Effects of immunostimulants targeting Ran GTPase on phagocytosis against virus infection in shrimp

The global shrimp aquaculture has been consistently beset by diseases that cause severe losses in production. To fight various harmful pathogens, the enhanced shrimp immunity by immunostimulants would play key roles against the invading pathogens. In aquaculture, however, the target proteins/genes which can be used for the screening of immunostimulants are very limited. Based on our previous study, in the present study, the shrimp Ran protein, which was required in shrimp antiviral phagocytosis, was used as the target protein to screen for immunostimulants. The GTPase activity assays showed that the IL-4 and lysophosphatidylcholine molecules could enhance the activity of Ran protein, suggesting that the two molecules might function in phagocytosis. When the IL-4 and lysophosphatidylcholine were respectively injected into shrimp, the results indicated that the two molecules enhanced the hemocytic phagocytosis against white spot syndrome virus (WSSV), suggesting that they improved the activity of phagocytosis through the activation of the Ran protein. It was evidenced that the enhancement of phagocytosis activity effectively inhibited the WSSV infection in shrimp, which further led to the decrease of mortalities of WSSV-infected shrimp. Therefore, our study presented a novel strategy for the screening of immunostimulants by using the key proteins in immune responses of aquatic organisms as the target proteins, which would be very helpful for the development of efficient approaches to prevent the aquatic organisms from pathogen infections.     

Contribution of the caspase gene sequence diversification to the specifically antiviral defense in invertebrate

Vertebrates achieve adaptive immunity of all sorts against pathogens through the diversification of antibodies. However the mechanism of invertebrates' innate immune defense against various pathogens remains largely unknown. Our study used shrimp and white spot syndrome virus (WSSV) to show that PjCaspase, a caspase gene of shrimp that is crucial in apoptosis, possessed gene sequence diversity. At present, the role of gene sequence diversity in immunity has not been characterized. To address this issue, we compared the PjCaspase gene sequence diversities from WSSV-free and WSSV-resistant shrimp. The sequence analysis indicated that the PjCaspase gene from the WSSV-resistant shrimp contained a special fragment, designated as fragment 3 (221-229 aa). Down-regulation or overexpression of the PjCaspase gene containing fragment 3 led to significant inhibition or enhancement of virus-induced apoptosis, but had no effect on bacterium challenge. We found evidence that the silencing or overexpression of this gene led to a 7-fold increase or 11-fold decrease of WSSV copies, respectively. Our results suggested that the PjCaspase gene containing fragment 3 provided the molecular basis for the antiviral defense of shrimp. This study represented the first report of the role of gene sequence diversity in the immunity of an invertebrate against virus infection. Invertebrates may employ this gene sequence diversity as a system to avoid pathogen interference with their immune response.     

Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins

A hallmark of many neurodegenerative diseases is accumulation of misfolded proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving protein disulfide isomerase (PDI), a protein chaperone that catalyzes isomerization, reduction and oxidation of disulfides. Through a small molecule screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated ER membranes and trigger apoptotic cell death via mitochondrial outer-membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon 1 and Aβ peptides processed from the amyloid precursor protein. This pro-apoptotic function of PDI represents a new mechanism linking protein misfolding and apoptotic cell death.     

Viral infections of aquatic animals with special reference to Asian aquaculture

Worldwide, the number of communicable diseases of animals raised in aquaculture continue to increase. Viral infections of cultivated shellfish, crustacea, and finfish have been frequently recognized in the past few years. In the Asian regions, penaeid shrimp and several teleost fish underwent epizootics associated with heavy losses in aquaculture. Baculoviruses are particularly harmful to shrimp and prawns. Herpes-, irido-, reo-, or rhabdovirus-like agents can cause outbreaks in fish farms. Viral diseases are important limiting factors in the expansion of aquaculture. However, studies on viral infections of aquatic animals have been focused primarily on economically important farmed fish. Therfore, certain viral diseases of teleost fish are relatively well understood. In contrast, our knowledge of viral infections of farmed aquatic invertebrates is still very spare. Although a great number of viruses have been detected in farmed molluscs and crustaceans, the pathogenicity and epizootiology of most of the agents is not known.     

Pattern recognition receptors acting in innate immune system of shrimp against pathogen infections

Invertebrates, including shrimp, have developed very complicated innate immune system against pathogens. Much work has been performed on the innate immunity of shrimp, including immune recognition, signal transduction, effector molecules and antiviral responses due to its great economic value. Pattern recognition is the first step of innate immunity. Pattern recognition receptors (PRRs) sense the presence of infection and activate immune responses. The studies on shrimp PRRs revealed the recognition mechanism of shrimp at a certain degree. To date, 11 types of pattern recognition receptors (PRRs) have been identified in shrimp, namely, β-1,3-glucanase-related proteins, β-1,3-glucan-binding proteins, C-type lectins, scavenger receptors, galectins, fibrinogen-related proteins, thioester-containing protein, Down syndrome cell adhesion molecule, serine protease homologs, trans-activation response RNA-binding protein and Toll like receptors. A number of PRRs have been functionally studied and have been found to have different binding specificities and immune functions. The present review aims to summarize the current knowledge on the PRRs of shrimp.     

几种热激蛋白在细胞凋亡信号通路中的调控作用

热激蛋白(heat shock proteins, HSPs)作为进化保守的蛋白家族 之一,普遍存在于各种生物体中,并在生物体内发挥着重要的生理功能.大 量的实验证据表明,热激蛋白与细胞凋亡密切相关,参与细胞凋亡信号通 路的多个环节. 近年来有关该领域的研究已获得了重要的突破与进展.一方 面,热激蛋白主要起着抑制细胞凋亡、促进细胞存活的作用;另一方面, 某些热激蛋白又能够作为凋亡蛋白的分子伴侣,促进细胞凋亡,比如HSP70 能够激活DNase来促使细胞凋亡,线粒体内HSP60能够促进caspase依赖的细 胞凋亡途径.本文在阐明细胞凋亡信号通路的基础上,综述了近年来几种不 同热激蛋白家族（HSP90、 HSP70 、HSP60和小分子HSPs）在细胞凋亡调控 中作用的研究进展,重点阐述了几种主要热激蛋白与细胞凋亡信号通路上 相关因子的相互作用,并绘制了热激蛋白在细胞凋亡信号通路中的调控图 ,为进一步完善细胞凋亡调控网络研究提供一定的参考.     

Chemical genetics: tailoring tools for cell biology

Chemical genetics is a research approach that uses small molecules as probes to study protein functions in cells or whole organisms. Here, I review the parallels between classical genetic and chemical-genetic approaches and discuss the merits of small molecules to dissect dynamic cellular processes. I then consider the pros and cons of different screening approaches and specify strategies aimed at identifying and validating cellular target proteins. Finally, I highlight the impact of chemical genetics on our current understanding of cell biology and its potential for the future.     

Immunostimulatory nature of beta-glucans and baker's yeast in gnotobiotic Artemia challenge tests

The use of new preventive approaches such as immunostimulants to reduce stress and mortalities, to maintain good health of cultured organisms and to stimulate the non-specific defence mechanism, is becoming increasingly important in aquaculture. Yet detailed analysis reveals that in most experiments the validity of some conclusions with respect to the benefit of immunostimulation is still doubtful, especially in invertebrates. The use of standardized trials under controlled rearing conditions, complemented with fundamental research on defence mechanisms can provide unequivocal evidence for the beneficial effects of immunostimulants in reducing invertebrate susceptibility to diseases or infections. This study investigated the use of small amounts of baker's yeast Saccharomyces cerevisiae and glucan particles (obtained from baker's yeast) in gnotobiotic Artemia to overcome the pathogenicity of two organisms: Vibrio campbellii and V proteolyticus. Artemia supplemented with small quantities of a yeast strain presenting higher concentrations of beta-glucans or with glucan particles seemed to completely resist the detrimental effects of both pathogens. The higher amount and/or availability of beta-glucans in that yeast might play an essential role in such protection, as most probably glucans stimulate the immune response of the nauplii.     

The use of probiotics in aquaculture

This study aims to present comprehensive notes for the use of probiotics in aquaculture. Probiotics have been proven to be positive promoters of aquatic animal growth, survival and health. In aquaculture, intestines, gills, the skin mucus of aquatic animals, and habitats or even culture collections and commercial products, can be sources for acquiring appropriate probiotics, which have been identified as bacteria (Gram‐positive and Gram‐negative) and nonbacteria (bacteriophages, microalgae and yeasts). While a bacterium is a pathogen to one aquatic animal, it can bring benefits to another fish species; a screening process plays a significant role in making a probiotic species specific. The administration of probiotics varies from oral/water routine to feed additives, of which the latter is commonly used in aquaculture. Probiotic applications can be either mono or multiple strains, or even in combination with prebiotic, immunostimulants such as synbiotics and synbiotism, and in live or dead forms. Encapsulating probiotics with live feed is a suitable approach to convey probiotics to aquatic animals. Dosage and duration of time are significant factors in providing desired results. Several modes of actions of probiotics are presented, while some others are not fully understood. Suggestions for further studies on the effects of probiotics in aquaculture are proposed.     

Identification of a type of human IgG-like protein in shrimp Penaeus vannamei by mass spectrometry

Recently, infectious diseases have delayed the growth of shrimp aquaculture. Interest has been focused on immune molecules and defense mechanisms to reduce these diseases in shrimp aquaculture. In invertebrates, various immunoglobulin superfamily (IgSF) molecules have been characterized in body tissues and fluids, which play a significant role in innate defense. In the current study, we found that a protein in shrimp serum, referred as an IgG-like protein, could be reacted with goat anti-human IgG, specifically. The IgG-like protein was purified from the serum of shrimp Penaeus vannamei by affinity chromatography using CNBr-activated sepharose 4B. The purified protein was subsequently analysed using one-dimensional sodium sulphate-polyacrylamide gel electrophoresis (1-DE), two-dimensional sodium sulphate-polyacrylamide gel electrophoresis (2-DE) and immunoblotting. Furthermore, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry methods were used for peptide mass fingerprint (PMF) and sequencing of the protein, respectively. Sequence information and PMF queried against the NCBI database confirmed the identity of the protein as hemocyanin. Conserved domain search showed that there was an Ig-like conserved domain of 252 amino acid residues in the C-terminus of arthropoda hemocyanins. In addition, four and one conserved regions were found between hemocyanin and human Ig heavy chain and Ig kappa chain, respectively. These results indicate that in addition to copper-binding domains hemocyanin has an Ig-like conserved domain, which would confer some new functions to multifunctional respiratory pigment of crustaceans.     

Phosphatases in apoptosis: to be or not to be,PP2A is in the heart of the question

Protein phosphatase type 2A (PP2A) is a major Ser/Thr phosphatase involved in several cellular signal transduction pathways. In this review, we will focus on recent progress concerning the role of PP2A in apoptotic signalling. Since PP2A activates pro-apoptotic and inhibits anti-apoptotic proteins of the Bcl-2 family, we conclude that PP2A has a positive regulatory function in apoptosis. However, in Drosophila, a specific subset of the PP2A holoenzyme family, containing B'/PR61 as third regulatory subunit, is inhibitory for apoptosis, suggesting different regulatory mechanisms and substrates in different species. Moreover, PP2A acts not only upstream as a regulator of the apoptotic signal transduction pathway but also downstream as a substrate of effector caspases. Hence, PP2A is involved in the regulation as well as in the cellular response of apoptosis. Probably, various PP2A holoenzymes with distinct regulatory subunits specifically target different apoptotic substrates. This could explain the implication of PP2A at several levels of the apoptotic signal transduction pathway. Finally, some viral proteins such as adenovirus E4orf4 and simian virus small t target PP2A to alter its activity, resulting in induction of apoptosis as a regulatory mechanism to enhance virus spread.     

Small molecule induction of MSH2-dependent cell death suggests a vital role of mismatch repair proteins in cell death

Avoidance of apoptosis is one of the hallmarks of cancer development and progression. Chemotherapeutic agents aim to initiate an apoptotic response, but often fail due to dysregulation. MSH proteins are capable of recognizing cisplatin damage in DNA and participate in the initiation of cell death. We have exploited this recognition and computationally simulated a MutS homolog (MSH) "death conformation". Screening and docking experiments based on this model determined that the MSH2-dependent cell-death pathway can be induced by a small molecule without DNA damage, reserpine. Reserpine was identified via virtual screening on structures obtained from molecular dynamics as a small molecule that selectively binds a protein "death" conformation. The virtual screening predicts that this small molecule binds in the absence of DNA. Cell biology confirmed that reserpine triggers the MSH2-dependent cell-death pathway. This result supports the hypothesis that the MSH2-dependent pathway is initiated by specific protein conformational changes triggered by binding to either DNA damage or small compound molecules. These findings have multiple implications for drug discovery and cell biology. Computational modeling may be used to identify and eventually design small molecules that selectively activate particular pathways through conformational control. Molecular dynamics simulations can be used to model the biologically relevant conformations and virtual screening can then be used to select for small molecules that bind specific conformations. The ability of a small molecule to induce the cell-death pathway suggests a broader role for MMR proteins in cellular events, such as cell-death pathways, than previously suspected.     

Heterogeneous interactome between Litopenaeus vannamei plasma proteins and Vibrio parahaemolyticus outer membrane proteins

A great loss has been suffered by microbial infectious diseases under intensive shrimp farming in recent years. In this background, the understanding of shrimp innate immunity becomes an importantly scientific issue, but little is known about the heterogeneous protein–protein interaction between pathogenic cells and hosts, which is a key step for the invading microbes to infect internet organs through bloodstream. In the present study, bacterial outer membrane (OM) protein array and pull-down approaches are used to isolate both Vibrio parahaemolyticus OM proteins that bind to shrimp serum proteins and the shrimp serum proteins that interact with bacterial cells, respectively. Three interacting shrimp serum proteins, hemocyanin, β-1,3-glucan binding protein and LV_HP_RA36F08r and thirty interacting OM proteins were determined. They form 63 heterogeneous protein–protein interactions. Nine out of the 30 OM proteins were randomly demonstrated to be up-regulated or down-regulated when bacterial cells were cultured with shrimp sera, indicating the biological significance of the network. The interesting findings uncover the complexity of struggle between host immunity and bacterial infection. Compared with our previous report on heterogeneous interactome between fish grill and bacterial OM proteins, the present study further extends the investigation from lower vertebrates to invertebrates and develops a bacterial OM protein array to identify the OM proteins bound with shrimp serum proteins, which elevates the frequencies of the bound OM proteins. Our results highlight the way to determine and understand the heterogeneous interaction between hosts and microbes.     

Ubiquitylation in apoptosis: a post-translational modification at the edge of life and death

The proper regulation of apoptosis is essential for the survival of multicellular organisms. Furthermore, excessive apoptosis can contribute to neurodegenerative diseases, anaemia and graft rejection, and diminished apoptosis can lead to autoimmune diseases and cancer. It has become clear that the post-translational modification of apoptotic proteins by ubiquitylation regulates key components in cell death signalling cascades. For example, ubiquitin E3 ligases, such as MDM2 (which ubiquitylates p53) and inhibitor of apoptosis (IAP) proteins, and deubiquitinases, such as A20 and ubiquitin-specific protease 9X (USP9X) (which regulate the ubiquitylation and degradation of receptor-interacting protein 1 (RIP1) and myeloid leukaemia cell differentiation 1 (MCL1), respectively), have important roles in apoptosis. Therapeutic agents that target apoptotic regulatory proteins, including those that are part of the ubiquitin-proteasome system, might afford clinical benefits.     

Development of paratransgenic Artemia as a platform for control of infectious diseases in shrimp mariculture

Aim: To study the accumulation and retention of recombinant proteins in Artemia gut for optimizing paratransgenic disease control in shrimp aquaculture. Methods and Results: Transgenic Escherichia coli expressing fluorescent marker proteins and the transgenic cyanobacterium Synechococcus bacillarus expressing a functional murine single chain antibody, DB3, were fed to Artemia franciscana. Stable expression and retention of several marker molecules (e.g. GFP, DS Red and DB3) up to 10 h after of feeding with E. coli were evident within the gut of Artemia. Engineered strains of S. bacillarus expressing DB3 accumulated within the gut of Artemia with detectable antibody activity for 8–10 h of feeding via ELISA, coincident with the time period of the highest density of transgenic S. bacillarus in the Artemia gut. Conclusions: Artemia fed transgenic bacteria or algae accumulated recombinant proteins for up to 10 h that retained biological activity. Co‐delivery of multiple recombinant proteins simultaneously in the gut of Artemia was also demonstrated. Significance and Impact of the Study: Expression of molecules that target infectious agents of mariculture in shrimp via commonly deployed feed organisms such as Artemia could potentially offer powerful new tools in the ongoing global effort to increase food supply.     

Bacteria-based in vivo peptide library screening using biopanning approach

Traditionally, library screening has been performed to identify biologically active agents including small molecules or peptides that inhibit target proteins or molecules with therapeutic interests. Due to its chemical nature, library screening is usually performed under in vitro environments using purified proteins and molecules. However, active agents identified from in vitro screenings often fail to exhibit biological activities in cells. To overcome this inherent limitation, we have developed an in vivo peptide library screening system that allows for the identification of dissociative inhibitors of protein interactions of interest. The screening is based on the reconstitution of the cI repressor from bacteriophage lambda with high-density expression peptide library and is entirely performed in bacteria cells. Furthermore, to enhance the efficacy and sensitivity of the screening, a multiple-round biopanning approach was employed for amplification and enrichment of positive peptides. Overall, this in vivo screening should provide a fast and efficient tool for identification of biologically active peptide molecules against target protein assembly.     

Mediators of endoplasmic reticulum stress-induced apoptosis

The efficient functioning of the endoplasmic reticulum (ER) is essential for most cellular activities and survival. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response (UPR) to restore normal ER function. If the stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, as ER stress-induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review, we examine the role of the molecules that are activated during the UPR in order to identify the molecular switch from the adaptive phase to apoptosis. We discuss how the activation of these molecules leads to the commitment of death and the mechanisms that are responsible for the final demise of the cell.     

A model for apoptotic interaction between white spot syndrome virus and shrimp

White spot syndrome virus (WSSV) is an enveloped, large dsDNA virus that mainly infects penaeid shrimp, causing serious damage to the shrimp aquaculture industry. Like other animal viruses, WSSV infection induces apoptosis. Although this occurs even in by-stander cells that are free of WSSV virions, apoptosis is generally regarded as a kind of antiviral immune response. To counter this response, WSSV has evolved several different strategies. From the presently available literature, we construct a model of how the host and virus both attempt to regulate apoptosis to their respective advantage. The basic sequence of events is as follows: first, when a WSSV infection occurs, cellular sensors detect the invading virus, and activate signaling pathways that lead to (1) the expression of pro-apoptosis proteins, including PmCasp (an effecter caspase), MjCaspase (an initiator caspase) and voltage-dependent anion channel (VDAC); and (2) mitochondrial changes, including the induction of mitochondrial membrane permeabilization and increased oxidative stress. These events initiate the apoptosis program. Meanwhile, WSSV begins to express its genes, including two anti-apoptosis proteins: AAP-1, which is a direct caspase inhibitor, and WSV222, which is an E3 ubiquitin ligase that blocks apoptosis through the ubiquitin-mediated degradation of shrimp TSL protein (an apoptosis inducer). WSSV also induces the expression of a shrimp anti-apoptosis protein, Pm-fortilin, which can act on Bax to inhibit mitochondria-triggered apoptosis. This is a life and death struggle because the virus needs to prevent apoptosis in order to replicate. If WSSV succeeds in replicating in sufficient numbers, this will result in the death of the infected penaeid shrimp host.