A Putative Cell Surface Receptor for White Spot Syndrome Virus Is a Member of a Transporter Superfamily

White spot syndrome virus (WSSV), a large enveloped DNA virus, can cause the most serious viral disease in shrimp and has a wide host range among crustaceans. In this study, we identified a surface protein, named glucose transporter 1 (Glut1), which could also interact with WSSV envelope protein, VP53A. Sequence analysis revealed that Glut1 is a member of a large superfamily of transporters and that it is most closely related to evolutionary branches of this superfamily, branches that function to transport this sugar. Tissue tropism analysis showed that Glut1 was constitutive and highly expressed in almost all organs. Glut1's localization in shrimp cells was further verified and so was its interaction with Penaeus monodon chitin-binding protein (PmCBP), which was itself identified to interact with an envelope protein complex formed by 11 WSSV envelope proteins. In vitro and in vivo neutralization experiments using synthetic peptide contained WSSV binding domain (WBD) showed that the WBD peptide could inhibit WSSV infection in primary cultured hemocytes and delay the mortality in shrimps challenged with WSSV. These findings have important implications for our understanding of WSSV entry

Comparative analysis of differentially expressed genes in normal and white spot syndrome virus infected Penaeus monodon

10.1186/1471-2164-8-120BMC Genomics8-BGME

Cloning and characterization of apoptosis-related proteins from Penaeus monodon: Inhibitor of apoptosis protein (IAP) and caspase

經由線蟲、果蠅和哺乳動物的研究，發現參與細胞凋亡保守性途徑的蛋白質主要包括：BCL-2蛋白家族, APAF-1同源蛋白和硫胱氨酸蛋白酶 (caspases)。但與其他重要的訊息傳遞途徑相似，愈高等的動物擁有越多的相關蛋白質和更複雜的網絡來控制細胞凋亡的進行。 硫胱氨酸蛋白酶(caspases)是一群在演化上具高保守性的細胞內半胱氨酸蛋白酶 (intracellular cysteine protease)，是細胞凋亡的執行者。這些蛋白質會經由一連串的蛋白水解作用而被活化。Caspases分成啟動者(initiator)和作用者(effector) 兩類；啟動者caspases接收細胞凋亡訊號而被活化，接著再活化下游的作用者caspases。活化的作用者caspases將各種不同的細胞蛋白質水解，引發細胞在型態和生化特性上的一連串變化，最後造成細胞的凋亡。 果蠅和哺乳動物的細胞凋亡抑制蛋白質 (inhibitor of apoptosis protein, IAP) 能與活化的caspases結合、抑制其活性。IAP 的特徵是在N端具有一或數個的BIR (baculoviral IAP repeat) domains，此domain是與caspases 相結合並抑制其活性的結構單位。而IAP抑制caspase的作用又受到另一類蛋白質的調控，這一類的蛋白質稱為 IBM (IAP binding motif)-containing或 IBM蛋白質。這些蛋白質雖然沒有任何演化上的關聯性或氨基酸序列上的相似性，它們的N端都具有一段保守的tetrapeptide motif。藉由這個motif，IBM蛋白質會與IAP 的BIR domain 結合，使得IAP無法結合並抑制caspases，因此這些蛋白質的大量表現會促使細胞凋亡。果蠅的IBM蛋白質，包括Rpr，Hid，Grim和Sickle，在果蠅的胚胎發生和發育上扮演重要的角色。 本論文針對選殖出之兩個與細胞凋亡有關的草蝦 (Penaeus monodon) 基因，PmIAP 和 Pm caspase，進行其蛋白質特性和功能之研究。氨基酸序列分析顯示PmIAP有三個BIR domains和一個RING domain，在整個結構上和果蠅DIAP2、人類cIAP1、cIAP2 和 XIAP相似；而Pm caspase和昆蟲的effector caspases的相似性高達百分之五十七。我的研究顯示在昆蟲細胞SF9中，PmIAP會和果蠅的Rpr結合，進而抑制Rpr所誘發的細胞凋亡現象。藉由刪除分析，我發現PmIAP 的BIR2和BIR3 domains 是結合並抑制Rpr活性的最小區域，雖然BIR1本身不會和Rpr結合並抑制其活性，但能促進PmIAP對Rpr的作用。大量表現的Pm caspase會誘發昆蟲細胞SF9進行細胞凋亡；進一步的分析發現雖然PmIAP 和蝦白點症病毒的抑制細胞凋亡蛋白質WSSV449都能與Pm caspase結合，但只有WSSV449能完全抑制Pm caspase所誘發的昆蟲細胞凋亡現象，PmIAP僅有些微抑制的作用。桿狀病毒P35蛋白質能抑制多種caspases的活性，其作用機制已很清楚；而本研究顯示WSSV449對Pm caspase 的作用機制與P35的相似。Our current understanding of apoptosis mainly comes from the studies of C. elegans, Drosophila and mammals. These studies have showed that this death program is evolutionarily conserved and that the participating core molecules in this conserved pathway are BCL-2 family proteins, APAF-1 homologues, and caspases. However, just like other signaling pathways or other regulatory networks, higher animals use more related proteins and have more complicated networks to regulate the apoptosis. Caspases, a family of evolutionarily conserved intracellular cysteine proteases, are the executioners of apoptosis. They are activated through proteolytic cleavage cascades during apoptosis. These proteases are classified into either initiator or effector caspases. The initiator caspases receive and are activated by apoptotic signals. The activated initiators then activate downstream effector caspases, which subsequently digest vast arrays of cellular proteins, leading to the morphological and biochemical changes of apoptotic cells and ultimately to the demise of apoptotic cells. In Drosophila and mammals, the activated caspases can be inhibited by members of inhibitor of apoptosis protein (IAP) family through direct interaction. The hallmark of IAP is the presence of one or more copies of baculoviral IAP repeat (BIR) domain at N-termini. The BIR domain is the structural unit for inhibiting caspase activity through direct binding. However, the caspase-inhibitory property of IAPs is in turn antagonized by another groups of proteins, the IAP binding motif (IBM)-containing proteins. Although these proteins show no relationship and sequence conservation to each other, their N-termini do share a conserved tetrapeptide motif, which is responsible for binding to BIR domain and releasing the suppression on caspases. These IBM-containing proteins are pro-apoptotic and when they were overexpressed in insect and mammalian cells, apoptosis is induced. The IBM-containing proteins, Rpr, Hid, Grim and Sickle, play very important roles in Drosophila embryogenesis and development. In this PhD thesis study, two apoptosis-related genes were cloned from Penaeus monodon, IAP and caspase, and the corresponding proteins, PmIAP and Pm caspase, were further characterized. PmIAP has 3 BIR domains and a RING domain, and is structurally similar to Drosophila IAP2, mammalian c-IAP1, c-IAP2 and XIAP. The Pm caspase cloned in this thesis is highly similar to insect effector caspase. My studies revealed that PmIAP could block Rpr-induced apoptosis in SF9 cells through direct interaction. Deletion analysis revealed that PmIAP BIR2 and BIR3 domains were the minimal regions to interact with Rpr and to block the Rpr-induced apoptosis, and that although BIR1 domain does not bind to and inhibit Rpr’s activity, it seems to be able to enhance PmIAP’s inhibition to Rpr. The expression of Pm caspase induced apoptosis in SF9 cells. This apoptosis could be completely blocked by the anti-apoptosis protein WSSV449 encoded by white spot syndrome virus, but was only minimally inhibited by PmIAP, even though both WSSV449 and PmIAP could all bind to Pm caspase. Baculovirus P35 is a well-understood pan caspases inhibitor, and further studies showed that WSSV449 blocked that activity of Pm caspase through a mechanism similar to that used by P35.口試委員會審定書………………………………………….i 誌謝…………………………………………………………ii 中文摘要…………….……………………………………iii Abstract……………….……………………….……...…v Chapter 1: Introduction 1.Overview of apoptosis…………………………..…..1 2.Apoptosis in C. elegans: The conserved core apoptosis regulators………….2 3.Apoptosis in mammals: increased complexity with intrinsic and extrinsic pathways………………………..…..3 4. Apoptosis in Drosophila: neither worm nor human……………….5 5. Inhibitor of apoptosis protein (IAP): inhibitors of caspases……..…………6 6. Caspases: the executioners of apoptosis………………………………….…10 7. Apoptosis in shrimp: induced by viral pathogens…………………….……11 8. References……………………………….……12 Chapter 2: Molecular cloning and characterization of an inhibitor of apoptosis protein (IAP) from the tiger shrimp, Penaeus monodon Abstract…………………………………….………19 1. Introduction…………… …………………….20 2. Materials and methods ……………….…..22 3 Results……………………………….………..31 4. Discussion………………………………..……40 5. References……………………………….……45 6. Tables………………………………………….…51 7. Figures and legends……………………….….53 Chapter 3: Penaeus monodon caspase is the target of the white spot syndrome virus anti-apoptosis protein WSSV449 Abstract……………..………………………….……...66 1. Introduction……………………………….……..…67 2. Materials and methods ……………………...….69 3. Results…………………………………….…….….77 4. Discussion……………………………………..……84 5 References………………………………..…….……89 6. Tables……………………………………….………94 7. Figures and legends………………….……………95 Chapter 4: Discussion…………………..……………104 References…………………………………………..…11