Proprotein-processing endopeptidases of the insulin secretory granule.

Enzymological studies have implicated two Ca2+ dependent endopeptidases in the conversion of proinsulin to insulin: a type 1 activity and a type 2 activity which cleave on the C-terminal side of R31R32 and K64R65 in proinsulin, respectively. These activities were further characterized and their relationship to the mammalian family of subtilisin-like proteases was investigated. PC2 was expressed in neuroendocrine tissues and in insulinoma secretory granule fractions predominantly as a 65kDa protein. On anion-exchange chromatography of solubilized granules, PC1/3 immunoreactivity comigrated with a peak of type 1 activity whereas PC2 immunoreactivity coeluted with the peak of type 2 endopeptidase activity. PC2 antiserum gave a specific immunoprecipitation of type 2 activity from insulin granule extracts. It was concluded that the PC2 gene-product has type 2 endopeptidase activity.     

Preferential cleavage of des-31,32-proinsulin over intact proinsulin by the insulin secretory granule type II endopeptidase. Implication of a favored route for prohormone processing.

Two Ca(2+)-dependent endopeptidase activities are involved in proinsulin to insulin conversion: type I cleaves COOH-terminal to proinsulin Arg31-Arg32 (B-chain/C-peptide junction); and type II preferentially cleaves at the Lys64-Arg65 site (C-peptide/A-chain junction). To further understand the mechanism of proinsulin processing, we have investigated types I and II endopeptidase processing of intact proinsulin in parallel to that of the conversion intermediates, des-31,32-proinsulin and des-64,65-proinsulin. The type I processed des-64,65-proinsulin and proinsulin at the same rate. In contrast, the type II endopeptidase processed des-31,32-proinsulin at a much faster rate (> 19-fold; p < 0.001) than it did intact proinsulin. Furthermore, unlabeled proinsulin concentrations required for competitive inhibition of 125I-labeled des-64,65-proinsulin and 125I-proinsulin processing by a purified insulin secretory granule lysate were similar (ID50 = 14-16 microM), whereas inhibition of 125I-labeled des-31,32-proinsulin processing required a higher nonradiolabeled proinsulin concentration (ID50 = 197 microM). Synthetic peptides corresponding to the sequences surrounding Lys64-Arg65 (AC-peptide/substrate) and Arg31-Arg32 (BC-peptide/substrate) of human proinsulin were synthesized for use as specific substrates or competitive inhibitors. Cleavage of the BC-substrate by type I and AC-substrate by type II was COOH-terminal of the dibasic sequence, with similar Ca(2+)-and pH requirements previously observed for proinsulin cleavage. Apparent Km and Vmax for type I processing of the BC-substrate was Km = 20 microM; Vmax = 22.8 pmol/min, and for type II processing of the AC-substrate was Km = 68 microM; Vmax = 97 pmol/min. In competitive inhibition assays, the BC-peptide similarly blocked insulin secretory granule lysate processing of des-64,65-proinsulin and proinsulin (ID50 = 45-55 microM), but did not inhibit des-31,32-proinsulin processing. However, the AC-peptide preferentially inhibited insulin secretory granule lysate processing of des-31,32-proinsulin (ID50 = microM) compared to proinsulin (ID50 = 330 microM), and not des-64,65-proinsulin. We conclude that the type I endopeptidase recognized des-64,65-proinsulin and proinsulin as similar substrates, whereas the type II endopeptidase has a stronger preference for des-31,32-proinsulin compared to intact proinsulin. Furthermore, we suggest that in intact proinsulin there exists a constraint to efficient processing that is relieved following type I processing. Structural flexibility, in addition to the presence of Lys64-Arg65, therefore appears to be important for type II endopeptidase specificity and may provide a molecular basis for a preferential route of proinsulin conversion via des-31,32-proinsulin.     

NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition

The proinsulin-insulin system provides a general model for the proteolytic processing of polypeptide hormones. Two proinsulin-specific endopeptidases have been defined, a type I activity that cleaves the B-chain/C-peptide junction (Arg31-Arg32) and a type II activity that cleaves the C-peptide/A-chain junction (Lys64-Arg65). These endopeptidases are specific for their respective dibasic target sites; not all such dibasic sites are cleaved, however, and studies of mutant proinsulins have demonstrated that additional sequence or structural features are involved in determining substrate specificity. To define structural elements required for endopeptidase recognition, we have undertaken comparative 1H NMR and photochemical dynamic nuclear polarization (photo-CIDNP) studies of human proinsulin, insulin, and split proinsulin analogues as models of prohormone processing intermediates. The overall conformation of proinsulin is observed to be similar to that of insulin, and the connecting peptide is largely unstructured. In the 1H NMR spectrum of proinsulin significant variation is observed in the line widths of insulin-specific amide resonances, reflecting exchange among conformational substates; similar exchange is observed in insulin and is not damped by the connecting peptide. The aromatic 1H NMR resonances of proinsulin are assigned by analogy to the spectrum of insulin, and assignments are verified by chemical modification. Unexpectedly, nonlocal perturbations are observed in the insulin moiety of proinsulin, as monitored by the resonances of internal aromatic groups. Remarkably, these perturbations are reverted by site-specific cleavage of the connecting peptide at the CA junction but not the BC junction. These results suggest that a stable local structure is formed at the CA junction, which influences insulin-specific packing interactions. We propose that this structure (designated the "CA knuckle") provides a recognition element for type II proinsulin endopeptidase.     

The post-translational processing and intracellular sorting of PC2 in the islets of Langerhans.

Proinsulin conversion in the insulin secretory granule is mediated by two sequence-specific endoproteases related to the Kex2 homologues, PC2 and PC3 (Bennett, D. L., Bailyes, E. M., Nielsen, E., Guest, P. C., Rutherford, N. G., Arden, S. D., and Hutton, J. C. (1992) J. Biol. Chem. 267, 15229-15236; Bailyes, E. M., Bennett, D. L., and Hutton, J. C. (1992) Enzyme, in press). Radiolabeling studies using isolated rat islets showed that PC2 was synthesized initially as a 76-kDa glycoprotein which was converted by limited proteolysis to the mature 64-66-kDa form. Conversion was initiated approximately 1 h after synthesis and proceeded via intermediates of 71, 68, and 66 kDa with a t1/2 of 140 min. Release of only the 66- and 64-66-kDa radiolabeled forms of PC2 was induced by glucose and then only at times more than 2 h following synthesis. Proinsulin conversion, by contrast, was more rapid (delay = 30 min, t1/2 = 60 min), and release commenced as soon as 1 h after synthesis with the secreted material being comprised of the precursor, intermediate, and mature forms of insulin. Ultrastructural analysis of islet beta cells showed that PC2 was concentrated in secretory granules. Subcellular fractionation combined with immunoblot analysis showed that insulinoma secretory granules contained only the mature 64-66-kDa form of PC2, whereas fractions enriched in Golgi and endoplasmic reticulum contained a mixture of the 76- and 66-kDa forms of the enzyme. These results indicate that post-translational proteolysis of PC2 is initiated before sorting into the regulated pathway of secretion and that the relative proportions of proinsulin and PC2 packaged into secretory granules will change with physiological conditions.     

Proalbumin to albumin conversion by a proinsulin processing endopeptidase of insulin secretory granules

A lysate of purified insulin secretory granules, which contains two types of proinsulin processing activity (type 1, Arg-Arg-directed and type II, Lys-Arg-directed (Davidson, H.W., Rhodes, C.J., and Hutton, J. C. (1988) Nature 333, 93-96), was found to process proalbumin by specific proteolytic cleavage of the COOH-terminal side of the Arg-2-Arg-1 sequence. The subcellular distribution of proalbumin processing activity in insulinoma tissue paralleled that for proinsulin conversion and occurred principally in a secretory granule fraction. Cleavage appeared to result from the Arg-Arg-directed type 1 proinsulin processing endo-peptidase. It was Ca2+-dependent (K0.5 activation = 1.0-1.5 mM Ca2+), unaffected by group-specific inhibitors of serine, cysteinyl, or aspartyl proteinases, and had an acidic pH optimum (5.5). Active-site inhibitor studies showed this activity had a preference for dibasic over monobasic amino acid sequences and indicated that the sequence of the dibasic site was an important determinant of the susceptibility of the substrate to cleavage. The activity did not process the proalbumin Christchurch mutant (Arg-2-Arg-1 to Arg-2-Gln-1). It was inhibited by the variant alpha 1-antitrypsin Pittsburgh (Met358 to Arg358; K0.5 = 100 nM) but not by other related proteins normally co-secreted with albumin from hepatocytes, namely alpha 1-antitrypsin M, alpha 2-macroglobulin, or antithrombin III. The insulin secretory granule proalbumin processing activity was indistinguishable from a proalbumin endopeptidase reported in rat liver membranes and similar to the yeast KEX-2 protease. These findings suggest that a highly conserved set of proprotein endopeptidases exists, which are specific for a dibasic sequence but broadly specific for proprotein substrates. Such enzymic activities appear to be active within both the constitutive and regulated pathways of secretion. Intraorganellar Ca2+ and pH appear to play a key role in regulating their activities.     

Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.

The biosynthesis and post-translational maturation of PC2, a neuroendocrine-specific Kex2-like endoprotease, following expression in Xenopus oocytes is described. The initial translation product was a 75-kDa membrane-associated protein which was released from the oocytes as a glycosylated 71-kDa protein. During extended chase periods, the extracellular 71-kDa protein was converted to a mature 68-kDa product. A deletion mutant lacking a putative COOH-terminal amphipathic helix was still membrane-associated, suggesting that this domain was not essential for attachment of PC2 to membranes. Two putative proregion cleavage site mutants were also constructed. Conversion of the 75-kDa peptide to the 71-kDa peptide involved cleavage at the sequence Lys-Arg-Arg-Arg (amino acids 78-81), since mutation of this sequence to Lys-Val-Arg-Leu resulted in the secretion of the 75-kDa peptide. Extracellular conversion of the 71-kDa peptide to the 68-kDa peptide involved cleavage at the sequence Arg-Lys-Lys-Arg (amino acids 106-109), since deletion of this tetrabasic sequence resulted in secretion of the 71-kDa peptide without further conversion to the 68-kDa form. Finally, a mutation which changed a catalytically important Asp to Asn did not affect processing of proPC2. These results may be relevant to our understanding of mechanisms in the intracellular sorting and maturation of proPC2 in neuroendocrine cells.     

Up-regulation of splenic prohormone convertases PC1 and PC2 in diabetic rats.

Organisms respond to infection in a complex manner involving bidirectional interactions between the neuroendocrine and immune systems. Many of the bioactive endocrine/immune factors are synthesized in a precursor form and are expected to be activated by prohormone convertases (PCs). Since patients with both type 1 and type 2 diabetes have an increased incidence and severity of infections, we hypothesized that in a condition of hyperglycemia, these processing enzymes would be activated in an immune tissue, the spleen. To test this hypothesis, we treated rats with intraperitoneal streptozotocin (STZ) (50 mg/kg/day) daily for 5 days and measured splenic PC1 and PC2 mRNA by ribonuclease protection assay. We found that PC1 mRNA was increased 6.0+/-0.02-fold (P<0.05) and PC2 mRNA was increased 1.80+/-0.01-fold (P<0.005) in the spleen of rats that received STZ compared to rats that received vehicle. Western blot indicated that the 75-kDa form of PC1 was the only form of PC1 present in the spleen and that this form increased with STZ treatment. Immunohistochemistry revealed that PC1 was found in both the white pulp (T-lymphocytes) and red pulp (monocytes and macrophages) and that its increase in immunoreactivity occurred primarily in the white pulp. PC2 and pro-opiomelanocortin (POMC, a possible splenic substrate for PC1/PC2) immunoreactivity was found predominantly in the red pulp. STZ induced an increase in splenic PC1 and POMC, but not PC2 protein levels. We conclude that in the STZ model of diabetes, splenic PCs are induced, which could lead to an increased activation of many immune-derived hormones. We speculate that this up-regulation of prohormone converting enzymes may be related to the increased infections seen in patients with both type 1 and type 2 diabetes.     

Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2

We have identified a human insulinoma cDNA (PC2) that encodes a protein homologous to the precursor processing Kex2 endoprotease of yeast by using a polymerase chain reaction to detect and amplify conserved sequences within the catalytic site. The 638-residue amino acid sequence of PC2 begins with a cleavable signal peptide, indicating that it enters the secretory pathway, and contains a 282-residue domain that is homologous to the catalytic modules of both Kex2 and the related bacterial subtilisins. Within this region 49 and 27% of the amino acids are identical to those in the aligned Kex2 and subtilisin BPN' sequences, respectively, and the catalytically essential Asp, His, and Ser residues are all conserved. Northern blot analysis revealed the presence of 2.8- and 5.0-kilobase hybridizing bands in mRNA from the insulinoma. The PC2 protein also shows great similarity to the incomplete NH2-terminal sequence of the human furin gene product, a putative membrane-inserted receptor-like molecule. We propose that PC2 is a member of a family of mammalian Kex2/subtilisin-like proteases that includes members involved in a number of specific proteolytic events within cells, including the processing of prohormones.     

A 36-residue peptide contains all of the information required for 7B2-mediated activation of prohormone convertase 2.

The prohormone convertases (PCs) are serine proteinases responsible for the processing of secretory protein precursors. PC2 is the only member of this family whose activation requires intracellular interaction with a helper protein, the neuroendocrine protein 7B2. In order to gain a better understanding of the mechanism of proPC2 activation, we have characterized the structural determinants of 7B2 required for proPC2 activation. We had already identified a proline-rich binding determinant in the 21-kDa domain, the portion of 7B2 responsible for proPC2 activation. We have now investigated the function of the weakly conserved amino-terminal portion of 21-kDa 7B2 by sequential deletions. Mutant proteins were analyzed in four assays: binding to proPC2, facilitation of proPC2 maturation, and activation of proPC2 in vivo and in vitro. We found that the amino-terminal half of 7B2 is not involved in proPC2 activation, and we identified an active 36-residue peptide that contains the previously characterized proline-rich sequence as well as an alpha-helix and the only disulfide bond of 7B2. Mutation of the alpha-helix and of the cysteines demonstrated that these determinants are absolutely required for PC2 activation. Thus, the 186-residue full-length 7B2 rat protein can be functionally reduced to an internal segment of only 36 residues.     

Inhibitory specificity and potency of proSAAS-derived peptides toward proprotein convertase 1.

Prohormone convertase 1 (PC1), mediating the proteolytic processing of neural and endocrine precursors, is thought to be regulated by the neuroendocrine protein proSAAS. The PC1 inhibitory sequence is mostly confined within a 10-12-amino acid segment near the C terminus of the conserved human proSAAS and contains the critical KR(244) dibasic motif. Our results show that the decapeptide proSAAS-(235-244)( 235)VLGALLRVKR(244) is the most potent reversible competitive PC1-inhibitor (K(i) approximately 9 nm). The C-terminally extended proSAAS-(235-246) exhibits a 5-6-fold higher K(i) ( approximately 51 nm). The additional LE sequence at P1'-P2', resulted in a competitive substrate cleaved by PC1 at KR(244) downward arrowLE(246). Systematic alanine scanning and in some cases lysine scanning tested the contribution of each residue within proSAAS-(235-246) toward the PC1-inhibition's specificity and potency. The amino acids P1 Arg, P2 Lys, and P4 Arg are all critical for inhibition. Moreover, the aliphatic P3 Val and P5, P6, and P1' Leu significantly affect the degree of enzyme inactivation and PC1 specificity. Interestingly, a much longer N- and C-terminally extended endogenous rat proSAAS-(221-254) called little PenLen, was found to be a 3-fold less potent PC1 inhibitor with reduced selectivity but a much better substrate than proSAAS-(235-246). Molecular modeling studies and circular dichroism analysis indicate an extended and poly-l-proline II type structural conformation for proSAAS-(235-244), the most potent PC1 inhibitor, a feature not present in poor PC1 inhibitors.     

Enzymatic inactivation of bradykinin by rat brain neuronal perikarya

1. Bradykinin (Bk; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg8) inactivation by bulk isolated neurons from rat brain is described. 2. Bk is rapidly inactivated by neuronal perikarya (4.2 +/- 0.6 fmol/min/cell body). 3. Sites of inactivating cleavages, determined by a kininase bioassay combined with a time-course Bk-product analysis, were the Phe5-Ser6, Pro7-Phe8, Gly4-Phe5, and Pro3-Gly4 peptide bonds. The cleavage of the Phe5-Ser6 bond inactivated Bk at least five fold faster than the other observed cleavages. 4. Inactivating peptidases were identified by the effect of inhibitors on Bk-product formation. The Phe5-Ser6 bond cleavage is attributed mainly to a calcium-activated thiol-endopeptidase, a predominantly soluble enzyme which did not behave as a metalloenzyme upon dialysis and was strongly inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate and endo-oligopeptidase A antiserum. Thus, neuronal perikarya thiol-endopeptidase seems to differ from endo-oligopeptidase A and endopeptidase 24.15. 5. Endopeptidase 24.11 cleaves Bk at the Gly4-Phe5 and, to a larger extent, at the Pro7-Phe8 bond. The latter bond is also cleaved by angiotensin-converting enzyme (ACE) and prolyl endopeptidase (PE). PE also hydrolyzes Bk at the Pro3-Gly4 bond. 6. Secondary processing of Bk inactivation products occurs by (1) a rapid cleavage of Ser6-Pro7-Phe8-Arg8 at the Pro7-Phe8 bond by endopeptidase 24.11, 3820ACE, and PE; (2) a bestatin-sensitive breakdown of Phe8-Arg9; and (3) conversion of Arg1-Pro7 to Arg1-Phe5, of Gly4-Arg9 to both Gly4-Pro7 and Ser6-Arg9, and of Phe5-Arg9 to Ser6-Arg9, Phe8-Arg9, and Ser6-Pro7, by unidentified peptidases. 7. A model for the enzymatic inactivation of bradykinin by rat brain neuronal perikarya is proposed.     

Expression of human prohormone convertase PC2 in a baculovirus-insect cell system.

PC2 is a member of the eukaryotic family of subtilisin-related proprotein convertases which are thought to be involved in the intracellular proteolytic processing of prohormones and proneuropeptides. The presence of only small amounts of PC2 in the secretory granules of certain mammalian neuroendocrine cell types has made the characterization and further study of this enzyme difficult. We report here the expression of proteolytically active human PC2 protein in the insect cell-baculovirus system. Human PC2 expressed in insect cells is a calcium-dependent intracellular protein active at neutral pH. In insect cells, human PC2 was found intracellularly as 75-kDa and 71-kDa proteins. Both 73-kDa and 68-kDa forms were found in the conditioned medium, but no PC2 proteolytic activity was detected. We demonstrated the presence of a soluble inhibitor in infected-cell medium which may block PC2 activity.     

Purification of the main somatostatin-degrading proteases from rat and pig brains, their action on other neuropeptides, and their identification as endopeptidases 24.15 and 24.16.

The main somatostatin-degrading proteases were purified from rat and pig brain homogenates and characterized as thiol- and metal-dependent endoproteases. Two types of proteases with apparent native and subunit molecular masses of 70 kDa and 68 kDa could be differentiated in both species. Beside somatostatin, both hydrolyzed several other neuropeptides with chain lengths between 8 and 30 amino acid residues. Cleavage sites were generally similar or identical, but some clear exceptions were observed for enzymes from both species which could be used to differentiate between the two proteases. The 68-kDa protease cleaved somatostatin at three bonds (Asn5-Phe6, Phe6-Phe7 and Thr10-Phe11) and neurotensin only at the Arg8-Arg9 bond, whereas the 70-kDa protease digested somatostatin at only two bonds (Phe6-Phe7 and Thr10-Phe11) and neurotensin as well as acetylneurotensin-(8-13) additionally (pig protease) or almost exclusively (rat protease) at the Pro10-Tyr11 bond. Relative rates for the digestions of various peptides were, however, more dependent on the species than on the type of protease. Cleavage sites for angiotensin II, bradykinin, dynorphin, gonadoliberin and substance P were, apart from different rates, identical for both proteases. In both species the 68-kDa protease was found to be mainly, but not exclusively, soluble and not membrane-associated, whereas the inverse was detected for the 70-kDa protease. Based on distinct molecular and catalytic properties, the 68-kDa protease is supposed to be congruent with the endopeptidase 24.15 (EC 3.4.24.15), the 70-kDa protease with endopeptidase 24.16 (EC 3.4.24.16, neurotensin-degrading endopeptidase). This investigation demonstrates that both proteases hydrolyze various neuropeptides with similar cleavage sites, but with species-dependent activity. Species-independent distinctions are the exclusive action of endopeptidase 24.16 on acetylneurotensin-(8-13) and liberation of free Phe from somatostatin only by endopeptidase 24.15.     

An endopeptidase associated with bovine neurohypophysis secretory granules cleaves pro-ocytocin/neurophysin peptide at paired basic residues

The octacosapeptide sequence [Tyr18] pro-ocytocin/neurophysin (1-18)NH2 [pro-OT/Np(1-18)NH2] was synthesized and used as substrate to detect endoprotease(s) possibly involved in the processing of this precursor in bovine hypothalamo-neurohypophyseal tract. An endopeptidase (58 Kda) was detected in Lysates made from highly purified neurosecretory granules. This protease which cleaves the peptide bond on the carboxyl side of the Lys-Arg doublet, and no single basic residue, generates both OT-Gly10-Lys11-Arg12+Ala13-Val-Leu-Asp-Leu-Tyr18 (NH2) from the octacosapeptide substrate. In addition, a carboxypeptidase B-like activity converting OT-Gly10-Lys11-Arg12 into OT-Gly10 was detected in the same granule Lysates. It is hypothesized that a combination of these endoprotease and carboxypeptidase B-like activities together with the amidating enzyme of secretory granules might participate in the cleavage and processing of pro-OT/Np in vivo.     

The protease core of the muscle-specific calpain,p94, undergoes Ca2+-dependent intramolecular autolysis

Limb girdle muscular dystrophy type 2A is linked to a skeletal muscle-specific calpain isoform known as p94. Isolation of the intact 94-kDa enzyme has been difficult to achieve due to its rapid autolysis, and uncertainty has arisen over its Ca2+-dependence for activity. We have expressed a C-terminally truncated form of the enzyme that comprises the protease core (domains I and II) along with its insertion sequence, IS1, and N-terminal leader sequence, NS. This 47-kDa p94I-II mini-calpain was stable during purification. In the presence of Ca2+, p94I-II cleaved itself within the NS and IS1 sequences. Mapping of the autolysis sites showed that NS and IS1 have the potential to be removed without damage to the protease core. Ca2+-dependent autolysis must be an intramolecular event because the inactive p94I-II C129S mutant was not cleaved by incubation with wild-type p94I-II. In addition, the rate of autolysis of p94I-II was independent of the concentration of the enzyme.     

The Arg617-Arg618 cleavage site in the C-terminal domain of PC1 plays a major role in the processing and targeting of the enzyme within the regulated secretory pathway

The C-terminal domain of the prohormone convertase PC1 is involved in targeting of the enzyme to secretory granules in neuroendocrine cells and is subsequently processed in this compartment at an Arg617-Arg618 site. Three other dibasics are found in the C-terminal domain of mouse PC1. Here, we examined the role of the four dibasics in targeting PC1 to secretory granules. All 15 possible combinations of dibasic mutations were performed. Wild-type (WT) and mutant PC1 were stably expressed in neuroendocrine PC12 cells that lacked endogenous PC1. Processing, secretion and intracellular localization of PC1 and its mutants were analyzed. Leaving intact Arg617-Arg618 and mutating any combination of the three other dibasics yielded proteins that were stored and processed in secretory granules, similarly to WT PC1. Mutating Arg617-Arg618 alone or with any one of the three remaining dibasics generated proteins that were efficiently stored in secretory granules but were not processed further. Mutating Arg617-Arg618 with more than one of the remaining dibasics produced proteins that reached the TGN but were not stored in secretory granules and exited the cells through the constitutive secretory pathway. These data demonstrate that the Arg617-Arg618 plays a prominent role in targeting PC1 to secretory granules.     

ProSAAS processing in mouse brain and pituitary

ProSAAS is a newly discovered protein with a neuroendocrine distribution generally similar to that of prohormone convertase 1 (PC1), a peptide-processing endopeptidase. Several proSAAS-derived peptides were previously identified in the brain and pituitary of the Cpe(fat)/Cpe(fat) mouse based on the accumulation of C-terminally extended peptides due to the absence of enzymatically active carboxypeptidase E, a peptide-processing exopeptidase. In the present study, antisera against different regions of proSAAS were used to develop radioimmunoassays and examine the processing profile of proSAAS in wild type and Cpe(fat)/Cpe(fat) mouse tissues following gel filtration and reverse phase high performance liquid chromatography. In wild type mouse brain and pituitary, the majority of proSAAS is processed into smaller peptides. These proSAAS-derived peptides elute from the reverse-phase column in the same positions as synthetic peptides that correspond to little SAAS, PEN, and big LEN. Mass spectrometry revealed the presence of peptides with the expected molecular masses of little SAAS and big LEN in the fractions containing immunoreactive peptides. The processing of proSAAS is slightly impaired in Cpe(fat)/Cpe(fat) mice, relative to wild-type mice, leading to the accumulation of partially processed peptides. One of these peptides, the C-terminally extended form of PEN, is known to inhibit PC1 activity and this could account for the reduction in enzymatically active PC1 seen in Cpe(fat)/Cpe(fat) mice. The observation that little SAAS and big LEN are the major forms of these peptides produced in mouse brain and pituitary raises the possibility that these peptides function as neurotransmitters or hormones.     

A novel human insulinoma-associated cDNA, IA-1, encodes a protein with "zinc-finger" DNA-binding motifs.

A subtraction library was constructed from human insulinoma (beta cell tumor) and glucagonoma (alpha cell tumor) cDNA phagemid libraries. Differential screening of 153 clones with end-labeled mRNAs from insulinoma, glucagonoma, and HeLa cells resulted in the isolation of a novel cDNA clone designated IA-1. This cDNA clone has a 2838-base pair sequence consisting of an open reading frame of 1530 nucleotides, which translates into a protein of 510 amino acids with a pI value of 9.1 and a molecular mass of 52,923 daltons. At the 3'-untranslated region there are seven ATTTA sequences between two polyadenylation signals (AATAAA). The IA-1 protein can be divided into two domains based upon the features of its amino acid sequence. The NH2-terminal domain of the deduced protein sequence (amino acids 1-250) has four classical pro-hormone dibasic conversion sites and an amidation signal sequence, Pro-Gly-Lys-Arg. The COOH-terminal domain (amino acids 251-510) contains five putative "zinc-finger" DNA-binding motifs of the form X3-Cys-X2-4-Cys-X12-His-X3-4-His-X4 which has been described as a consensus sequence for members of the Cys2-His2 DNA-binding protein class. Northern blot analysis revealed IA-1 mRNA in five of five human insulinoma and three of three murine insulinoma cell lines. Expression of this gene was undetectable in normal tissues. Additional tissue studies revealed that the message is expressed in several tumor cell lines of neuroendocrine origin including pheochromocytoma, medullary thyroid carcinoma, insulinoma, pituitary tumor, and small cell lung carcinoma. The restricted tissue distribution and unique sequence motifs suggest that this novel cDNA clone may encode a protein associated with the transformation of neuroendocrine cells.     

Jagn1 Is Induced in Response to ER Stress and Regulates Proinsulin Biosynthesis

The Jagn1 protein was indentified in a SILAC proteomic screen of proteins that are increased in insulinoma cells expressing a folding-deficient proinsulin. Jagn1 mRNA was detected in primary rodent islets and in insulinoma cell lines and the levels were increased in response to ER stress. The function of Jagn1 was assessed in insulinoma cells by both knock-down and overexpression approaches. Knock-down of Jagn1 caused an increase in glucose-stimulated insulin secretion resulting from an increase in proinsulin biosynthesis. In contrast, overexpression of Jagn1 in insulinoma cells resulted in reduced cellular proinsulin and insulin levels. Our results identify a novel role for Jagn1 in regulating proinsulin biosynthesis in pancreatic β-cells. Under ER stress conditions Jagn1 is induced which might contribute to reducing proinsulin biosynthesis, in part by helping to relieve the protein folding load in the ER in an effort to restore ER homeostasis.