Trans-dominant inhibitory human immunodeficiency virus type 1 protease monomers prevent protease activation and virion maturation.

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coli gpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIV-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed p55gag and p160gag-pol polyproteins. In the presence of HIV-1 gp160 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.

Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.

Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders.

Autoantibodies to calpastatin (an endogenous inhibitor for calcium-dependent neutral protease, calpain) in systemic rheumatic diseases.

We identified an autoantibody that reacts with calpastatin [an inhibitor protein of the calcium-dependent neutral protease calpain (EC 3.4.22.17)]. In early immunoblot studies, sera from patients with rheumatoid arthritis (RA) recognized unidentified 60-, 45-, and 75-kDa proteins in HeLa cell extracts. To identify these autoantigens, we used patient sera to clone cDNAs from a lambda gt11 expression library. We isolated clones of four genes that expressed fusion proteins recognized by RA sera. The 1.2-kb cDNA insert (termed RA-6) appeared to encode a polypeptide corresponding to the 60-kDa antigen from HeLa cells, since antibodies bound to the RA-6 fusion protein also reacted with a 60-kDa HeLa protein. The deduced amino acid sequence of the RA-6 cDNA was completely identical with the C-terminal 178 amino acids of human calpastatin except for one amino acid substitution. Patient sera that reacted with the RA-6 also bound pig muscle calpastatin, and a monoclonal antibody to human calpastatin recognized the RA-6 fusion protein, confirming the identity of RA-6 with calpastatin. Moreover, the purified RA-6 fusion protein inhibited the proteolytic activity of calpain, and IgG from a serum containing anti-calpastatin antibodies blocked the calpastatin activity of the RA-6 fusion protein. Immunoblots of the RA-6 product detected autoantibodies to calpastatin in 57% of RA patients; this incidence was significantly higher than that observed in other systemic rheumatic diseases, including systemic lupus erythematosus (27%), polymyositis/dermatomyositis (24%), systemic sclerosis (38%), and overlap syndrome (29%). Thus, anti-calpastatin antibodies are present most frequently in patients with RA and may participate in pathogenic mechanisms of rheumatic diseases.

Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.

Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB. However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB. Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex. We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40. Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40. The close proximity of the VP16 and TAF40 binding sites on the surface of TFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.

Ancylostoma caninum anticoagulant peptide: a hookworm-derived inhibitor of human coagulation factor Xa.

Human hookworm infection is a major cause of gastrointestinal blood loss and iron deficiency anemia, affecting up to one billion people in the developing world. These soil-transmitted helminths cause blood loss during attachment to the intestinal mucosa by lacerating capillaries and ingesting extravasated blood. We have isolated the major anticoagulant used by adult worms to facilitate feeding and exacerbate intestinal blood loss. This 8.7-kDa peptide, named the Ancylostoma caninum anticoagulant peptide (AcAP), was purified by using a combination of ion-exchange chromatography, gel-filtration chromatography, and reverse-phase HPLC. N-terminal sequencing of AcAP reveals no homology to any previously identified anticoagulant or protease inhibitor. Single-stage chromogenic assays reveal that AcAP is a highly potent and specific inhibitor of human coagulation, with an intrinsic K*i for the inhibition of free factor Xa of 323.5 pM. In plasma-based clotting time assays, AcAP was more effective at prolonging the prothrombin time than both recombinant hirudin and tick anticoagulant peptide. These data suggest that AcAP, a specific inhibitor of factor Xa, is one of the most potent naturally occurring anticoagulants described to date.

Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier.

Scrapie is a transmissible neurodegenerative disease that appears to result from an accumulation in the brain of an abnormal protease-resistant isoform of prion protein (PrP) called PrPsc. Conversion of the normal, protease-sensitive form of PrP (PrPc) to protease-resistant forms like PrPsc has been demonstrated in a cell-free reaction composed largely of hamster PrPc and PrPsc. We now report studies of the species specificity of this cell-free reaction using mouse, hamster, and chimeric PrP molecules. Combinations of hamster PrPc with hamster PrPsc and mouse PrPc with mouse PrPsc resulted in the conversion of PrPc to protease-resistant forms. Protease-resistant PrP species were also generated in the nonhomologous reaction of hamster PrPc with mouse PrPsc, but little conversion was observed in the reciprocal reaction. Glycosylation of the PrPc precursors was not required for species specificity in the conversion reaction. The relative conversion efficiencies correlated with the relative transmissibilities of these strains of scrapie between mice and hamsters. Conversion experiments performed with chimeric mouse/hamster PrPc precursors indicated that differences between PrPc and PrPsc at residues 139, 155, and 170 affected the conversion efficiency and the size of the resultant protease-resistant PrP species. We conclude that there is species specificity in the cell-free interactions that lead to the conversion of PrPc to protease-resistant forms. This specificity may be the molecular basis for the barriers to interspecies transmission of scrapie and other transmissible spongiform encephalopathies in vivo.

Addiction protein Phd of plasmid prophage P1 is a substrate of the ClpXP serine protease of Escherichia coli.

Plasmid-encoded addiction genes augment the apparent stability of various low copy number bacterial plasmids by selectively killing plasmid-free (cured) segregants or their progeny. The addiction module of plasmid prophage P1 consists of a pair of genes called phd and doc. Phd serves to prevent host death when the prophage is retained and, should retention mechanisms fail, Doc causes death on curing. Doc acts as a cell toxin to which Phd is an antidote. In this study we show that host mutants with defects in either subunit of the ClpXP protease survive the loss of a plasmid that contains a P1 addiction module. The small antidote protein Phd is fully stable in these two mutant hosts, whereas it is labile in a wild-type host. We conclude that the role of ClpXP in the addiction mechanism of P1 is to degrade the Phd protein. This conclusion situates P1 among plasmids that elicit severe withdrawal symptoms and are able to do so because they encode both a cell toxin and an actively degraded macromolecule that blocks the synthesis or function of the toxin.

Protein structure-based design of potent orally bioavailable, nonpeptide inhibitors of human immunodeficiency virus protease.

A class of potent nonpeptidic inhibitors of human immunodeficiency virus protease has been designed by using the three-dimensional structure of the enzyme as a guide. By employing iterative protein cocrystal structure analysis, design, and synthesis the binding affinity of the lead compound was incrementally improved by over four orders of magnitude. An inversion in inhibitor binding mode was observed crystallographically, providing information critical for subsequent design and highlighting the utility of structural feedback in inhibitor optimization. These inhibitors are selective for the viral protease enzyme, possess good antiviral activity, and are orally available in three species.

ABT-538 is a potent inhibitor of human immunodeficiency virus protease and has high oral bioavailability in humans.

Examination of the structural basis for antiviral activity, oral pharmacokinetics, and hepatic metabolism among a series of symmetry-based inhibitors of the human immunodeficiency virus (HIV) protease led to the discovery of ABT-538, a promising experimental drug for the therapeutic intervention in acquired immunodeficiency syndrome (AIDS). ABT-538 exhibited potent in vitro activity against laboratory and clinical strains of HIV-1 [50% effective concentration (EC50) = 0.022-0.13 microM] and HIV-2 (EC50 = 0.16 microM). Following a single 10-mg/kg oral dose, plasma concentrations in rat, dog, and monkey exceeded the in vitro antiviral EC50 for > 12 h. In human trials, a single 400-mg dose of ABT-538 displayed a prolonged absorption profile and achieved a peak plasma concentration in excess of 5 micrograms/ml. These findings demonstrate that high oral bioavailability can be achieved in humans with peptidomimetic inhibitors of HIV protease.

Gene cloning, molecular modeling, and phylogenetics of serine protease P32 and serine carboxypeptidase SCP1 from nematophagous fungi Pochonia rubescens and Pochonia chlamydosporia

The fungi Pochonia chlamydosporia and Pochonia rubescens are parasites of nematode eggs and thus are biocontrol agents of nematodes. Proteolytic enzymes such as the S8 proteases VCP1 and P32, secreted during the pathogenesis of nematode eggs, are major virulence factors in these fungi. Recently, expression of these enzymes and of SCP1, a new putative S10 carboxypeptidase, was detected during endophytic colonization of barley roots by these fungi. In our study, we cloned the genomic and mRNA sequences encoding P32 from P. rubescens and SCP1 from P. chlamydosporia. P32 showed a high homology with the serine proteases Pr1A from the entomopathogenic fungus Metarhizium anisopliae and VCP1 from P. chlamydosporia (86% and 76% identity, respectively). However, the catalytic pocket of P32 showed differences in the amino acids of the substrate-recognition sites compared with the catalytic pockets of Pr1A and VCP1 proteases. Phylogenetic analysis of P32 suggests a common ancestor with protease Pr1A. SCP1 displays the characteristic features of a member of the S10 family of serine proteases. Phylogenetic comparisons show that SCP1 and other carboxypeptidases from filamentous fungi have an origin different from that of yeast vacuolar serine carboxypeptidases. Understanding protease genes from nematophagous fungi is crucial for enhancing the biocontrol potential of these organisms.

Mecanismos celulares, stress oxidativo e balanço protease-antiprotease na DPOC : revisão de literatura

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Rhomboid intramembrane protease RHBDL4 triggers ER-export and non-canonical secretion of membrane-anchored TGFα

Rhomboid intramembrane proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian rhomboid protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this rhomboid protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes.

Coordination of protein and mRNA abundances of stromal enzymes and mRNA abundances of the Clp protease subunits during senescence of Phaseolus vulgaris (L.) leaves

Our objective was to determine the coordination of transcript and/or protein abundances of stromal enzymes during leaf senescence. First trifolioliate leaves of Phaseolus vulgaris L. plants were sampled beginning at the time of full leaf expansion; at this same time, half of the plants were switched to a nutrient solution lacking N. Total RNA and soluble protein abundances decreased after full leaf expansion whereas chlorophyll abundance remained constant; N stress enhanced the decline in these traits. Abundances of ribulose-1,5-bisposphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39), Rubisco activase and phosphoribulokinase (Ru5P kinase; EC 2.7.1.19) decreased after full leaf expansion in a coordinated manner for both treatments. In contrast, adenosine diphosphate glucose (ADPGlc) pyrophosphorylase (EC 2.7.7.27) abundance was relatively constant during natural senescence but did decline similar to the other enzymes under N stress. Northern analyses indicated that transcript abundances for all enzymes declined markedly on a fresh-weight basis just after full leaf expansion. This rapid decline was particularly strong for the Rubisco small subunit (rbcS) transcript. The decline was enhanced by N stress for rbcS and Rubisco activase (rca), but not for Ru5P kinase (prk) and ADPGlc pyrophosphorylase (agp). Transcripts of the Clp protease subunits clpC and clpP declined in abundance just after full leaf expansion, similar to the other mRNA species. When Northern blots were analyzed using equal RNA loads, rbcS transcripts still declined markedly just after full leaf expansion whereas rca and clpC transcripts increased over time. The results indicated that senescence was initiated near the time of full leaf expansion, was accelerated by N stress, and was characterized by large decline in transcripts of stromal enzymes. The decreased mRNA abundances were in general associated with steadily declining stromal protein abundances, with ADPGlc pyrophosphorylase being the notable exception. Transcript analyses for the Clp subunits supported a recent report (Shanklin et al., 1995, Plant Cell 7: 1713--1722) indicating that the Clp protease subunits were constitutive throughout development and suggested that ClpC and ClpP do not function as a senescence-specific proteolytic system in Phaseolus.

Enzymatic cyclization of a potent Bowman-Birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide

The most potent known naturally occurring Bowman-Birk inhibitor, sunflower trypsin inhibitor-1 (SFTI-1), is a bicyclic 14-amino acid peptide from sunflower seeds comprising one disulfide bond and a cyclic backbone. At present, little is known about the cyclization mechanism of SFTI-1. We show here that an acyclic permutant of SFTI-1 open at its scissile bond, SFTI-1[ 6,5], also functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine beta-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI1[6,5] is similar to9:1 regardless of whether trypsin is incubated with SFTI-1[ 6,5] or SFTI-1. Enzymatic resynthesis of the scissile bond to form cyclic SFTI-1 is a novel mechanism of cyclization of SFTI-1[ 6,5]. Such a reaction could potentially occur on a trypsin affinity column as used in the original isolation procedure of SFTI-1. We therefore extracted SFTI-1 from sunflower seeds without a trypsin purification step and confirmed that the backbone of SFTI-1 is indeed naturally cyclic. Structural studies on SFTI-1[ 6,5] revealed high heterogeneity, and multiple species of SFTI-1[ 6,5] were identified. The main species closely resembles the structure of cyclic SFTI-1 with the broken binding loop able to rotate between a cis/trans geometry of the I7-P8 bond with the cis conformer being similar to the canonical binding loop conformation. The non-reactive loop adopts a beta-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an isoaspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.

Characterisation of human kallikrein 6 protease M expression in ovarian cancer

Kallikrein 6 (hK6, also known as protease M/zyme/neurosin) is a member of the human kallikrein gene family. We have previously cloned the cDNA for this gene by differential display and shown the overexpression of the mRNA in breast and ovarian primary tumour tissues and cell lines. To thoroughly characterise the expression of this kallikrein in ovarian cancer, we have developed a novel monoclonal antibody specific to hK6 and employed it in immunohistochemistry with a wide range of ovarian tumour samples. The expression was found elevated in 67 of 80 cases of ovarian tumour samples and there was a significant difference in the expression levels between normal and benign ovarian tissues and the borderline and invasive tumours (P<0.001). There was no difference of expression level between different subtypes of tumours. More significantly, high level of kallikrein 6 expression was found in many early-stage and low-grade tumours, and elevated hK6 proteins were found in benign epithelia coexisting with borderline and invasive tissues, suggesting that overexpression of hK6 is an early phenomenon in the development of ovarian cancer. Quantitative real-time reverse transcription-polymerase chain reactions also showed elevated kallikrein 6 mRNA expression in ovarian tumours. Genomic Southern analysis of 19 ovarian tumour samples suggested that gene amplification is one mechanism for the overexpression of hK6 in ovarian cancer.