Structure and expression of a shrimp prohormone convertase 2.

Although many crustacean neuroendocrine hormones have been reported, the enzymes responsible for post-translational modification of neuroendocrine hormones have rarely been characterized. A prohormone convertase 2 (PC2)-like enzyme has been isolated from the optic lobe of the giant tiger shrimp, Penaeus monodon and referred as PmPC2. The full length cDNA sequence of PmPC2 has been identified and found to resemble evolutionarily conserved PC2 enzymes of vertebrates and invertebrates. PmPC2 was expressed in all larval developmental stages and in neuroendrocrine cells in the adult optic lobe. Its expression was found to be negatively related with shrimp body weight by qPCR (P<0.05). Immunohistochemistry results using an anti-rPmPC2 antibody with adult shrimp revealed high staining intensity in specific neurosecretory cells including the sinus gland, the organ of Hanström (also referred to as the medullar terminalis X-organ) and the organ of Bellonci (also referred to as the sensory or X-organ). By using the yeast two hybrid technique, PmPC2 was found to bind with P. monodon hyperglycemic hormone (Pem-CHH1) that plays an important role in glucose metabolism. Since PmPC2 is a subtilisin-like serine proteinase, it is expected to cleave the synthetic substrate, pyr-RTKR-MCA, but the expressed recombinant catalytic domain of PmPC2 (rPmPC2-cat) showed no enzymatic activity as expected. In vivo injection of dsRNA-PmPC2 resulted in reduced transcripts for both PmPC2 and Pem-CHH1 on day 3 post injection, but there was no accompanying reduction of glucose level in the hemolymph. Taken together, PmPC2 localization, expression and activity suggest that it has a function(s) in the shrimp neuroendrocrine system and that it may not only activate Pem-CHH1 but also affect its expression. However, there is no obvious explanation for the negative correlation between PmPC2 expression level and shrimp body weight.

Molecular cloning, ontogeny and tissue distribution of zebrafish (Danio rerio) prohormone convertases: pcsk1 and pcsk2.

Prohormone convertase subtilisin/kexin (PCSK) enzymes are a family of nine related serine proteases, found in a multitude of tissues, and responsible for the maturation of a variety of protein and peptide precursors. Pcsk1 and Pcsk2 are found within dense core secretory granules in endocrine and neuroendocrine cells and are responsible for cleaving several hormones and neuropeptide precursors. In this work, we cloned and sequenced the cDNA of pcsk1 and pcsk2 from zebrafish (Danio rerio). pcsk1 is a 2268bp ORF, whose 755 amino acid protein product is identical to that predicted from the genome sequence. pcsk2 is a 1941bp ORF, encoding a 646 amino acid peptide. Both Pcsk1 and Pcsk2 display high degrees of similarity to their counterparts in other species, including the conservation of the catalytic triad and other essential residues. The brain contained the highest expression levels of both pcsk1 (1.49+/-0.21) (displayed as ratio to EF-1a), and pcsk2 (0.23+/-0.04). Both transcripts were also detectable in the fore, mid and distal gut. pcsk1 and 2 were detectable at 4.5h post-fertilization, and while pcsk1 expression increased throughout development (0.12+/-0.01 maximum at 3 days post-fertilization), pcsk2 expression was highest at day 5 post-fertilization (0.03+/-0.01), and decreased prior. For the first time, we have identified and characterized a pcsk1 transcript in fish. We have also identified and characterized the pcsk2 transcript in zebrafish, and have assessed the tissue distribution and ontogeny of both.

Structure prediction of neuroendocrine convertase -2: a potential target in various cancers.

Prohormone or proprotein convertases (PC2) are members of the subtilisin family of serine proteases. They are involved in the activation of precursor molecules by endoproteolytic cleavage at basic amino acid residues within the general motif (K/R)-(X)n- (K/R)2, where n is 0, 2, 4 or 6 and X is usually not Cys. Among the members of this prohormone convertase family, Neuroendocrine Convertase-2 (NEC-2) is regarded as one of the important proteins involved in the maturation of many precursor proteins. Being widely distributed in the neuroendocrine cells, these proteins play a vital role in causing malignant gliomas. They can serve as important drug targets in the treatment of cancers. In the present study, a 3D model of NEC-2 was generated using homology modeling. The model was optimized by a brief energy minimization in CHARMM and dynamics simulation of 250ps in MOE. The validation results of PROCHECK and Profile 3D show that the stereochemical quality of the model is good. The Calpha backbone of the template and the target (NEC-2) when superimposed showed RMSD of 0.39A. The model showed Asp51, His92 and Ser268 in the active site as seen in most of the PC2 members. The NEC-2 structure differs from that of furin at the catalytic pocket region with relevance to the amino acid composition which can be exploited for the design of specific inhibitors towards NEC-2.

Knock-out mouse models of proprotein convertases: unique functions or redundancy?

The members of the proprotein convertase family play a central role in the processing and/or activation of various protein precursors involved in many physiological processes and various pathologies. The proteolysis of these precursors that occur at basic residues within the general motif (K/R)-(X)-(K/R) is mediated by the proprotein convertases PC1/3, PC2, Furin, PACE4, PC4, PC5 and PC7, whereas the proteolysis of precursors within hydrophobic residues performed by the convertase S1P/SKI-1 and the convertase NARC-1/PCSK9 seems to prefer cleavages at the motif LVFAQSIP. Here we provide a comprehensive overview of their remarkable complex roles as revealed by disruption of their genes individually using generalized or conditional approaches.

7B2 prevents unfolding and aggregation of prohormone convertase 2.

Prohormone convertase 2 (PC2) requires interaction with the neuroendocrine protein 7B2 for the production of an activatable zymogen; the mechanism for this effect is unknown. 7B2 could act proactively to generate an activation-competent form of pro-PC2 during synthesis, or block spontaneous generation of activation-incompetent forms. We here demonstrate that addition of exogenous recombinant 7B2 to CHO cells expressing pro-PC2 prevented the unfolding and aggregation of secreted PC2 forms in a dose-dependent manner, as assessed by aggregation assays, activity assays, cross-linking experiments, and sucrose density gradients. Intracellular pro-PC2 was also found to exist in part as higher-order oligomers that were reduced in the presence of coexpressed 7B2. 7B2 addition did not result in the acquisition of enzymatic competence unless added before or very rapidly after pro-PC2 secretion, indicating that an activation-competent structure cannot be maintained in the absence of 7B2. Velocity sedimentation experiments showed that addition of extracellular 7B2 solubilized three different PC2 species from a precipitable aggregate: two activatable pro-PC2 species, the intact zymogen and a zymogen with a partially cleaved propeptide, and an inactive 66-kDa form. Our results suggest that 7B2 possesses chaperone activity that blocks partially unfolded pro-PC2 forms from losing catalytic competence and then aggregating. The loss of the catalytically competent conformer appears to represent the earliest indicator of pro-PC2 unfolding and is followed on a slower time scale by the appearance of aggregates. Because 7B2 expression is not confined to areas expressing pro-PC2, 7B2 may represent a general intracellular and extracellular secretory chaperone.

PC1/3, PC2 and PC5/6A are targeted to dense core secretory granules by a common mechanism.

There are seven members of the proprotein convertase (PC) family of secreted serine proteases that cleave their substrates at basic amino acids, thereby activating a variety of hormones, growth factors, and viruses. PC1/3, PC2 and PC5/6A are the only members of the PC family that are targeted to dense core secretory granules, where they carry out the processing of proteins that are secreted from the cell in a regulated manner. Previous studies have identified alpha-helices in the C-termini of the PC1/3 and PC2 proteases that are required for this subcellular targeting. In the current study, we demonstrate that a predicted alpha-helix in the C-terminus of PC5/6A is also critical for the ability of this domain to target a heterologous protein to the regulated secretory pathway of mouse endocrine AtT-20 cells. Analysis of the subcellular distribution of fusion proteins containing the C-terminal domains of PC1/3, PC2 and PC5/6A confirmed that all three domains have the capacity to redirect a constitutively secreted protein to the granule-containing cytoplasmic extensions. Analysis of the predicted structures formed by these three granule-sorting helices shows a correlation between their granule-sorting efficiency and the clustering of hydrophobic amino acids in their granule-targeting helices.

Processing and trafficking of a prohormone convertase 2 active site mutant.

Processing of most PC zymogens is required for successful folding and/or passage through the secretory pathway; active site mutants are retained in the ER and degraded. We here report that the active site serine mutant of PC2 (PC2-S383A) was efficiently secreted as the intact zymogen in CHO-K1 cells, suggesting that its propeptide can productively insert into the mutated binding pocket without causing misfolding. In AtT-20 cells, PC2-S383A was cleaved at the secondary cleavage site within the propeptide; this cleavage event was pH-dependent and was inhibited by a proprotein convertase inhibitor. In vitro digestion of PC2-S383A with various convertases indicates that this site is accessible to in trans cleavage. Abundant immunoreactive S383A PC2 was found in secretory granules, supporting the idea that this protein is efficiently trafficked through the secretory pathway.

Prohormone-convertase 1 processing enhances post-Golgi sorting of prothyrotropin-releasing hormone-derived peptides.

Rat prothyrotropin-releasing hormone (pro-TRH) is endoproteolyzed within the regulated secretory pathway of neuroendocrine cells yielding five TRH peptides and seven to nine other unique peptides. Endoproteolysis is performed by two prohormone convertases, PC1 and PC2. Proteolysis of pro-TRH begins in the trans-Golgi network and forms two intermediates that are then differentially processed as they exit the Golgi and are packaged into immature secretory granules. We hypothesized that this initial endoproteolysis may be necessary for downstream sorting of pro-TRH-derived peptides as it occurs before Golgi exit and thus entry into the regulated secretory pathway. We now report that when pro-TRH is transiently expressed in GH4C1 cells, a neuroendocrine cell line lacking PC1, under pulse-chase conditions release is constitutive and composed of more immature processing intermediates. This is also observed by radioimmunoassay under steady-state conditions. When a mutant form of pro-TRH, which has the dibasic sites of initial processing mutated to glycines, is expressed in AtT20 cells, a neuroendocrine cell line endogenously expressing PC1, both steady-state and pulse-chase experiments revealed that peptides derived from this mutant precursor are secreted in a constitutive fashion. A constitutively secreted form of PC1 does not target pro-TRH peptides to the constitutive secretory pathway but results in sorting to the regulated secretory pathway. These results indicated that initial processing action of PC1 on pro-TRH in the trans-Golgi network, and not a cargo-receptor relationship, is important for the downstream sorting events that result in storage of pro-TRH-derived peptides in mature secretory granules.

Recombinant prohormone convertase 1 and 2 cleave purified pro cholecystokinin (CCK) and a synthetic peptide containing CCK 8 Gly Arg Arg and the carboxyl-terminal flanking peptide.

Purified recombinant prohormone convertase 1 and 2 (PC1 and PC2) cleave a peptide containing cholecystokinin (CCK) 8 Gly Arg Arg and the carboxyl-terminal peptide liberating CCK 8 Gly Arg Arg. PC1 and PC2 also cleave purified pro CCK, liberating the amino terminal pro-peptide while no carboxyl-terminal cleavage was detected. Under the conditions of the in vitro cleavage assay, it appears that the carboxyl-terminal cleavage site of pro CCK is not accessible to the enzymes while this site is readily cleaved in a synthetic peptide. Additional cellular proteins that unfold the prohormone may be required to expose the carboxyl-terminal site for cleavage.

Significance of prohormone convertase 2, PC2, mediated initial cleavage at the proglucagon interdomain site, Lys70-Arg71, to generate glucagon.

To define the biological significance of the initial cleavage at the proglucagon (PG) interdomain site, K70-R71 downward arrow, we created two interdomain mutants, K70Q-R71Q and R71A. Cotransfection studies in GH4C1 cells show significant amounts of glucagon production by PC2 along with some glicentin, glicentin-related polypeptide-glucagon (GRPP-glucagon) and oxyntomodulin from wild-type PG. In contrast, a larger peptide, PG 33-158, and low amounts of GRPP-glucagon are predominantly generated from interdomain mutants. HPLC analysis shows a 5-fold increase in glucagon production by PC2 from wild-type PG and a corresponding 4-fold lower accumulation and secretion of unprocessed precursor relative to interdomain mutants. PC2 generates significant levels of glucagon from a glicentin (PG 1-69) expression plasmid, whereas PC1/3 produces only modest amounts of oxyntomodulin. Employing a major PG fragment (PG 72-158) expression plasmid, we show that PC1/3 predominantly generates glucagon-like peptide (GLP)-1, whereas PC2 produces only N-terminally extended GLP-1. Surprisingly, production of GLP-1 and GLP-2 by PC1/3 from interdomain mutants, compared with wild-type PG, is not significantly impaired. In addition to PC2 and PC1/3, PC5/6A and furin are also able to cleave the sites, K70-R71 downward arrow and R107-X-R-R110 downward arrow in PG. We show a much greater ability of furin to cleave the monobasic site, R77 downward arrow, than at the dibasic site, R124-R125 downward arrow, which is also weakly processed by PC5/6A, indicating overlapping specificities of these two convertases mainly with PC1/3. We propose here a trimer-like model of the spatial organization of the hormonal sequences within the PG molecule in which the accessibility to prohormone convertase action of most cleavage sites is restricted with the exception of the interdomain site, K70-R71, which is maximally accessible.

The C-terminus of prohormone convertase 2 is sufficient and necessary for Raft association and sorting to the regulated secretory pathway.

Prohormone convertase 2 (PC2) is a member of the subtilisin family of proteases involved in prohormone maturation in the granules of the regulated secretory pathway (RSP). It has been suggested that targeting of this enzyme to the RSP is dependent on its association with lipid rafts in membranes at the trans-Golgi network. Here, we investigate the orientation of PC2 in granule membranes and the role of the C-terminus in sorting of the enzyme to the RSP. Molecular modeling and circular dichroism showed that this domain of PC2 forms an alpha-helix and inserts into artificial membranes. Furthermore, we show that the C-terminus of PC2 can be biotinylated at the C-terminus in intact chromaffin granules, indicating that it is a transmembrane protein. To determine if the PC2 C-terminus is necessary for raft association and sorting, we transfected a chimera of CPEDelta15 (carboxypeptidase E without the last 15 residues) and the last 25 residues of PC2 (CPEDelta15-PC2), and a truncated PC2 mutant with the last 6 residues deleted (PC2Delta6) into Neuro2a cells. Whereas CPEDelta15 was not raft-associated or sorted to the RSP, addition of the 25 residues of PC2 C-terminus to CPEDelta15 restored raft association and localization to the RSP granules, as determined by immunocytochemistry. Deletion of the last 6 residues of PC2 eliminated lipid raft association and sorting of PC2Delta6 to the RSP. These results showed that the PC2 C-terminus confers raft association and is sufficient and necessary for sorting PC2 to the RSP.

Cleavage of recombinant proenkephalin and blockade mutants by prohormone convertases 1 and 2: an in vitro specificity study.

Proenkephalin (PE) derived-peptides are thought to be generated predominantly through endoproteolytic cleavage by prohormone convertases 1 and 2 (PC1 and PC2). In order to compare cleavage site preferences of these convertases, we studied the processing of recombinant wild-type rat PE and of two mutant PEs by recombinant purified mouse PC1 and PC2. Western blot analyses of timed digestions showed that both mouse PC1 and PC2 were able to produce a variety of large and intermediate sized-peptides from wild-type PE as well as from the precursors mutated at initial blockade sites. PC2 exhibited a broader specificity against PE than PC1, generating a much greater number of peptide products. Mass spectrometric identification of cleavage products showed that PC2 appeared to be the principal enzyme involved in the generation of smaller active opioids. Both enzymes were able to cleave various KR- and KK-containing sites, but PC2 was also able to cleave efficiently at an RR-V site and a KK-M site not cleaved by PC1, suggesting the exclusion of large aliphatic residues at the P1' position in PC1 cleavage. Alternative cleavage sites were readily chosen by convertases in blockade mutants, confirming in vivo results that cleavages do not follow an obligatory order. Furthermore, glycosylated PE was less efficiently processed by PC2, indicating that glycosylation may serve as a mechanism to hinder processing.