The subtilisin-like protease SBT3 contributes to insect resistance in tomato.

Subtilisin-like proteases (SBTs) constitute a large family of extracellular plant proteases, the function of which is still largely unknown. In tomato plants, the expression of SBT3 was found to be induced in response to wounding and insect attack in injured leaves but not in healthy systemic tissues. The time course of SBT3 induction resembled that of proteinase inhibitor II and other late wound response genes suggesting a role for SBT3 in herbivore defense. Consistent with such a role, larvae of the specialist herbivore Manduca sexta performed better on transgenic plants silenced for SBT3 expression (SBT3-SI). Supporting a contribution of SBT3 to systemic wound signaling, systemic induction of late wound response genes was attenuated in SBT3-SI plants. The partial loss of insect resistance may thus be explained by a reduction in systemic defense gene expression. Alternatively, SBT3 may play a post-ingestive role in plant defense. Similar to other anti-nutritive proteins, SBT3 was found to be stable and active in the insect's digestive system, where it may act on unidentified proteins of insect or plant origin. Finally, a reduction in the level of pectin methylesterification that was observed in transgenic plants with altered levels of SBT3 expression suggested an involvement of SBT3 in the regulation of pectin methylesterases (PMEs). While such a role has been described in other systems, PME activity and the degree of pectin methylesterification did not correlate with the level of insect resistance in SBT3-SI and SBT3 overexpressing plants and are thus unrelated to the observed resistance phenotype.

Prodomain of the proprotein convertase subtilisin/kexin Furin (ppFurin) protects from tumor progression and metastasis.

Proteolytic maturation of various precursor proteins by the proprotein convertase Furin is now considered as a crucial step in tumor progression and metastasis. Here, we report the repression of the malignant and metastatic potential of carcinoma cells by the prodomain region of Furin (ppFurin), a naturally occurring inhibitor of this convertase. Overexpression of ppFurin in carcinoma cells in a stable manner significantly reduced their convertase activity and ability to mediate processing of the Furin cancer-related substrates platelet-derived growth factor (PDGF)-A and insulin-like growth factor-I receptor precursors. Unprocessed platelet-derived growth factor-A produced by ppFurin expressing cells failed to induce the activation of Akt in the platelet-derived growth factor receptor-expressing cells NIH BALB/c-3T3 and treatment of ppFurin expressing cells with insulin-like growth factor-I failed to induce Akt phosphorylation, compared with controls. The malignant potential of ppFurin expressing cells was significantly reduced as revealed by the loss of anchorage-independent growth and survival that associated their increased chemosensitivity. In vivo, comparative studies revealed that expression of ppFurin in the carcinoma cells MDA-MB-231 and CT-26 cells inhibited tumor growth when subcutaneously inoculated in nude mice. The use of an experimental liver colorectal metastasis model revealed the reduced ability of metastatic carcinoma CT-26 cells to colonize the liver in response to intrasplenic/portal inoculation. Further analyses revealed reduced Furin activity in tumors derived from intrasplenic inoculated mice with ppFurin expressing CT-26 cells. This finding highlights the role of Furin in the malignant and metastatic potential of tumor cells and suggests the possible consideration of using its naturally occurring inhibitor ppFurin in anticancer therapy.

The malarial serine protease SUB1 plays an essential role in parasite liver stage development.

Transmission of the malaria parasite to its vertebrate host involves an obligatory exoerythrocytic stage in which extensive asexual replication of the parasite takes place in infected hepatocytes. The resulting liver schizont undergoes segmentation to produce thousands of daughter merozoites. These are released to initiate the blood stage life cycle, which causes all the pathology associated with the disease. Whilst elements of liver stage merozoite biology are similar to those in the much better-studied blood stage merozoites, little is known of the molecular players involved in liver stage merozoite production. To facilitate the study of liver stage biology we developed a strategy for the rapid production of complex conditional alleles by recombinase mediated engineering in Escherichia coli, which we used in combination with existing Plasmodium berghei deleter lines expressing Flp recombinase to study subtilisin-like protease 1 (SUB1), a conserved Plasmodium serine protease previously implicated in blood stage merozoite maturation and egress. We demonstrate that SUB1 is not required for the early stages of intrahepatic growth, but is essential for complete development of the liver stage schizont and for production of hepatic merozoites. Our results indicate that inhibitors of SUB1 could be used in prophylactic approaches to control or block the clinically silent pre-erythrocytic stage of the malaria parasite life cycle.

Mycobacterium tuberculosis MycP1 protease plays a dual role in regulation of ESX-1 secretion and virulence.

Mycobacterium tuberculosis uses the ESX-1 secretion system to deliver virulence proteins during infection of host cells. Here we report a mechanism of posttranscriptional control of ESX-1 mediated by MycP1, a M. tuberculosis serine protease. We show that MycP1 is required for ESX-1 secretion but that, unexpectedly, genetic inactivation of MycP1 protease activity increases secretion of ESX-1 substrates. We demonstrate that EspB, an ESX-1 substrate required for secretion, is a target of MycP1 in vitro and in vivo. During macrophage infection, an inactive MycP1 protease mutant causes hyperactivation of ESX-1-stimulated innate signaling pathways. MycP1 is required for growth in mice during acute infection, while loss of its protease activity leads to attenuated virulence during chronic infection. As the key ESX-1 substrates ESAT-6 and CFP-10 are highly immunogenic, fine-tuning of their secretion by MycP1 may balance virulence and immune detection and be essential for successful maintenance of long-term M. tuberculosis infection.

Post-endoplasmic reticulum rescue of unstable MHC class I requires proprotein convertase PC7.

The function of the peptide-loading complex (PLC) is to facilitate loading of MHC class I (MHC I) molecules with antigenic peptides in the endoplasmic reticulum and to drive the selection of these ligands toward a set of high-affinity binders. When the PLC fails to perform properly, as frequently observed in virus-infected or tumor cells, structurally unstable MHC I peptide complexes are generated, which are prone to disintegrate instead of presenting Ags to cytotoxic T cells. In this study we show that a second quality control checkpoint dependent on the serine protease proprotein convertase 7 (PC7) can rescue unstable MHC I, whereas the related convertase furin is completely dispensable. Cells with a malfunctioning PLC and silenced for PC7 have substantially reduced MHC I surface levels caused by high instability and significantly delayed surface accumulation of these molecules. Instead of acquiring stability along the secretory route, MHC I appears to get largely routed to lysosomes for degradation in these cells. Moreover, mass spectrometry analysis provides evidence that lack of PLC quality control and/or loss of PC7 expression alters the MHC I-presented peptide profile. Finally, using exogenously applied peptide precursors, we show that liberation of MHC I epitopes may directly require PC7. We demonstrate for the first time an important function for PC7 in MHC I-mediated Ag presentation.

Selective roles for the PC2 processing enzyme in the regulation of peptide neurotransmitter levels in brain and peripheral neuroendocrine tissues of PC2 deficient mice.

The prohormone convertase 2 (PC2) is hypothesized to convert multiple pro-neuropeptides into active peptides that function as neurotransmitters. To examine the in vivo role of PC2 in neuropeptide production, the tissue contents of six different neuropeptides in brain and peripheral nervous tissues were examined in PC2 deficient mice. Specific neuropeptide radioimmunoassays and RP-HPLC (reverse-phase HPLC) provided evaluation of processed, active neuropeptides in brain and neuroendocrine tissues of PC2 deficient mice. Results demonstrated three features with regard to the selective roles of PC2 in determining the production of NPY, somatostatin-28, enkephalin, VIP, galanin, and CRF in neuroendocrine tissues. Firstly, PC2 deficient mice showed changes in several neuropeptides, but not all neuropeptides examined. The absence of active PC2 resulted in altered cellular levels of NPY, somatostatin-28, and (Met)enkephalin; few changes in VIP or galanin occurred in the tissues examined. CRF content was not altered in brains of PC2 deficient mice. Secondly, comparison of a single neuropeptide among different tissues of PC2 deficient mice demonstrated tissue-selective roles for PC2 in production of the neuropeptide. For example, NPY levels were decreased in ileum of PC2 deficient mice, but NPY content was not altered in hypothalamus that is abundant in NPY. In addition, (Met)enkephalin levels in hypothalamus and cortex were decreased in PC2 deficient mice, but no changes were observed in adrenal or intestine. Thirdly, a single tissue region often showed selective alterations among different neuropeptides. For example, the neuropeptide-rich hypothalamus region showed decreased (Met)enkephalin in PC2 deficient mice, but NPY, VIP, galanin, and CRF were not altered. These results demonstrate the selective role of PC2 in neuropeptide production that provides active peptide neurotransmitter or hormones for biological functions in brain and neuroendocrine systems.

Processing of alpha4 integrin by the proprotein convertases: histidine at position P6 regulates cleavage.

The proprotein convertases (PCs) participate in the limited proteolysis of integrin alpha4 subunit at the H(592)VISKR(597) downward arrow ST site (where underlined residues indicate positively charged amino acids important for PC-mediated cleavage and downward arrow indicates the cleavage site), since this cleavage is inhibited by the serpin alpha1-PDX (alpha1-antitrypsin Portland). Co-expression of alpha4 with each convertase in LoVo (furin-deficient human colon carcinoma) cells revealed that furin and proprotein convertase 5A (PC5A) are the best pro-alpha4 convertases. In agreement, processing of endogenous pro-alpha4 in human lymphoblastoid CEM-T4 cells was enhanced greatly in stable transfectants overexpressing either enzyme. In many leucocyte cell lines, the expression of furin closely correlated with the endogenous processing efficacy, suggesting that furin is a candidate pro-alpha4 convertase. Mutational analysis showed that replacement of P1 Arg(597) with alanine (R597A) abrogated cleavage, whereas the P6 mutant H592R is even better processed by the endogenous convertases of Chinese-hamster ovary CHO-K1 cells. In vitro kinetic studies using synthetic peptides confirmed the importance of a positively charged residue at P6 and showed that wild-type alpha4 processing is performed best by furin and PC5A at acidic and neutral pHs, respectively. Biosynthetic analysis of pro-alpha4 and its H592R and H592K mutants in the presence or absence of the weak base, NH(4)Cl, revealed that the P6 histidine residue renders its processing by furin sensitive to cellular pH. This suggests that pro-alpha4 cleavage occurs preferentially in acidic compartments. In conclusion, although the accepted furin processing motif is Arg-Xaa-(Lys/Arg)-Arg downward arrow, our data further extend it to include a regulatory histidine residue at P6 in precursors that lack a basic residue at P4.

Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme.

The tumor necrosis factor alpha converting enzyme (TACE) activity is required for the shedding of a variety of biologically active membrane bound precursors. The activation of TACE necessitates the proteolytic cleavage of its prodomain, a process that was suggested to be catalyzed by the proprotein convertase furin. However, the involvement of furin in this activation process has never been experimentally demonstrated. We have shown that the furinlike cleavage site (R-V-K-R(214)) localized between the prodomain and the metalloprotease domain of TACE is the sole site that can be in vitro cleaved by furin. In Cos7 cells, the release of TACE-processed substrates was reduced by the overexpression of the furin-specific proprotein convertase inhibitor Portland alpha1-antitrypsin inhibitor, but the release of TACE-processed substrates was increased by overexpression of furin in LoVo cells (deficient in furin activity) in which a mature form of TACE was identified. The immature form of TACE was detected at the surface of LoVo cells and at the surface of Cos7 and HT29 cells upon proprotein convertase inhibition. These results suggest that furin is the major proprotein convertase involved in the maturation/activation of TACE which is not a prerequisite for its cell-surface expression.

Precursor convertases in the secretory pathway, cytosol and extracellular milieu.

Precursor proteins that transit through the secretory pathway often require processing at specific sites in order to release their bioactive entities. The most prevalent limited proteolysis occurs at single or paired basic residues, and is achieved by one or more of the seven subtilisin-like proprotein convertases (PCs); Furin, PC1, PC2, PACE4 (paired basic amino acid converting enzyme 4), PC4, PC5 and PC7. Other types of cleavages occur at hydophobic residues, some of which are performed by subtilisin/kexin-like isozyme-1 (SKI-1), which is also known as site-1 protease. Together, the PCs and SKI-1 regulate the activity of a large variety of cellular proteins, including growth factors, neuropeptides, receptors, enzymes and even toxins and glycoproteins from infectious retroviruses. These processing events are exquisitely regulated by multiple zymogen-activation steps, as well as by specific subcellular localization signals. The above mentioned convertases are implicated in a number of pathologies such as cancer, neurodegenerative diseases, endocrine disorders and inflammation. Recently, it was recognized that the metalloendopeptidase N-arginine dibasic convertase (NRDc; nardilysin), which cleaves at the N-terminus side of basic residues in dibasic pairs, is localized both in the cytosol and at the cell surface or in the extracellular milieu. While NRDc binds heparin-binding epidermal growth factor (HB-EGF) at the cell surface and potentiates its physiological effect, HB-EGF potently inhibits the NRDc's activity. NRDc could represent the equivalent of the PCs in the cytosol or the extracellular space.

Processing of the HTLV-II envelope precursor glycoprotein gp63 by furin is essential for cell fusion activity.

To investigate the relationship between the fusogenic properties of HTLV-II and the processing of the envelope precursor glycoprotein gp63, recombinant cowpox virus expressing this protein was used to infect a range of cell lines derived from different species. Syncytium formation and gp63 processing were observed in all cells with the exception of LoVo cells, which are known to have a dysfunctional form of the endoprotease, furin. Furin has been shown to be necessary for the processing of a number of viral envelope glycoproteins, and gp63 contains a consensus sequence (305)Arg-Arg-Arg-Arg, which is a furin substrate motif. Pulse-chase studies demonstrated gp63 processing in Vero but not in LoVo cells. In addition it could be shown that expression of recombinant furin restored the processing of gp63 to gp46 in LoVo cells, and this resulted in syncytium formation. Our findings suggest that furin plays a pivotal role in cleavage of the HTLV-II envelope gp63, which in turn is a prerequisite for the fusogenic properties of the virus.

Prohormone convertase furin has a role in gastric cancer cell proliferation with parathyroid hormone-related peptide in a reciprocal manner.

We investigated the expression of parathyroid hormone-related peptide (PTHrP) and the relationship between PTHrP and its endoprotease furin in gastric cancer. PTHrP was colocalized with furin in 75% of gastric cancer tissues (six of eight) from patients with high serum PTHrP levels. PTHrP mRNA expression was confirmed in 67% of gastric cancer cell lines (four of six), whereas furin mRNA was detected in all six gastric cancer cell lines. In a cultured gastric cancer cell line, MKN28, mature PTHrP protein expression was markedly increased by transfection of furin cDNA. Furin cDNA-transfected MKN28 cells grew faster than did the mock controls. Moreover, furin mRNA expression in cultured gastric cancer cells was enhanced when PTHrP was added to the culture medium. These results suggest a link between PTHrP and furin in the regulation of gastric cancer cell growth. Furin might be involved not only in the production of the mature form of PTHrP, but also in promoting growth in gastric cancer cells.

Extraembryonic proteases regulate Nodal signalling during gastrulation.

During gastrulation, a cascade of inductive tissue interactions converts pre-existing polarity in the mammalian embryo into antero-posterior pattern. This process is triggered by Nodal, a protein related to transforming growth factor-beta (TFG-beta) that is expressed in the epiblast and visceral endoderm, and its co-receptor Cripto, which is induced downstream of Nodal. Here we show that the proprotein convertases Spc1 and Spc4 (also known as Furin and Pace4, respectively) are expressed in adjacent extraembryonic ectoderm. They stimulate Nodal maturation after its secretion and are required in vivo for Nodal signalling. Embryo explants deprived of extraembryonic ectoderm phenocopy Spc1(-/-); Spc4(-/-) double mutants in that endogenous Nodal fails to induce Cripto. But recombinant mature Nodal, unlike uncleaved precursor, can efficiently rescue Cripto expression. Cripto is also expressed in explants treated with bone morphogenetic protein 4 (BMP4). This indicates that Nodal may induce Cripto through both a signalling pathway in the embryo and induction of Bmp4 in the extraembryonic ectoderm. A lack of Spc1 and Spc4 affects both pathways because these proteases also stimulate induction of Bmp4.

Proteolytic processing of chromogranins is modified in brains of transgenic mice.

In normal brain, secretogranin II and chromogranins A and S are extensively converted by endopeptidases to the peptides secretoneurin, GE-19, and PE-11, respectively. After genetic knockout of PC2 or its helper protein 7B2, but not after mutation of carboxypeptidase E, endoproteolytic processing decreased, as indicated by appearance of intermediate-sized processing products.

Role of proprotein convertases in the pathogenic processing of the amyloidosis-associated form of secretory gelsolin.

Familial amyloidosis of the Finnish type (FAF) is caused by two proteolytic cleavages of mutant gelsolin leading to the accumulation of FAF amyloid in the patients' tissues. Here, we demonstrate that, in mouse pituitary corticotropic AtT20 cells, the enzyme responsible for the first cleavage of mutant secretory FAF gelsolin to FAF amyloid precursor is present in reasonable amounts. Furthermore, in At T20 cells stably expressing alpha1-PDX a potent inhibitor of most proprotein convertases, this cleavage was inhibited The present data provide strong evidence that proprotein convertases, possibly furin or PC5, are involved in the initialpathological cleavage of mutant secretory FAF gelsolin leading ultimately to the amyloid disease.

Furin at the cutting edge: from protein traffic to embryogenesis and disease.

Furin catalyses a simple biochemical reaction--the proteolytic maturation of proprotein substrates in the secretory pathway. But the simplicity of this reaction belies furin's broad and important roles in homeostasis, as well as in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever. This review summarizes various features of furin--its structural and enzymatic properties, intracellular localization, trafficking, substrates, and roles in vivo.

Proprotein convertases in tumor progression and malignancy: novel targets in cancer therapy.

The mammalian subtilisin/kexin-like proprotein convertase (PC) family has been implicated in the activation of a wide spectrum of proteins. These proteins are usually synthesized as inactive precursors before their conversion to fully mature bioactive forms. A large majority of these active proteins such as matrix metalloproteases, growth factors, and adhesion molecules are crucial in the processes of cellular transformation, acquisition of the tumorigenic phenotype, and metastases formation. Inhibition of PCs significantly affects the malignant phenotype of various tumor cells. In addition to direct tumor cell proliferation and migration blockade, PC inhibitors can also be used to target tumor angiogenesis. In this Review article we discuss a number of recent findings on the clinical relevance of PCs in cancer patients, their implication in the regulation of multiple cellular functions that impact on the invasive/metastatic potential of cancer cells. Thus, PC inhibitors may constitute new promising agents for the treatment of multiple tumors and/or in adjuvant therapy to prevent recurrence.

Activation of membrane-type matrix metalloproteinase 3 zymogen by the proprotein convertase furin in the trans-Golgi network.

Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases implicated in tumor invasion and metastasis, must undergo zymogen activation prior to expressing any proteolytic activity. Although the cysteine-switch model predicts the well-established autoactivation process, approximately 40% of the known MMPs possess a conserved RXKR motif between their pro- and catalytic domains and, thus, could be activated directly by members of the proprotein convertase family. To further understand this process, we analyzed the activation of proMT3-MMP as a model system. We demonstrated that the conversion of MT3-MMP zymogen into active form is dependent on both the furin-type convertase activity and the R(116)RKR motif. Consistently, MT3-MMP was colocalized with furin in the trans-Golgi network by confocal microscopy. However, neither furin activity nor its recognition site in MT3-MMP is required for the observed colocalization. In fact, the colocalization pattern remains intact, even in the presence of brefeldin A, an agent known to block endoplasmic reticulum to Golgi trafficking. Yet, brefeldin A completely blocked the activation of MT3-MMP. A23187, a calcium ionophore known to block furin maturation, also blocked proMT3-MMP activation but had minimal effect on the colocalization between MT3-MMP and furin. Thus, furin processes MT3-MMP zymogen in the trans-Golgi network, where they colocalize independently of their apparent enzyme-substrate relationship.

Mortality in 7B2 null mice can be rescued by adrenalectomy: involvement of dopamine in ACTH hypersecretion.

The serine protease prohormone convertase 2 (PC2), principally involved in the processing of polypeptide hormone precursors in neuroendocrine tissues, requires interaction with the neuroendocrine protein 7B2 to generate an enzymatically active form. 7B2 null mice express no PC2 activity and release large quantities of uncleaved ACTH, resulting in a lethal endocrine condition that resembles pituitary Cushing's (Westphal, C. H., Muller, L., Zhou, A., Bonner-Weir, S., Schambelan, M., Steiner, D. F., Lindberg, I. & Leder, P. (1999) Cell 96, 689). Here, we have compared the 7B2 and PC2 null mouse models to determine why the 7B2 null, but not the PC2 null, exhibits a lethal disease state. Both 7B2 and PC2 nulls contained highly elevated pituitary adrenocorticotropic hormone (ACTH); the neurointermediate lobe content of ACTH in 7B2 nulls was 13-fold higher than in WT mice; that of the PC2 null was 65-fold higher. However, circulating ACTH levels were much higher in the 7B2 null than in the PC2 null. Because hypothalamic inhibitory dopaminergic control represents the major influence on intermediate lobe proopiomelanocortin-derived peptide secretion, dopamine levels were measured, and they revealed that 7B2 null pituitaries contained only one-fourth of WT pituitary dopamine. Adrenalectomized 7B2 null animals survived past the usual time of death at 5 weeks; a month after adrenalectomy, they exhibited normal levels of pituitary dopamine, circulating ACTH, and corticosterone. Elevated corticosterone, therefore, seems to play a central role in the lethal phenotype of the 7B2 null, whereas a 7B2-mediated dopaminergic deficiency state may be involved in the actual ACTH hypersecretion phenomenon. Interestingly, adrenalectomized 7B2 nulls also developed unexpectedly severe obesity.

Post-translational processing of bovine chondromodulin-I.

Chondromodulin-I (ChM-I) is a small glycoprotein that is abundant in fetal cartilage. Mature chondromodulin-I is processed from a larger precursor form, presumably at a proteolytic site RERR-ELVR. The precursor, mature chondromodulin-I and two processed products, the remnant left after removal of mature chondromodulin-I and a smaller, unglycosylated form, were identified using antipeptide antisera. The products of chondromodulin-I precursor processing were seen in cultured chondrocytes, a stable long-term culture chondrosarcoma cell line, as well as Chinese hamster ovary (CHO) cells transfected with an expression plasmid that contained cDNA coding for the chondromodulin-I precursor. Pulse-chase analysis allowed a processing pathway to be analyzed for chondromodulin-I. To further dissect the processing events, three constructs that express recombinant wild-type or mutant chondromodulin-I were transfected into CHO cells. We showed that chondromodulin-I is cleaved intracellularly at the predicted cleavage site, and that the mature glycopeptide is rapidly secreted immediately after processing. The chondromodulin-1 precursor has a short half-life and is not readily apparent in tissue samples, suggesting that chondromodulin is not a member of the juxtacrine family of growth factors, despite some similarities. The smaller unglycosylated form of chondromodulin-I was only observed in cartilage and not in short-term cultures or transfected cells, suggesting an extracellular processing event. No processing occurred when the precursor cleavage site was mutated to RERQ-SLVR or when precursor chondromodulin-I was expressed in the furin-deficient CHO cell line, suggesting the involvement of furin in processing.

The prohormone convertase enzyme 2 (PC2) is essential for processing pro-islet amyloid polypeptide at the NH2-terminal cleavage site.

Impaired processing of pro-islet amyloid polypeptide (proIAPP), the precursor of the beta-cell peptide islet amyloid polypeptide (IAPP) (amylin), has been implicated in islet amyloid formation in type 2 diabetes. The prohormone convertase enzymes PC3 (also known as PC1) and PC2 are localized to beta-cell secretory granules with proIAPP and proinsulin and are responsible for proinsulin processing. To determine whether PC2 might be essential for proIAPP processing, we performed Western blot analysis of freshly isolated islets from normal mice and mice lacking active PC2. As expected, the primary species of IAPP immunoreactivity in islets from wild-type mice was fully processed (4-kDa) IAPP, with only small amounts of the 8-kDa precursor (unprocessed proIAPP) present. Islets from heterozygous PC2 null mice were identical to wild-type animals, suggesting that half the normal complement of PC2 is sufficient for normal proIAPP processing. By contrast, in islets from homozygous PC2 null mice, the predominant IAPP-immunoreactive form was of intermediate size (approximately 6 kDa), with no detectable mature IAPP and slightly elevated amounts of the 8-kDa precursor form present. Thus, in the absence of PC2, proIAPP processing appears to be blocked at the level of a proIAPP conversion intermediate. Immunofluorescence of pancreas sections and immunoblotting using antisera raised to the NH2- and COOH-terminal flanking regions of mouse proIAPP demonstrated that the 6-kDa intermediate form was an NH2-terminally extended proIAPP conversion intermediate (processed only at the COOH-terminus). These data indicate that PC2 is essential for processing of proIAPP at the NH2-terminal cleavage site in vivo and that PC3 is likely only capable of processing proIAPP at the COOH-terminal cleavage site.