Purified recombinant human Chromogranin A N46 peptide with remarkable anticancer effect on human colon cancer cells.

Human Chromogranin A N46 (CGA-N46) is a weak cationic α-helical peptide with wide-spectrum antibacterial, fungal, and anticancer activities. In this study, the recombinant human CGA-N46 peptide was expressed successfully in Escherichia coli. The gene of CGA-N46 was cloned into the expression vector pET-15b without a fusion tag at the N terminus and the peptide was expressed using Isopropyl-ß-d-thiogalactoside (IPTG) as an inducer. Using 8 M guanidinium HCl, inclusion bodies containing the peptide were purified and solubilized. The rhCGA-N46 peptide was purified using Q-FF anion exchange column. The cytotoxicity of the purified rhCGA-N46 peptide was investigated on WI-38 human lung normal cell line. The anticancer activity was studied on human colon cancer cell line; HCT-116 cell line. Besides, the possible involvement of rhCGA-N46 peptide in regulating apoptotic signal pathways was analyzed by detecting the expression levels of BCL2, BID, and CAS-8 in the treated cells. The results concluded that the active peptide recovery was up to 41.98%. The purified rhCGA-N46 was safe on normal cells with IC50 = 227.74 µg/ml (40.67 µM) and had an obvious anticancer effect on colon cancer cells with IC50 = 1.997 µg/ml (356.6 nM). The expression level of BCL2 was down-regulated and BID and CAS-8 were up-regulated significantly in treated HCT-116 cells compared to untreated. In conclusion, the rhCGA-N46 peptide was produced successfully in the native form with promising anti-colon cancer activity.

CGA-N9, an antimicrobial peptide derived from chromogranin A: direct cell penetration of and endocytosis by Candida tropicalis.

CGA-N9 is a peptide derived from the N-terminus of human chromogranin A comprising amino acids 47-55. Minimum inhibitory concentration (MIC) assays showed that CGA-N9 had antimicrobial activity and exhibited time-dependent inhibition activity against Candida tropicalis, with high safety in human red blood cells (HRBCs) and mouse brain microvascular endothelial cells (bEnd.3). According to the results of transmission electron microscopy (TEM), flow cytometry and confocal microscopy, CGA-N9 accumulated in cells without destroying the integrity of the cell membrane; the peptide was initially localized to the cell membrane and subsequently internalized into the cytosol. An investigation of the cellular internalization mechanism revealed that most CGA-N9 molecules entered the yeast cells, even at 4°C and in the presence of sodium azide (NaN3), both of which block all energy-dependent transport mechanisms. In addition, peptide internalization was affected by the endocytic inhibitors 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), cytochalasin D (CyD) and heparin; chlorpromazine (CPZ) also had some effect on CGA-N9 internalization. Similar results were obtained in the MIC assays, whereby the anticandidal activity of CGA-N9 was blocked to different degrees in the presence of EIPA, CyD, heparin or CPZ. Therefore, most CGA-N9 passes through the C. tropicalis cell membrane via direct cell penetration, whereas the remainder enters through macropinocytosis and sulfate proteoglycan-mediated endocytosis, with a slight contribution from clathrin-mediated endocytosis.

The chromogranin A-derived antifungal peptide CGA-N9 induces apoptosis in Candida tropicalis.

CGA-N9, a peptide derived from human chromogranin A (CGA), was found to have antimicrobial activity in our previous investigation, but its mechanism of action remains unclear. Herein, the mechanism of action of CGA-N9 was investigated. We found that CGA-N9 induced the depolarization of the cell membrane and uptake of calcium ions into the cytosol and mitochondria. With the disruption of the mitochondrial membrane potential, the generation of intracellular reactive oxygen species (ROS) increased. Accordingly, we assessed apoptotic processes in Candida tropicalis cells post-treatment with CGA-N9 and found cytochrome c leakage, chromatin condensation and DNA degradation. The interaction of CGA-N9 with DNA in vitro showed that CGA-N9 did not degrade DNA but bound to DNA via an electrostatic interaction. In conclusion, CGA-N9 exhibits antifungal activity by inducing apoptosis in C. tropicalis.

Circulating chromogranin A and its fragments as diagnostic and prognostic disease markers.

Chromogranin A (CgA), a secretory protein released in the blood by neuroendocrine cells and neurons, is the precursor of various bioactive fragments involved in the regulation of the cardiovascular system, metabolism, innate immunity, angiogenesis, and tissue repair. After the original demonstration that circulating CgA can serve as a biomarker for a wide range of neuroendocrine tumors, several studies have shown that increased levels of CgA can be present also in the blood of patients with cardiovascular, gastrointestinal, and inflammatory diseases with, in certain cases, important diagnostic and prognostic implications. Considering the high structural and functional heterogeneity of the CgA system, comprising precursor and fragments, it is not surprising that the different immunoassays used in these studies led, in some cases, to discrepant results. Here, we review these notions and we discuss the importance of measuring total-CgA, full-length CgA, specific fragments, and their relative levels for a more thorough assessment of the pathophysiological function and diagnostic/prognostic value of the CgA system.

N-terminal additions to the WE14 peptide of chromogranin A create strong autoantigen agonists in type 1 diabetes.

Chromogranin A (ChgA) is an autoantigen for CD4(+) T cells in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). The natural ChgA-processed peptide, WE14, is a weak agonist for the prototypical T cell, BDC-2.5, and other ChgA-specific T-cell clones. Mimotope peptides with much higher activity share a C-terminal motif, WXRM(D/E), that is predicted to lie in the p5 to p9 position in the mouse MHC class II, IA(g7) binding groove. This motif is also present in WE14 (WSRMD), but at its N terminus. Therefore, to place the WE14 motif into the same position as seen in the mimotopes, we added the amino acids RLGL to its N terminus. Like the other mimotopes, RLGL-WE14, is much more potent than WE14 in T-cell stimulation and activates a diverse population of CD4(+) T cells, which also respond to WE14 as well as islets from WT, but not ChgA(-/-) mice. The crystal structure of the IA(g7)-RLGL-WE14 complex confirmed the predicted placement of the peptide within the IA(g7) groove. Fluorescent IA(g7)-RLGL-WE14 tetramers bind to ChgA-specific T-cell clones and easily detect ChgA-specific T cells in the pancreas and pancreatic lymph nodes of NOD mice. The prediction that many different N-terminal amino acid extensions to the WXRM(D/E) motif are sufficient to greatly improve T-cell stimulation leads us to propose that such a posttranslational modification may occur uniquely in the pancreas or pancreatic lymph nodes, perhaps via the mechanism of transpeptidation. This modification could account for the escape of these T cells from thymic negative selection.

Identification of the Vasoconstriction-Inhibiting Factor (VIF), a Potent Endogenous Cofactor of Angiotensin II Acting on the Angiotensin II Type 2 Receptor.

BACKGROUND: The renin-angiotensin system and especially the angiotensin peptides play a central role in blood pressure regulation. Here, we hypothesize that an as-yet unknown peptide is involved in the action of angiotensin II modulating the vasoregulatory effects as a cofactor. METHODS AND RESULTS: The peptide with vasodilatory properties was isolated from adrenal glands chromatographically. The effects of this peptide were evaluated in vitro and in vivo, and the receptor affinity was analyzed. The plasma concentration in humans was quantified in patients with chronic kidney disease, patients with heart failure, and healthy control subjects. The amino acid sequence of the peptide from bovine adrenal glands was HSSYEDELSEVL EKPNDQAE PKEVTEEVSSKDAAE, which is a degradation product of chromogranin A. The sequence of the peptide isolated from human plasma was HSGFEDELSEVLENQSSQAELKEAVEEPSSKDVME. Both peptides diminished significantly the vasoconstrictive effect of angiotensin II in vitro. Therefore, we named the peptide vasoconstriction-inhibiting factor (VIF). The vasoregulatory effects of VIF are mediated by the angiotensin II type 2 receptor. VIF impairs angiotensin II-induced phosphorylation of the p38 mitogen-activated protein kinase pathway but not of extracellular-regulated kinase 1/2. The vasodilatory effects were confirmed in vivo. The plasma concentration was significantly increased in renal patients and patients with heart failure. CONCLUSIONS: VIF is a vasoregulatory peptide that modulates the vasoconstrictive effects of angiotensin II by acting on the angiotensin II type 2 receptor. It is likely that the increase in VIF may serve as a counterregulatory effect to defend against hypertension. The identification of this target may help us to understand the pathophysiology of renal and heart failure and may form a basis for the development of new strategies for the prevention and treatment of cardiovascular disease.

Regulation of intestinal SGLT1 by catestatin in hyperleptinemic type 2 diabetic mice.

The small intestine is the major site for nutrient absorption that is critical in maintenance of euglycemia. Leptin, a key hormone involved in energy homeostasis, directly affects nutrient transport across the intestinal epithelium. Catestatin (CST), a 21-amino acid peptide derived from proprotein chromogranin A, has been shown to modulate leptin signaling. Therefore, we reasoned that leptin and CST could modulate intestinal Na(+)-glucose transporter 1 (SGLT1) expression in the context of obesity and diabetes. We found that hyperleptinemic db/db mice exhibit increased mucosal mass, associated with an enhanced proliferative response and decreased apoptosis in intestinal crypts, a finding absent in leptin-deficient ob/ob mice. Intestinal SGLT1 abundance was significantly decreased in hyperleptinemic but not leptin-deficient mice, indicating leptin regulation of SGLT1 expression. Phlorizin, a SGLT1/2 inhibitor, was without effect in an oral glucose tolerance test in db/db mice. The alterations in architecture and SGLT1 abundance were not accompanied by changes in the localization of intestinal alkaline phosphatase, indicating intact differentiation. Treatment of db/db mice with CST restored intestinal SGLT1 abundance and intestinal turnover, suggesting a cross-talk between leptin and CST, without affecting plasma leptin levels. Consistent with this hypothesis, we identified structural homology between CST and the AB-loop of leptin and protein-protein docking revealed binding of CST and leptin with the Ig-like binding site-III of the leptin receptor. In summary, downregulation of SGLT1 in an obese type 2 diabetic mouse model with hyperleptinemia is presumably mediated via the short form of the leptin receptor and reduces overt hyperglycemia.

Label-Free Detection of Human Glycoprotein (CgA) Using an Extended-Gated Organic Transistor-Based Immunosensor.

Herein, we report on the fabrication of an extended-gated organic field-effect transistor (OFET)-based immunosensor and its application in the detection of human chromogranin A (hCgA). The fabricated OFET device possesses an extended-gate electrode immobilized with an anti-CgA antibody. The titration results of hCgA showed that the electrical changes in the OFET characteristics corresponded to the glycoprotein recognition ability of the monoclonal antibody (anti-CgA). The observed sensitivity (detection limit: 0.11 µg/mL) and selectivity indicate that the OFET-based immunosensor can be potentially applied to the rapid detection of the glycoprotein concentration without any labeling.

MicroRNA-22 and promoter motif polymorphisms at the Chga locus in genetic hypertension: functional and therapeutic implications for gene expression and the pathogenesis of hypertension.

Hypertension is a common hereditary syndrome with unclear pathogenesis. Chromogranin A (Chga), which catalyzes formation and cargo storage of regulated secretory granules in neuroendocrine cells, contributes to blood pressure homeostasis centrally and peripherally. Elevated Chga occurs in spontaneously hypertensive rat (SHR) adrenal glands and plasma, but central expression is unexplored. In this report, we measured SHR and Wistar-Kyoto rat (control) Chga expression in central and peripheral nervous systems, and found Chga protein to be decreased in the SHR brainstem, yet increased in the adrenal and the plasma. By re-sequencing, we systematically identified five promoter, two coding and one 3'-untranslated region (3'-UTR) polymorphism at the SHR (versus WKY or BN) Chga locus. Using HXB/BXH recombinant inbred (RI) strain linkage and correlations, we demonstrated genetic determination of Chga expression in SHR, including a cis-quantitative trait loci (QTLs) (i.e. at the Chga locus), and such expression influenced biochemical determinants of blood pressure, including a cascade of catecholamine biosynthetic enzymes, catecholamines themselves and steroids. Luciferase reporter assays demonstrated that the 3'-UTR polymorphism (which disrupts a microRNA miR-22 motif) and promoter polymorphisms altered gene expression consistent with the decline in SHR central Chga expression. Coding region polymorphisms did not account for changes in Chga expression or function. Thus, we hypothesized that the 3'-UTR and promoter mutations lead to dysregulation (diminution) of Chga in brainstem cardiovascular control nuclei, ultimately contributing to the pathogenesis of hypertension in SHR. Accordingly, we demonstrated that in vivo administration of miR-22 antagomir to SHR causes substantial (∼18 mmHg) reductions in blood pressure, opening a novel therapeutic avenue for hypertension.

Vasostatin-1 antigenic epitope mapping for induction of cellular and humoral immune responses to chromogranin A autoantigen in NOD mice.

The chromogranin A (ChgA) 29-42 sequence is the antigenic epitope for the BDC2.5 CD4(+) T-cell receptor in NOD mice (H-2(g7) ). We have now characterized the binding register of the ChgA 29-42 peptide for the I-A(g7) molecule. Truncation of the peptide demonstrated that the KCVLEVISD sequence 34-42 is the binding register and extension of this sequence by flanking residues increased its binding affinity and antigenic capacity. We employed anti-ChgA peptide antibodies generated against different fragments of ChgA for immunostaining of pancreatic islet sections from NOD mice. A strong immuno-staining pattern was observed for the ChgA 17-38 peptide antibodies that overlap with the ChgA 29-42 sequence. Moreover, sera from diabetic NOD mice showed elevated titers of autoantibodies to the ChgA 29-42 peptide. These findings indicate that peptides from the N-terminal region of ChgA are able to induce cellular and humoral immune responses in NOD mice.

A new chromogranin A-dependent angiogenic switch activated by thrombin.

Angiogenesis, the formation of blood vessels from pre-existing vasculature, is regulated by a complex interplay of anti and proangiogenic factors. We found that physiologic levels of circulating chromogranin A (CgA), a protein secreted by the neuroendocrine system, can inhibit angiogenesis in various in vitro and in vivo experimental models. Structure-activity studies showed that a functional anti-angiogenic site is located in the C-terminal region, whereas a latent anti-angiogenic site, activated by cleavage of Q76-K77 bond, is present in the N-terminal domain. Cleavage of CgA by thrombin abrogated its anti-angiogenic activity and generated fragments (lacking the C-terminal region) endowed of potent proangiogenic activity. Hematologic studies showed that biologically relevant levels of forms of full-length CgA and CgA1-76 (anti-angiogenic) and lower levels of fragments lacking the C-terminal region (proangiogenic) are present in circulation in healthy subjects. Blood coagulation caused, in a thrombin-dependent manner, almost complete conversion of CgA into fragments lacking the C-terminal region. These results suggest that the CgA-related circulating polypeptides form a balance of anti and proangiogenic factors tightly regulated by proteolysis. Thrombin-induced alteration of this balance could provide a novel mechanism for triggering angiogenesis in pathophysiologic conditions characterized by prothrombin activation.

A molecular dynamics study of catestatin docked on nicotinic acetylcholine receptors to identify amino acids potentially involved in the binding of chromogranin A fragments.

Catestatin, a cationic and hydrophobic 21-amino acid fragment of chromogranin A, is known to be a non-competitive nicotinic antagonist acting through nicotinic acetylcholine receptors (nAChRs) to inhibit catecholamine release. Since this receptor is the target of several neuronal and non-neuronal disorder prophylaxes and treatments, this study aims at the elucidation of the binding of human catestatin to the entire nAChR reconstructed in lipid bilayers by means of docking followed by full atomistic molecular dynamics simulations. The obtained results show that the minimum free energy for the binding of the peptide and the receptor attains minimal values for locations at the pore site and in the outer beta subunit. This result is consistent with previous studies showing that catestatin occludes the pore opening. A new finding is an additional even stronger binding seat at the beta subunit and that membrane presence could be an important factor. Specific amino acids involved in catestatin binding have been identified, indicating targets for point mutation studies. In addition to improving the understanding of the interaction between the peptide and muscle-type and even other nAChR subtypes, the results of this study provide directions for future peptidomimetic research.

The important role of catestatin in cardiac remodeling.

Catestatin (CST) was first discovered as a potent non-competitive and reversible inhibitor of catecholamine secretion. Recent reports on plasma CST level in heart diseases suggested a cardioprotective role for this peptide. Given that cardiac remodeling is the dominant pathologic process in cardiac dysfunction, we propose that CST participates in the regulation of concern pathways and contributes to the inhibition of cardiac remodeling. In this minireview, the potential mechanism of cardiac remodeling involving CST will be discussed from three aspects: hypertrophy, fibrosis, and apoptosis.

Antioxidant properties of a human neuropeptide and its protective effect on free radical-induced DNA damage.

Human catestatin CgA352-372 (SL21) is an endogenous neuropeptide with multiple biological functions. The present study aimed to evaluate the antioxidant, antibacterial, cytotoxic, and DNA damage protective effects of SL21 neuropeptide. SL21 neuropeptide generated from the C-terminus of chromogranin A (CgA) was synthesized by solid-phase method. Synthetic peptide was subjected to various in vitro antioxidant assays including the scavenging of 1,1-diphenyl-2-pycryl-hydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(·+) ), and hydroxyl free radicals, metal ion chelation, inhibition of lipid peroxidation, and reducing power. Moreover, protective effect of SL21 on H2 O2 -induced DNA damage was analyzed using pTZ57/RT plasmid. Methylthiazoltetrazolium assay was also performed to study the cytotoxic effect of SL21 neuropeptide on human peripheral blood mononuclear cells. Furthermore, antibacterial and hemolysis assays were conducted. The results demonstrated high activities of SL21 in scavenging free radicals (DPPH, ABTS(·+) , and hydroxyl), chelating of Cu(2+) /Fe(2+) metal ions, reducing power, and inhibition of lipid peroxidation in a concentration-dependent manner. SL21 neuropeptide revealed a protective effect on DNA damage caused by hydroxyl radicals. Interestingly, the peptide exhibited no significant cytotoxicity towards peripheral blood mononuclear cells. Furthermore, SL21 peptide displayed antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa without any hemolytic activity on human red blood cells. Conclusively, the present study established SL21 (catestatin) as a novel antioxidative peptide that could further be investigated for its potential use as a pharmaceutical agent.

Functional genetic variants of the catecholamine-release-inhibitory peptide catestatin in an Indian population: allele-specific effects on metabolic traits.

Catestatin (CST), a chromogranin A (CHGA)-derived peptide, is a potent inhibitor of catecholamine release from adrenal chromaffin cells and postganglionic sympathetic axons. We re-sequenced the CST region of CHGA in an Indian population (n = 1010) and detected two amino acid substitution variants: G364S and G367V. Synthesized CST variant peptides (viz. CST-Ser-364 and CST-Val-367) were significantly less potent than the wild type peptide (CST-WT) to inhibit nicotine-stimulated catecholamine secretion from PC12 cells. Consistently, the rank-order of blockade of nicotinic acetylcholine receptor (nAChR)-stimulated inward current and intracellular Ca(2+) rise by these peptides in PC12 cells was: CST-WT > CST-Ser-364 > CST-Val-367. Structural analysis by CD spectroscopy coupled with molecular dynamics simulations revealed the following order of α-helical content: CST-WT > CST-Ser-364 > CST-Val-367; docking of CST peptides onto a major human nAChR subtype and molecular dynamics simulations also predicted the above rank order for their binding affinity with nAChR and the extent of occlusion of the receptor pore, providing a mechanistic basis for differential potencies. The G364S polymorphism was in strong linkage disequilibrium with several common CHGA genetic variations. Interestingly, the Ser-364 allele (detected in ∼15% subjects) was strongly associated with profound reduction (up to ∼2.1-fold) in plasma norepinephrine/epinephrine levels consistent with the diminished nAChR desensitization-blocking effect of CST-Ser-364 as compared with CST-WT. Additionally, the Ser-364 allele showed strong associations with elevated levels of plasma triglyceride and glucose levels. In conclusion, a common CHGA variant in an Indian population influences several biochemical parameters relevant to cardiovascular/metabolic disorders.

The novel chromogranin A-derived serpinin and pyroglutaminated serpinin peptides are positive cardiac ß-adrenergic-like inotropes.

Three forms of serpinin peptides, serpinin (Ala26Leu), pyroglutaminated (pGlu)-serpinin (pGlu23Leu), and serpinin-Arg-Arg-Gly (Ala29Gly), are derived from cleavage at pairs of basic residues in the highly conserved C terminus of chromogranin A (CgA). Serpinin induces PN-1 expression in neuroendocrine cells to up-regulate granule biogenesis via a cAMP-protein kinase A-Sp1 pathway, while pGlu-serpinin inhibits cell death. The aim of this study was to test the hypothesis that serpinin peptides are produced in the heart and act as novel ß-adrenergic-like cardiac modulators. We detected serpinin peptides in the rat heart by HPLC and ELISA methods. The peptides included predominantly Ala29Gly and pGlu-serpinin and a small amount of serpinin. Using the Langendorff perfused rat heart to evaluate the hemodynamic changes, we found that serpinin and pGlu-serpinin exert dose-dependent positive inotropic and lusitropic effects at 11-165 nM, within the first 5 min after administration. The pGlu-serpinin-induced contractility is more potent than that of serpinin, starting from 1 nM. Using the isolated rat papillary muscle preparation to measure contractility in terms of tension development and muscle length, we further corroborated the pGlu-serpinin-induced positive inotropism. Ala29Gly was unable to affect myocardial performance. Both pGlu-serpinin and serpinin act through a ß1-adrenergic receptor/adenylate cyclase/cAMP/PKA pathway, indicating that, contrary to the ß-blocking profile of the other CgA-derived cardiosuppressive peptides, vasostatin-1 and catestatin, these two C-terminal peptides act as ß-adrenergic-like agonists. In cardiac tissue extracts, pGlu-serpinin increased intracellular cAMP levels and phosphorylation of phospholamban (PLN)Ser16, ERK1/2, and GSK-3ß. Serpinin and pGlu-serpinin peptides emerge as novel ß-adrenergic inotropic and lusitropic modulators, suggesting that CgA and the other derived cardioactive peptides can play a key role in how the myocardium orchestrates its complex response to sympathochromaffin stimulation.

Development of a pharmacophore model for the catecholamine release-inhibitory peptide catestatin: virtual screening and functional testing identify novel small molecule therapeutics of hypertension.

The endogenous catecholamine release-inhibitory peptide catestatin (CST) regulates events leading to hypertension and cardiovascular disease. Earlier we studied the structure of CST by NMR, molecular modeling, and amino acid scanning mutagenesis. That structure has now been exploited for elucidation of interface pharmacophores that mediate binding of CST to its target, with consequent secretory inhibition. Designed pharmacophore models allowed screening of 3D structural domains. Selected compounds were tested on both cultured catecholaminergic cells and an in vivo model of hypertension; in each case, the candidates showed substantial mimicry of native CST actions, with preserved or enhanced potency and specificity. The approach and compounds have thus enabled rational design of novel drug candidates for treatment of hypertension or autonomic dysfunction.

Inhibition of Candida albicans in vivo and in vitro by antimicrobial peptides chromogranin A-N12 through microRNA-155/suppressor of cytokine signaling 1 axis.

Antimicrobial peptides (AMPs) have proven to inhibit a variety of pathogens. Chromogranin A-N12 (CGA-N12) is a kind of AMP, and it is characterized by stable structure, high anti-Candida activity, and good safety. However, it remains unclear whether CGA-N12 could effectively inhibit the growth of Candida albicans (C. albicans). Colony forming assays were used to measure minimal inhibitory concentration (MIC), minimal fungicidal concentration (MFC), and time-kill curve. Disseminated C. albicans rabbit model was established to investigate the influence of CGA-N12 on histological damage. The protein and mRNA levels of suppressor of cytokine signaling 1 (SOCS1) after treatment were investigated. The MIC and MFC of CGA-N12 against C. albicans was 6 mg/mL. CGA-N12 considerably inhibited germ tube formation of C. albicans. The fungal load in the tissues and inflammatory factors in the serum were suppressed by CGA-N12. CGA-N12 significantly reduced the histological changes caused by C. albicans, and the protein and mRNA levels of SOCS1 were markedly inhibited. The inhibition effect of CGA-N12 on C. albicans and significant improvement of histological damage by CGA-N12 through microRNA-155/SOCS1 axis were proved in this study. This study proposes a novel therapeutic strategy for the treatment and prevention of C. albicans.Abbreviations: AMPs: Antimicrobial peptides; MIC: Minimal inhibitory concentration; MFC: Minimal fungicidal concentration; AIDS: Acquired immune deficiency syndrome; PBS: Phosphate buffer saline; FBS: Fetal bovine serum; ROS: Reactive oxygen species; CFU: Colony formation unit; CGA: Chromogranin A; SOCS1: Suppressor of cytokine signaling 1; SDA: Sabouraud Dextrose Agar; GRAVY: Grand average of hydropathicity; C. parapsilosis: Candida parapsilosis; C. albicans: Candida albicans.

Functional Gly297Ser Variant of the Physiological Dysglycemic Peptide Pancreastatin Is a Novel Risk Factor for Cardiometabolic Disorders.

Pancreastatin (PST), a chromogranin A-derived potent physiological dysglycemic peptide, regulates glucose/insulin homeostasis. We have identified a nonsynonymous functional PST variant (p.Gly297Ser; rs9658664) that occurs in a large section of human populations. Association analysis of this single nucleotide polymorphism with cardiovascular/metabolic disease states in Indian populations (n = 4,300 subjects) displays elevated plasma glucose, glycosylated hemoglobin, diastolic blood pressure, and catecholamines in Gly/Ser subjects as compared with wild-type individuals (Gly/Gly). Consistently, the 297Ser allele confers an increased risk (∼1.3-1.6-fold) for type 2 diabetes/hypertension/coronary artery disease/metabolic syndrome. In corroboration, the variant peptide (PST-297S) displays gain-of-potency in several cellular events relevant for cardiometabolic disorders (e.g., increased expression of gluconeogenic genes, increased catecholamine secretion, and greater inhibition of insulin-stimulated glucose uptake) than the wild-type peptide. Computational docking analysis and molecular dynamics simulations show higher affinity binding of PST-297S peptide with glucose-regulated protein 78 (GRP78) and insulin receptor than the wild-type peptide, providing a mechanistic basis for the enhanced activity of the variant peptide. In vitro binding assays validate these in silico predictions of PST peptides binding to GRP78 and insulin receptor. In conclusion, the PST 297Ser allele influences cardiovascular/metabolic phenotypes and emerges as a novel risk factor for type 2 diabetes/hypertension/coronary artery disease in human populations.

Catestatin, a neuroendocrine antimicrobial peptide, induces human mast cell migration, degranulation and production of cytokines and chemokines.

Catestatin, a neuroendocrine peptide with effects on human autonomic function, has recently been found to be a cutaneous antimicrobial peptide. Human catestatin exhibits three single nucleotide polymorphisms: Gly364Ser, Pro370Leu and Arg374Gln. Given reports indicating that antimicrobial peptides and neuropeptides induce mast cell activation, we postulated that catestatin might stimulate numerous functions of human mast cells, thereby participating in the regulation of skin inflammatory responses. Catestatin and its naturally occurring variants caused the human mast cell line LAD2 and peripheral blood-derived mast cells to migrate, degranulate and release leukotriene C(4) and prostaglandins D(2) and E(2). Moreover, catestatins increased intracellular Ca(2+) mobilization in mast cells, and induced the production of pro-inflammatory cytokines/chemokines such as granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1/CCL2, macrophage inflammatory protein-1α/CCL3 and macrophage inflammatory protein-1ß/CCL4. Our evaluation of possible cellular mechanisms suggested that G-proteins, phospholipase C and the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) are involved in catestatin-induced mast cell activation as evidenced by the inhibitory effects of pertussis toxin (G-protein inhibitor), U-73122 (phospholipase C inhibitor) and U0126 (ERK inhibitor), respectively. We also found that human mast cells express the α7 subunit of the nicotinic acetylcholine receptor at both the mRNA and protein levels. Given that silencing the α7 receptor mRNA and an α7-specific inhibitor did not affect catestatin-mediated activation of mast cells, however, we concluded that this receptor is not likely to be functional in human mast cell stimulation by catestatins. Our finding that the neuroendocrine antimicrobial peptide catestatin activates human mast cells suggests that this peptide might have immunomodulatory functions, and provides a new link between neuroendocrine and cutaneous immune systems.