Proteolytic processing of the Aplysia egg-laying hormone prohormone

By using matrix-assisted laser desorption/ionization time-of-flight MS, individual peptidergic neurons from Aplysia are assayed. A semiquantitative method is developed for comparing single-cell profiles by using spectral normalization, and peptides are localized to specific cells by mass spectrometric cell mapping. In addition to all previously identified products of the egg-laying hormone (ELH) gene, other peptides are formed from proteolytic hydrolysis of Leu-Leu residues within ELH and acidic peptide (AP). AP exhibits further processing to yield AP1-20 and AP9-27. These peptides appear to be colocalized in vesicles with ELH, transported to specific neuronal targets, and released in a Ca2+-dependent manner. A differential peptide distribution is observed at a specific target cell, and a low-frequency variation of AP, [Thr21]AP, is detected in a single animal.     

Immunocytochemical localization of chromogranin A and secretogranin II in female rat gonadotropes

The family of CD44 glycoproteins has been suggested to be involved in lymphocyte homing, maturation and activation. Using in vitro blocking studies with a monoclonal antibody, we here addressed the question of functional activity of CD44 variant exon v10 (CD44v10) in B-cell activation. We became interested in this question by the observation that CD44v10O was transiently expressed on activated T cells, B cells and monocytes as well as on a subpopulation of bone marrow cells. A potential ligand, as revealed by staining with a CD44v10 receptor globulin, was only detected on monocytes. Anti-CD44v10 had no major impact on T-cell activation and no influence on primed B cells, but interfered with the mounting of a primary B-cell response to T-independent and T-dependent antigens. Addition of anti-CD44v10 at different stages during the activation process revealed that CD44v10 was not engaged in B-cell-T-cell interactions. The antibody exerted some effect on monocyte activation as defined by a slight decrease in IL-1 production, but most efficiently inhibited antigen-specific as well as mitogen-induced B-cell activation when present during the coculture of virgin B cells with monocytes. These findings, together with the observation that a CD44v10 ligand was only detected on monocytes but not on lymphocytes, point towards a requirement for CD44v10 in a B-cell-monocyte interaction. Furthermore, since activation of B cells by engagement both of the B-cell receptor and of mitogen receptors was inhibited by anti-CD44v10, the data suggest that a costimulatory function of CD44v10 proceeds independent of the B-cell receptor.     

Proteolytic processing regulates receptor specificity and activity of VEGF-C

The recently identified vascular endothelial growth factor C (VEGF-C) belongs to the platelet-derived growth factor (PDGF)/VEGF family of growth factors and is a ligand for the endothelial-specific receptor tyrosine kinases VEGFR-3 and VEGFR-2. The VEGF homology domain spans only about one-third of the cysteine-rich VEGF-C precursor. Here we have analysed the role of post-translational processing in VEGF-C secretion and function, as well as the structure of the mature VEGF-C. The stepwise proteolytic processing of VEGF-C generated several VEGF-C forms with increased activity towards VEGFR-3, but only the fully processed VEGF-C could activate VEGFR-2. Recombinant 'mature' VEGF-C made in yeast bound VEGFR-3 (K[D] = 135 pM) and VEGFR-2 (K[D] = 410 pM) and activated these receptors. Like VEGF, mature VEGF-C increased vascular permeability, as well as the migration and proliferation of endothelial cells. Unlike other members of the PDGF/VEGF family, mature VEGF-C formed mostly non-covalent homodimers. These data implicate proteolytic processing as a regulator of VEGF-C activity, and reveal novel structure-function relationships in the PDGF/VEGF family.     

Cellular localization of the prohormone convertases in the hypothalamic paraventricular and supraoptic nuclei: selective regulation of PC1 in corticotrophin-releasing hormone parvocellular neurons mediated by glucocorticoids

The prohormone convertases (PCs) are processing enzymes that activate proproteins via cleavage at specific single or pairs of basic residues. The hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) are primary sites of biosynthesis of several neuroendocrine hormone precursors, including provasopressin (pro-AVP), pro-oxytocin (pro-OT), and procorticotrophin-releasing hormone (pro-CRH), which require post-translational processing to yield active products. Using in situ hybridization, we observed PC1 and PC5 mRNAs in PVN and SON magnocellular neurons, while PC2 mRNA was observed in both magnocellular and parvocellular PVN neurons as well as magnocellular SON neurons. Similar to furin, PC7 mRNA was expressed throughout the PVN and SON, whereas PACE4 mRNA levels were undetectable. Both immunohistochemical and Western blot studies were performed to demonstrate the presence of PC proteins and forms in the PVN and SON. Using double-labeling in situ hybridization, we examined the cellular colocalization of each PC mRNA with pro-AVP, pro-OT, and pro-CRH mRNAs in PVN and SON. PC1 mRNA was colocalized with both AVP and OT mRNA in PVN and SON magnocellular neurons. All AVP, OT, and CRH neurons expressed PC2. In contrast, PC5 mRNA was colocalized only with OT mRNA. We examined the effects of adrenalectomy (ADX) on PVN PC mRNA levels. PC1 mRNA levels were increased selectively within CRH/AVP parvocellular neurons but were unchanged in PVN magnocellular AVP or OT neurons. These results established the anatomical organization of each convertase and proneuropeptide substrates in the PVN and SON and suggested potential roles for each enzyme under resting and stimulated conditions.     

Differences in pH optima and calcium requirements for maturation of the prohormone convertases PC2 and PC3 indicates different intracellular locations for these events

PC2 and PC3, which is also known as PC1, are subtilisin-like proteases that are involved in the intracellular processing of prohormones and proneuropeptides. Both enzymes are synthesized as propolypeptides that undergo proteolytic maturation within the secretory pathway. An in vitro translation/translocation system from Xenopus egg extracts was used to investigate mechanisms in the maturation of pro-PC3 and pro-PC2. Pro-PC3 underwent rapid (t1/2 < 10 min) processing of the 88-kDa propolypeptide at the sequence RSKR83 to generate the 80-kDa active form of the enzyme. This processing was blocked when the active site aspartate was changed to asparagine, suggesting that an autocatalytic mechanism was involved. In this system, processing of pro-PC3 was optimal between pH 7.0 and 8.0 and was not dependent on additional calcium. These results are consistent with pro-PC3 maturation occurring at an early stage in the secretory pathway, possibly within the endoplasmic reticulum, where the pH would be close to neutral and the calcium concentration less than that observed in later compartments. Processing of pro-PC2 in the Xenopus egg extract was much slower than that of pro-PC3 (t1/2 = 8 h). It exhibited a pH optimum of 5.5-6.0 and was dependent on calcium (K0.5 = 2-4 mM). The enzymatic properties of pro-PC2 processing were similar to that of the mature enzyme. Further studies using mutant pro-PC2 constructs suggested that cleavage of pro-PC2 was catalyzed by the mature 68-kDa PC2 molecule. The results were consistent with pro-PC2 maturation occurring within a late compartment of the secretory pathway that contains a high calcium concentration and low pH.     

Proteolytic processing of ricin A chain is not required for cytotoxicity

PURPOSE: To evaluate prospectively the use of peripherally inserted central catheters in a large pediatric population. MATERIALS AND METHODS: During a 3-year period, data were collected prospectively on 523 consecutive attempts to place peripherally inserted central catheters in children. Patients underwent radiologically guided placement because attempts were unsuccessful on the inpatient units or a patient request was made. Fluoroscopy with use of contrast material and venography were used to place catheters and document the position of the catheter tip. Follow-up data were collected until treatment cessation or catheter removal. RESULTS: Among 523 attempts, 486 (92.9%) catheters were successfully placed. In the 37 (7.1%) unsuccessful cases, more than half of these children were younger than 24 months of age or weighed less than 5 kg. Ages of patients in whom 523 placement attempts were made ranged from 3 weeks to 18 years (mean, 6.9 years). Catheters were in place from 1 to 390 days (mean, 20 days). Frequency of infection was 1.9% (nine cases); incidence of infection was 0.93 per 1,000 catheter-placement days. There were two cases (0.4%) of central venous thrombosis. Most patients were discharged within 2 days of catheter placement. CONCLUSION: Fluoroscopically guided placement of peripherally inserted central catheters is a safe and effective method for establishing intermediate- and long-term central venous access in the pediatric population.     

Enzymatic characterization of immunopurified prohormone convertase 2: potent inhibition by a 7B2 peptide fragment

Prohormone convertase (PCs) are thought to mediate the controlled proteolysis of prohormones and neuropeptide precursors. While recombinant PC1 and furin are currently available, thus far it has not been possible to produce recombinant PC2. We have used conditioned medium obtained from the mouse insulinoma cell line beta TC3 to generate a working preparation of enzymatically active PC2 through immunopurification. Immunopurified PC2 cleaved the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC in a time- and calcium-dependent manner. It was half-maximally stimulated at 75 microM Ca2+, had an optimum pH of 5, and exhibited PCMS and EDTA sensitivity similar to that reported for furin and PC1. The tight-binding inhibitor 27 kDa 7B2 was used to calculate the Kd for this inhibitor and the active enzyme concentration. The Kd was 7.3 +/- 1.7 nM, and the turnover rate of PC2 was 5.2 molecules substrate per enzyme molecule per minute. The specific activity was 4.9 nmol/micrograms/h (assuming a molecular mass for PC2 of 64 kDa). The enzyme preparation was able to cleave recombinant proenkephalin at at least four of the expected paired basic sites in the absence, but not in the presence, of 27 kDa 7B2. Since 21 kDa 7B2 is functionally inactive as a proteinase inhibitor, we examined the inhibitory activity of the carboxy-terminal portion of 27 kDa 7B2 (7B2 CT-peptide). Synthetic peptides were used to demonstrate that the 7B2 CT-peptide (a) represents a potent inhibitor of PC2 (Ki = 57 nM), (b) can block the conversion of proPC2 to PC2, and (c) can block the PC2-mediated conversion of proenkephalin to smaller peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolytic processing of the adherens junctions components beta-catenin and gamma-catenin/plakoglobin during apoptosis

Apoptotic cells undergo specific morphological changes that include loss of cell-cell interactions. Cellular adhesiveness is dependent on members of the cadherin family of adhesion receptors and on the cytoplasmic adaptor proteins alpha-catenin, beta-catenin and gamma-catenin/plakoglobin. The caspase family of cystein proteases play a key role during the execution phase of the apoptotic program. These proteolytic enzymes, once activated, cleave cellular proteins which are important for the maintenance of cell integrity. Here we report that gamma-catenin is cleaved at different sites during apoptosis in various cell lines. The major apoptotic product of gamma-catenin still retains the ability to bind alpha-catenin but loses the carboxy-terminal region. We also show that gamma-catenin is cleaved by caspase-3 in vitro although with lower affinity when compared to PARP or beta-catenin. These findings indicate that multiple proteolytic events regulate the dismantling of the cell-cell junctional complexes during apoptosis.     

Role of the prohormone convertase PC3 in the processing of proglucagon to glucagon-like peptide 1

Proglucagon is processed differentially in pancreatic alpha-cells and intestinal endocrine L cells to release either glucagon or glucagon-like peptide-1-(7-36amide) (tGLP-1), two peptide hormones with opposing biological actions. Previous studies have demonstrated that the prohormone convertase PC2 is responsible for the processing of proglucagon to glucagon, and have suggested that the related endoprotease PC3 is involved in the formation of tGLP-1. To understand better the biosynthetic pathway of tGLP-1, proglucagon processing was studied in the mouse pituitary cell line AtT-20, a cell line that mimics the intestinal pathway of proglucagon processing and in the rat insulinoma cell line INS-1. In both of these cell lines, proglucagon was initially cleaved to glicentin and the major proglucagon fragment (MPGF) at the interdomain site Lys70-Arg71. In both cell lines, MPGF was cleaved successively at the monobasic site Arg77 and then at the dibasic site Arg109-Arg110, thus releasing tGLP-1, the cleavages being less extensive in INS-1 cells. Glicentin was completely processed to glucagon in INS-1 cells, but was partially converted to oxyntomodulin and very low levels of glucagon in AtT-20 cells in the face of generation of tGLP-1. Adenovirus-mediated co-expression of PC3 and proglucagon in GH4C1 cells (normally expressing no PC2 or PC3) resulted in the formation of tGLP-1, glicentin, and oxyntomodulin, but no glucagon. When expressed in alphaTC1-6 (transformed pancreatic alpha-cells) or in rat primary pancreatic alpha-cells in culture, PC3 converted MPGF to tGLP-1. Finally, GLP-1-(1-37) was cleaved to tGLP-1 in vitro by purified recombinant PC3. Taken together, these results indicate that PC3 has the same specificity as the convertase that is responsible for the processing of proglucagon to tGLP-1, glicentin and oxyntomodulin in the intestinal L cell, and it is concluded that this enzyme is thus able to act alone in this processing pathway.     

Proteolytic processing of the replicase ORF1a protein of equine arteritis virus

To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire ORF1a protein, results in cleavage products of approximately 29, 61, 22, 31, 41, and 3 kDa, which were named nonstructural proteins (nsps) 1 through 6, respectively. Pulse-chase experiments revealed that the cleavages at the nsp1/2 and nsp2/3 junctions are the most rapid processing steps. The remaining nsp3456 precursor is first cleaved at the nsp4/5 site. Final processing of the nsp34 and nsp56 intermediates is extremely slow. As predicted from previous in vitro translation experiments (E. J. Snijder, A. L. M. Wassenaar, and W. J. M. Spaan, J. Virol. 66:7040-7048, 1992), a cysteine protease domain in nsp1 was shown to be responsible for the nsp1/2 cleavage. The other processing steps are carried out by the putative EAV serine protease in nsp4 and by a third protease, which remains to be identified.     

Attenuation of the polypeptide 7B2, prohormone convertase PC2, and vasopressin in the hypothalamus of some Prader-Willi patients: indications for a processing defect

7B2 is a neuroendocrine chaperone interacting with the prohormone convertase PC2 in the regulated secretory pathway. Its gene is located near the Prader-Willi syndrome (PWS) region on chromosome 15. In a previous study we were able to show 7B2 immunoreactivity in the supraoptic nucleus (SON) or the paraventricular nucleus (PVN) in only three of five PWS patients. Here we report that in contrast with five other PWS patients, the neurons in the hypothalamic SON and PVN of the two 7B2-immunonegative PWS patients also failed to show any reaction using two antibodies directed against processed vasopressin (VP). On the other hand, even these two cases reacted normally with five antibodies that recognize different parts of the VP precursor. This finding pointed to a processing defect. Indeed, the same patients had no PC2 immunoreactivity in the SON or PVN, whereas PC1 immunoreactivity was only slightly diminished. In conclusion, in the VP neurons of two PWS patients, greatly reduced amounts of 7B2 and PC2 are present, resulting in diminished VP precursor processing.     

Neuroendocrine cell type-specific and inducible expression of the secretogranin II gene: crucial role of cyclic adenosine monophosphate and serum response elements

Secretogranin II, an acidic protein in the chromogranin/secretogranin family, is widely distributed in neuroendocrine secretory granules. What factors govern such widespread, yet selective, expression? The 5' deletions localized neuroendocrine cell type-specific expression to the proximal mouse secretogranin II promoter: such expression was abolished after deletion past the cAMP response element (CRE; [-67 bp]TGACGTCA[-60 bp]), and transfer of the CRE to a neutral promoter conferred 3.4- to 5.3-fold neuroendocrine selectivity. Thus, the CRE is, at least partly, sufficient to confer tissue-specific expression. Substantial (48-59%) loss of cell type-specific expression also occurred upon deletion past the serum response element (SRE; [-302 bp]GATGTCC[-296 bp]), and transfer of the SRE to a neutral promoter also conferred neuroendocrine selectivity. Expression of both the endogenous gene and the transfected secretogranin II promoter was up-regulated after secretagogues, and the degree of trans-activation of the transfected promoter (2.2- to 5.4-fold) paralleled activation of the endogenous gene (1.8- to 3.2-fold). The 5' promoter deletions revealed complete loss of secretagogue responses after deletion past the CRE. Transfer of the CRE to a neutral promoter conferred secretagogue responses (by 2.2- to 18.6-fold). Substantial (59-74%) falls in secretagogue responses also occurred after deletion past the promoter's SRE. Transfer of the SRE to a neutral promoter conferred secretagogue responses (by 2.7- to 8.3-fold). We conclude that the CRE is a crucial determinant of cell type-specific constitutive and secretagogue-inducible expression of the secretogranin II gene and that the SRE also plays a substantial role in both processes.     

Proteolytic activation of tick-borne encephalitis virus by furin

Flaviviruses are assembled intracellularly in an immature form containing heterodimers of two envelope proteins, E and prM. Shortly before the virion exits the cell, prM is cleaved by a cellular enzyme, and this processing step can be blocked by treatment with agents that raise the pH of exocytic compartments. We carried out in vivo and in vitro studies with tick-borne encephalitis (TBE) virus to investigate the possible role of furin in this process as well as the functional consequences of prM cleavage. We found that prM in immature virions can be correctly cleaved in vitro by recombinant bovine furin but that efficient cleavage occurs only after exposure of the virion to mildly acidic pH. The data suggest that exposure to an acidic environment induces an irreversible structural change that renders the cleavage site accessible to the enzyme. Cleavage by furin in vitro resulted in biological activation, as shown by a 100-fold increase in specific infectivity, the acquisition of membrane fusion and hemagglutination activity, and the ability of the envelope proteins to undergo low-pH-induced structural rearrangements characteristic of mature virions. In vivo, prM cleavage was blocked by a furin inhibitor, and infection of the furin-deficient cell line LoVo yielded only immature virions, suggesting that furin is essential for cleavage activation of flaviviruses.     

Stimulation of prohormone convertase-1 mRNA expression by second messenger signaling systems

The purpose of this study was to investigate the effects of activation of various second messenger signaling systems on gene expression (i.e. mRNA levels) of a peptide hormone processing enzyme called prohormone convertase-1 (PC-1, also called PC-3) in a human pancreatic carcinoid cell line (BON) that expresses several endocrine peptides (chromogranin A, pancreastatin, neurotensin). Pharmacologic activation of adenylate cyclase-cAMP, protein kinase-C and Ca2+ mobilization pathways increased PC-1 mRNA levels and neurotensin secretion. Elevations in PC-1 mRNA levels were dose and time-related. Secretagogue-induced cellular depletion of neurotensin was followed by a replenishment of cellular neurotensin stores and an upregulation of PC-1 mRNA levels. Together, these data indicate that PC-1 mRNA expression is increased with peptide secretion and coordinated with maintenance of cellular stores of peptides.     

Proteolytic degradation of microtubule associated protein tau by thrombin

Deposits in the brain of beta-amyloid and tau proteins constitute the two major characteristics of Alzheimer's disease. It is unknown how the deposits are formed, but several studies have suggested that proteases might play a crucial role. Consequently, the search for proteases responsible for processing tau and amyloid precursor protein has become relevant. Here, the ability of thrombin to process tau in vitro is examined. Thrombin, which is found in blood but presumably also in the nervous system, cleaves tau and generates a stable 25 kDa fragment. Immunoblot and amino acid sequencing reveals that the fragment is derived from the C-terminal of tau, and a microtubule assembly assay shows that it has a reduced capacity to promote microtubule assembly compared with full length tau.     

Proteolytic cleavage of ICAM-1 by human neutrophil elastase

Human leukocyte elastase (HLE) participates in tissue destruction in a number of inflammatory disorders, including rheumatoid arthritis and cystic fibrosis. Since HLE has been shown to bind to Mac-1, and ICAM-1 plays a key role during the recruitment and the activation of leukocytes at inflamed sites, we investigated the capacity of HLE to cleave ICAM-1. Flow-cytometric analyses showed a dose-dependent cleavage of ICAM-1 by HLE on different human cell lines. The cleavage was completely inhibited by alpha1-antitrypsin, a natural HLE protease inhibitor. The ability of HLE to degrade ICAM-1 was further confirmed by electrophoretic analysis using a soluble form of ICAM-1 (D1-D5). Enzymatic removal of N-linked glycosylation did not significantly modulate ICAM-1 cleavage by HLE, while removal of sialic acid residues partially reduced the sensitivity of ICAM-1 to HLE. We further showed that sputum of cystic fibrosis patients contains high levels of HLE activity capable of cleavage of cell surface ICAM-1. The cleavage induced by incubation of cells with the sputum sample was totally inhibited by alpha1-antitrypsin and the specific peptidic HLE inhibitor N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone. Moreover, the cleavage of ICAM-1 was concomitant to that of CD4 at the surface of the same cell, at the same amplitude, and at all HLE concentrations. The capacity of HLE to modulate the expression of ICAM-1 on the surface of leukocytes by proteolytic cleavage brings support to the hypothesis that overproduction of HLE can cause severe immunologic lung disorders by affecting intercellular adhesion.     

Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and Is essential for virus replication

The open reading frame (ORF) 1b-encoded part of the equine arteritis virus (EAV) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an ORF1ab fusion protein (345 kDa). Four ORF1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in EAV-infected cells (L. C. van Dinten, A. L. M. Wassenaar, A. E. Gorbalenya, W. J. M. Spaan, and E. J. Snijder, J. Virol. 70:6625-6633, 1996). In the present study, the generation of these four nonstructural proteins was shown to be mediated by the nsp4 serine protease, which is the main viral protease (E. J. Snijder, A. L. M. Wassenaar, L. C. van Dinten, W. J. M. Spaan, and A. E. Gorbalenya, J. Biol. Chem. 271:4864-4871, 1996). Mutagenesis of candidate cleavage sites revealed that Glu-2370/Ser, Gln-2837/Ser, and Glu-3056/Gly are the probable nsp9/10, nsp10/11, and nsp11/12 junctions, respectively. Mutations which abolished ORF1b protein processing were introduced into a recently developed infectious cDNA clone (L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991-997, 1997). An analysis of these mutants showed that the selective blockage of ORF1b processing affected different stages of EAV reproduction. In particular, the mutant with the nsp10/11 cleavage site mutation Gln-2837-->Pro displayed an unusual phenotype, since it was still capable of RNA synthesis but was incapable of producing infectious virus.