Protease Profiles Research Articles

Protease cleavage site fingerprinting by label-free in-gel degradomics reveals pH-dependent specificity switch of legumain.

Determination of protease specificity is of crucial importance for understanding protease function. We have developed the first gel-based label-free proteomic approach (DIPPS-direct in-gel profiling of protease specificity) that enables quick and reliable determination of protease cleavage specificities under large variety of experimental conditions. The methodology is based on in-gel digestion of the gel-separated proteome with the studied protease, enrichment of cleaved peptides by gel extraction, and subsequent mass spectrometry analysis combined with a length-limited unspecific database search. We applied the methodology to profile ten proteases ranging from highly specific (trypsin, endoproteinase GluC, caspase-7, and legumain) to broadly specific (matrix-metalloproteinase-3, thermolysin, and cathepsins K, L, S, and V). Using DIPPS, we were able to perform specificity profiling of thermolysin at its optimal temperature of 75°C, which confirmed the applicability of the method to extreme experimental conditions. Moreover, DIPPS enabled the first global specificity profiling of legumain at pH as low as 4.0, which revealed a pH-dependent change in the specificity of this protease, further supporting its broad applicability.

Population-associated heterogeneity of the digestive Cys protease complement in Colorado potato beetle, Leptinotarsa decemlineata

Herbivorous insects use complex protease complements to process plant proteins, useful to adjust their digestive functions to the plant diet and to elude the antidigestive effects of dietary protease inhibitors. We here assessed whether basic profiles and diet-related adjustments of the midgut protease complement may vary among populations of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). Two laboratory colonies of this insect were used as models, derived from insect samples collected in potato fields ∼1200km distant from each other in North America. Synchronized 4th-instar larvae reared on potato were kept on this plant, or switched to tomato or eggplant, to compare their midgut cathepsin activities and content of intestain Cys proteases under different diet regimes. Cathepsin D activity, cathepsin L activity, cathepsin B activity and total intestain content shortly after larval molting on potato leaves were about two times lower in one population compared to the other. By comparison, cathepsin D activity, cathepsin B activity, total intestain content and relative abundance of the most prominent intestain families were similar in the two populations after three days regardless of the plant diet, unlike cathepsin L activity and less prominent intestain families showing population-associated variability. Variation in Cys protease profiles translated into the differential efficiency of a Cys protease inhibitor, tomato cystatin SlCYS8, to inhibit cathepsin L activity in midgut extracts of the two insect groups. Despite quantitative differences, SlCYS8 single variants engineered to strongly inhibit Cys proteases showed improved potency against cathepsin L activity of either population. These data suggest the feasibility of designing cystatins to control L. decemlineata that are effective against different populations of this insect. They underline, on the other hand, the practical relevance of considering natural variability of the protease complement among L. decemlineata target populations, eventually determinant in the success or failure of cystatin-based control strategies on a large-scale basis.

Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum.

Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5–75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases.

Biological Soil Quality Indicators and Conditioners in a Plant-Assisted Remediation of Crude Oil Polluted Farmland

Owing to vital roles played by enzymes in the preservation of the make-up of soil ecosystem and functional diversity, the influence of organic manure on the resultant biological quality of a crude oil polluted agricultural soil from a 90 d phytoremediation pot experiment was investigated. A 4-factor phyto-assisted clean-up of crude oil polluted agricultural soil was designed with options of manure amendments, to boost micobial activities. Profiles of β-glucosidases, proteases, dehydrogenases, phosphomonoesterases and respiration were investigated. Analysis of variance of triplicate experiment was carried out. Application of soil conditioner gave no marked dehydrogenase activity, which increased with depletion of available phosphorus. Marked increases in CO2 release and alkaline phosphatase activity with soil conditioning may implicate beneficial relationship with the abundance of microbial populations. Flooding in some pots correlated with β-glucosidase and respiratory acitivities. A direct relationship between cellulose breakdown, measurable with β-glucosidase activity, organic matter and CO2 release, measurable with respiratory activity within all soils was found in the present study. Use of organic manure significantly improved CO2 release by soil biota in hydrocarbon-impacted soil and may be explored for phytoremediation technique.

Protease substrate profiling using bacterial display of self-blocking affinity proteins and flow-cytometric sorting.

Proteases are involved in fundamental biological processes and are important tools in both biotechnological and biomedical research. An important property of proteases is to discriminate among potential substrates. Here, a new method for substrate profiling of proteases is presented. The substrates are displayed between two anti-idiotypic affinity domains on the Gram-positive bacterium Staphylococcus carnosus. The first domain functions as a reporter tag and has affinity for a labeled reporter protein, whereas the second domain blocks the reporter tag from interacting with the reporter protein. Site-specific proteolysis of the substrate results in release of the blocking domain, enabling the reporter tag to bind the labeled reporter protein. Proteolysis is therefore reflected in reporter binding, which is quantified by flow cytometry. First, the method with tobacco etch virus protease (TEVp) is evaluated and then the substrate preference of matrix metalloprotease-1 (MMP-1) is determined using two libraries of around three million substrates each. Identified substrate peptides contained the previously reported motif (PXXXHy ) and on-cell determination of apparent kcat /KM revealed that the enriched substrate peptides are hydrolyzed six to eight-fold more efficiently than a previously reported substrate peptide. The method thus works as intended and the authors believe it has potential as an efficient tool for substrate profiling.

Single Cell Analysis of Leukocyte Protease Activity Using Integrated Continuous-Flow Microfluidics

Leukocytes are the essential cells of the immune system that protect the human body against bacteria, viruses, and other foreign invaders. Secretory products of individual leukocytes, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAMs), are critical for regulating the inflammatory response and mediating host defense. Conventional single cell analytical methods, such as flow cytometry for cellular surface biomarker studies, are insufficient for performing functional assays of the protease activity of individual leukocytes. Here, an integrated continuous-flow microfluidic assay is developed to effectively detect secretory protease activity of individual viable leukocytes. Leukocytes in blood are first washed on-chip with defined buffer to remove background activity, followed by encapsulating individual leukocytes with protease sensors in water-in-oil droplets and incubating for 1 h to measure protease secretion. With this design, single leukocyte protease profiles under naive and phorbol 12-myristate 13-acetate (PMA)-stimulated conditions are reliably measured. It is found that PMA treatment not only elevates the average protease activity level but also reduces the cellular heterogeneity in protease secretion, which is important in understanding immune capability and the disease condition of individual patients.

Spatiotemporal expression profile of proteases and immunological, angiogenic, hormonal and apoptotic mediators in rat placenta before and during intrauterine trophoblast migration.

The gene and/or protein expression of proteases and immunological, angiogenic, hormonal and apoptotic mediators was evaluated in rat placenta before and during intrauterine trophoblast migration. The depth of interstitial and endovascular intrauterine trophoblast invasion and the immunohistochemical expression of vascular endothelial growth factor (VEGF), fetal liver kinase 1 (Flk1), interferon (IFN)-γ, migration inhibitory factor (MIF), and inducible nitric oxide synthase (iNOS; also known as nitric oxide synthase (NOS) 2) were evaluated. In addition, the expression of the Vegf, Flk1, placental growth factor (Pigf), soluble fms-like tyrosine kinase 1 (sFlt1), placental lactogen 1 (Pl1), proliferin-related protein (rPlf), placental leptin (Lep), Toll-like receptor 2 (Tlr2), Toll-like receptor 4 (Tlr4), Infg, Mif, tumour necrosis factor-α (Tnf), interleukin-10 (Il10), Nos2, caspase 3 (Casp3), Bax, Bcl2, matrix metalloproteinase 2 (Mmp2) and matrix metalloproteinase 9 (Mmp9) genes was determined by real-time reverse transcription-polymerase chain reaction. At 10 days gestation, gene expression of Tlr2, Tlr4, Tnf, Infg, Il10, Casp3, Pigf, sFlt1 and Lep (P<0.05) were higher than at 14 and/or 19 days of gestation. The beginning of intrauterine trophoblast invasion, i.e., at 14 days of gestation, coincided with higher gene and/or protein expression of MMP9, VEGF, Flk1, NOS2, MIF, BAX and rPlf compared to days 10 and 19 (P<0.05). In contrast, gene expression of Mmp2 and Pl1 was higher at the end of trophoblast invasion compared to 10 and 14 days of gestation (P<0.05). In conclusion, before intrauterine trophoblast migration, expression of TLRs and immunological and pro-apoptotic mediators is higher, whereas the beginning of trophoblast migration is characterised by higher expression of the pro-angiogenic factors NOS2 and MMP9. In contrast, MMP2 and PL1 expression is higher at the end of intrauterine trophoblast migration.

Cell death patterns in Arabidopsis cells subjected to four physiological stressors indicate multiple signalling pathways and cell cycle phase specificity.

Corpse morphology, nuclear DNA fragmentation, expression of senescence-associated genes (SAG) and cysteine protease profiles were investigated to understand cell death patterns in a cell cycle-synchronised Arabidopsis thaliana cell suspension culture treated with four physiological stressors in the late G2 phase. Within 4h of treatment, polyethylene glycol (PEG, 20%), mannose (100mM) and hydrogen peroxide (2mM) caused DNA fragmentation coinciding with cell permeability to Evans Blue (EB) and produced corpse morphology corresponding to apoptosis-like programmed cell death (AL-PCD) with cytoplasmic retraction from the cell wall. Ethylene (8mL per 250-mL flask) caused permeability of cells to EB without concomitant nuclear DNA fragmentation and cytoplasmic retraction, suggesting necrotic cell death. Mannose inducing glycolysis block and PEG causing dehydration resulted in relatively similar patterns of upregulation of SAG suggesting similar cell death signalling pathways for these two stress factors, whereas hydrogen peroxide caused unique patterns indicating an alternate pathway for cell death induced by oxidative stress. Ethylene did not cause appreciable changes in SAG expression, confirming necrotic cell death. Expression of AtDAD, BoMT1 and AtSAG2 genes, previously shown to be associated with plant senescence, also changed rapidly during AL-PCD in cultured cells. The profiles of nine distinct cysteine protease-active bands ranging in size from ca. 21.5 to 38.5kDa found in the control cultures were also altered after treatment with the four stressors, with mannose and PEG again producing similar patterns. Results also suggest that cysteine proteases may have a role in necrotic cell death.

A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation.

A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (E a), quotient energy (Q 10), K m, and V max were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively.

Genome-wide identification and expression profiling of serine proteases and homologs in the diamondback moth, Plutella xylostella (L.).

BackgroundSerine proteases (SPs) are crucial proteolytic enzymes responsible for digestion and other processes including signal transduction and immune responses in insects. Serine protease homologs (SPHs) lack catalytic activity but are involved in innate immunity. This study presents a genome-wide investigation of SPs and SPHs in the diamondback moth, Plutella xylostella (L.), a globally-distributed destructive pest of cruciferous crops.ResultsA total of 120 putative SPs and 101 putative SPHs were identified in the P. xylostella genome by bioinformatics analysis. Based on the features of trypsin, 38 SPs were putatively designated as trypsin genes. The distribution, transcription orientation, exon-intron structure and sequence alignments suggested that the majority of trypsin genes evolved from tandem duplications. Among the 221 SP/SPH genes, ten SP and three SPH genes with one or more clip domains were predicted and designated as PxCLIPs. Phylogenetic analysis of CLIPs in P. xylostella, two other Lepidoptera species (Bombyx mori and Manduca sexta), and two more distantly related insects (Drosophila melanogaster and Apis mellifera) showed that seven of the 13 PxCLIPs were clustered with homologs of the Lepidoptera rather than other species. Expression profiling of the P. xylostella SP and SPH genes in different developmental stages and tissues showed diverse expression patterns, suggesting high functional diversity with roles in digestion and development.ConclusionsThis is the first genome-wide investigation on the SP and SPH genes in P. xylostella. The characterized features and profiled expression patterns of the P. xylostella SPs and SPHs suggest their involvement in digestion, development and immunity of this species. Our findings provide a foundation for further research on the functions of this gene family in P. xylostella, and a better understanding of its capacity to rapidly adapt to a wide range of environmental variables including host plants and insecticides.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2243-4) contains supplementary material, which is available to authorized users.

Characterizing Protease Specificity: How Many Substrates Do We Need?

Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points). Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4’) with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design.

ADAM33 and ADAM12 genetic polymorphisms and their expression in Egyptian children with asthma

BackgroundThe ADAM family is involved in some pathologic processes, such as inflammation and asthma. ObjectivesTo assess the association between ADAM33 and ADAM12 single-nucleotide polymorphisms (SNPs) with asthma risk and severity and to investigate the effect of ADAM33 and ADAM12 polymorphisms on expression of these proteases in sputum. MethodsTwo SNPs of the ADAM33 gene, F+1 (rs511898) G/A and ST+4 (rs44707) A/C, and 2 SNPs of the ADAM12 gene, rs3740199 and rs1871054, were analyzed in 400 asthma cases and 200 controls aged 3 to 14 years using the polymerase chain reaction–restriction fragment length polymorphism method. Messenger RNA expression profile of ADAM33 and ADAM12 proteases in sputum from studied groups was determined by reverse transcription polymerase chain reaction. ResultsADAM33 F+1 homozygous mutant genotype (AA) and ST+4 heterozygous and homozygous mutant genotype (AC and CC) and mutant alleles of both polymorphisms were significantly associated with asthma risk and severity in moderate and severe subgroups. Patients with the ADAM12 (rs3740199) CC genotype were at increased risk for moderate and severe asthma. Messenger RNA levels of ADAM12 were significantly increased in asthmatic children compared with controls, whereas we were not able to detect the expression of ADAM33 in the sputum of the groups studied. The ADAM12 expression was significantly higher in homozygous CC (variant type) compared with homozygous GG (wild type) of both ADAM12 rs3740199 and rs1871054 in the asthmatic group. ConclusionOur analysis suggests a likely role for ADAM33 and ADAM12 in the development of asthma in Egyptian children. Furthermore, ADAM12 polymorphisms may affect ADAM12 expression in asthma.

Demonstration of elevated levels of active cathepsin S in dextran sulfate sodium colitis using a new activatable probe.

Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue samples. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis. We designed and synthesized a new fluorescent activatable probe, NB200, for the detection of active cathepsin S. Colitis was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). Homogenized mouse colons, with or without the addition of the specific cathepsin S inhibitor MV026031, were incubated with NB200 in a fluorescent plate reader. NB200 selectively detected purified cathepsin S and not other common inflammatory proteases. Homogenates of colon from mice with DSS colitis induced a significant fluorescent increase when compared to control animals (control vs DSS: p<0.05 at 200min and p<0.01 at 220-240min), indicating cathepsin S activation. The cathepsin S inhibitor abolished this increase in fluorescence (DSS vs DSS + MV026031: p<0.05 at 140min, p<0.01 at 180min, p<0.001 at 200-240min), which confirms cathepsin S activation. Cathepsin S activity correlated with the disease activity index (Spearman r=0.77, p=0.017). Our investigation has demonstrated the utility of activatable probes for detecting protease activity in intestinal inflammation. Panels of such probes may allow 'signature' protease profiles to be established for a range of inflammatory diseases and disorders.

Commentary: Extracellular peptidase hunting for improvement of protein production in plant cells and roots.

Commentary: Extracellular peptidase hunting for improvement of protein production in plant cells and roots.

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (kcat/KM) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1' substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.

Profiling of proteins and proteases in the products of the salivary gland, digestive tract and excretions from larvae of the camel nasal botfly, Cephalopina titillator (Clark).

Proteins and proteolytic activities in the contents of the salivary gland (SGc), digestive tract (DTc) and excretory-secretory products (ESP) from larvae of the camel nasal botfly Cephalopina titillator were separated electrophoretically, and characterized. The protein profiles of the different samples were qualitatively quite similar in the larval stages L2 and L3. Zymogram analysis of proteases in the samples indicated that the digestive tract contained a greater variety of proteases than the salivary gland or the excretory-secretory products. They are mainly serine proteases. Proteases of ESP and DTc (especially of 3rd instar) contain trypsin- and chymotrypsin-like serine proteases, while the serine proteases of SGc are not of the trypsin- or chemotrypsin-type.

Extracellular proteases are released by ciliates in defined seawater microcosms

The biodegradation of proteins in seawater requires various proteases which are commonly thought to be mainly derived from heterotrophic bacteria. We, however, found that protists showed a high protease activity and continuously produced trypsin-type enzymes. The free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium was isolated and used for microcosm incubation with different concentrations of killed bacteria as food for 10 days. The results showed that the co-existence of the ciliate with its associated bacterium produced a significant protease activity in both cell-associated and cell-free fractions while that in the associated bacterium only microcosm was negligible. The protease profiles are different between cell-associated and cell-free fractions, and a trypsin-type enzyme hydrolyzing Boc-Val-Leu-Lys-MCA was detected throughout the period in the presence of ciliates. This suggests that ciliates release proteases into the surrounding environment which could play a role in protein digestion outside cells. It has been previously suggested that bacteria are the major transformers in seawater. We here present additional data which indicates that protists, or at least ciliates with their specific enzymes, are a potential player in organic matter degradation in water columns.

Activity-Based Protein Profiling of Rhomboid Proteases in Liposomes.

Although activity-based protein profiling (ABPP) has been used to study a variety of enzyme classes, its application to intramembrane proteases is still in its infancy. Intramembrane proteolysis is an important biochemical mechanism for activating proteins residing within the membrane in a dormant state. Rhomboid proteases (intramembrane serine proteases) are embedded in the lipid bilayers of membranes and occur in all phylogenetic domains. The study of purified rhomboid proteases has mainly been performed in detergent micelle environments. Here we report on the reconstitution of rhomboids in liposomes. Using ABPP, we have been able to detect active rhomboids in large and giant unilamellar vesicles. We have found that the inhibitor profiles of rhomboids in micelles and liposomes are similar, thus validating previous inhibitor screenings. Moreover, fluorescence microscopy experiments on the liposomes constitute the first steps towards activity-based imaging of rhomboid proteases in membrane environments.

Toward the definition of a peptidome signature and protease profile in chronic periodontitis.

Chronic periodontitis (CP) is a complex immuno-inflammatory disease that results from preestablished gingivitis. We investigated potential differences in salivary peptidome in health and CP. Saliva was collected from nine CP patients and ten healthy subjects, from which five CP and five healthy were enriched following endoProteoFASP approach, separated and identified by nanoHPLC-MALDI-TOF/TOF. Protease prediction was carried out in silico with Proteasix. Parallel gelatin and collagen (I) zymographies were performed to study proteolytic activity in CP. An association of CP with increased gelatinolytic and collagenolytic activity was observed, which is mainly attributed to metalloproteases, remarkably MMP9. Protease prediction revealed distinct protease profiles in CP and in health. Peptidomic data corroborated the inflammatory status, and demonstrated that intact histatin 1 may play an important role in the defense response against oral pathogens. The application of the endoProteoFASP approach to study the salivary peptidome of CP subjects resulted in the identification of eight surrogate peptide markers, which may be used in multiplex to identify CP. These peptides belong to acidic PRP and to P-B peptide. Particularly, P-B peptide fragments exhibited domains with potential predicted antimicrobial activity, corroborating an antimicrobial function. The comparison between the salivary peptidome obtained by control and CP samples showed a specific association of eight peptides to CP, with remarkable predicted antimicrobial activity, which should be further validated in studies with large number of subjects.

Temporal analysis of molecular changes in shrimp (Penaeus vannamei) tissues with respect to white spot disease

In spite of lot of attention and significant research efforts, White spot disease (WSD) is the major cause for shrimp mortality in aquaculture industry. This is due to the limited understanding in White Spot Syndrome Virus (WSSV) pathogenesis. To understand shrimp molecular responses towards WSSV infection, proteome and protease profiles of various shrimp tissues (gill, muscle, gut and hepatopancreas) were studied at different time-intervals post-infection (pi) using SDS-PAGE analysis and In-gel gelatin zymography, respectively. Expression of new proteins along with up-regulation, down-regulation and varied expression of many host proteins were observed. These variations were observed as early as 6 h pi and the maximum variations were observed at the time-intervals 6 h pi and 12 h pi representing the early stage of infection. Protease profile analysis had revealed that most of the host proteases were down-regulated during WSSV infection. Among the tested shrimp tissues, gill, gut and hepatopancreas are the most affected due to WSSV infection, while muscle is the least affected one with minimum proteolytic activity, whereas hepatopancreas is highly enriched with active proteases. These results suggest that during WSSV infection, both protein and protease profiles of shrimp tissues gets drastically altered and down-regulation of the host proteases is the major step in WSSV-pathogenesis. These observations are significant for intervening with the early stages and delaying the morbidity and mortality, so that shrimps could be harvested at profitable incubation time and reduce the impact on shrimp aquaculture industry.