Chitin-Binding or Active-Site Domains in Viral Cathepsin Processing
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DOI：
10.1128/jvi.01937-12
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发表时间：
2013-03-01
影响因子：
5.4
通讯作者：
Krell, Peter J.
中科院分区：
文献类型：
--
作者：
Hodgson, Jeffrey J.;Arif, Basil M.;Krell, Peter J.
多核多角体病毒几丁质酶(ChIA)与病毒组织蛋白酶前体(Prov-Cath)的结合调控细胞/内质网(ER)共滞留ChIA和Prov-Cath，从而在宿主细胞死亡时协调两种宿主组织降解酶的同时释放。Chia是一种被建议的Prov-Cath折叠伴侣，因为Chia的插入失活会导致Prov-Cath聚集体的产生，而这些聚集体不能将蛋白质分解成熟为活性的V-Cath酶。我们想要确定Chia的N-末端几丁质结合结构域(CBD，149个残基)和C-末端Chia活性部位结构域(ASD，402个残基)是否独立地与Prov-Cath结合，以及这两个结构域是否对Chia推测的Prov-Cath折叠伴侣活性是必需的。我们证明了N-末端绿色荧光蛋白(GFP)融合的CHIA、ASD和CBD都以类似ER的模式与Prov-Cath-RFP共存，并且ASD和CBD在体内使用双分子荧光互补(BIFC)和体外使用镍组氨酸下拉实验独立地与Prov-Cath结合。总之，来自共定位、BIFC和互惠互通化分析的数据表明，Prov-Cath和CHIA的两个结构域之间存在特定和独立的相互作用。这些数据也表明，对于正常的Prov-Cath处理，任何一个Chia结构域都是必不可少的。此外，与先前的证据表明缺乏chia表达导致Prov-Cath聚集、不能溶解并且不能成熟为V-Cath相反，我们设计的仅表达v-cath的chia缺失杆状病毒产生了可溶性的Prov-Cath，它过早地从细胞中分泌出来，并蛋白水解性成熟为活性的V-Cath酶。
The binding of Autographa californica multiple nucleopolyhedrovirus chitinase (CHIA) to viral cathepsin protease progenitor (proV-CATH) governs cellular/endoplasmic reticulum (ER) coretention of CHIA and proV-CATH, thus coordinating simultaneous cellular release of both host tissue-degrading enzymes upon host cell death. CHIA is a proposed proV-CATH folding chaperone because insertional inactivation of chiA causes production of proV-CATH aggregates that are incompetent for proteolytic maturation into active V-CATH enzyme. We wanted to determine whether the N-terminal chitin-binding domain (CBD, 149 residues) and C-terminal CHIA active-site domain (ASD, 402 residues) of CHIA bind to proV-CATH independently of one another and whether either domain is dispensable for CHIA's putative proV-CATH folding chaperone activity. We demonstrate that N-terminally green fluorescent protein (GFP)-fused CHIA, ASD, and CBD each colocalize with proV-CATH-RFP in ER-like patterns and that both ASD and CBD independently associate with proV-CATH in vivo using bimolecular fluorescence complementation (BiFC) and in vitro using reciprocal nickel-histidine pulldown assays. Altogether, the data from colocalization, BiFC, and reciprocal copurification analyses suggest specific and independent interactions between proV-CATH and both domains of CHIA. These data also demonstrate that either CHIA domain is dispensable for normal proV-CATH processing. Furthermore, in contrast to prior evidence suggesting that a lack of chiA expression causes proV-CATH to become aggregated, insoluble, and unable to mature into V-CATH, a chiA deletion bacmid virus we engineered to express just v-cath produced soluble proV-CATH that was prematurely secreted from cells and proteolytically matured into active V-CATH enzyme.