Structure and Mechanism of Rhomboid Protease

Rhomboid protease was first discovered in Drosophila. Mutation of the fly gene interfered with growth factor signaling and produced a characteristic phenotype of a pointed head skeleton. The name rhomboid has since been widely used to describe a large family of related membrane proteins that have diverse biological functions but share a common catalytic core domain composed of six membrane-spanning segments. Most rhomboid proteases cleave membrane protein substrates near the N terminus of their transmembrane domains. How these proteases function within the confines of the membrane is not completely understood. Recent progress in crystallographic analysis of the Escherichia coli rhomboid protease GlpG in complex with inhibitors has provided new insights into the catalytic mechanism of the protease and its conformational change. Improved biochemical assays have also identified a substrate sequence motif that is specifically recognized by many rhomboid proteases.     

Rhomboids of Mycobacteria: Characterization Using an aarA Mutant of Providencia stuartii and Gene Deletion in Mycobacterium smegmatis

Background

Rhomboids are ubiquitous proteins with unknown roles in mycobacteria. However, bioinformatics suggested putative roles in DNA replication pathways and metabolite transport. Here, mycobacterial rhomboid-encoding genes were characterized; first, using the Providencia stuartii null-rhomboid mutant and then deleted from Mycobacterium smegmatis for additional insight in mycobacteria.

Methodology/Principal Findings

Using in silico analysis we identified in M. tuberculosis genome the genes encoding two putative rhomboid proteins; Rv0110 (referred to as “rhomboid protease 1”) and Rv1337 (“rhomboid protease 2”). Genes encoding orthologs of these proteins are widely represented in all mycobacterial species. When transformed into P. stuartii null-rhomboid mutant (ΔaarA), genes encoding mycobacterial orthologs of “rhomboid protease 2” fully restored AarA activity (AarA is the rhomboid protein of P. stuartii). However, most genes encoding mycobacterial “rhomboid protease 1” orthologs did not. Furthermore, upon gene deletion in M. smegmatis, the ΔMSMEG_4904 single mutant (which lost the gene encoding MSMEG_4904, orthologous to Rv1337, “rhomboid protease 2”) formed the least biofilms and was also more susceptible to ciprofloxacin and novobiocin, antimicrobials that inhibit DNA gyrase. However, the ΔMSMEG_5036 single mutant (which lost the gene encoding MSMEG_5036, orthologous to Rv0110, “rhomboid protease 1”) was not as susceptible. Surprisingly, the double rhomboid mutant ΔMSMEG_4904–ΔMSMEG_5036 (which lost genes encoding both homologs) was also not as susceptible suggesting compensatory effects following deletion of both rhomboid-encoding genes. Indeed, transforming the double mutant with a plasmid encoding MSMEG_5036 produced phenotypes of the ΔMSMEG_4904 single mutant (i.e. susceptibility to ciprofloxacin and novobiocin).

Conclusions/Significance

Mycobacterial rhomboid-encoding genes exhibit differences in complementing aarA whereby it''s only genes encoding “rhomboid protease 2” orthologs that fully restore AarA activity. Additionally, gene deletion data suggests inhibition of DNA gyrase by MSMEG_4904; however, the ameliorated effect in the double mutant suggests occurrence of compensatory mechanisms following deletion of genes encoding both rhomboids.     

Cloning,over expression and functional attributes of serine proteases from Oceanobacillus iheyensis O.M.A18 and Haloalkaliphilic bacterium O.M.E12

Cloning, over-expression, characterization and structural and functional analysis of two alkaline proteases from the newly isolated haloalkaliphilic bacteria: Oceanobacillus iheyensis O.M.A₁8 and Haloalkaliphilic bacterium O.M.E₁2 were carried out. The cloned protease genes were over-expressed in Escherichia coli within 6 h of the IPTG induction. The protease genes were sequenced and the sequence submitted to the GenBank with the accession numbers, HM219179 and HM219182. The recombinant proteases were active in the range of pH 8–11 and temperature 30–50 °C. The amino acid sequences of the alkaline proteases displayed hydrophobic character and stable configurations. The amino acids Asp 141, His 171 and Ser 324 formed the catalytic triad, while Ile, Leu and Ser were other amino acid moieties present in the active site. The characteristics of the recombinant proteases were compared and found to be similar to their native counterparts. On the basis of the in-silico analysis and inhibitor studies, the enzymes were confirmed as serine proteases. The study hold significance as only limited enzymes from the haloalkaliphilic bacteria have been cloned, sequenced and analyzed for the structure and function analysis.     

Degradation of 2,4-dichlorophenol by Bacillus sp. isolated from an aeration pond in the Baikalsk pulp and paper mill (Russia)

A pure culture of 2,4-dichlorophenol (2,4-DCP)-degrading bacteria was isolated from a natural enrichment that had been adapted to chlorophenols in the aeration pond of the Baikalsk pulp and paper mill (Russia). The bacteria were identified by 16S rDNA intergenic region analysis, using PCR with universal primers. Comparative analysis of the 16S rDNA sequence (1545 bp) in the GenBank database revealed that these bacteria are related to Bacillus cereus GN1. Degradation of 2,4-DCP was studied using this culture in liquid medium under aerobic conditions, at initial concentrations of 20–560 μM 2,4-DCP. The 2,4-DCP degradation rates by B. cereus GN1 could be determined at concentrations up to 400 μM. However, higher concentrations of 2,4-DCP (560 μM) were inhibitory to cell growth.     

Heterogeneity in thylakoid membrane proteome of Synechocystis 6803

Cell-free extracts of Synechocystis 6803 were fractionated by successive ultracentrifugation at 40,000 × g, 90,000 × g and 150,000 × g to obtain the three thylakoid fractions designated as 40 k, 90 k and 150 k fractions respectively. These fractions showed differences in absorption and emission spectra. Nano-LC-ESI-Q-TOF MS analysis identified 123 proteins belonging to membrane as well as cytosolic fraction. Out of these proteins, there were 22 proteins with transmembrane helices and 12 proteins with signal peptide. There were 77 proteins common across all the three fractions. Most of these proteins were subunits of photosynthetic complexes, CF₀–CF₁ ATP synthase or ribosomal proteins. Among the rest of the proteins, 8 were exclusive to 40 k fraction, 3 were exclusive to 90 k fraction and 13 were exclusive to 150 k fraction. There were 10 proteins common between 40 k and 90 k fractions and 12 proteins common between 90 k and 150 k fractions. There were no common proteins detected between 40 k and 150 fractions. The results suggested existence of heterogeneity in thylakoids of Synechocystis 6803, which may lead to micro-compartmentation and functional heterogeneity in the thylakoids of this organism as seen previously.     

Bacillus subtillis RTSBA6 6.00, a new strain isolated from gut of Helicoverpa armigera (Lepidoptera: Noctuidae) produces chymotrypsin-like proteases

Exploring bacterial communities with proteolytic activity from the gut of the Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) insect pests was the purpose of this study. As initial efforts to achieve this goal here we report the isolation of new Bacillus subtillis RTSBA6 6.00 strain from the gut of H. armigera and demonstrated as proteases producer. Zymographic analysis revealed 12 proteolytic bands with apparent molecular weights ranging from 20 to 185 kDa. Although some activity was detected at acidic pH, the major activity was observed at slight alkaline pH (7.8). The optimum temperature was found to be 35 °C with complete loss of activity at 70 °C. All proteases were completely inactivated by PMSF (phenylmethylsulfonyl fluoride) and TPCK (N-tosyl-l-phenylalanine chloromethyl ketone), suggesting that proteases secreted by B. subtillis RTSBA6 6.00 belong to serine proteases class with chymotrypsin-like activity. The occurrence of protease producing bacterial community in the gut of the H. armigera advocates its probable assistance to insect in proteinaceous food digestion and adaptation to protease inhibitors of host plants.     

A transcriptomic analysis on gene expressions in the infective third and pathogenic fifth larval stages of Angiostrongylus cantonensis

Although Angiostrongylus cantonensis is a parasite of rats, it is an important etiologic agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. This study was designed to compare the gene expression in the third- and fifth-stage (L3 and L5) by analysis of expressed sequence tags (ESTs). After removing low quality sequences, vector trimming, clustering and contig assembly, there remained 1437 clusters (285 contigs and 1152 singletons). Among these clusters, 779 (54.2%) showed significant similarity to the known proteins in the non-redundant protein database of GenBank (E-value < 1 × 10^− 10) and species of the best hit sequences mainly belonged to nematodes. These clusters included 869 (60.5%) that were entirely comprised of ESTs from L3 (L3-biased clusters), 518 (36.0%) entirely from L5 (L5-biased clusters) and 50 (3.5%) from both stages (stage-shared clusters). Functional annotations by the Gene Ontology (GO) comparing with the eukaryotic clusters of orthologous groups of proteins (KOG) indicate that the L3-biased clusters significantly related to metabolism and the L5-biased clusters to growth, development, sexual differentiation and reproduction. Moreover, L3 were found to have expressions of metalloproteases, aspartic proteases, and cysteine proteases whereas expressions of cysteine, aspartic and serine proteases were revealed in L5. The results indicate that earlier developmental stages of nematodes may have a gene expression profile towards metabolism and later stages towards growth and development.     

Substrate specificity of rhomboid intramembrane proteases is governed by helix-breaking residues in the substrate transmembrane domain

Rhomboid intramembrane proteases initiate cell signaling during Drosophila development and Providencia bacterial growth by cleaving transmembrane ligand precursors. We have determined how specificity is achieved: Drosophila Rhomboid-1 is a site-specific protease that recognizes its substrate Spitz by a small region of the Spitz transmembrane domain (TMD). This substrate motif is necessary and sufficient for cleavage and is composed of residues known to disrupt helices. Rhomboids from diverse organisms including bacteria and vertebrates recognize the same substrate motif, suggesting that they use a universal targeting strategy. We used this information to search for other rhomboid substrates and identified a family of adhesion proteins from the human parasite Toxoplasma gondii, the TMDs of which were efficient substrates for rhomboid proteases. Intramembrane cleavage of these proteins is required for host cell invasion. These results provide an explanation of how rhomboid proteases achieve specificity, and allow some rhomboid substrates to be predicted from sequence information.     

Proteomic analysis of the adaptation to warming in the Antarctic bacteria Shewanella frigidimarina

Antarctica is subjected to extremely variable conditions, but the importance of the temperature increase in cold adapted bacteria is still unknown. To study the molecular adaptation to warming of Antarctic bacteria, cultures of Shewanella frigidimarina were incubated at temperatures ranging from 0 °C to 30 °C, emulating the most extreme conditions that this strain could tolerate. A proteomic approach was developed to identify the soluble proteins obtained from cells growing at 4 °C, 20 °C and 28 °C. The most drastic effect when bacteria were grown at 28 °C was the accumulation of heat shock proteins as well as other proteins related to stress, redox homeostasis or protein synthesis and degradation, and the decrease of enzymes and components of the cell envelope. Furthermore, two main responses in the adaptation to warm temperature were detected: the presence of diverse isoforms in some differentially expressed proteins, and the composition of chaperone interaction networks at the limits of growth temperature. The abundance changes of proteins suggest that warming induces a stress situation in S. frigidimarina forcing cells to reorganize their molecular networks as an adaptive response to these environmental conditions.     

Proteases supplementation to high gravity worts enhances fermentation performance of brewer's yeast

Nitrogen limitation, particularly prevailing in the case of high gravity beer brewing, results in poor yeast viability and even stuck or sluggish fermentations. Although wort contains abundant proteins and longer chain peptides, brewer's yeast does not assimilate them due to the fact that cells hardly secrete proteases during fermentation. The objective of this study was to investigate the possibility for utilizing unavailable nitrogen from two types of high gravity worts (20 °P and 24 °P) by adding three food-grade commercial proteases (Neutrase, Flavorzyme and Protamex) at the beginning of fermentations, respectively. Results showed that proteases supplementation significantly increased the FAN level and thus the amount of cell suspension in the later stages of fermentations (ca. 10 days later for 20 °P and 25 days later for 24 °P) (p < 0.05). Among the studied three proteases, we found that fermentations with Flavorzyme supplementation exhibited the best fermentation performance in terms of significantly improved wort fermentability, higher ethanol yield and flavor volatiles formation (p < 0.05). Furthermore, the foam of final beers produced by adding proteases was as stable as that of the control at each of the corresponding gravities.     

Structural–functional insights of single and multi-domain Capsicum annuum protease inhibitors

Pin-II protease inhibitors (PIs) are the focus of research interest because of their large structural–functional diversity and relevance in plant defense. Two representative Capsicum annuum PI genes (CanPI-15 and -7) comprising one and four inhibitory repeat domains, respectively, were expressed and recombinant proteins were characterized. β-Sheet and unordered structure was found predominant in CanPI-15 while -7 also displayed the signatures of polyproline fold, as revealed by circular dichroism studies. Inhibition kinetics against bovine trypsin indicated three times higher potency of CanPI-7 (K_i ～ 57 μM) than -15 (～184 μM). Activity and structural stability of these CanPIs were revealed under various conditions of pH, temperature and denaturing agent. Structure prediction, docking studies with proteases and mass spectroscopy revealed the organization of multiple reactive site loops of multi domain PIs in space as well as the steric hindrances imposed while binding to proteases due to their close proximity.     

Synthesis and antimicrobial activity of polyhalo isophthalonitrile derivatives

A series of polyhalo isophthalonitrile derivatives (3 and 4) that incorporate a variety of substituents at the 2-, 4-, 5- and/or 6-positions of the isophthalonitrile moieties have been designed and synthesized. These derivatives were evaluated for their antimicrobial activity against Staphylococcus aureus, Bacillus cereus (Gram-positive bacteria), Escherichia coli, Pseudomonas aeruginosa (Gram-negative bacteria); and Candida albicans (Fungi). Compounds 3 and 4 showed stronger inhibition of Gram-positive bacteria and fungi growth, and the antimicrobial ability of compound 3j (a 4-(benzylamino)-5-chloro-2,6-difluoro analog, MIC[SA] = 0.5 μg/mL; MIC[BC] = 0.4 μg/mL; MIC[CA] = 0.5 μg/mL) were close to nofloxacin and fluconazole and identified as the most potent antimicrobial agents in the series. The preliminary analysis of structure–activity relationships is also discussed.     

Cloning,heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1

The keratin-degrading strain Stenotrophomonas maltophilia BBE11-1 secretes two keratinolytic proteases, KerSMD and KerSMF. However, the genes encoding these proteases remain unknown. Here, we have isolated these two genes with a modified TAIL-PCR (thermal asymmetric interlaced PCR) method based on the N-terminal amino acid sequences of mature keratinases. These two keratinase genes encode serine proteases with PPC (bacterial pre-peptidase C-terminal) domain, which are successfully expressed with the help of pelB leader in Escherichia coli cells. Recombinant KerSMD (48 kDa) shows a better activity in feather degradation, higher thermostability and substrate specificity than KerSMF (40 kDa). KerSMD has a t_1/2 of 90 min at 50 °C and 64 min at 60 °C, and a better tolerance to surfactants SDS and triton X-100. The predicted model of KerSMD helps to explain the phenomenon of auto-catalytic C-terminal propeptide truncation, the special function of PPC domain, and the molecular weight of the C-terminal-processed mature keratinase KerSMD. This work not only provides a new way to overproduce keratinases but also helps to explore keratinases folding mechanism.     

One- and two-dimensional copper benzenedicarboxylate coordination polymers incorporating the flexible tethering diimine N,N′-bis(3-pyridylmethyl)piperazine: Synthesis,structure, and thermal properties

A family of three copper benzenedicarboxylate coordination polymers has been constructed using the conformationally flexible and hydrogen-bonding capable tethering ligand N,N′-bis(3-pyridylmethyl)piperazine (3-bpmp). These three coordination polymers have been characterized via single crystal X-ray diffraction, infrared spectroscopy and elemental and thermogravimetric analysis. {[Cu(ph)(Hph)(H3-bpmp)(H₂O)] · 3H₂O} (1, ph = phthalate) manifests a 1-D chain motif held into a pseudo 3-D supramolecular structure through hydrogen bonding. While both {[Cu(ip)(3-bpmp)(H₂O)] · 2H₂O} (2, ip = isophthalate) and [Cu(tp)(3-bpmp)] (3, tp = terephthalate) exhibit 2-D (4,4) rhomboid grid-like layers, they possess different layer stacking patterns and supramolecular interactions due to coordination geometry variances.     

Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions

BackgroundThe intestinal epithelium forms a barrier that food allergens must cross in order to induce sensitization. The aim of this study was to evaluate the impact of the plant-derived food cysteine protease — actinidin (Act d1) on the integrity of intestinal epithelium tight junctions (TJs).MethodsEffects of Act d1 on the intestinal epithelium were evaluated in Caco-2 monolayers and in a mouse model by measuring transepithelial resistance and in vivo permeability. Integrity of the tight junctions was analyzed by confocal microscopy. Proteolysis of TJ protein occludin was evaluated by mass spectrometry.ResultsActinidin (1 mg/mL) reduced the transepithelial resistance of the cell monolayer by 18.1% (after 1 h) and 25.6% (after 4 h). This loss of barrier function was associated with Act d 1 disruption of the occludin and zonula occludens (ZO)-1 network. The effect on intestinal permeability in vivo was demonstrated by the significantly higher concentration of 40 kDa FITC-dextran (2.33 μg/mL) that passed from the intestine into the serum of Act d1 treated mice in comparison to the control group (0.5 μg/mL). Human occludin was fragmented, and putative Act d1 cleavage sites were identified in extracellular loops of human occludin.ConclusionAct d1 caused protease-dependent disruption of tight junctions in confluent Caco-2 cells and increased intestinal permeability in mice.General significanceIn line with the observed effects of food cysteine proteases in occupational allergy, these results suggest that disruption of tight junctions by food cysteine proteases may contribute to the process of sensitization in food allergy.     

Chitin and chitosan preparation from shrimp shells using optimized enzymatic deproteinization

Different crude microbial proteases were applied for chitin extraction from shrimp shells. A Box–Behnken design with three variables and three levels was applied in order to approach the prediction of optimal enzyme/substrate ratio, temperature and incubation time on the deproteinization degree with Bacillus mojavensis A21 crude protease. These optimal conditions were: an enzyme/substrate ratio of 7.75 U/mg, a temperature of 60 °C and an incubation time of 6 h allowing to predict 94 ± 4% deproteinization. Experimentally, in these optimized conditions, a deproteinization degree of 88 ± 5% was obtained in good agreement with the prediction and larger than values generally given in literature. The deproteinized shells were then demineralized to obtain chitin which was converted to chitosan by deacetylation and its antibacterial activity against different bacteria was investigated. Results showed that chitosan dissolved at 50 mg/ml markedly inhibited the growth of most Gram-negative and Gram-positive bacteria tested.     

Development of an immunochromatographic assay for diagnosing the production of IMP-type metallo-β-lactamases that mediate carbapenem resistance in Pseudomonas

Rapid and reliable detection of carbapenem-resistant bacteria is an important infection-control measure and a crucial aspect of antimicrobial chemotherapy. IMP-type metallo-β-lactamase (MBL) is an emzyme that mediate carbapenem resistance in bacteria. Here, an immunochromatographic assay was newly developed using novel monoclonal antibodies (mAbs) recognizing IMP-type MBL. Epitope mapping of mAbs and mutational analysis of the epitope region in IMP antigen suggested that the mAbs could react to all known subtypes of IMP-type MBL. Evaluation of the assay using Pseudomonas aeruginosa strains (n = 248) showed that the results of the immunochromatographic detection of the IMP-type MBLs were fully consistent with those of the PCR analysis for bla_IMP genes, showing false positives and negatives. All positive strains were resistant to carbapenem (MIC ≥ 16 μg/ml). The assay also accurately distinguished the production of IMP-type MBLs in Pseudomonas putida, Acinetobacter baumannii, and Alcaligenes xylosoxidans. The detection limit of the assay was 5.7 × 10⁴ cfu per test. Taken together, these data suggest that the developed assay can be used for rapid and reliable diagnosis of the production of IMP-type MBLs in Gram-negative bacteria.     

Secretory expression and characterization of the recombinant myofibril-bound serine proteinase of crucian carp (Carassius auratus) in Pichia pastoris

The myofibril-bound serine proteinase (MBSP) is effective in the degradation of myofibrillar proteins, including myosin heavy chain (MHC), α-actinin, actin, and tropomyosin and was thus regarded as an important proteinase responsible for the metabolism of fish muscle in vivo. In order to better understand the characteristic differences between native MBSP and recombinant MBSP (rMBSP) and to obtain large quantity of MBSP for its application in protein science study, the crucian carp MBSP gene was cloned (669 bp) and expressed in Pichia pastoris (P. pastoris). The recombinant P. pastoris strain was cultured in shake flasks, and 66.85 mg rMBSP/L in the fermentation supernatant was obtained. SDS-polyacrylamide gel electrophoresis (PAGE) showed a main protein band with molecular weight of approximately 36 kDa. Substrate specificity analysis revealed that the rMBSP specifically cleaved substrates at the carboxyl side of lysine residue which differed from native MBSP that cleaved substrates at the carboxyl side of arginine and lysine residues. The optimum temperature and optimum pH range of the rMBSP were 55 °C and pH 7.5, respectively. Furthermore, similar to native MBSP, the rMBSP also revealed high thermostability and pH stability and is effective in degradation of myofibrillar proteins from the skeletal muscle of crucian carp.