Prokaryote-derived protein inhibitors of peptidases: A sketchy occurrence and mostly unknown function

In metazoan organisms protein inhibitors of peptidases are important factors essential for regulation of proteolytic activity. In vertebrates genes encoding peptidase inhibitors constitute up to 1% of genes reflecting a need for tight and specific control of proteolysis especially in extracellular body fluids. In stark contrast unicellular organisms, both prokaryotic and eukaryotic consistently contain only few, if any, genes coding for putative peptidase inhibitors. This may seem perplexing in the light of the fact that these organisms produce large numbers of proteases of different catalytic classes with the genes constituting up to 6% of the total gene count with the average being about 3%. Apparently, however, a unicellular life-style is fully compatible with other mechanisms of regulation of proteolysis and does not require protein inhibitors to control their intracellular and extracellular proteolytic activity. So in prokaryotes occurrence of genes encoding different types of peptidase inhibitors is infrequent and often scattered among phylogenetically distinct orders or even phyla of microbiota. Genes encoding proteins homologous to alpha-2-macroglobulin (family I39), serine carboxypeptidase Y inhibitor (family I51), alpha-1-peptidase inhibitor (family I4) and ecotin (family I11) are the most frequently represented in Bacteria. Although several of these gene products were shown to possess inhibitory activity, with an exception of ecotin and staphostatins, the biological function of microbial inhibitors is unclear. In this review we present distribution of protein inhibitors from different families among prokaryotes, describe their mode of action and hypothesize on their role in microbial physiology and interactions with hosts and environment.     

Regulation of expression of genes encoding digestive proteases in the gut of a polyphagous lepidopteran larva in response to dietary protease inhibitors

Abstract. Larvae of Helicoverpa armigera (Hübner), a polyphagous lepidopteran crop pest, adapt to the presence of protease inhibitors in their diet by differential regulation of multiple genes encoding digestive proteases. The time‐course of their response to dietary soybean Kunitz trypsin inhibitor (SKTI) involves several stages; an initial up‐regulation of all protease genes assayed (up to 12 h after exposure to inhibitor) is succeeded by a longer‐term down‐regulation of expression of specific genes that encode proteases most sensitive to the inhibitor, whereas genes encoding putative inhibitor‐insensitive proteases continue to be up‐regulated (after 24 h of exposure). Consequently, insect protease activity changes from being sensitive to the inhibitor, to being largely insensitive. The insect response is comparable in its timescale with that of the synthesis of protease inhibitors in the plant wounding response. SKTl causes similar effects on protease gene expression and gut protease activity when fed in diets containing casein or hydrolysed casein as sources of amino acid, suggesting that the insect response is not mediated through inhibition of digestive proteolysis. Soybean Bowman–Birk inhibitor, which has a broader range of inhibitory activity against gut proteases in H. armigera, but is a less effective inhibitor on an I₅₀ basis, proves to be a more effective antimetabolite than SKTI, but does not induce inhibitor‐insensitive protease activity because it causes a general up‐regulation of protease‐encoding genes. A possible mechanism to account for these different responses is discussed.     

Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: identification of proteins binding the delta-endotoxin, Cry1Ac of Bacillus thuringiensis

Helicoverpa armigera midgut proteins that bind the Bacillus thuringiensis (Bt) delta-endotoxin Cry1Ac were purified by affinity chromatography. SDS-PAGE showed that several proteins were eluted with N-acetylgalactosamine and no further proteins were detected after elution with urea. Tandem mass spectral data for tryptic peptides initially indicated that the proteins resembled aminopeptidases (APNs) from other lepidopterans and cDNA sequences for seven APNs were isolated from H. armigera through a combination of cloning with primers derived from predicted peptide sequences and established EST libraries. Phylogenetic analysis showed lepidopteran APN genes in nine clades of which five were part of a lepidopteran-specific radiation. The Cry1Ac-binding proteins were then identified with four of the seven HaAPN genes. Three of those four APNs are likely orthologs of APNs characterised as Cry1Ac-binding proteins in other lepidopterans. The fourth Cry1Ac-binding APN has orthologs not previously identified as Cry1Ac-binding partners. The HaAPN genes were expressed predominantly in the midgut through larval development. Each showed consistent expression along the length of the midgut but five of the genes were expressed at levels about two orders of magnitude greater than the remaining two. The remaining mass spectral data identified sequences encoding polycalin proteins with multiple lipocalin-like domains. A polycalin has only been previously reported in another lepidopteran, Bombyx mori, but polycalins in both species are now linked with binding of Bt Cry toxins. This is the first report of hybrid, lipocalin-like domains in shorter polycalin sequences that are not present in the longest sequence. We propose that these hybrid domains are generated by alternative splicing of the mRNA.     

Molecular cloning and characterization of two rat renal kallikrein genes

Kallikreins compose a multigene family coding for a subgroup of serine proteases, which are involved in the processing of bioactive peptides. Two rat kallikrein-related genes, RSKG-7 (rat submandibular gland kallikrein gene 7) and RSKG-3, have been cloned and their sequences analyzed. RSKG-7 is approximately 4200 bases in length and consists of five exons and four introns. The 5' end region contains the variant CATAT box and TTTAAA box; the 3' end region contains the polyadenylation signal AATAAA. This gene encodes a putative 28,935-dalton preproenzyme of 261 amino acids (aa). The active enzyme consists of 237 aa and is preceded by a deduced signal peptide of 18 aa and a profragment of 6 aa. RSKG-3 is highly homologous to RSKG-7 in terms of its sequence and structure; it encodes a 28,730-dalton prepropeptide consisting of a signal peptide of 18 aa, a profragment of 6 aa, and an active peptide of 235 aa. Sequence comparisons of RSKG-7, RSKG-3, and other kallikrein-related enzymes reveal the key amino acid residues needed for both serine protease activity (His/Asp/Ser) and kallikrein-like cleavage specificity at basic amino acids. Northern blot analyses using specific oligonucleotide probes demonstrate that, among the 12 tissues studied, RSKG-7 and RSKG-3 are expressed in the rat kidney and submandibular gland. Castration of male rats results in a decrease in submandibular gland RSKG-7 mRNA, which can be restored to the normal level by treatment with thyroxine or testosterone. On the other hand, neither castration nor hormonal manipulation affects RSKG-7 mRNA levels in the kidney.     

Regulation of the Escherichia coli sigma-dependent envelope stress response

The Escherichia colisigma(E)-dependent stress response pathway controls the expression of genes encoding periplasmic folding catalysts, proteases, biosynthesis enzymes for lipid A (a component of lipopolysaccharide or LPS) and other proteins known or predicted to function in or produce components of the envelope. When E. coli is subjected to heat or other stresses that generate unfolded envelope proteins, sigma(E) activity is induced. Four key players in this signal transduction pathway have been identified: RseA, an inner membrane sigma(E) antisigma factor; RseB, a periplasmic protein that binds to the periplasmic face of RseA; and the DegS and YaeL proteases. The major point of regulation, the interaction between sigma(E) and RseA, is primarily controlled by the stability of RseA. Envelope stress promotes RseA degradation, which occurs by a proteolytic cascade initiated by DegS. There is evidence that one sigma(E)-inducing stress (OmpC overexpression) directly activates DegS to cleave RseA. Secondarily, envelope stress may relieve RseB-mediated enhancement of RseA activity. Additional levels of control upon sigma(E) activity may become evident upon further study of this stress response pathway.     

Thiol-dependent proteases of African trypanosomes. Analysis by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels co-polymerized with fibrinogen

The proteases of several species of African trypanosomes were analysed by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels containing fibrinogen or collagen. After electrophoresis the gels were incubated in the presence of enzyme activators and/or inhibitors and then stained with Coomassie brilliant blue. The areas where the proteolytic activity had degraded the fibrinogen did not stain and so formed clear bands against a blue background. The proteases were found to have pH optima between 5 and 6, and required dithiothreitol or 2-mercaptoethanol for full expression of their activity. They were inhibited by amino acid chloromethanes, iodoacetamide, p-chloromercuribenzoate and other inhibitors of the thiol-dependent proteases, as well as by the trypanocidal drugs berenil (4,4'-diamidinodiazoaminobenzene-diacetamidoacetate) and pentamidine [1,5-di-(4-amidinophenoxy)pentane-di-(2- hydroxyethanesulphonate)]. Trypanosoma evansi, Trypanosoma brucei brucei and Trypanosoma brucei gambiense each have a protease with a relative molecular mass, Mr, of 28 000. In addition they occasionally exhibit activity at higher Mr values (up to 105000). Trypanosoma congolense has a low-Mr protease (31 000) and may exhibit higher-Mr proteases (up to 150000). The protease profiles of Trypanosoma vivax differ from the other species, T. brucei or T. congolense, and are present in lesser amounts. The proteases of the cultured procyclic forms are present in much smaller amounts than those of the metacyclic or mammalian blood stream forms of these parasites. The catalytic properties and inhibition characteristics of these thiol-dependent enzymes suggest that they resemble the mammalian lysosomal cathepsins B and L.     

Expression Profile of the Schistosoma japonicum Degradome Reveals Differential Protease Expression Patterns and Potential Anti-schistosomal Intervention Targets

Blood fluke proteases play pivotal roles in the processes of invasion, nutrition acquisition, immune evasion, and other host-parasite interactions. Hundreds of genes encoding putative proteases have been identified in the recently published schistosome genomes. However, the expression profiles of these proteases in Schistosoma species have not yet been systematically analyzed. We retrieved and culled the redundant protease sequences of Schistosoma japonicum, Schistosoma mansoni, Echinococcus multilocularis, and Clonorchis sinensis from public databases utilizing bioinformatic approaches. The degradomes of the four parasitic organisms and Homo sapiens were then comparatively analyzed. A total of 262 S. japonicum protease sequences were obtained and the expression profiles generated using whole-genome microarray. Four main clusters of protease genes with different expression patterns were identified: proteases up-regulated in hepatic schistosomula and adult worms, egg-specific or predominantly expressed proteases, cercaria-specific or predominantly expressed proteases, and constantly expressed proteases. A subset of protease genes with different expression patterns were further validated using real-time quantitative PCR. The present study represents the most comprehensive analysis of a degradome in Schistosoma species to date. These results provide a firm foundation for future research on the specific function(s) of individual proteases and may help to refine anti-proteolytic strategies in blood flukes.     

The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria

Background  

The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide.     

Gene expression in autumn leaves

Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula x tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.     

A genomic survey of proteases in Aspergilli

Background

Proteases can hydrolyze peptides in aqueous environments. This property has made proteases the most important industrial enzymes by taking up about 60% of the total enzyme market. Microorganisms are the main sources for industrial protease production due to their high yield and a wide range of biochemical properties. Several Aspergilli have the ability to produce a variety of proteases, but no comprehensive comparative study has been carried out on protease productivity in this genus so far.

Results

We have performed a combined analysis of comparative genomics, proteomics and enzymology tests on seven Aspergillus species grown on wheat bran and sugar beet pulp. Putative proteases were identified by homology search and Pfam domains. These genes were then clusters based on orthology and extracellular proteases were identified by protein subcellular localization prediction. Proteomics was used to identify the secreted enzymes in the cultures, while protease essays with and without inhibitors were performed to determine the overall protease activity per protease class. All this data was then integrated to compare the protease productivities in Aspergilli.

Conclusions

Genomes of Aspergillus species contain a similar proportion of protease encoding genes. According to comparative genomics, proteomics and enzymatic experiments serine proteases make up the largest group in the protease spectrum across the species. In general wheat bran gives higher induction of proteases than sugar beet pulp. Interesting differences of protease activity, extracellular enzyme spectrum composition, protein occurrence and abundance were identified for species. By combining in silico and wet-lab experiments, we present the intriguing variety of protease productivity in Aspergilli.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-523) contains supplementary material, which is available to authorized users.     

Sea urchin metalloproteases: a genomic survey of the BMP-1/tolloid-like, MMP and ADAM families

Analysis of the Strongylocentrotus purpuratus genome has revealed approximately 240 metalloprotease genes, and they represent all 23 families expressed in vertebrates. EST/cDNA sequencing and microarray analysis show that nearly 70% are represented in embryo RNA. Among them are many metalloproteases with demonstrated developmental roles in other systems-BMP-1/TLD (tolloid) (astacins), MMPs (matrix metalloproteases) and the ADAMs (disintegrin/metalloproteases). The developmental functions of these kinds of metalloproteases include modifying the extracellular matrix, regulating signaling pathways or modulating cellular adhesive properties. The unexpectedly large number of BMP-1/TLD-like protease genes (23) results primarily from expansion of a set encoding an unusual domain conserved in structure and primary sequence only in nematode astacins. Such proteases may have interesting developmental functions because the expression patterns of several are highly regulated along the primary axis at times when cell differentiation and morphogenesis begin. The size of the sea urchin MMP family and the clustered arrangement of many of its members are similar to vertebrates, but phylogenetic analyses suggest that different ancestral genes were independently amplified in sea urchins and vertebrates. One expansion appears to be genes encoding MMPs that have putative transmembrane domains and may be membrane-tethered (MT). Interestingly, the genes encoding TIMPs, inhibitors of MMPs, have also been amplified and the 10 genes are tandemly arranged in a single cluster. In contrast, there are fewer ADAM and ADAMTS genes in sea urchins, but they represent all but one of the chordate-specific groups. The genome sequence now opens the door to experimental manipulations designed to understand how modulation of the extracellular environment affects development.     

Apicomplexan rhomboids have a potential role in microneme protein cleavage during host cell invasion

Apicomplexan parasites secrete transmembrane (TM) adhesive proteins as part of the process leading to host cell attachment and invasion. These microneme proteins are cleaved in their TM domains by an unidentified protease termed microneme protein protease 1 (MPP1). The cleavage site sequence (IA downward arrowGG), mapped in the Toxoplasma gondii microneme proteins TgMIC2 and TgMIC6, is conserved in microneme proteins of other apicomplexans including Plasmodium species. We report here the characterisation of novel T. gondii proteins belonging to the rhomboid family of intramembrane-cleaving serine proteases. T. gondii possesses six genes encoding rhomboid-like proteins. Four are localised along the secretory pathway and therefore constitute possible candidates for MPP1 activity. Toxoplasma rhomboids TgROM1, TgROM2 and TgROM5 cleave the TM domain of Drosophila Spitz, an established substrate for rhomboids from several species, demonstrating that they are active proteases. In addition, TgROM2 cleaves chimeric proteins that contain the TM domains of TgMIC2 and TgMIC12.     

Cloning and characterization of chymotrypsin- and trypsin-like cDNAs from the gut of the Hessian fly [Mayetiola destructor (Say)

Fifteen unique cDNA clones encoding trypsin- or chymotrypsin-like proteins were cloned and characterized from a gut cDNA library derived from Hessian fly [Mayetiola destructor (Say)] larvae. Based on sequence similarities, the cDNAs were sorted into five gene groups, which were named MDP1 to MDP5. Two of the gene groups, MDP1 and MDP2, encoded chymotrypsin-like proteins; the other three encoded putative trypsins. All deduced proteins have conserved His(87), Asp(136), and Ser(241) residues for the catalytic triad and three pairs of cysteine residues for disulfide bridge configurations. The substrate specificity determination residue at position 235 was also conserved in the putative trypsins and chymotrypsins. In addition, all the deduced protein precursors had a typical secretion signal peptide and activation peptide. Northern blot analysis revealed that all these gene groups were exclusively expressed in the larval stage. The expression profiles for each gene group differed significantly in different ages of the larva, as well as in different tissues. Protease activity analysis of gut extract, using specific inhibitors, demonstrated that serine proteases were the major digestive enzymes in the gut of M. destructor larvae. Serine protease inhibitors inhibited as much as 90% proteolytic activities of gut extract, whereas inhibitors specific to other proteases, including cysteine proteases, aspartic proteases, and metallo-proteases, inhibited only 10-24% of gut protease activity.     

The ability of plants to secrete proteases by roots.

The aim of our study was to find out if the culture medium of aseptically cultivated seedlings exhibits proteolytic activity and if this event is universal in angiospermous plants. Seedlings of 15 agricultural and wild-living plant species were cultivated for 14days without any addition of nutrients. Our studies showed that roots of higher plants could secrete proteases and that levels of proteolytic activity in the culture medium of individual species (and cultivars of the same species) could be significantly different. The differences between quantities of the secreted proteases were connected neither with the fresh weight of the growing seedlings nor with the surface of the root system. No proteins were required to induce secretion of proteases. The culture medium of a few studied species (Allium porrum, Zea mays, Helianthus annuus) showed the highest proteolytic activity at pH 7. Studies of the influence of standard protease inhibitors showed that examined proteases belong to the cysteine protease family. The results suggest that the apical parts of roots exuded proteases more intensively than mature parts. Our studies suggest that some plant species could develop a strategy to actively increase the level of free amino acids in the soil solution as a source of N. Our results may contribute to studying plant N nutrition in natural ecosystems and to increasing yield after organic fertilization of agricultural species.     

Genetic manipulation of Bacillus amyloliquefaciens.

Application of modern gene technology to strain improvement of the industrially important bacterium Bacillus amyloliquefaciens is reported. Several different plasmid constructions carrying the alpha-amylase gene (amyE) from B. amyloliquefaciens were amplified in this species either extrachromosomally or intrachromosomally. The amyE gene cloned on a pUB110-derived high copy plasmid pKTH10 directed the highest yields both in rich laboratory medium and in crude industrial medium. The alpha-amylase activity, when compared with the parental strain, was enhanced up to 20-fold in the pKTH 10 transformant. This strain showed decreased activities for other exoenzymes, such as proteases and beta-glucanase suggesting common limiting resources in the processing of these enzymes. Deletions were made in vitro in genes encoding neutral (nprE), alkaline (aprE) protease and beta-glucanase (bglA). The engineered genes were cloned into the thermosensitive plasmid pE194, and the resulting plasmids were used to replace the corresponding wild type chromosomal genes in B. amyloliquefaciens by integration-excision at non-permissive temperature. The double mutant deficient in the major proteases (delta nprE delta aprE) showed about a 2-fold further enhancement in alpha-amylase production in the industrial medium compared with the relevant wild type backgroud, both when plasmid-free and when transformed with pKTH10; this strain also produced elevated levels of the chromosomally-encoded beta-glucanase; pKTH10 was stably maintained both in the wild type strain and in the delta nprE delta aprE mutant. We suggest that the higher yields in alpha-amylase and beta-glucanase in the delta nprE delta aprE strain are primarily due to improved access to limiting resources, and that decreased proteolytic degradation may have had a secondary role in retaining the high activity obtained.     

Mouse DESC1 is located within a cluster of seven DESC1-like genes and encodes a type II transmembrane serine protease that forms serpin inhibitory complexes

We report the identification and functional analysis of a type II transmembrane serine protease encoded by the mouse differentially expressed in squamous cell carcinoma (DESC) 1 gene, and the definition of a cluster of seven homologous DESC1-like genes within a 0.5-Mb region of mouse chromosome 5E1. This locus is syntenic to a region of human chromosome 4q13.3 containing the human orthologues of four of the mouse DESC1-like genes. Bioinformatic analysis indicated that all seven DESC1-like genes encode functional proteases. Direct cDNA cloning showed that mouse DESC1 encodes a multidomain serine protease with an N-terminal signal anchor, a SEA (sea urchin sperm protein, enterokinase, and agrin) domain, and a C-terminal serine protease domain. The mouse DESC1 mRNA was present in epidermal, oral, and male reproductive tissues and directed the translation of a membrane-associated 60-kDa N-glycosylated protein with type II topology. Mouse DESC1 was synthesized in insect cells as a zymogen that could be activated by exposure to trypsin. The purified activated DESC1 hydrolyzed synthetic peptide substrates, showing a preference for Arg in the P1 position. DESC1 proteolytic activity was abolished by generic inhibitors of serine proteases but not by other classes of protease inhibitors. Most interestingly, DESC1 formed stable inhibitory complexes with both plasminogen activator inhibitor-1 and protein C inhibitor that are expressed in the same tissues with DESC1, suggesting that type II transmembrane serine proteases may be novel targets for serpin inhibition. Together, these data show that mouse DESC1 encodes a functional cell surface serine protease that may have important functions in the epidermis, oral, and reproductive epithelium.