The rhomboid-like superfamily: molecular mechanisms and biological roles.

The rhomboid proteases were first discovered as regulators of Drosophila EGF receptor signaling; soon after, it was recognized that they represented the founder members of a widespread family of intramembrane serine proteases conserved in all kingdoms. More recently still, the family was promoted to a superfamily, encompassing a wide variety of distantly related proteins. One of the surprises has been that many members of the rhomboid-like superfamily are not active proteases. Given the size of this clan, and its relatively recent discovery, there is still much to learn. Nevertheless, we already understand much about how rhomboid proteases perform their surprising function of cleaving transmembrane domains. We also already know that members of the rhomboid-like superfamily participate in biological functions as diverse as growth factor signaling, mitochondrial dynamics, inflammation, parasite invasion, and the machinery of protein quality control. Their potential medical significance is now becoming apparent in several areas.

Hemagglutinin Cleavability, Acid Stability, and Temperature Dependence Optimize Influenza B Virus for Replication in Human Airways.

Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population.IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses.

Comprehensive Study of the Proteome and Transcriptome of the Venom of the Most Venomous European Viper: Discovery of a New Subclass of Ancestral Snake Venom Metalloproteinase Precursor-Derived Proteins.

The nose-horned viper, its nominotypical subspecies Vipera ammodytes ammodytes ( Vaa), in particular, is, medically, one of the most relevant snakes in Europe. The local and systemic clinical manifestations of poisoning by the venom of this snake are the result of the pathophysiological effects inflicted by enzymatic and nonenzymatic venom components acting, most prominently, on the blood, cardiovascular, and nerve systems. This venom is a very complex mixture of pharmacologically active proteins and peptides. To help improve the current antivenom therapy toward higher specificity and efficiency and to assist drug discovery, we have constructed, by combining transcriptomic and proteomic analyses, the most comprehensive library yet of the Vaa venom proteins and peptides. Sequence analysis of the venom gland cDNA library has revealed the presence of messages encoding 12 types of polypeptide precursors. The most abundant are those for metalloproteinase inhibitors (MPis), bradykinin-potentiating peptides (BPPs), and natriuretic peptides (NPs) (all three on a single precursor), snake C-type lectin-like proteins (snaclecs), serine proteases (SVSPs), P-II and P-III metalloproteinases (SVMPs), secreted phospholipases A2 (sPLA2s), and disintegrins (Dis). These constitute >88% of the venom transcriptome. At the protein level, 57 venom proteins belonging to 16 different protein families have been identified and, with SVSPs, sPLA2s, snaclecs, and SVMPs, comprise ∼80% of all venom proteins. Peptides detected in the venom include NPs, BPPs, and inhibitors of SVSPs and SVMPs. Of particular interest, a transcript coding for a protein similar to P-III SVMPs but lacking the MP domain was also found at the protein level in the venom. The existence of such proteins, also supported by finding similar venom gland transcripts in related snake species, has been demonstrated for the first time, justifying the proposal of a new P-IIIe subclass of ancestral SVMP precursor-derived proteins.

Shotgun Proteomics Analysis of Saliva and Salivary Gland Tissue from the Common Octopus Octopus vulgaris.

The salivary apparatus of the common octopus ( Octopus vulgaris) has been the subject of biochemical study for over a century. A combination of bioassays, behavioral studies and molecular analysis on O. vulgaris and related species suggests that its proteome should contain a mixture of highly potent neurotoxins and degradative proteins. However, a lack of genomic and transcriptomic data has meant that the amino acid sequences of these proteins remain almost entirely unknown. To address this, we assembled the posterior salivary gland transcriptome of O. vulgaris and combined it with high resolution mass spectrometry data from the posterior and anterior salivary glands of two adults, the posterior salivary glands of six paralarvae and the saliva from a single adult. We identified a total of 2810 protein groups from across this range of salivary tissues and age classes, including 84 with homology to known venom protein families. Additionally, we found 21 short secreted cysteine rich protein groups of which 12 were specific to cephalopods. By combining protein expression data with phylogenetic analysis we demonstrate that serine proteases expanded dramatically within the cephalopod lineage and that cephalopod specific proteins are strongly associated with the salivary apparatus.

Understanding serine proteases implications on Leishmania spp lifecycle.

Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment.

Characterization of an antigenic serine protease in the Trichinella spiralis adult.

Serine proteases have been identified as important molecules that are involved in many parasitic infections, and these molecules have also been suggested to play important roles in Trichinella spiralis infections. In the present study, the antigenic serine protease gene Ts-ADSp-7, which was screened from a cDNA library of Trichinella spiralis Adults at 3 days post-infection (p.i.), was cloned and expressed in Escherichia coli. The encoded protein, Ts-ADSp-7, revealed a potential trypsin-like serine protease domain but lacked substrate banding site at position 227 and protease activity. Transcription could be detected in the Adult and muscle larval stage but not in the newborn larval stage, where no fluorescent signal was detected. Western blot analysis revealed that the 3 days p.i. Adults and muscle larvae could secrete Ts-ADSp-7. Interestingly, strong fluorescent signal of Ts-ADSp-7 could be detected in the nucleoli of the enlarged muscle cell nuclei from 12 to 16 days p.i. and in the ß-stichosomes of the muscle larvae from 16 to 35 days p.i.. The coagulation assay indicated that Ts-ADSp-7 could inhibit intrinsic coagulation pathway. Regarding the putatively important function of the serine protease in the helminth infection to hosts, a total of 81 serine proteases were found in the parasite and mainly comprised eight subfamilies. These subfamilies exhibited high similarity to transmembrane serine protease, coagulation factor XI, lipocalin, guanylin, ceropin, kallikrein, and plasminogen. Moreover, stage specificity was detected in several subfamilies. In summary, the putatively inactive serine protease-like protein Ts-ADSp-7 could inhibit blood coagulation, and the protein is located in the enlarged nuclei of nurse cells during capsule formation. Furthermore, members of the serine protease family in the parasite might be important molecules in the parasite-host interaction.

Bacterial serine proteases secreted by the autotransporter pathway: classification, specificity, and role in virulence.

Serine proteases exist in eukaryotic and prokaryotic organisms and have emerged during evolution as the most abundant and functionally diverse group. In Gram-negative bacteria, there is a growing family of high molecular weight serine proteases secreted to the external milieu by a fascinating and widely employed bacterial secretion mechanism, known as the autotransporter pathway. They were initially found in Neisseria, Shigella, and pathogenic Escherichia coli, but have now also been identified in Citrobacter rodentium, Salmonella, and Edwardsiella species. Here, we focus on proteins belonging to the serine protease autotransporter of Enterobacteriaceae (SPATEs) family. Recent findings regarding the predilection of serine proteases to host intracellular or extracellular protein-substrates involved in numerous biological functions, such as those implicated in cytoskeleton stability, autophagy or innate and adaptive immunity, have helped provide a better understanding of SPATEs' contributions in pathogenesis. Here, we discuss their classification, substrate specificity, and potential roles in pathogenesis.

The high prevalence of serine protease autotransporters of Enterobacteriaceae (SPATEs) in Escherichia coli causing neonatal septicemia.

Serine protease autotransporters of Enterobacteriaceae (SPATEs) are secreted proteins demonstrating diverse virulence functions. The distribution of SPATEs is studied among diarrheagenic and extraintestinal pathogenic Escherichia coli. However, the contribution of SPATEs to the virulence of neonatal septicemic Escherichia coli (NSEC) has not yet been elucidated. This study was undertaken to evaluate the prevalence and phylogenetic distribution of different subtypes of SPATEs among NSEC. The presence of virulence factors and subtypes of SPATEs among different E. coli isolates was determined by polymerase chain reaction (PCR). E. coli phylogrouping was done by triplex PCR. Clonality of the isolates was assessed by pulsed-field gel electrophoresis (PFGE). The presence of SPATEs was significantly higher among the septicemic isolates (89 %) than the fecal (7.5 %) and environmental isolates (2.5 %). Vat (vacuolating autotransporter toxin) and Sat (secreted autotransporter toxin) were found to be the two most predominant SPATEs. The incidence of SPATEs was high in septicemic isolates of phylogroups A and B1 (87 %), lacking other virulence factors. The high prevalence of SPATEs in the non-B2 phylogroups of septicemic isolates in comparison with fecal and environmental isolates indicates an association of SPATEs with NSEC. The NSEC isolates were found to be clonally distinct, suggesting that the high prevalence of SPATEs was not due to clonal relatedness of the isolates. This study is the first to show the association of SPATEs with NSEC. The presence of SPATEs in the septicemic/NSEC isolates may be considered as the most discriminatory trait studied here.

Serine protease autotransporters from Shigella flexneri and pathogenic Escherichia coli target a broad range of leukocyte glycoproteins.

The serine protease autotransporters of Enterobacteriaceae (SPATEs) are secreted by pathogenic Gram-negative bacteria through the autotransporter pathway. We previously classified SPATE proteins into two classes: cytotoxic (class 1) and noncytotoxic (class 2). Here, we show that Pic, a class 2 SPATE protein produced by Shigella flexneri 2a, uropathogenic and enteroaggregative Escherichia coli strains, targets a broad range of human leukocyte adhesion proteins. Substrate specificity was restricted to glycoproteins rich in O-linked glycans, including CD43, CD44, CD45, CD93, CD162 (PSGL-1; P-selectin glycoprotein ligand 1), and the surface-attached chemokine fractalkine, all implicated in leukocyte trafficking, migration, and inflammation. N-terminal sequencing of proteolytic products revealed Pic (protease involved in colonization) cleavage sites to occur before Thr or Ser residues. The purified carbohydrate sLewis-X implied in inflammation and malignancy inhibited cleavage of PSGL-1 by Pic. Exposure of human leukocytes to purified Pic resulted in polymorphonuclear cell activation, but impaired chemotaxis and transmigration; Pic-treated T cells underwent programmed cell death. We also show that the Pic-related protease Tsh/Hbp, implicated in extraintestinal infections, exhibited a spectrum of substrates similar to those cleaved by Pic. In the guinea pig keratoconjunctivitis model, a Shigella pic mutant induced greater inflammation than its parent strain. We suggest that the class-2 SPATEs represent unique immune-modulating bacterial virulence factors.

Characterization of Bactrocera dorsalis serine proteases and evidence for their indirect role in insecticide tolerance.

The oriental fruit fly Bactrocera dorsalis (Hendel) causes devastating losses to agricultural crops world-wide and is considered to be an economically important pest. Little is known about the digestive enzymes such as serine proteases (SPs) in B. dorsalis, which are important both for energy supply and mitigation of fitness cost associated with insecticide tolerance. In this study, we identified five SP genes in the midgut of B. dorsalis, and the alignments of their deduced amino acid sequences revealed the presence of motifs conserved in the SP superfamily. Phylogenetic analyses with known SPs from other insect species suggested that three of them were trypsin-like proteases. Analyses of the expression profiles among the different developmental stages showed that all five genes were most abundant in larvae than in other stages. When larvae were continuously fed on diet containing 0.33 µg/g ß-Cypermethrin, expression of all five genes were upregulated in the midgut but the larval development was delayed. Biochemical assays were consistent with the increased protease activity exhibited by SPs in the midgut after treatment with ß-Cypermethrin. Taken together, these findings provide evidence for the hypothesis that enhanced SP activity may play an indirect role in relieving the toxicity stress of insecticide in B. dorsalis.

[Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper].

Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.

Biochemical and functional characterization of BmooSP, a new serine protease from Bothrops moojeni snake venom.

In this work, we describe the purification and characterization of a new serine protease enzyme from Bothrops moojeni snake venom (BmooSP). On SDS-PAGE, BmooSP was found to be a single-chain protein with an apparent molecular mass of 36,000 and 32,000 under reduced and non-reduced conditions, respectively. Mass spectrometry analysis showed that the BmooSP is composed by two isoforms with molecular mass of 30,363 and 30,070, respectively. The purified enzyme consists of 277 amino acid residues, disregarding the cysteine and tryptophan residues that have been degraded by acid hydrolysis, and its N-terminal sequence showed similarity with other serine protease enzymes. BmooSP induced blood-clotting in vitro, defibrination in vivo, caseinolytic and fibrin(ogen)olytic activities. The enzyme is stable at high temperatures (up to 100 °C) and shows maximum activity at pH around 7.0. Preliminary results show that BmooSP can induce the formation of a stable fibrin clot for more than 10 days. BmooSP presents medical interest because it can be used as biodegradable fibrin glue and for the treatment and prevention of cardiovascular disorders because of its ability to promote the defibrination in vivo, decreasing blood viscosity and improving blood circulation.

VaSP1, catalytically active serine proteinase from Vipera ammodytes ammodytes venom with unconventional active site triad.

VaSP1, a serine proteinase from Vipera ammodytes ammodytes venom, is a glycosylated monomer of 31.5 kDa, as determined by MALDI mass spectrometry, showing multiple isoelectric points between pH 6.5 and pH 8.5. Partial amino acid sequencing of VaSP1 by Edman degradation and MS/MS analysis identified sequences which allowed its classification among the so-called snake venom serine proteinase homologues, members of the peptidase S1 family, however being devoid of the canonical catalytic triad. Only few representatives of this group have been identified so far with just two of them characterised in detail at the protein level. Despite substitution of His57 with Arg, VaSP1 possesses proteolytic activity which can be inhibited by Pefabloc, benzamidine, Zn²âº ions, DTT and trypsin inhibitor II, a Kunitz/BPTI group member. It hydrolyses N(α)-benzoyl-Phe-Val-Arg-p-NA, exhibiting Michaelis-Menten behaviour with K(m) = 48.2 µM and V(m) = 0.019 nM s⁻¹. The pH for optimal activity on tested substrate is around 9.0. VaSP1 also cleaves insulin B-chain, digesting it at positions His¹°-Leu¹¹, Ala¹4-Leu¹5 and Tyr¹6-Leu¹7. Furthermore, the novel serine proteinase is active towards wide array of proteins involved in haemostasis where its degradation of fibrinogen, fibrin, prothrombin, factor X and plasminogen in vivo probably results in depletion of coagulation factors in blood circulation. The possibility that VaSP1 possesses anticoagulant properties has been further indicated by its ability to prolong prothrombin time and activated partial thromboplastin time.

What are the proteolytic enzymes of honey and what they do tell us? A fingerprint analysis by 2-D zymography of unifloral honeys.

Honey is a sweet and healthy food produced by honeybees (Apis mellifera L.) from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification.

Characterization of the molecular features and expression patterns of two serine proteases in Hermetia illucens (Diptera: Stratiomyidae) larvae.

To investigate the molecular scavenging capabilities of the larvae of Hermetia illucens, two serine proteases (SPs) were cloned and characterized. Multiple sequence alignments and phylogenetic tree analysis of the deduced amino acid sequences of Hi-SP1 and Hi-SP2 were suggested that Hi-SP1 may be a chymotrypsin- and Hi-SP2 may be a trypsin-like protease. Hi-SP1 and Hi-SP2 3-D homology models revealed that a catalytic triad, three disulfide bonds, and a substrate-binding pocket were highly conserved, as would be expected of a SP. E. coli expressed Hi-SP1 and Hi-SP2 showed chymotrypsin or trypsin activities, respectively. Hi-SP2 mRNAs were consistently expressed during larval development. In contrast, the expression of Hi-SP1 mRNA fluctuated between feeding and molting stages and disappeared at the pupal stages. These expression pattern differences suggest that Hi-SP1 may be a larval specific chymotrypsin-like protease involved with food digestion, while Hi-SP2 may be a trypsin-like protease with diverse functions at different stages.

Expression of four trypsin-like serine proteases from the Chinese shrimp, Fenneropenaeus chinensis, as regulated by pathogenic infection.

Four trypsin-like serine proteases, designated as Fctry1, Fctry2, Fctry3, and Fctry4, were obtained from the hepatopancreas of the Chinese shrimp, Fenneropenaeus chinensis. Three trypsin-like serine proteases, specifically Fctry1, 2, and 3 were observed to have the conserved catalytic triad (H, D, and S). Regarding Fctry4, the catalytic triad S was substituted by F, and the mature peptide was found to be inactive. Further analysis for similarities indicated that Fctry1, 2 and 4 were 89-92% identical to trypsins from Pacific white shrimp (Litopenaeus vannamei); while Fctry3 was only 42% identical to trypsins from salmon louse (Lepeophtheirus salmonis). The genomic organizations of Fctry1, 2 and 4 are also quite different from Fctry3. So, Fctry3 may be a new member of the trypsin-like serine protease family. Moreover, a semi-quantitative reverse transcription polymerase chain reaction (PCR) and quantitative real-time PCR was carried out to analyze the distribution profiles and expression patterns after they were subjected to a bacterial and viral challenge. The results showed that the four trypsin-like serine proteases were upregulated in the hepatopancreas of shrimp infected with the white spot syndrome virus (WSSV), and Fctry3 increased after a bacteria challenge. Therefore, these trypsin-like serine proteases might be involved in the innate defense reactions against different pathogens in Chinese shrimp.