Prohormone Convertase Research Articles

Development of Novel DNA-Encoded PCSK9 Monoclonal Antibodies as Lipid-Lowering Therapeutics.

Elevated low-density lipoprotein cholesterol (LDL-C) is one of the major contributors to cardiovascular heart disease (CHD), the leading cause of death worldwide. Due to severe side effects of statins, alternative treatment strategies are required for statin-intolerant patients. Monoclonal antibodies (mAbs) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) have shown great efficacy in LDL-C reduction. Limitations for this approach include the need for multiple injections as well as increased costs associated with patient management. Here, we engineered a DNA-encoded mAb (DMAb) targeting PCSK9 (daPCSK9), as an alternative approach to protein-based lipid-lowering therapeutics, and we characterized its expression and activity. A single intramuscular administration of mouse daPCSK9 generated expression in vivo for over 42 days that corresponded with a substantial decrease of 28.6% in non-high-density lipoprotein cholesterol (non-HDL-C) and 10.3% in total cholesterol by day 7 in wild-type mice. Repeated administrations of the DMAb plasmid led to increasing expression, with DMAb levels of 7.5 μg/mL at day 62. daPCSK9 therapeutics may provide a novel, simple, less frequent, cost-effective approach to reducing LDL-C, either as a stand-alone therapy or in combination with other LDL-lowering therapeutics for synergistic effect.

Proapelin is processed extracellularly in a cell line-dependent manner with clear modulation by proprotein convertases.

Apelin is a peptide hormone that binds to a class A GPCR (the apelin receptor/APJ) to regulate various bodily systems. Upon signal peptide removal, the resulting 55-residue isoform, proapelin/apelin-55, can be further processed to 36-, 17-, or 13-residue isoforms with length-dependent pharmacological properties. Processing was initially proposed to occur intracellularly. However, detection of apelin-55 in extracellular fluids indicates that extracellular processing may also occur. To test for this, apelin-55 was applied exogenously to HEK293A cells overexpressing proprotein convertase subtilisin kexin 3 (PCSK3), the only apelin processing enzyme identified thus far, and to differentiated 3T3-L1 adipocytes, which endogenously express apelin, PCSK3 and other proprotein convertases. Analysis of culture media constituents from each cell type by high performance liquid chromatography–mass spectrometry and western blot demonstrated a time-dependent decrease in apelin-55 levels. This decrease was partially, but not fully, attenuated by PCSK inhibitor treatment in both cell lines. Comparison of the resulting apelin-55-derived peptide profile between the two cell lines demonstrated distinct processing patterns, with apelin-36 production apparent in 3T3-L1 adipocytes vs. detection of the prodomain of a shorter isoform (likely the apelin-13 prodomain, observed after additional proteolytic processing) in PCSK3-transfected HEK293A cells. Extracellular processing of apelin, with distinct cell type dependence, provides an alternative mechanism to regulate isoform-mediated physiological effects of apelin.Electronic supplementary materialThe online version of this article (10.1007/s00726-018-2674-8) contains supplementary material, which is available to authorized users.

EGL-3 and EGL-21 are required to trigger nocifensive response of Caenorhabditis elegans to noxious heat

Caenorhabditis elegans (C. elegans) is a widely used model organism to examine nocifensive response to noxious stimuli, including heat avoidance. Recently, comprehensive analysis of the genome sequence revealed several pro-neuropeptide genes, encoding a series of bioactive neuropeptides. C. elegans neuropeptides are involved in the modulation of essentially all behaviors including locomotion, mechanosensation, thermosensation and chemosensation. The maturation of pro-neuropeptide to neuropeptide is performed by ortholog pro-protein convertases and carboxypeptidase E (e.g. EGL-3 and EGL-21). We hypothesized that C. elegans egl-3 or egl-21 mutants will have a significant decrease in mature neuropeptides and they will display an impaired heat avoidance behavior. Our data has shown that thermal avoidance behavior of egl-3 and egl-21 mutants was significantly hampered compared to WT(N2) C. elegans. Moreover, flp-18, flp-21 and npr-1 mutant C. elegans displayed a similar phenotype. EGL-3 pro-protein convertase and EGL-21 carboxypeptidase E are essential enzymes for the maturation of pro-neuropeptides to active neuropeptides in C. elegans. Quantitative mass spectrometry analyses with egl-3 and egl-21 mutant C. elegans homogenates demonstrated that proteolysis of ProFLP-18 and ProFLP-21 are severely impeded, leading to a lack of mature bioactive neuropeptides. Not only FLP-21 but also FLP-18 related mature neuropeptides, both are ligands of NPR-1 and are needed to trigger nocifensive response of C. elegans to noxious heat.

Normoglycemia in Mice Lacking Beta-Cell Prohormone Convertase 1/3

Introduction: In islet beta cells, the endoprotease activity of prohormone convertases (PC) PC1/3 and PC2 are believed necessary for efficient processing of proinsulin to mature insulin. PC1/3 expression is a marker of beta-cell maturity and function, and polymorphisms in PC1/3 are associated with obesity, impaired glucose tolerance and elevated circulating proinsulin:insulin. Given PC1/3's key role in beta-cell function, we hypothesized that beta-cell PC1/3 deficiency would lead to beta-cell dysfunction and glucose intolerance.

POMC: The Physiological Power of Hormone Processing.

Pro-opiomelanocortin (POMC) is the archetypal polypeptide precursor of hormones and neuropeptides. In this review, we examine the variability in the individual peptides produced in different tissues and the impact of the simultaneous presence of their precursors or fragments. We also discuss the problems inherent in accurately measuring which of the precursors and their derived peptides are present in biological samples. We address how not being able to measure all the combinations of precursors and fragments quantitatively has affected our understanding of the pathophysiology associated with POMC processing. To understand how different ratios of peptides arise, we describe the role of the pro-hormone convertases (PCs) and their tissue specificities and consider the cellular processing pathways which enable regulated secretion of different peptides that play crucial roles in integrating a range of vital physiological functions. In the pituitary, correct processing of POMC peptides is essential to maintain the hypothalamic-pituitary-adrenal axis, and this processing can be disrupted in POMC-expressing tumors. In hypothalamic neurons expressing POMC, abnormalities in processing critically impact on the regulation of appetite, energy homeostasis, and body composition. More work is needed to understand whether expression of the POMC gene in a tissue equates to release of bioactive peptides. We suggest that this comprehensive view of POMC processing, with a focus on gaining a better understanding of the combination of peptides produced and their relative bioactivity, is a necessity for all involved in studying this fascinating physiological regulatory phenomenon.

Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery.

The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.

In Situ Imaging of Furin Activity with a Highly Stable Probe by Releasing of Precipitating Fluorochrome.

Furin, a kind of trans-Golgi proprotein convertases, plays important role in various physiological processes. It is overexpressed in many cancers and relates to tumor growth and migration. In situ detection and imaging of furin is of great significance for obtaining real-time information about its activity. However, the previously reported fluorescent probes for furin usually failed to realize in situ detection and long-term bioimaging, because these probes are based on water-soluble fluorophores, which tend to diffuse away from the reaction sites after converted by furin. Such a problem can be addressed by designing a probe, which releases a precipitating fluorophore upon furin conversion. Herein, we developed a probe HPQF for in situ detection of endogenous furin activity and long-term bioimaging by integrating a strictly insoluble solid-state fluorophore 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (Cl-HPQ) with a furin specific peptide substrate (RVRR) through a self-immolative linker. The HPQF probe shows high selectivity and sensitivity to furin. Upon converted by furin, HPQF releases free Cl-HPQ, which precipitates near the enzyme active site. The precipitates emit bright solid-state fluorescence for in situ imaging. HPQF could truly visualize the location of intracellular furin, which was further confirmed by colocalization and immunofluorescence experiments. Excitingly, the long-term bioimaging was also achieved benefiting from its outstanding signal-stability and antidiffusion ability. HPQF was further utilized to monitor the level change of furin under stabilizing of hypoxia-inducible factor (HIF) regulated by cobalt chloride (CoCl2) as well as visualization of furin in MDA-MB-468 cell tumor tissues.

Semaphorin-3A inhibits multiple myeloma progression in a mouse model.

Previous studies revealed that progression of multiple myeloma (MM) is associated with downregulation of semaphorin-3A (sema3A) expression in bone marrow endothelial cells. We therefore determined if serum sema3A concentrations are correlated with MM progression and if sema3A can affect MM progression. We find that the concentration of sema3A in sera of MM patients is strongly reduced and that the decrease is correlated with disease progression. A similar depletion is found in patients having acute myeloid leukemia and acute lymphoblastic leukemia but not in cancer forms that do not involve the bone marrow such as in colon cancer. Expression of a modified sema3A [furin-resistant sema3A (FR-sema3A)] stabilized against cleavage by furin-like proprotein convertases in CAG MM cells did not affect their behavior in-vitro. CAG cells injected into the tail vein of severe combined immunodeficient (SCID) mice home to the bone marrow and proliferate, mimicking MM disease progression. Disease progression in mice injected with CAG cells expressing FR-sema3A was inhibited, resulting in prolonged survival and a lower incidence of bone lesions. Histological examination and fluorescence-activated cell sorting analysis revealed that FR-sema3A expression reduced the infiltration of the CAG cells into the bone marrow, reduced bone marrow necrosis and reduced angiogenesis induced by the MM cells in the bone marrow. Our results suggest that measurement of sema3A serum concentrations may be of use for the diagnosis and for the monitoring of malignancies of the bone marrow such as MM. Furthermore, our results suggest that FR-sema3A may perhaps find use as an inhibitor of MM disease progression.

Insulin regulates glucagon-like peptide-1 secretion by pancreatic alpha cells.

Proglucagon is expressed in both pancreatic alpha cells and intestinal epithelial L cells and is cleaved into glucagon and glucagon-like peptide-1 (GLP-1) by different prohormone convertases (PCs). Recent studies have shown that α-cells can also secrete GLP-1, which may improve islet function. However, little is known about the factors influencing GLP-1 secretion by α cells. In this study, we investigated whether insulin promotes GLP-1 secretion by α cells, as well as the mechanisms underlying this phenomenon. We cultured the alpha-cell line In-R1-G9 in low- or high-glucose medium in the presence or absence of insulin to determine the influence of glucose concentrations on the actions of insulin. We also treated In-R1-G9 cells with insulin for different times and at different doses. Then GLP-1 and glucagon protein expression levels were estimated. Moreover, ERK and phosphatidylinositol-3-kinase/AKT (PI3K/AKT) pathway activity levels and prohormone convertase expression levels were evaluated to elucidate the mechanism underlying the effects of insulin on GLP-1 secretion by α-cells. Insulin promoted GLP-1 secretion in a time- and dose-dependent manner under high-glucose conditions. Inhibiting the PI3K/AKT pathway with LY294002 and the Ras/mitogen-activated protein kinase (RAS/MAPK) pathway with PD98059 reduced GLP-1 secretion, respectively, in inhibitor-treated cells compared with insulin-treated cells. Moreover, insulin increased prohormone convertase 1/3 expression levels in the corresponding group of IN-R1-G9 cells compared with the control group of cells. Insulin facilitates GLP-1 secretion by pancreatic alpha cells by inducing PC1/3 expression under high-glucose conditions, a phenomenon that may be associated mainly with PI3K/AKT pathway activation.

Unexpected subcellular distribution of a specific isoform of the Coxsackie and adenovirus receptor, CAR-SIV, in human pancreatic beta cells

Aims/hypothesisThe Coxsackie and adenovirus receptor (CAR) is a transmembrane cell-adhesion protein that serves as an entry receptor for enteroviruses and may be essential for their ability to infect cells. Since enteroviral infection of beta cells has been implicated as a factor that could contribute to the development of type 1 diabetes, it is often assumed that CAR is displayed on the surface of human beta cells. However, CAR exists as multiple isoforms and it is not known whether all isoforms subserve similar physiological functions. In the present study, we have determined the profile of CAR isoforms present in human beta cells and monitored the subcellular localisation of the principal isoform within the cells.MethodsFormalin-fixed, paraffin-embedded pancreatic sections from non-diabetic individuals and those with type 1 diabetes were studied. Immunohistochemistry, confocal immunofluorescence, electron microscopy and western blotting with isoform-specific antisera were employed to examine the expression and cellular localisation of the five known CAR isoforms. Isoform-specific qRT-PCR and RNA sequencing (RNAseq) were performed on RNA extracted from isolated human islets.ResultsAn isoform of CAR with a terminal SIV motif and a unique PDZ-binding domain was expressed at high levels in human beta cells at the protein level. A second isoform, CAR-TVV, was also present. Both forms were readily detected by qRT-PCR and RNAseq analysis in isolated human islets. Immunocytochemical studies indicated that CAR-SIV was the principal isoform in islets and was localised mainly within the cytoplasm of beta cells, rather than at the plasma membrane. Within the cells it displayed a punctate pattern of immunolabelling, consistent with its retention within a specific membrane-bound compartment. Co-immunofluorescence analysis revealed significant co-localisation of CAR-SIV with zinc transporter protein 8 (ZnT8), prohormone convertase 1/3 (PC1/3) and insulin, but not proinsulin. This suggests that CAR-SIV may be resident mainly in the membranes of insulin secretory granules. Immunogold labelling and electron microscopic analysis confirmed that CAR-SIV was localised to dense-core (insulin) secretory granules in human islets, whereas no immunolabelling of the protein was detected on the secretory granules of adjacent exocrine cells. Importantly, CAR-SIV was also found to co-localise with protein interacting with C-kinase 1 (PICK1), a protein recently demonstrated to play a role in insulin granule maturation and trafficking.Conclusions/interpretationThe SIV isoform of CAR is abundant in human beta cells and is localised mainly to insulin secretory granules, implying that it may be involved in granule trafficking and maturation. We propose that this subcellular localisation of CAR-SIV contributes to the unique sensitivity of human beta cells to enteroviral infection.

Variation in Phenotypic Features of Two Siblings with PC1/3 Deficiency—Obesity and Hyperproinsulinemia

Background: Inactivating mutations in the PCSK1 gene encoding prohormone convertase 1/3 (PC1/3) are associated with failure to thrive (FTT) and diarrhea during infancy, a tendency toward childhood obesity, and the potential for development of endocrinopathies throughout life, including hyperproinsulinemia. We describe two siblings with the same inactivating compound heterozygous mutations (c.625G>A and c.473G>A) in PCSK1, but with phenotypes of differing severity affecting both body composition and glucose homeostasis. Methods: After sequencing confirmed inactivating PCSK1 mutations, we assessed the siblings for resultant endocrinopathies of PC1/3 deficiency. Results: The siblings, a 9y male (M) and an 11y female (F), had severe early onset FTT with diarrhea. Currently, M has extreme obesity (BMI=38.2 kg/m2, >99%), diabetes insipidus, central hypothyroidism (FT4 0.7 ng/dL; TSH 4.9 mIU/L with little diurnal variation), and adrenal insufficiency (peak cortisol post-ACTH 10.8 ug/dL). After an overnight fast, glucose was 64 mg/dL and he had dramatic hyperproinsulinemia (5030 pmol/L, normal <8), far exceeding his fasting insulin concentration (360 pmol/L). Glucose tolerance was better than predicted for his degree of obesity (OGTT 2h glucose 79 mg/dL) and proinsulin remained markedly elevated during the OGTT (6610 pmol/L; normal <200). F was in the 86th percentile for BMI. She had central hypothyroidism (FT4 0.7 ng/dL; TSH 4.7 mIU/L with little diurnal variation) and diabetes insipidus, but had normal adrenal function (peak cortisol post-ACTH 20.3 ug/dL). F also had high fasting proinsulin (1340 pmol/L), out of proportion to fasting insulin (70 pmol/L) and glucose (87 mg/dL) and good glucose tolerance (OGTT 2h glucose 89 mg/dL). Discussion: PC1/3 deficiency reduces processing of proinsulin to insulin and may thus predispose to fasting or post-prandial hypoglycemia. There is substantial heterogeneity in PC1/3 deficiency even within families. Disclosure E.K. Davis: None. M. Broadney: None. R. Persky: None. A. Torky: None. J.A. Yanovski: Research Support; Self; Rhythm Pharmaceuticals Inc., Zafgen.

Melanocortin 4 Receptor Pathway Dysfunction in Obesity: Patient Stratification Aimed at MC4R Agonist Treatment.

ContextThe hypothalamic melanocortin 4 receptor (MC4R) pathway serves a critical role in regulating body weight. Loss of function (LoF) mutations in the MC4R pathway, including mutations in the pro-opiomelanocortin (POMC), prohormone convertase 1 (PCSK1), leptin receptor (LEPR), orMC4R genes, have been shown to cause early-onset severe obesity.MethodsThrough a comprehensive epidemiological analysis of known and predicted LoF variants in thePOMC, PCSK1, andLEPR genes, we sought to estimate the number of US individuals with biallelic MC4R pathway LoF variants.ResultsWe predict ~650α-melanocyte-stimulating hormone (MSH)/POMC, 8500PCSK1, and 3600LEPR homozygous and compound heterozygous individuals in the United States, cumulatively enumerating >12,800 MC4R pathway–deficient obese patients. Few of these variants have been genetically diagnosed to date. These estimates increase when we include a small subset of less rare variants:β-MSH/POMC,PCSK1 N221D, and aPCSK1 LoF variant (T640A). To further define the MC4R pathway and its potential impact on obesity, we tested associations between body mass index (BMI) and LoF mutation burden in thePOMC, PCSK1, andLEPR genes in various populations. We show that the cumulative allele burden in individuals with two or more LoF alleles in one or more genes in the MC4R pathway are predisposed to a higher BMI than noncarriers or heterozygous LoF carriers with a defect in only one gene.ConclusionsOur analysis represents a genetically rationalized study of the hypothalamic MC4R pathway aimed at genetic patient stratification to determine which obese subpopulations should be studied to elucidate MC4R agonist (e.g., setmelanotide) treatment responsiveness.

The ever-expanding saga of the proprotein convertases and their roles in body homeostasis: emphasis on novel proprotein convertase subtilisin kexin number 9 functions and regulation.

The nine members of the proprotein convertase family play major physiological roles during development and in the adult, and their dysregulation leads to various diseases. The primary objective of this article is to review recent findings on the clinical importance of some of these convertases concentrating mostly on PCSK9, the ninth member of the convertase family. This includes the transcriptional and translational regulation of PCSK9, its ability to enhance the degradation of LDL receptor (LDLR), and the implication of PCSK9 in inflammation and sepsis. PCSK9 levels are upregulated by E2F1 and reduced by specific miRNAs and by Annexin A2 that bind the 3' end of its mRNA. The implication of the LDLR in the clearance of pathogenic bacterial debris in mice and human puts in perspective a new role for PCSK9 in the regulation of sepsis. The specific implication of the LDLR in the clearance of Lp(a) is now confirmed by multiple studies of PCSK9 inhibition in human cohorts. Emerging data suggest that PCSK9 can be regulated at the transcriptional and translational levels by specific factors and miRNAs. The identification of a novel pocket in the catalytic domain of PCSK9 represents a harbinger for a new class of small inhibitor drugs. The implication of the LDLR in reducing the effects of bacterially induced sepsis has been supported by both human and mouse data. Outcome studies confirmed the clinical importance of reducing PCSK9 levels. The present review puts in perspective new developments in the PCSK9 biology and its regulation of the LDLR. VIDEO ABSTRACT: http://links.lww.com/COL/A17.

β Cell GLP-1R Signaling Alters α Cell Proglucagon Processing after Vertical Sleeve Gastrectomy in Mice

SUMMARYBariatric surgery, such as vertical sleeve gastrectomy (VSG), causes high rates of type 2 diabetes remission and remarkable increases in postprandial glucagon-like peptide-1 (GLP-1) secretion. GLP-1 plays a critical role in islet function by potentiating glucose-stimulated insulin secretion; however, the mechanisms remain incompletely defined. Therefore, we applied a murine VSG model to an inducible β cell-specific GLP-1 receptor (GLP-1R) knockout mouse model to investigate the role of the β cell GLP-1R in islet function. Our data show that loss of β cell GLP-1R signaling decreases α cell GLP-1 expression after VSG. Furthermore, we find a β cell GLP-1R-dependent increase in α cell expression of the prohormone convertase required for the production of GLP-1 after VSG. Together, the findings herein reveal two concepts. First, our data support a paracrine role for α cell-derived GLP-1 in the metabolic benefits observed after VSG. Second, we have identified a role for the β cell GLP-1R as a regulator of α cell proglucagon processing.

Furin inhibitor protects against neuronal cell death induced by activated NMDA receptors

The proprotein convertases (PCs) act as serine proteases and are known to convert diverse precursor proteins into their active forms. Among the PCs, furin has been considered to play a crucial role not only in embryogenesis, but also in the initiation and progression of certain pathologic conditions. However, the roles played by furin with respect to neuronal cell injuries remain to be determined. An excessive influx of Ca2+ through the N-methyl-d-aspartate (NMDA) receptor has been associated with diverse neurological and neurodegenerative disorders. The aim of this study was to achieve further insight into the pathophysiologic roles of furin in cultured cortical neurons. We demonstrated that furin inhibitors dose-dependently prevented neuronal injury induced by NMDA treatment. Neuronal injury induced by NMDA treatment was attenuated by the calpain inhibitor calpeptin. And the increase observed in the activity of calpain after NMDA treatment was significantly inhibited by these furin inhibitors. Furthermore, calpain-2 activity, which was evaluated by means of the immunoblotting assay, was increased by NMDA treatment. It was noteworthy that this increased activity was almost completely inhibited by a furin inhibitor. Our findings suggested that furin is involved in NMDA-induced neuronal injury by acting upstream of calpain.

Characterization of neuropeptide K processing in rat spinal cord S9 fractions using high-resolution quadrupole-Orbitrap mass spectrometry.

Tachykinins are a family of pronociceptive neuropeptides with a specific role in pain and inflammation. Several mechanisms regulate endogenous tachykinins levels, including the differential expression of protachykinin mRNA and the controlled secretion of tachykinin peptides from neurons. We suspect that proteolysis regulates extracellular neuropeptide K (NPK) and neurokinin A (NKA) concentrations and NPK is a precursor of NKA. Here, we provide evidence that proteolysis controls NPK and NKA levels in the spinal cord, leading to the formation of active C-terminal peptide fragments. Using high-resolution mass spectrometry, specific tachykinin fragments were identified and characterized. The metabolic stability in rat spinal cord fractions of NPK and NKA was very short, resulting in half-lives of 1.9 and 2.2 min respectively. Following the degradation of NPK, several C-terminal fragments were identified, including NPK1-26 , NKA, NKA2-10 , NKA3-10 , NKA5-10 and NKA6-10 , which conserve affinity for the neurokinin 2 receptor but also for the neurokinin 1 receptor. Interestingly, the same fragments were identified following the degradation of NKA. A specific proprotein convertases inhibitor was used and showed a significant reduction in the rate of formation of NKA, providing strong evidence that proprotein convertase is involved in C-terminal processing of NPK in the spinal cord, leading to the formation of NKA.

GPR142 prompts glucagon-like Peptide-1 release from islets to improve β cell function

ObjectiveGPR142 agonists are being pursued as novel diabetes therapies by virtue of their insulin secretagogue effects. But it is undetermined whether GPR142's functions in pancreatic islets are limited to regulating insulin secretion. The current study expands research on its action. Methods and ResultsWe demonstrated by in situ hybridization and immunostaining that GPR142 is expressed not only in β cells but also in a subset of α cells. Stimulation of GPR142 by a selective agonist increased glucagon secretion in both human and mouse islets. More importantly, the GPR142 agonist also potentiated glucagon-like peptide-1 (GLP-1) production and its release from islets through a mechanism that involves upregulation of prohormone convertase 1/3 expression. Strikingly, stimulation of insulin secretion and increase in insulin content via GPR142 engagement requires intact GLP-1 receptor signaling. Furthermore, GPR142 agonist increased β cell proliferation and protected both mouse and human islets against stress-induced apoptosis. ConclusionsCollectively, we provide here evidence that local GLP-1 release from α cells defines GPR142's beneficial effects on improving β cell function and mass, and we propose that GPR142 agonism may have translatable and durable efficacy for the treatment of type 2 diabetes.

Stepwise processing analyses of the single-turnover PCSK9 protease reveal its substrate sequence specificity and link clinical genotype to lipid phenotype

Proprotein convertase subtilisin/kexin type 9 (PCSK9) down-regulates the low-density lipoprotein (LDL) receptor, elevating LDL cholesterol and accelerating atherosclerotic heart disease, making it a promising cardiovascular drug target. To achieve its maximal effect on the LDL receptor, PCSK9 requires autoproteolysis. After cleavage, PCSK9 retains its prodomain in the active site as a self-inhibitor. Unlike other proprotein convertases, however, this retention is permanent, inhibiting any further protease activity for the remainder of its life cycle. Such inhibition has proven a major challenge toward a complete biochemical characterization of PCSK9's proteolytic function, which could inform therapeutic approaches against its hypercholesterolemic effects. To address this challenge, we employed a cell-based, high-throughput method using a luciferase readout to evaluate the single-turnover PCSK9 proteolytic event. We combined this method with saturation mutagenesis libraries to interrogate the sequence specificities of PCSK9 cleavage and proteolysis-independent secretion. Our results highlight several key differences in sequence identity between these two steps, complement known structural data, and suggest that PCSK9 self-proteolysis is the rate-limiting step of secretion. Additionally, we found that for missense SNPs within PCSK9, alterations in both proteolysis and secretion are common. Last, we show that some SNPs allosterically modulate PCSK9's substrate sequence specificity. Our findings indicate that PCSK9 proteolysis acts as a commonly perturbed but critical switch in controlling lipid homeostasis and provide a new hope for the development of small-molecule PCSK9 inhibitors.

Compartment-Specific Biosensors Reveal a Complementary Subcellular Distribution of Bioactive Furin and PC7

Furin trafficking, and that of related proprotein convertases (PCs), may regulate which substrates are accessible for endoproteolysis, but tools to directly test this hypothesis have been lacking. Here, we develop targeted biosensors that indicate Furin activity in endosomes is 10-fold less inhibited by decanoyl-RVKR-chloromethylketone and enriched >3-fold in endosomes compared to the trans-Golgi network (TGN). Endogenous PC7, which resists this inhibitor, was active in distinct vesicles. Only overexpressed PC7 activity reached the cell surface, endosomes, and the TGN. A PLC motif in the cytosolic tail of PC7 was dispensable for endosomal activity, but it was specifically required for TGN recycling and to rescue proActivin-A cleavage in Furin-depleted B16F1 melanoma cells. In sharp contrast, PC7 complemented Furin in cleaving Notch1 independently of PLC-mediatedTGN access. Our study provides a proof in principle that compartment-specific biosensors canbe used to gain insight into the regulation of PC trafficking and to map the tropism of PC-specific inhibitors.

Profiles of pro-opiomelanocortin and encoded peptides, and their processing enzymes in equine pituitary pars intermedia dysfunction.

Equine pituitary pars intermedia dysfunction (PPID) is characterized by hyperplasia of the pars intermedia (PI) melanotrophs of the pituitary gland (PG), and increased production of proopiomelanocortin (POMC). POMC is cleaved by prohormone convertase 1 (PC1) to produce adrenocorticotropic hormone (ACTH), and further processing of ACTH by PC2 to produce alpha-melanocyte stimulating hormone (α-MSH) and corticotropin-like intermediate peptide (CLIP). High plasma ACTH concentrations in horses with PPID might be related to reduced conversion of ACTH to α-MSH by PCs. The hypothesis of this study was that PC1 and PC2 expression in the pituitary gland are altered in PPID, resulting in an abnormal relative abundance of POMC derived proteins. The objectives of this study were to identify the partial sequences of equine POMC, PC1, and PC2 mRNAs; and to determine whether the expression of POMC, PC1, and PC2 mRNAs in whole pituitary extracts, and POMC-protein in the cavernous sinus blood of horses are altered in PPID. We confirmed (RT-PCR and sequencing) that the partial sequences obtained match the corresponding regions of predicted equine POMC, PC1 and PC2 sequences. The expression (quantification by RT-qPCR) of POMC, PC1 and PC2 mRNAs were found upregulated in the pituitary of horses with PPID. Plasma (measured using RIA/ELISA) ACTH and α-MSH were elevated in PPID horses. These results indicate distinct differences in gene and protein expression of POMC and its intermediates, and processing enzymes in PPID. It provides evidence to support the notion that local, pituitary-specific inadequacies in prohormone processing likely contribute to equine PPID.