Peptides Derived From Insulin Granule Proteins Are Targeted by CD8+ T Cells Across MHC Class I Restrictions in Humans and NOD Mice.

The antigenic peptides processed by ß-cells and presented through surface HLA class I molecules are poorly characterized. Each HLA variant (e.g., the most common being HLA-A2 and HLA-A3) carries some peptide-binding specificity. Hence, features that, despite these specificities, remain shared across variants may reveal factors favoring ß-cell immunogenicity. Building on our previous description of the HLA-A2/A3 peptidome of ß-cells, we analyzed the HLA-A3-restricted peptides targeted by circulating CD8+ T cells. Several peptides were recognized by CD8+ T cells within a narrow frequency (1-50/106), which was similar in donors with and without type 1 diabetes and harbored variable effector/memory fractions. These epitopes could be classified as conventional peptides or neoepitopes, generated either via peptide cis-splicing or mRNA splicing (e.g., secretogranin-5 [SCG5]-009). As reported for HLA-A2-restricted peptides, several epitopes originated from ß-cell granule proteins (e.g., SCG3, SCG5, and urocortin-3). Similarly, H-2Kd-restricted CD8+ T cells recognizing the murine orthologs of SCG5, urocortin-3, and proconvertase-2 infiltrated the islets of NOD mice and transferred diabetes into NOD/scid recipients. The finding of granule proteins targeted in both humans and NOD mice supports their disease relevance and identifies the insulin granule as a rich source of epitopes, possibly reflecting its impaired processing in type 1 diabetes.

Conventional and Neo-antigenic Peptides Presented by ß Cells Are Targeted by Circulating Naïve CD8+ T Cells in Type 1 Diabetic and Healthy Donors.

Although CD8+ T-cell-mediated autoimmune ß cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by ß cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known ß cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8+ T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by ß cells and for the development of T cell biomarkers and tolerogenic vaccination strategies.

7B2 chaperone knockout in APP model mice results in reduced plaque burden.

Impairment of neuronal proteostasis is a hallmark of Alzheimer's and other neurodegenerative diseases. However, the underlying molecular mechanisms leading to pathogenic protein aggregation, and the role of secretory chaperone proteins in this process, are poorly understood. We have previously shown that the neural-and endocrine-specific secretory chaperone 7B2 potently blocks in vitro fibrillation of Aß42. To determine whether 7B2 can function as a chaperone in vivo, we measured plaque formation and performed behavioral assays in 7B2-deficient mice in an hAPPswe/PS1dE9 Alzheimer's model mouse background. Surprisingly, immunocytochemical analysis of cortical levels of thioflavin S- and Aß-reactive plaques showed that APP mice with a partial or complete lack of 7B2 expression exhibited a significantly lower number and burden of thioflavin S-reactive, as well as Aß-immunoreactive, plaques. However, 7B2 knockout did not affect total brain levels of either soluble or insoluble Aß. While hAPP model mice performed poorly in the Morris water maze, their brain 7B2 levels did not impact performance. Since 7B2 loss reduced amyloid plaque burden, we conclude that brain 7B2 can impact Aß disposition in a manner that facilitates plaque formation. These results are reminiscent of prior findings in hAPP model mice lacking the ubiquitous secretory chaperone clusterin.

Posttranslational processing of FGF23 in osteocytes during the osteoblast to osteocyte transition.

FGF23 is an O-glycosylated circulating peptide hormone with a critical role in phosphate homeostasis; it is inactivated by cellular proprotein convertases in a pre-release degradative pathway. We have here examined the metabolism of FGF23 in a model bone cell line, IDG-SW3, prior to and following differentiation, as well as in regulated secretory cells. Labeling experiments showed that the majority of (35)S-labeled FGF23 was cleaved to smaller fragments which were constitutively secreted by all cell types. Intact FGF23 was much more efficiently stored in differentiated than in undifferentiated IDG-SW3 cells. The prohormone convertase PC2 has recently been implicated in FGF23 degradation; however, FGF23 was not targeted to forskolin-stimulatable secretory vesicles in a regulated cell line, suggesting that it lacks a targeting signal to PC2-containing compartments. In vitro, PC1/3 and PC2, but not furin, efficiently cleaved glycosylated FGF23; surprisingly, PC5/6 accomplished a small amount of conversion. FGF23 has recently been shown to be phosphorylated by the kinase FAM20C, a process which was shown to reduce FGF23 glycosylation and promote its cleavage; our in vitro data, however, show that phosphorylation does not directly impact cleavage, as both PC5/6 and furin were able to efficiently cleave unglycosylated, phosphorylated FGF23. Using qPCR, we found that the expression of FGF23 and PC5/6, but not PC2 or furin, increased substantially following osteoblast to osteocyte differentiation. Western blotting confirmed the large increase in PC5/6 expression upon differentiation. FGF23 has been linked to a variety of bone disorders ranging from autosomal dominant hypophosphatemic rickets to chronic kidney disease. A better understanding of the biosynthetic pathway of this hormone may lead to new treatments for these diseases.

Phosphorylation and Alternative Splicing of 7B2 Reduce Prohormone Convertase 2 Activation.

FAM20C is a secretory kinase responsible for the phosphorylation of multiple secreted proteins in mammalian cells; it has been shown to phosphorylate serine residues within a variety of different bone proteins. In this work we demonstrate that FAM20C also phosphorylates threonines, specifically those within the N-terminal domain of the neuroendocrine chaperone 7B2. Analysis of the primary sequence of 7B2 revealed that three threonine residues in its N-terminal domain are located within FAM20C consensus motifs: Thr73, Thr99, and Thr111. The individual substitution of Thr73 and Thr111 residues by neutral alanines caused a marked decrease in the total phosphorylation of 7B2. Furthermore, the phosphomimetic substitution of Thr111 by Glu clearly diminished the ability of 7B2 to activate pro-prohormone convertase 2 (PC2) in 7B2-lacking SK-N-MC neuroblastoma cells, suggesting that the phosphorylation of this residue critically impacts the 7B2-proPC2 interaction. However, the phosphomimetic mutation did not alter 7B2's ability to function as an antiaggregant for human islet amyloid polypeptide. FAM20C-mediated phosphorylation of a common alternatively spliced variant of human 7B2 that lacks Ala100 (thus eliminating the Thr99 phosphorylation consensus site) was similar to the Ala-containing protein, but this variant did not activate proPC2 as efficiently as the Ala-containing protein. Although threonines within 7B2 were phosphorylated efficiently, FAM20C was incapable of performing the well-known regulatory threonine phosphorylation of the molecular chaperone binding immunoglobulin protein. Taken together, these results indicate that FAM20C plays a role in 7B2-mediated proPC2 activation by phosphorylating residue Thr111; and that 7B2 function is regulated by alternative splicing.

A three-gene panel that distinguishes benign from malignant thyroid nodules.

Reliable preoperative diagnosis of malignant thyroid tumors remains challenging because of the inconclusive cytological examination of fine-needle aspiration biopsies. Although numerous studies have successfully demonstrated the use of high-throughput molecular diagnostics in cancer prediction, the application of microarrays in routine clinical use remains limited. Our aim was, therefore, to identify a small subset of genes to develop a practical and inexpensive diagnostic tool for clinical use. We developed a two-step feature selection method composed of a linear models for microarray data (LIMMA) linear model and an iterative Bayesian model averaging model to identify a suitable gene set signature. Using one public dataset for training, we discovered a three-gene signature dipeptidyl-peptidase 4 (DPP4), secretogranin V (SCG5) and carbonic anhydrase XII (CA12). We then evaluated the robustness of our gene set using three other independent public datasets. The gene signature accuracy was 85.7, 78.8 and 85.7%, respectively. For experimental validation, we collected 70 thyroid samples from surgery and our three-gene signature method achieved an accuracy of 94.3% by quantitative polymerase chain reaction (QPCR) experiment. Furthermore, immunohistochemistry in 29 samples showed proteins expressed by these three genes are also differentially expressed in thyroid samples. Our protocol discovered a robust three-gene signature that can distinguish benign from malignant thyroid tumors, which will have daily clinical application.

Blockade of islet amyloid polypeptide fibrillation and cytotoxicity by the secretory chaperones 7B2 and proSAAS.

The deposition of fibrillated human islet ß-cell peptide islet amyloid polypeptide (hIAPP) into amyloid plaques is characteristic of the pathogenesis of islet cell death during type 2 diabetes. We investigated the effects of the neuroendocrine secretory proteins 7B2 and proSAAS on hIAPP fibrillation in vitro and on cytotoxicity. In vitro, 21-kDa 7B2 and proSAAS blocked hIAPP fibrillation. Structure-function studies showed that a central region within 21-kDa 7B2 is important in this effect and revealed the importance of the N-terminal region of proSAAS. Both chaperones blocked the cytotoxic effects of exogenous hIAPP on Rin5f cells; 7B2 generated by overexpression was also effective. ProSAAS and 7B2 may perform a chaperone role as secretory anti-aggregants in normal islet cell function and in type 2 diabetes.

Network analysis of endogenous gene expression profiles after polyethyleneimine-mediated DNA delivery.

BACKGROUND: DNA delivery systems, which transport exogenous DNA to cells, have applications that include gene therapy, tissue engineering and medical devices. Although the cationic nonviral DNA carrier polyethyleneimine (PEI) has been widely studied, the molecular factors and pathways underlying PEI-mediated DNA transfer remain largely unknown, preventing the design of more efficient delivery systems. METHODS: HEK 293 T cells were treated with polyplexes formed with PEI and pEGFPLuc encoding for green fluorescent protein (GFP). Transfected cells expressing GFP were flow-separated from treated, untransfected cells. Gene expression profiles were obtained using Affymetrix HG-U133 2.0 microarrays and differentially expressed genes were identified using R/Bioconductor. Gene network analysis using EGAN (exploratory gene association network) bioinformatics tools was then used to find interaction among genes and enriched gene ontology (GO) terms related to transfection. Genes identified by this method were perturbed using pharmacologic activators or inhibitors to assess their effect on DNA transfer. RESULTS: Microarray analysis comparing transfected cells to untransfected cells revealed 215 genes to be differentially expressed, with the majority enriched to GO processes including metabolism, response to stimulus, cell cycle, biological regulation and cellular component organization or biogenesis pathways. Gene network analysis revealed a coordinated induction of RAP1A, SCG5, PGAP1, ATF3 and NEB genes implicated in cell stress, cell cycle and cytoskeletal processes. Altering pathways with pharmacologic agents confirmed the potential role of RAP1A, SCG5 and ATF3 in transfection. CONCLUSIONS: Microarray and gene network analyses of the sorted, transfected cell population can identify potential mediators of transfection, providing a basis for the design of improved delivery systems.

Hexa-D-arginine treatment increases 7B2•PC2 activity in hyp-mouse osteoblasts and rescues the HYP phenotype.

Inactivating mutations of the "phosphate regulating gene with homologies to endopeptidases on the X chromosome" (PHEX/Phex) underlie disease in patients with X-linked hypophosphatemia (XLH) and the hyp-mouse, a murine homologue of the human disorder. Although increased serum fibroblast growth factor 23 (FGF-23) underlies the HYP phenotype, the mechanism(s) by which PHEX mutations inhibit FGF-23 degradation and/or enhance production remains unknown. Here we show that treatment of wild-type mice with the proprotein convertase (PC) inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (Dec), increases serum FGF-23 and produces the HYP phenotype. Because PC2 is uniquely colocalized with PHEX in osteoblasts/bone, we examined if PC2 regulates PHEX-dependent FGF-23 cleavage and production. Transfection of murine osteoblasts with PC2 and its chaperone protein 7B2 cleaved FGF-23, whereas Signe1 (7B2) RNA interference (RNAi) transfection, which limited 7B2 protein production, decreased FGF-23 degradation and increased Fgf-23 mRNA and protein. The mechanism by which decreased 7B2•PC2 activity influences Fgf-23 mRNA was linked to reduced conversion of the precursor to bone morphogenetic protein 1 (proBMP1) to active BMP1, which resulted in limited cleavage of dentin matrix acidic phosphoprotein 1 (DMP1), and consequent increased Fgf-23 mRNA. The significance of decreased 7B2•PC2 activity in XLH was confirmed by studies of hyp-mouse bone, which revealed significantly decreased Sgne1 (7B2) mRNA and 7B2 protein, and limited cleavage of proPC2 to active PC2. The expected downstream effects of these changes included decreased FGF-23 cleavage and increased FGF-23 synthesis, secondary to decreased BMP1-mediated degradation of DMP1. Subsequent Hexa-D-Arginine treatment of hyp-mice enhanced bone 7B2•PC2 activity, normalized FGF-23 degradation and production, and rescued the HYP phenotype. These data suggest that decreased PHEX-dependent 7B2•PC2 activity is central to the pathogenesis of XLH.

The neuroendocrine protein 7B2 suppresses the aggregation of neurodegenerative disease-related proteins.

Neurodegenerative diseases such as Alzheimer (AD) and Parkinson (PD) are characterized by abnormal aggregation of misfolded ß-sheet-rich proteins, including amyloid-ß (Aß)-derived peptides and tau in AD and α-synuclein in PD. Correct folding and assembly of these proteins are controlled by ubiquitously expressed molecular chaperones; however, our understanding of neuron-specific chaperones and their involvement in the pathogenesis of neurodegenerative diseases is limited. We here describe novel chaperone-like functions for the secretory protein 7B2, which is widely expressed in neuronal and endocrine tissues. In in vitro experiments, 7B2 efficiently prevented fibrillation and formation of Aß(1-42), Aß(1-40), and α-synuclein aggregates at a molar ratio of 1:10. In cell culture experiments, inclusion of recombinant 7B2, either in the medium of Neuro-2A cells or intracellularly via adenoviral 7B2 overexpression, blocked the neurocytotoxic effect of Aß(1-42) and significantly increased cell viability. Conversely, knockdown of 7B2 by RNAi increased Aß(1-42)-induced cytotoxicity. In the brains of APP/PSEN1 mice, a model of AD amyloidosis, immunoreactive 7B2 co-localized with aggregation-prone proteins and their respective aggregates. Furthermore, in the hippocampus and substantia nigra of human AD- and PD-affected brains, 7B2 was highly co-localized with Aß plaques and α-synuclein deposits, strongly suggesting physiological association. Our data provide insight into novel functions of 7B2 and establish this neural protein as an anti-aggregation chaperone associated with neurodegenerative disease.

Frequent epigenetic inactivation of the chaperone SGNE1/7B2 in human gliomas.

In a genome-wide screen using DMH (differential methylation hybridization) we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in low- and high-grade astrocytomas compared to normal brain tissue. Pyrosequencing was performed to confirm the methylation status of this CpG island in 89 astrocytic gliomas of different malignancy grades and six glioma cell lines. Hypermethylation of SGNE1/7B2 was significantly more frequent in diffuse low-grade astrocytomas as well as secondary glioblastomas and anaplastic astrocytomas as compared to primary glioblastomas. mRNA expression analysis by real-time RT-PCR indicates that SGNE1/7B2 expression is downregulated in astrocytic gliomas compared to white matter samples. Treatment of glioma cells with the demethylating agent 5-aza-2'-deoxycytidine restores the transcription of SGNE1/7B2. Overexpression of SGNE1/7B2 in T98G, A172 and U373MG glioblastoma cells significantly suppressed focus formation and led to a significant increase in apoptotic cells as determined by flow cytometric analysis in T98G cells. In summary, we have identified SGNE1/7B2 as a novel target silenced by DNA methylation in astrocytic gliomas. The high incidence of this alteration and the significant effects of SGNE1/7B2 on the growth and apoptosis of glioblastoma cells provide a first proof for a functional implication of SGNE1/7B2 inactivation in the molecular pathology of gliomas.

Dynamic modulation of prohormone convertase 2 (PC2)-mediated precursor processing by 7B2 protein: preferential effect on glucagon synthesis.

The small neuroendocrine protein 7B2 is required for the production of active prohormone convertase 2 (PC2), an enzyme involved in the synthesis of peptide hormones, such as glucagon and proopiomelanocortin-derived α-melanocyte-stimulating hormone. However, whether 7B2 can dynamically modulate peptide production through regulation of PC2 activity remains unclear. Infection of the pancreatic alpha cell line α-TC6 with 7B2-encoding adenovirus efficiently increased production of glucagon, whereas siRNA-mediated knockdown of 7B2 significantly decreased stored glucagon. Furthermore, rescue of 7B2 expression in primary pituitary cultures prepared from 7B2 null mice restored melanocyte-stimulating hormone production, substantiating the role of 7B2 as a regulatory factor in peptide biosynthesis. In anterior pituitary and pancreatic beta cell lines, however, overexpression of 7B2 affected neither production nor secretion of peptides despite increased release of active PC2. In direct contrast, 7B2 overexpression decreased the secretion and increased the activity of PC2 within α-TC6 cells; the increased intracellular concentration of active PC2 within these cells may therefore account for the enhanced production of glucagon. In line with these findings, we found elevated circulating glucagon levels in 7B2-overexpressing cast/cast mice in vivo. Surprisingly, when proopiomelanocortin and proglucagon were co-expressed in either pituitary or pancreatic alpha cell lines, proglucagon processing was preferentially decreased when 7B2 was knocked down. Taken together, these results suggest that proglucagon cleavage has a greater dependence on PC2 activity than other precursors and moreover that 7B2-dependent routing of PC2 to secretory granules is cell line-specific. The manipulation of 7B2 could therefore represent an effective way to selectively regulate synthesis of certain PC2-dependent peptides.

Regulation of 7B2 mRNA translation: dissecting the role of its 5'-untranslated region.

7B2 is a chaperone for the prohormone/proneuropeptide convertase PC2. Its mRNA is readily detectable in most neuronal and endocrine cells; the protein, in contrast, is often found at relatively low levels, suggesting that translation of the corresponding mRNA may be repressed. Because the 5' untranslated region (5'-UTR) of this mRNA is relatively long and burdened with multiple AUGs, it has been speculated that it contributes to this repression. In this report, the influence of this region was assessed using in vitro and ex vivo approaches. The results showed that, in a cell-free system, full-length 7B2 mRNA was a poor template for translation. Its translatability dramatically improved when its 5'-UTR was truncated or when it was replaced with the 5'-UTR of carboxypeptidase E mRNA. These observations were confirmed in transfected mouse insulinoma MIN6 cells and human embryonic kidney HEK293 cells. Acute exposure of MIN6 cells to high glucose increased endogenous 7B2 biosynthesis without affecting the levels of its mRNA, suggesting that translation repression of this mRNA can be relieved by physiological stimuli.

V-ATPase-mediated granular acidification is regulated by the V-ATPase accessory subunit Ac45 in POMC-producing cells.

The vacuolar (H(+))-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells the following: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral pH-dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing.

Distribution of 7B2 (secretogranin V)-like immunoreactivity in the Japanese red-bellied newt (Cynops pyrrhogaster) pituitary.

In this study, we examined 7B2 (secretogranin V)-like immunoreactivity (IR) in the Japanese red-bellied newt (Cynops pyrrhogaster) pituitary. Results showed that the pars nervosa was filled with immunoreactive granules. In the pars intermedia, all melanotrophs showed 7B2-IR. In the pars distalis, immunoreactive cells were dispersed, and the 7B2-immunoreactive cells were also immunopositive for the beta-subunit of bullfrog luteinizing hormone (fLHbeta). 7B2-IR co-localized with fLHbeta-IR in the same secretory granules. Our results suggest that 7B2 may participate in the secretion processes of gonadotropins in the pars distalis.

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.

Prohormone convertases 1/3, 2, furin and protein 7B2 (Secretogranin V) in endocrine cells of the human pancreas.

Prohormone convertases (PCs) are proteinases that cleave inactive prohormones to biologically active peptides. Seven PCs have been identified; two of them, PC1/3 and PC2, have only been localized in neuroendocrine (NE) tissues; a third, furin, in both endocrine and exocrine tissues. We have studied the immunoreactivity of PC1/3, PC2 and furin in the four major NE cell types of the human pancreas by using double immunofluorescence techniques. The study also included the expression of NE secretory protein 7B2 (secretogranin V), a member of the granin family, which influences the function of PC2. The results showed that the three PCs and 7B2 were expressed only in endocrine pancreas, furin also in exocrine cells. Insulin (B) cells harboured PC1/3 and PC2, but not furin. Glucagon (A) cells were immunoreactive to all three PCs; all glucagon cells expressed PC2, but one subpopulation showed PC1/3 immunoreactivity and another furin. Only a few somatostatin (D) cells contained PC2, but no other proconvertase. Pancreatic polypeptide (PP) cells were non-reactive to all three PCs. 7B2 occurred only in insulin and glucagon cells. A varying co-localization pattern was observed between PCs and between PCs and 7B2, with the exception of PC1/3 and furin which were not co-localized. In conclusion, our study shows that PCs are localized in insulin and glucagon cells and do seem to be important in these cell types for processing of hormone and other protein precursors, especially chromogranins, but for the two other major cell types probably other enzymes are of importance.

Differences of pancreatic expression of 7B2 between C57BL/6J and C3H/HeJ mice and genetic polymorphisms at its locus (Sgne1).

C57BL/6 (B6) mice develop glucose intolerance with age, whereas C3H/He (C3H) mice do not. In this study, we examined whether this differential glucose homeostasis was associated with differences of proteolytic activation of pancreatic prohormones. Radioimmunoassays showed comparable levels of fasting plasma insulin between the two strains but a significantly lower glucagon level in B6 mice. Pulse-chase analysis of glucagon biosynthesis in isolated pancreatic islets revealed that proglucagon was less efficiently processed in B6 mice. Because proprotein convertase (PC)2 and its 7B2 helper protein are required for this processing, we quantified islet mRNA levels by RT-PCR and protein levels by immunoblotting. The levels of proPC2 mRNA were similar between the two strains, but B6 protein extracts contained less of the mature PC2. In contrast, 7B2 mRNA and protein levels were both significantly lower in B6 pancreas. Sequencing of the 7B2 gene promoter and cDNA in the two strains revealed seven single nucleotide polymorphisms and one dinucleotide insertion/deletion in the cDNA as well as a single nucleotide polymorphism and two insertions/deletions in the promoter. Differential expression of 7B2 may contribute to the difference between B6 and C3H mice not only in glucagon production and secretion but also in glucose tolerance.

Neuroendocrine protein 7B2 can be inactivated by phosphorylation within the secretory pathway.

The prohormone convertases play important roles in the maturation of neuropeptides and peptide hormone precursors. Prohormone convertase-2 (PC2) is the only convertase that requires the expression of another neuroendocrine protein, 7B2, for expression of enzyme activity. In this study, we determined that 7B2 can be phosphorylated in Rin cells (a rat insulinoma cell line) and cultured chromaffin cells, but not in AtT-20 cells (derived from mouse anterior pituitary). Phosphoamino acid analysis of Rin cell 7B2 indicated the presence of phosphorylated serine and threonine. Phosphorylation of Ser115 (located within the minimally active 36-residue peptide) was confirmed by mutagenesis, although Ser115 did not represent the sole residue phosphorylated. Two independent assays were used to investigate the effect of phosphorylated 7B2 on PC2 activation: the ability of 7B2 to bind to pro-PC2 was assessed by co-immunoprecipitation, and activation of pro-PC2 was assessed in a cell-free assay. Phosphorylated 7B2 was unable to bind pro-PC2, and the phosphorylated 7B2 peptide (residues 86-121, known to be the minimally active peptide for pro-PC2 activation) was impaired in its ability to facilitate the generation of PC2 activity in membrane fractions containing pro-PC2. In vitro phosphorylation experiments using Golgi membrane fractions showed that 7B2 could be phosphorylated by endogenous Golgi kinases. Golgi kinase activity was strongly inhibited by the broad-range kinase inhibitor staurosporine and partially inhibited by the protein kinase C inhibitor bisindolylmaleimide I, but not by the other protein kinase A, Ca2+/calmodulin-dependent kinase II, myosin light chain kinase, and protein kinase G inhibitors tested. We conclude that phosphorylation of 7B2 functionally inactivates this protein and suggest that this may be analogous to the phosphorylating inactivation of BiP, which impairs its ability to bind substrate.

Detection of functionally active melanocortin receptors and evidence for an immunoregulatory activity of alpha-melanocyte-stimulating hormone in human dermal papilla cells.

Proopiomelanocortin (POMC)-derived peptides and their receptors have been identified in many peripheral organs including the skin in which they exert a diversity of biological actions. We investigated the expression and potential role of the POMC system in human dermal papilla cells (DPCs), a specialized cutaneous mesenchymal cell type regulating hair follicle activity. In culture, these cells expressed POMC and displayed immunoreactivity for ACTH, alphaMSH, and beta-endorphin. Among the prohormone convertases (PCs) tested, only PC2, its chaperone 7B2, and furin convertase but not PC1 and paired basic amino acid cleaving enzyme 4 gene were detected. Human DPCs in vitro expressed both the melanocortin-1 receptor (MC-1R) and MC-4R, and immunoreactivity for these receptors was also present in cells of the human dermal papilla in situ. In contrast to the dermal papilla of agouti mice, agouti signaling protein, a natural and highly selective MC-1R and MC-4R antagonist, was undetectable in human DPCs. The MC-Rs detected in human DPCs were functionally active because alphaMSH increased intracellular cAMP and calcium. Preincubation of the cells with a synthetic peptide corresponding to the C-terminal domain of agouti signaling protein abrogated cAMP induction by alphaMSH. Furthermore, alphaMSH was capable of antagonizing the expression of intercellular adhesion molecule-1 induced by the proinflammatory cytokine interferon-gamma. Our data suggest a regulatory function of alphaMSH within the dermal papilla whose disruption may lead to deregulation of immune and inflammatory responses of the hair follicle, thereby possibly contributing to the development of inflammatory forms of alopecia.