Secretory aspartyl peptidase activity from mycelia of the human fungal pathogen Fonsecaea pedrosoi: effect of HIV aspartyl proteolytic inhibitors

Fonsecaea pedrosoi is the principal causative agent of chromoblastomycosis, which is a chronic, often debilitating, suppurative and granulomatous mycosis. Very little is known about the hydrolytic enzymes produced by this human fungal pathogen. In the present study, we have identified extracellular proteolytic activity from F. pedrosoi mycelial forms when grown in chemically defined conditions. Secretory aspartyl peptidase activity was measured during 15 days of fungal growth in vitro using bovine serum albumin (BSA) as the soluble substrate and extreme acidic pH (2.0). This activity was totally inhibited by pepstatin A, a classic aspartyl peptidase inhibitor. Conversely, metallo (o-phenanthroline), cysteine (E-64) and serine (PMSF) proteolytic inhibitors failed to restrain proteolytic activity. We also evaluated the effect of four distinct HIV aspartyl peptidase inhibitors on the secretory proteolytic activity of F. pedrosoi mycelia. Indinavir, ritonavir and nelfinavir powerfully inhibited extracellular aspartyl proteolytic activity by approximately 97, 96 and 87%, respectively, whereas saquinavir did not significantly interfere with BSA hydrolysis. Mycelial-derived secretory aspartyl peptidase activity cleaved other proteinaceous substrates, including human albumin, fibrinogen, fibronectin, laminin and type I collagen. As previously reported by our group, conidia also produce secretory aspartyl peptidase. In this sense, we investigated the effect of pepstatin A on F. pedrosoi development. Pepstatin A was able to inhibit the growth of conidium and its transformation into mycelium. Taken together, our results suggest a possible participation of aspartyl peptidases in the essential fungal processes, such as growth, differentiation, nutrition and cleavage of relevant host proteinaceous components.     

Human U937 cell surface peptidase activities: characterization and degradative effect on tumor necrosis factor-alpha.

Surface peptidase activities on the human monocytic lineage cell line U937 were characterized. Two diisopropyl phosphofluoridate (DFP)-inhibitable serine peptidases were identified by differences in their hydrolytic activities on chromogenic peptides: one removed tripeptides from the free NH2-terminal end of the synthetic peptide Ala-Ala-Phe-p-nitroanilide (pNA) and was not inhibited by inhibitors of metallo-, cysteic-, and aspartic-proteinases, or by those of elastase-, trypsin- and chymotrypsin-like enzymes, suggesting the presence of a hitherto unidentified serine tripeptidyl endopeptidase; the other peptidase catalyzed the release of Gly-Pro from Gly-Pro-pNA and was inhibited by DFP, phenylmethyl sulfonyl fluoride and diprotin A, thus resembling dipeptidyl peptidase IV (DPP IV) with respect to its substrate specificity and inhibitor profile. A group of N-exo-aminopeptidase activities specifically inhibited by bestatin, was also detected when Ala-, Leu-, Arg- and Lys-pNA were used a substrates. The activities were surface associated and not secreted as determined by extracellular location of product and enzymatic recovery in highly purified U937 cell membranes. Peripheral monocytes and macrophages were found to virtually exhibit identical levels of these two classes of peptidase activities when compared to those detected on U937 cells. The relative contributions of these hydrolytic enzymes to the cleavage of bioactive and radioiodinated cytokines including tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha and interferon-gamma was next examined. The results indicated that N-aminopeptidases do not appear to participate in the catabolism of any tested cytokine. In contrast, the most interesting finding was that both serine peptidases participate in TNF-alpha degradation. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native 17-kDa molecule TNF-alpha, and the concomitant release of biologically inactive fragments of less than or equal to 2 kDa. Together, these observations indicate new roles for both the DPP IV-like enzyme and the tripeptidyl endopeptidase located at the surface of human monocytic cells, including the regulation of the extracellular TNF-alpha concentration. Thus, the identification of functional ectopeptidases provides insight into their potential role in both normal and malignant monocytic function.     

Leishmania (L.) amazonensis peptidase activities inside the living cells and in their lysates

In this study we investigated the peptidase activity in Leishmania (L.) amazonensis live amastigote by confocal microscopy using peptidyl-MCA as substrates, the hydrolysis of which releases the MCA fluorophore inside the cells. Cell pre-treatment with peptidase inhibitors indicated the presence of cysteine and serine peptidases. It was noteworthy that Leishmania amastigotes incorporate only substrates (Z-FR-MCA, Z-RR-MCA) or inhibitors (E64, TLCK) containing positively charged groups. The peptidase activities in the supernatants of amastigotes and promastigotes lysates were also evaluated with the same peptidyl-MCA substrates and inhibitors in the pH range 4.5-9.0. The effects of temperature and different salts were also included in this study. The hydrolytic activities of supernatants on Z-FR-MCA clearly indicate the presence of different cysteine peptidases that adapted to work in different environment conditions. Intact Leishmania cells incorporated Z-RR-MCA, the hydrolysis of which was inhibited only by TLCK indicating the presence of at least one serine peptidase. The pH profile of Z-RR-MCA hydrolysis by amastigotes and promastigotes lysate supernatants, and the hydrolysis time course of the FRET peptide Abz-AGRRRAQ-EDDnp at RA bond, followed by removal of the two C-termini R to yield Abz-AGR-OH that is a unique characteristic of oligopeptidase B, indicate its presence in the parasite.     

Degradation of substance P by membrane peptidases in the rat substantia nigra: effect of selective inhibitors

The hydrolysis of substance P by membrane peptidases prepared from the rat substantia nigra was studied in the presence of selective inhibitors. Substance P degradation by synaptic and mitochondrial membranes was completely inhibited by 1,10-phenanthroline (1 mM), a non-specific metallopeptidase inhibitor. Captopril and bestatine, selective inhibitors of angiotensin converting enzyme and aminopeptidases respectively, were without effects. However, phosphoramidon (1 microM), a putative 'enkephalinase' inhibitor, selectively inhibited substance P degradation by synaptic membranes. These results suggest that a phosphoramidon-sensitive endopeptidase may be the principal enzyme responsible for substance P degradation in substantia nigra.     

Metallopeptidases produced by group B Streptococcus: influence of proteolytic inhibitors on growth and on interaction with human cell lineages

Group B Streptococcus (GBS) is a major etiologic agent of neonatal bacterial infections and is the most common cause of sepsis and pneumonia in newborns. Surface and secreted molecules of GBS are often essential virulence factors which are involved in the adherence of the bacteria to host cells or are required to suppress the defense mechanisms of hosts. We analyzed the peptidase profiles of GBS by detection of proteolytic activities on SDS-PAGE containing copolymerized gelatin as substrate. Based on the inhibition by o-phenathroline and EGTA, three distinct peptidases of 220, 200 and 180 kDa were identified in the culture medium, besides one major cell-associated proteolytic activity, a 200-kDa metallopeptidase, suggesting that all were zinc-metallopeptidases. GBS culture supernatants, rich in metallotype peptidases, also cleaved fibronectin, laminin, type IV collagen, fibrinogen and albumin. Cleavage of the host extracellular matrix by GBS may be a relevant factor in the process of bacterial dissemination and/or invasion. Notably, metallopeptidase inhibitors strongly blocked GBS growth as well as its interaction with human cell lineages. Understanding the contribution of peptidases to the pathogenesis of GBS disease may broaden our perception of how this significant pathogen causes severe infections in newborn infants.     

Electrophoretic detection of Trypanosoma cruzi peptidases

Peptidases of Trypanosoma cruzi epimastigotes were examined by polyacrylamide gel electrophoresis in gels containing gelatin as peptidase substrate. Mini-gels were far superior to large gels in their sensitivity of peptidase detection. Patterns of peptidases were similar between different strains of T. cruzi, although some inter-strain heterogeneity was found. In strain Y, at least five peptidases were detected: four of these enzymes were shown to be cysteine-type peptidases with acidic pH optima. The other peptidase was a 60-kDa membrane-associated peptidase that was sensitive to o-phenanthroline; it was tentatively characterised as a metallopeptidase, and was optimally active at alkaline pH. This membrane-associated peptidase was conserved between strains of T. cruzi.     

Oligopeptidases of brush border membranes of rat small intestinal mucosal cells

1. The properties of peptidases located in the brush borders of rat small intestinal mucosal cells have been investigated using a new method for the assay of peptidase activity. In this method amino acids produced by hydrolysis of peptides are oxidized by ophidian L-amino acid oxidase and the hydrogen peroxide produced during reoxidation of the reduced enzyme is estimated spectrophotometrically.2. 10-20% of the total cellular peptidase activity and 50% of the leucylnaphthylamidase (LNAase) activity were found to be tightly bound to the brush borders and to possess different substrate specificity from the supernatant peptidase activity.3. Evidence from kinetic and competition studies indicates the presence of more than one peptidase in the brush borders. The peptidases exhibit pH optima of 8.0-8.5, are inhibited by EDTA, but are not usually activated by divalent metal ions. The brush border peptidases hydrolyse di- and tripeptides, some oligopeptides, leucinamide and leucyl-beta-naphthylamine. On the basis of the Michaelis constants, these substrates differ in their affinities for the enzymes.4. It is proposed that the brush border peptidases serve an analogous function in the terminal stages of protein digestion to that of the disaccharidases in the case of carbohydrate digestion and absorption.     

Regulation of the processing and release of tumor necrosis factor alpha in a human macrophage cell line

The proteolytic cleavage of a variety of membrane proteins, including pro-tumor necrosis factor alpha (pro-TNF-alpha), is induced by various reagents such as phorbol 12-myristate 13-acetate (PMA). In this study we generated a human macrophage cell line that constitutively produces TNF-alpha, and examined how the processing and release of TNF-alpha are regulated. The processing and release of TNF-alpha were enhanced by PMA through a protein kinase C-dependent pathway. Although only hydroxamate matrix metalloproteinase (MMP) inhibitors inhibited the basal processing and release of TNF-alpha, the PMA-induced processing and release of TNF-alpha were inhibited not only by MMP inhibitors but also by 1,10-phenanthroline, 3,4-dichloroisocoumarin (3,4-DCI), iodoacetamide, and Nalpha-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK). Hydroxamate MMP inhibitors and 1,10-phenanthroline inhibited the processing of TNF-alpha on the cell surface, whereas 3,4-DCI, iodoacetamide, and TPCK inhibited the transport of TNF-alpha to the cell surface. These results suggest that serine and/or cysteine protease(s) may be involved in PMA-induced processing and/or transport to the cell surface.     

Membrane peptidases in the peripheral nervous system of the pig: their localization by immunohistochemistry at light and electron microscopic levels.

The presence and cellular localization of five membrane peptidases has been investigated in peripheral nerves, including those of the autonomic nervous system, in the pig. Endopeptidase-24.11 ("enkephalinase") peptidyl dipeptidase A, aminopeptidase N, aminopeptidase W and dipeptidyl peptidase IV were studied by both enzymic assays of membranes prepared from samples of nerve and by immunoperoxidase histochemistry at light and in two cases, endopeptidase-24.11 and aminopeptidase W, at electron microscopic levels. All five peptidases could be quantified by enzymic assay, though the activities were about 1% of those in renal microvilli and less than those of choroid plexus membranes. Endopeptidase-24.11 was associated with Schwann cell membranes in all types of nerve examined, including major nerves containing predominantly myelinated fibres as well as autonomic nerves, such as the vagus and splenic nerves and the sympathetic chain, staining being observed in membranes associated with myelinated and unmyelinated fibres. The Schwann cell location of endopeptidase-24.11 was confirmed by correlation with immunostaining for glial fibrillary acidic protein and by electron microscopy. This peptidase is known to have a wide repertoire of susceptible substrates among neuropeptides which was here shown to include vasoactive intestinal polypeptide (Km 268 microM, kcat 568 min-1), one of a number of neuropeptides present in peripheral nerve fibres. Three of the peptidases, peptidyl dipeptidase A, aminopeptidase N and dipeptidyl peptidase IV, were associated with microvessels of peripheral nerves. Aminopeptidase N was also observed in connective tissue elements, including the perineurium. Aminopeptidase W was unique among the five peptidases in having a neuronal localization. This was observed in unmyelinated and myelinated nerves and was supported by comparison with the pattern of staining observed for neurofilament protein and by electron microscopic immunoperoxidase staining. This observation was unexpected since aminopeptidase W has not been detected as a neuronal marker in the brain. Some possible roles for the membrane peptidases in peripheral nerves are discussed.     

Evidence of gelatinase secretion by the submandibular gland in prepubescent rats

The gelatin cleaving activities in secretions of cultured fragments of male rat submandibular glands were studied using zymography. Gelatinolytic activities of 88-, 64-, and 57-kDa proteins detected in the tissues from 22-28-day old animals were undetectable in 31-70-day old rats. The traces of gelatinolytic activity associated with 28-kDa protein were detectable from 22-day old rats in serum-free media, and this activity of the enzyme markedly increased with aging from 38-days old. At 52-days and the subsequent stages, in addition to 28-kDa, activities associated with 60-, 32-, and 29-kDa proteins were strong. When the conditioned media were treated with 1,10-phenanthroline and diisopropyl fluorophosphate (DFP), both products inhibited activity of 88-kDa enzyme, indicating that this enzyme is Cls-like enzyme. The 64- and 57-kDa activities were inhibited by 1,10-phemanthroline, but not by DFP; when the conditioned medium of the tissue from 24-day old rats was treated with p-aminophenylmercuric acetate, gelatinolytic activity associated with 64-kDa converted to 57-kDa. Therefore, 64- and 57-kDa activities were concluded to be progelatinase A and gelatinase A, respectively. On the other hand, the gelatinolytic activities associated with 60-, 32-, 29- and 28-kDa proteins were inhibited by DFP but not by 1,10-phenanthroline, indicating that these enzymes belong to the family of serine proteinase, most probably kallikrein-related enzymes. From these findings, it was suggested that gelatinase A, along with Cls-like enzyme, participates in the maturation of the submandibular gland before it becomes active as an exocrine organ.     

A major role for TPPII in trimming proteasomal degradation products for MHC class I antigen presentation

Intracellular proteins are degraded by the proteasome, and resulting peptides surviving cytoplasmic peptidase activity can be presented by MHC class I molecules. Here, we show that intracellular aminopeptidases degrade peptides within seconds, almost irrespectively of amino acid sequence. N- but not C-terminal extension increases the half-life of peptides until they are 15 amino acids long. Beyond 15 amino acids, peptides are exclusively trimmed by the peptidase TPPII, which displays both exo- and endopeptidase activity. Surprisingly, most proteasomal degradation products are handled by TPPII before presentation by MHC class I molecules. We define three distinct proteolytic activities during antigen processing in vivo. Proteasome-generated peptides relevant for antigen presentation are mostly 15 amino acids or longer. These require TPPII activity for further trimming before becoming substrates for other peptidases and MHC class I. The heterogeneous pool of aminopeptidases will process TPPII products into MHC class I peptides and beyond.     

Tissue distributions, substrate specificities and molecular sizes of human peptidases determined by separate gene loci

1. The distribution of seven different peptidases thought to be determined by separate gene loci has been examined in a variety of human tissues. Although most of them are widely distributed, their relative activities vary from tissue to tissue. 2. The enzymes have been characterized in terms of their electrophoretie mobilities, their patterns of specificity with thirty-five different substrates, and their molecular sizes as estimated by gel nitration. 3. One of the peptidases (peptidase S) which is not found in red cells but is present in most other tissues has not been previously described. It appears to hydrolyse a somewhat wider range of substrates than the other peptidases, and to have a larger molecular size (c. 245,000).     

Peptidase activities in serum and bronchoalveolar lavage fluid from allergic asthmatics — comparison with healthy non-smokers and smokers and effects of inhaled glucocorticoids

BACKGROUND: Neuropeptides may be involved in the pathogenesis of asthma by evoking neurogenic inflammation. Since the effects of neuropeptides are limited by peptidases, reduced activity of peptidases may contribute to the inflammatory process. OBJECTIVE: We hypothesized that soluble peptidase activities are decreased in asthmatics and that inhaled glucocorticoids exert part of their anti-inflammatory action by increasing soluble peptidase activities. METHODS: Serum and bronchoalveolar lavage (BAL) fluid was obtained from non-smoking and smoking volunteers and from allergic asthmatics both before and after treatment for 12 weeks with placebo or inhaled fluticasone propionate. Activities of neutral endopeptidase (NEP), aminopeptidase N (APN) and dipeptidyl peptidase IV (DPP IV) were determined using colourometric assays. RESULTS: Reduced DPP IV activity in serum and reduced NEP activity in BAL fluid were found in healthy smokers compared with non-smokers. In contrast, no differences in peptidase activities in serum or BAL fluid were observed between allergic asthmatics and healthy non-smokers. Fluticasone propionate treatment did not affect peptidase activities in the asthmatic patients. CONCLUSIONS: We conclude that reduced peptidase activities in serum or BAL fluid can be found in healthy smokers, but not in allergic asthmatics, and that inhaled glucocorticoids do not affect peptidase activities in BAL fluid or serum of asthmatics. Our results do not support the hypothesized dysfunction of peptidases in the asthmatic airways.     

Processing of TCP pilin by TcpJ typifies a common step intrinsic to a newly recognized pathway of extracellular protein secretion by gram-negative bacteria.

Biogenesis of the Vibrio cholerae toxin-coregulated pilus (TCP) requires the activities of at least seven accessory proteins. We demonstrate that a portion of this pathway involves a novel processing step in which a hydrophilic leader peptide is proteolytically removed from TcpA by the gene product characterized in this report, TcpJ, to yield the mature, export-competent form of the pilin. Cleavage of the pilin leader peptide is independent of known signal peptidases as demonstrated by pilin-processing profiles in Escherichia coli strains conditionally defective for production of leader peptidase or grown in the presence of the antibiotic globomycin. Additionally, pilin cleavage did not rely on the SecA protein, as evidenced by TcpA processing in azide-treated cells. These results suggest that TcpJ is representative of a new class of proteins involved in SecA-independent proteolytic cleavage of a set of atypical leader peptides during extracellular export.     

Characterization of genes encoding novel peptidases in the biocontrol fungus <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> CECT 2413 using the TrichoEST functional genomics approach

Proteolytic enzymes (EC 3.4) secreted by Trichoderma strains are receiving increasing attention because of their potential implication in the Trichoderma biocontrol abilities. We have used an expressed sequence tag (EST) approach to identify genes encoding extracellular peptidases in T. harzianum CECT 2413 grown under several biocontrol-related conditions. Based on BlastX results and Gene Ontology annotation, a total of 61 (among 3478) unique sequences (unisequences) were predicted to encode enzymes with peptidase activity, three corresponding to secreted peptidases already known from this Trichoderma strain (PAPA, PRA1 and P6281). Further manual screening based on the functional identity and cellular location of the best matches revealed ten unisequences encoding novel extracellular peptidases. We report the characterization of the corresponding genes as well as a potential orthologous gene of the intracellular peptidase PAPB from T. asperellum. In each case, full-length coding sequences were obtained, and deduced proteins were compared at phylogenetic level with peptidases from other organisms. T. harzianum CECT 2413 novel peptidases included six serine endopeptidases (EC 3.4.21) belonging to the families S1, S8 and S53, three aspartic endopeptidases (EC 3.4.23) of the family A1, one metallo-endopeptidase (EC 3.4.24) of the family M35, and one aminopeptidase (EC 3.4.11) of the family M28. Results obtained by Northern blot analyses demonstrated that the genes within a family are differentially regulated in response to different culture conditions, suggesting that they have diverse functional roles.     

A method for the determination of cytosolic aminopeptidase in serum and its usefulness for clinical application. Selection of inhibitors for membrane-bound aminopeptidase and cystyl aminopeptidase

A method for the determination of cytosolic aminopeptidase (EC 3.4.11.1; C-AP) in serum was developed. At first, more specific and adequate inhibitor for other serum peptidases, such as membrane-bound aminopeptidase (EC 3.4.11.2; arylamidase, AA) and cystyl aminopeptidase (EC 3.4.11.3; CAP) was selected from 1,10-phenanthroline derivatives. The compound, 4,7-dimethyl-1,10-phenanthroline (4,7-DMP) is one of the most effective inhibitor for AA and CAP, and it inhibits completely these enzymes at the concentration of less than 0.4 mmol/l. C-AP in serum at an optimum pH of 8.0 in the assay using L-leucine amide (LA) as the substrate had no inhibitory effects at the concentration of more than 0.4 mmol/l of 4,7-DMP. The results with the proposed method correlated with those with a conventional electrophoretic method. The proposed method hence is a specific and easily available assay for C-AP in serum. The further analysis of C-AP using this method would promise the establishment of clinical assessment of the enzyme.     

Cellular reorganisation of membrane peptidases in Wallerian degeneration of pig peripheral nerve

Summary Immunohistochemical techniques have been used to study a group of membrane peptidases in the distal segment of the ulnar nerve of piglets 7 and 14 days after surgical section. Five peptidases were studied, all of which have a wide distribution on the surfaces of many cell types and have roles in metabolising neuropeptides. In normal pig nerves, endopeptidase-24.11 is expressed by both myelin- and nonmyelin-forming Schwann cells. Peptidyl dipeptidase A (angiotensin converting enzyme), aminopeptidase-N and dipeptidyl peptidase IV are present in the microvessels, and aminopeptidase-N is also seen in the perineurial connective tissue. Of this group of peptidases, only aminopeptidase-W is a neuronal marker in normal nerve. Macrophages were identified by two antibodies, 74-22-15 and 40D (which recognises Ia). Short-term cultures of macrophages obtained by alveolar lavage were positively stained by both antibodies and about half of the cells also expressed aminopeptidase-N and dipeptidyl peptidase IV. Staining by 40D and 74-22-15 revealed the presence of significant numbers of macrophages in normal nerve, but none of the membrane peptidases colocalized with these cells. Seven days after section of the nerve, the distal segment showed morphological changes typical of Wallerian degeneration. Endopeptidase-24.11 was no longer visible in myelin sheaths, but remained a marker for the surface of Schwann cells (defined also by staining for glial fibrillary acidic protein). The macrophage markers revealed marked changes in the morphology of these cells, often consistent with their phagocytic activity. Two peptidases, aminopeptidase-N and aminopeptidase-W, also appeared at this time to be associated with cells exhibiting the morphology of activated macrophages. This association could be confirmed in many instances by double staining with 74-22-15 and antibodies to the peptidases. Angiotensin converting enzyme retained its single location in microvessels at 7 days after section, but at 14 days a new pattern emerged as it, too, was expressed by macrophages. Dipeptidyl peptidase IV was not shown to be a macrophage marker in the degenerating nerve. Thus Wallerian degeneration leads to remarkable changes in the cellular expression of membrane peptidases; endopeptidase-24.11 reflects the changed morphology of Schwann cells while aminopeptidase-N, aminopeptidase-W and angiotensin converting enzyme become expressed by the actively phagocytosing macrophages.     

Fibroblast activation protein-α promotes tumor growth and invasion of breast cancer cells through non-enzymatic functions

Fibroblast activation protein-α (FAP) is a cell surface, serine protease of the post-prolyl peptidase family that is expressed in human breast cancer but not in normal tissues. Previously, we showed that FAP expression increased tumor growth rates in a mouse model of human breast cancer. Here the role of the proteolytic activities of FAP in promoting tumor growth, matrix degradation and invasion was investigated. Mammary fat pads of female SCID mice were inoculated with breast cancer cells that express FAP and the mice treated with normal saline or Val-boroPro (talabostat); Glu-boroPro (PT-630); or 1-[[(3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine (LAF-237) that inhibit prolyl peptidases. Other mice were injected with breast cancer cells expressing a catalytically inactive mutant of FAP and did not receive inhibitor treatment. PT-630 and LAF-237 did not slow growth of tumors produced by any of the three cell lines expressing FAP. Talabostat slightly decreased the growth rates of the FAP-expressing tumors but because PT-630 and LAF-237 did not, the growth retardation was likely not related to the inhibition of FAP or the related post-prolyl peptidase dipeptidyl peptidase IV. Breast cancer cells expressing a catalytically inactive mutant of FAP (FAP^S624A) also produced tumors that grew rapidly. In vitro studies revealed that cells expressing wild type FAP or FAP^S624A degrade extracellular matrix (ECM) more extensively, accumulate higher levels of matrix metalloproteinase-9 (MMP-9) in conditioned medium, are more invasive in type I collagen gels, and have altered signaling compared to control transfectants that do not express FAP and form slow growing tumors. We conclude that the proteolytic activity of FAP participates in matrix degradation, but other functions of the protein stimulate increased tumor growth.     

Chronic sino-naso-orbital fungal infection due to Pseudallescheria boydii in a nonimmunocompromised host--a case report

A case of recurrent sino-naso-orbital fungal infection due to Pseudallescheria boydii described in a 28 yrs. old man, who appeared immunocompetent, and was found negative for HIV I and II by ELISA tested on two occasions. The fungal culture was negative. It is very essential to identify P boydii as Miconazole is the only antifungal drug of choice for this fungus. The pathologist plays an important role in identifying this fungus when fungal culture fails to yield the growth. The pathologist has to look for clinching clues such as "intercalary conidia" and "chlamydoconidia" to distinguish P boydii from Aspergillus.     

Interactions between mature Der p 1 and its free prodomain indicate membership of a new family of C1 peptidases

BACKGROUND: Studies in vivo have shown that the cysteine peptidase activity of group 1 house dust mite allergens contributes to their allergenicity. These allergens are synthesized initially as proenzymes and removal of the propiece is necessary to unmask their proteolytic activity. In related C1 family cysteine peptidases of enzyme clan CA, liberated propieces continue to inhibit the mature peptidase as tight binding inhibitors. As it is not known whether mite peptidase allergens behave similarly, our objective was to investigate the effect of the Der p 1 propiece on the catalytic activity of Der p 1 and Der f 1. METHODS: Enzymatic activity of natural Der p 1 and Der f 1 was assessed using a specific substrate and the effect of the recombinant propiece on its enzyme kinetics defined. The integrity of the propiece during these interactions was studied functionally and by analysis of the reaction mixtures. RESULTS: Der p 1 propiece was a potent competitive inhibitor of Der p 1 and Der f 1. In contrast to other cysteine peptidase prodomains, which are cognate tight binding inhibitors, the Der p 1 propiece behaves as a substrate and is fully degraded during this interaction. CONCLUSION: Mature Der p 1-prodomain interactions differ from other C1 family cysteine peptidases, suggesting that group 1 mite allergens are a new subgroup among C1 family cysteine peptidases. The rapid inactivation of Der p 1 prodomain is a newly identified mechanism that may contribute to the potency of this allergen.