Proteolytic activation of influenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium

Host cell proteases that cleave the hemagglutinin (HA) of influenza viruses in the human respiratory tract are still not identified. Here we cloned two human type II transmembrane serine proteases with known airway localization, TMPRSS2 and HAT, into mammalian expression vector. Cotransfection of mammalian cells with plasmids encoding HA and either protease resulted in HA cleavage in situ. Transient expression of either protease in MDCK cells enabled multicycle replication of influenza viruses in these cells in the absence of exogenous trypsin. These data suggest that TMPRSS2 and HAT are candidates for proteolytic activation of influenza viruses in vivo.     

Structure and energetics of protein-protein interactions: the role of conformational heterogeneity in OMTKY3 binding to serine proteases

Proteins with flexible binding surfaces can interact with numerous binding partners. However, this promiscuity is more difficult to understand in "rigid-body" proteins, whose binding results in little, or no, change in the position of backbone atoms. The binding of Kazal inhibitors to serine proteases is considered a classic case of rigid-body binding, although they bind to a wide range of proteases. We have studied the thermodynamics of binding of the Kazal serine protease inhibitor, turkey ovomucoid third domain (OMTKY3), to the serine protease subtilisin Carlsberg using isothermal titration calorimetry and have determined the crystal structure of the complex at very high resolution (1.1A). Comparison of the binding energetics and structure to other OMTKY3 interactions demonstrates that small changes in the position of side-chains can make significant contributions to the binding thermodynamics, including the enthalpy of binding. These effects emphasize that small, "rigid-body" proteins are still dynamic structures, and these dynamics make contributions to both the enthalpy and entropy of binding interactions.     

Bacillus thuringiensis crystal delta-endotoxin: role of proteases in the conversion of protoxin to toxin

The conversion of delta-endoprotoxins of Bacillus thuringiensis to active toxins is mediated by trypsin, insect gut (exogenous) and bacterial (endogenous) proteases. The biochemical aspects of exogenous and endogenous proteases involved in the conversion of protoxin to toxin are reviewed. Perhaps, these proteases also play a role in influencing the host range of toxin and in the development of resistance to toxin.     

Proteolytic conversion of rat thymus DNA polymerase alpha to a more active form.

The relationship between two DNA polymerase alpha species from mammalian tissues has been resolved with the isolation of a protease from rat thymus which converts the larger alpha polymerase (7.3S) to a smaller (5.4S) size. The proteolytic activity is present only in the chromatin fraction and the limited proteolysis is accompanied by an increase in activity of the DNA polymerase, possibly consistent with a biological control function for this phenomenon.     

Electrostatic recognition in substrate binding to serine proteases

Serine proteases of the Chymotrypsin family are structurally very similar but have very different substrate preferences. This study investigates a set of 9 different proteases of this family comprising proteases that prefer substrates containing positively charged amino acids, negatively charged amino acids, and uncharged amino acids with varying degree of specificity. Here, we show that differences in electrostatic substrate preferences can be predicted reliably by electrostatic molecular interaction fields employing customized GRID probes. Thus, we are able to directly link protease structures to their electrostatic substrate preferences. Additionally, we present a new metric that measures similarities in substrate preferences focusing only on electrostatics. It efficiently compares these electrostatic substrate preferences between different proteases. This new metric can be interpreted as the electrostatic part of our previously developed substrate similarity metric. Consequently, we suggest, that substrate recognition in terms of electrostatics and shape complementarity are rather orthogonal aspects of substrate recognition. This is in line with a 2‐step mechanism of protein‐protein recognition suggested in the literature.     

Characterization of serine proteases of Lumbriculus variegatus and their role in regeneration

Serine proteases, ubiquitous enzymes known to function in digestion and immune protection in both vertebrates and invertebrates and implicated in regeneration in some species, were investigated in the California blackworm, Lumbriculus variegatus. Several serine proteases, rather than a single enzyme with broad specificity, were present in tissue extracts from the worms. Extracts were treated with a fluorescein‐labeled peptide chloromethyl ketone that specifically binds to trypsin/thrombin‐like proteases. Denaturing gel electrophoresis of labeled extracts showed several serine proteases with their molecular weight ranging 28,000–38,000 daltons. The trypsin/thrombin‐like activity was localized, using the fluorescein‐conjugated reagent, to the pharynx and digestive tract of L. variegatus. Movement of cells labeled by the reagent into regenerating tissues suggests that some differentiated endodermal tissues were used for reformation of digestive structures during regeneration in L. variegatus. The types of serine proteases in the extracts were further characterized by inhibitor studies. Presence of plasmin‐like activity was indicated by degradation of fibrin by tissue homogenates from the worms and the inhibitory effect of aprotinin on enzymes in these extracts. The ability of L. variegatus extracts to generate clots when incubated with rabbit plasma and partial inhibition of extract activity by phenylmethylsulfonyl fluoride and hirudin indicated presence of thrombin‐like activity. Consistent with the detection of trypsin, chymotrypsin, and plasmin‐like enzymes in the extracts was partial inhibition of L. variegatus serine protease activity by aminoethyl benzenesulfonyl fluoride and soybean trypsin inhibitor. Selective inhibition of chymotrypsin‐like activity by N‐tosyl‐l ‐phenylalanine chloromethyl ketone and chymostatin as well as trypsin‐like activity by N‐tosyl‐l ‐lysine chloromethyl ketone was observed. A potential role during regeneration for serine proteases is suggested by blockage of formation of head and tail structures by aminoethyl benzenesulfonyl fluoride, an inhibitor of these proteases.     

Inhibition of human serine proteases by substituted 2-azetidinones.

trans-4-Ethoxycarbonyl-3-ethyl-1-(4-nitrophenyl-sulfonyl)-azetidin -3-one described by Firestone et al. (1990, Tetrahedron 46, 2255) as an inhibitor of human leucocyte elastase (HLE) displayed potent, time-dependent inhibition of both HLE and human cathepsin G (Cat-G). The cis-isomer was 7- and 180-fold less active, respectively. The mechanism likely involves opening of the beta-lactam ring by the active site serine to form an acyl-enzyme intermediate(s). This intermediate partitions with ratios of 4:1 between turnover of the inhibitor and formation of relatively stable enzyme-inhibitor complexes from both enzymes. The final HLE-inhibitor complex reactivated with a half-life of 48 h at 25 degrees C and was 16-fold more stable than the Cat-G-inhibitor complex. The stability of the acyl-enzymes supports a "double hit" chemical mechanism involving both serine acylation and alkylation of the histidine. These observations suggest that beta-lactams may be developed as a class of serine protease inhibitors.     

Subcellular localization of Gi alpha in human neutrophils

Subcellular fractions were prepared from human neutrophils by sucrose density gradient centrifugation and analyzed for Gi-like proteins by pertussis toxin-catalyzed [32P]ADP-ribosylation and by immunoblotting with rabbit antiserum AS/6 which recognizes purified transducin and Gi, but not Gs or Go alpha-subunits. In resting cells, approximately equal to 60% of pertussis toxin substrate retrieved from the sucrose density gradient localized to the plasma membrane-enriched fraction, approximately equal to 35% to the specific granule-enriched fraction, and approximately equal to 5% to cytosol. The azurophil granule-enriched fraction did not contain pertussis toxin substrate. In contrast to plasma membrane, the specific granule-enriched fraction demonstrated increased AS/6 immunoreactivity of a approximately equal to 41-kDa protein relative to a approximately equal to 40-kDa protein. Within the specific granule-enriched fraction, the peak of pertussis toxin substrate detected immunochemically or by [32P]ADP-ribosylation sedimented at a lighter density (rho = 1.6 g/ml) than did lactoferrin (rho = 1.19 g/ml), suggesting that the intracellular compartment bearing pertussis toxin substrate may not be the lactoferrin containing specific granule, per se. Furthermore, in neutrophils exposed to 10(-8) M N-formylmethionylleucylphenylalanine, a weak degranulating stimulus (7% lactoferrin degranulation), there was a 31-42% decline in pertussus toxin-catalyzed [32P]ADP-ribosylation of approximately equal to 40-41-kDa proteins in the specific granule-enriched fraction accompanied by a near-quantitative increase in labeling of plasma membrane. The pool of intracellular formyl peptide receptors localized to the specific granule-enriched fraction appeared functionally coupled to a cosedimenting G-protein in experiments demonstrating modulation of high affinity N-formylmethionylleucyl[3H]phenylalanine binding by guanosine 5'-(3-O-thio)triphosphate or pertussis toxin. The data indicate that neutrophils contain a surface translocatable pool of intracellular G-protein sedimenting in the specific granule-enriched fraction and support the view that mobilization of intracellular G-protein represents a mechanism by which cells can regulate receptor activity.     

A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation

Desquamation of the stratum corneum is a serine protease-dependent process. Two members of the human tissue kallikrein (KLK) family of (chymo)tryptic-like serine proteases, KLK5 and KLK7, are implicated in desquamation by digestion of (corneo)desmosomes and inhibition by desquamation-related serine protease inhibitors (SPIs). However, the epidermal localization and specificity of additional KLKs also supports a role for these enzymes in desquamation. This study aims to delineate the probable contribution of KLK1, KLK5, KLK6, KLK13, and KLK14 to desquamation by examining their interactions, in vitro, with: 1) colocalized SPI, lympho-epithelial Kazal-type-related inhibitor (LEKTI, four recombinant fragments containing inhibitory domains 1-6 (rLEKTI(1-6)), domains 6-8 and partial domain 9 (rLEKTI(6-9')), domains 9-12 (rLEKTI(9-12)), and domains 12-15 (rLEKTI(12-15)), secretory leukocyte protease inhibitor, and elafin and 2) their ability to digest the (corneo)desmosomal cadherin, desmoglein 1. KLK1 was not inhibited by any SPI tested. KLK5, KLK6, KLK13, and KLK14 were potently inhibited by rLEKTI(1-6), rLEKTI(6-9'), and rLEKTI(9-12) with Ki values in the range of 2.3-28.4 nm, 6.1-221 nm, and 2.7-416 nm for each respective fragment. Only KLK5 was inhibited by rLEKTI(12-15) (Ki = 21.8 nm). No KLK was inhibited by secretory leukocyte protease inhibitor or elafin. Apart from KLK13, all KLKs digested the ectodomain of desmoglein 1 within cadherin repeats, Ca2+ binding sites, or in the juxtamembrane region. Our study indicates that multiple KLKs may participate in desquamation through cleavage of desmoglein 1 and regulation by LEKTI. These findings may have clinical implications for the treatment of skin disorders in which KLK activity is elevated.     

The human T3 gamma chain is phosphorylated at serine 126 in response to T lymphocyte activation

The gamma subunit of the human T lymphocyte T3 antigen is rapidly phosphorylated on serine residues in vivo during the initiation of T cell activation by a polyclonal mitogen (Phaseolus vulgaris phytohemagglutinin), an activator of protein kinase C (phorbol 12,13-dibutyrate), and an elevator of intracellular calcium (ionomycin). The sites of phosphorylation were identified by comparing tryptic peptide analyses of T3 gamma chains labeled in vivo with various synthetic peptides, corresponding to portions of the cytoplasmic domain of the gamma chain that had been labeled in vitro using purified protein kinase C. Two sites, serines 123 and 126, were phosphorylated in response to ionomycin, whereas a single site, serine 126, was phosphorylated when T lymphocytes were stimulated by P. vulgaris phytohemagglutinin or when protein kinase C was directly activated by phorbol 12,13-dibutyrate. Immune activation of T cells via the protein kinase C pathway thus induces phosphorylation of a single site on the T3 gamma chain, namely serine 126.     

IL-2 activation of a PI3K-dependent STAT3 serine phosphorylation pathway in primary human T cells

Interleukin-2 (IL-2) is the major growth factor of activated T lymphocytes. By inducing cell cycle progression and protection from apoptosis in these cells, IL-2 is involved in the successful execution of an immune response. Upon binding its receptor, IL-2 activates a variety of signal transduction pathways, including the Ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and Janus kinase (JAK)/STAT cascades. In addition, activation of phosphatidylinositol 3-kinase (PI3K) and several of its downstream targets has also been shown. However, the coupling of STAT3 serine phosphorylation to PI3K in response to IL-2 has yet to be shown in either T cell lines or primary human T cells. This report shows that the PI3K inhibitors LY294002 and wortmannin block activation of MEK and ERK by IL-2 in primary human T cells. Moreover, these inhibitors significantly reduce IL-2-triggered STAT3 serine phosphorylation without affecting STAT5 serine phosphorylation. Analysis of the effects of these inhibitors on cell cycle progression and apoptosis strongly suggests that PI3K-mediated events, which includes STAT3 activation, are involved in IL-2-mediated cell proliferation but not cell survival. Finally, results presented illustrate that in primary human T cells, activation of Akt is insufficient for IL-2-induced anti-apoptosis. Thus, these results demonstrate that IL-2 stimulates PI3K-dependent events that correlate with cell cycle progression, but not anti-apoptosis, in activated primary human T cells.     

Proteolytic clipping of histone tails: the emerging role of histone proteases in regulation of various biological processes

Chromatin is a dynamic DNA scaffold structure that responds to a variety of external and internal stimuli to regulate the fundamental biological processes. Majority of the cases chromatin dynamicity is exhibited through chemical modifications and physical changes between DNA and histones. These modifications are reversible and complex signaling pathways involving chromatin-modifying enzymes regulate the fluidity of chromatin. Fluidity of chromatin can also be impacted through irreversible change, proteolytic processing of histones which is a poorly understood phenomenon. In recent studies, histone proteolysis has been implicated as a regulatory process involved in the permanent removal of epigenetic marks from histones. Activities responsible for clipping of histone tails and their significance in various biological processes have been observed in several organisms. Here, we have reviewed the properties of some of the known histone proteases, analyzed their significance in biological processes and have provided future directions.     

Ontogeny of proteolytic immunity: IgM serine proteases

We report the chemical activity of immunoglobulin micro and kappa/lambda subunits expressed on the surface of B cells and in secreted IgM antibodies (Abs) found in the preimmune repertoire. Most of the nucleophilic reactivity of B cells measured by formation of covalent adducts of a hapten amidino phosphonate diester was attributed to micro and kappa/lambda subunits of the B cell receptor. Secreted IgM Abs displayed superior nucleophilic reactivity than IgG Abs. IgM Abs catalyzed the cleavage of model peptide substrates at rates up to 344-fold greater than IgG Abs. Catalytic activities were observed in polyclonal IgM Abs from immunologically na?ve mice and humans without immunological disease, as well as monoclonal IgM Abs to unrelated antigens. Comparison of several IgM Abs indicated divergent activity levels and substrate preferences, with the common requirement of a basic residue flanking the cleavage site. Fab fragments of a monoclonal IgM Ab expressed catalytic activity, confirming the V domain location of the catalytic site. The catalytic reaction was inhibited by the covalently reactive hapten probe and diisopropylfluorophosphate, suggesting a serine protease-like mechanism. These observations indicate the existence of serine protease-like BCRs and secreted IgM Abs as innate immunity components with potential roles in B cell development and Ab effector functions.     

Neutrophil serine proteases: specific regulators of inflammation

Neutrophils are essential for host defence against invading pathogens. They engulf and degrade microorganisms using an array of weapons that include reactive oxygen species, antimicrobial peptides, and proteases such as cathepsin G, neutrophil elastase and proteinase 3. As discussed in this Review, the generation of mice deficient in these proteases has established a role for these enzymes as intracellular microbicidal agents. However, I focus mainly on emerging data indicating that, after release, these proteases also contribute to the extracellular killing of microorganisms, and regulate non-infectious inflammatory processes by activating specific receptors and modulating the levels of cytokines.     

Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63

Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.     

The repertoire of serine rhomboid proteases of piroplasmids of importance to animal and human health

Babesia, Theileria and Cytauxzoon are tick-borne apicomplexan protozoans of the order Piroplasmida, notorious for the diseases they cause in livestock, pets and humans. Host cell invasion is their Achilles heel, allowing for the development of drug or vaccine-based therapies. In other apicomplexans, cleavage of the transmembrane domain of adhesins by the serine rhomboid proteinase ROM4 is required for successful completion of invasion. In this study, we record and classify the rhomboid repertoire encoded in the genomes of 10 piroplasmid species pertaining to the lineages Babesia sensu stricto (s.s., Clade VI), Theileria sensu stricto (Clade IV), Theileria equi (Clade IV), Cytauxzoon felis (Clade IIIb) and Babesia microti (Clade I), as defined by Schnittger et al. (2012). Fifty-six piroplasmid rhomboid-like proteins were assigned by phylogenetic analysis and bidirectional best hit to the ROM4, ROM6, ROM7 or ROM8 groups, and their crucial motifs for conformation and function were identified. Forty-four of these rhomboids had either been incorrectly classified or misannotated. Babesia s.s. encode five or three ROM4 proteinase paralogs, whereas the remaining piroplasmids encode two ROM4 paralogs. All piroplasmids encode a single ROM6, ROM7 and ROM8. Thus, an increased paralog number of ROM4 is the only feature distinguishing Babesia s.s. from other piroplasmid lineages. Piroplasmid ROM6 is related to the mammalian mitochondrial rhomboid and, accordingly, N-terminal mitochondrial targeting signal sequences was found in some cases. ROM6 is the only rhomboid encoded by piroplasmids that is ubiquitous in other organisms. ROM8 represents a pseudoproteinase that is highly conserved between studied piroplasmids, suggesting that it is important in regulatory functions. ROM4, ROM6, ROM7 and ROM8 are exclusively present in Aconoidasida, which comprises piroplasmids and Plasmodium, suggesting a relevant functional role in erythrocyte invasion. The correct classification and designation of piroplasmid rhomboids presented in this study facilitates an informed choice for future in-depth study of their functions.     

Thermodynamics of Na+ binding to coagulation serine proteases

The sodium binding to serine proteases triggers a conformational change in the proteins that enhances the catalytic activity of the enzymes. The interaction of the cation with the protein is mediated by the hydrogen-bonding network of water molecules that embed the Na+ site. We pointed out the crucial role of the insertion loop 186a-d and the I16-D194 ion pair in the stabilization of sodium binding pocket in thrombin. This paper contributes to better explain the molecular mechanism of sodium binding for different serine proteases leading to the identification of the structural changes necessary to engineer a functional Na+ site and regulate catalytic activity in serine proteases.