Vasostatins: new molecular targets for atherosclerosis, post-ischaemic angiogenesis, and arteriogenesis.

The chromogranin-secretogranin secretory proteins-granins-are acidic proteins localized in granules of endocrine cells and neurons. The chromogranin family includes chromogranins A (CgA) and B, as well as secretogranin II (once called chromogranin C). Members of this family undergo catalytic proteolysis to produce active peptides. The CgA-derived peptides vasostatin-1 and vasostatin-2, in particular, appear to protect against atherosclerosis, suppressing the expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, as well as exerting vasodilatory effects by enhancing nitric oxide bioavailability. Vasostatin-1 also suppresses vasoconstriction and abnormal angiogenesis. Vasostatin-1 and vasostatin-2 may be novel therapeutic targets for atherosclerosis and coronary heart disease, also protecting the myocardium against ischaemic damage.

Biomarker potential of lncRNA GNAS-AS1 in osteosarcoma prognosis and effect on cellular function.

BACKGROUND: Osteosarcoma (OS) is a type of bone cancer that occurs in children and adolescents at a rate of 5%. The purpose of this study is to explore the lncRNA GNAS-AS1 expression profile, prognosis significance in OS, and biological effect on OS cell function. METHODS: One hundred eight pairs of tissues were collected, and OS cell lines were purchased. lncRNA GNAS-AS1 expression in these tissues and cells were analyzed by qRT-PCR. Clinical data were analyzed using chi-square tests, Kaplan-Meier curves (log-rank test), and Cox regression. CCK-8 and transwell assay were conducted to analyze the effect of lncRNA GNAS-AS1 on cell proliferation, invasion, and migration. The downstream miRNA was presumed. RESULTS: The expression of lncRNA GNAS-AS1 was significantly increased in OS cells and tissues, and related to Enneking staging and distant metastasis. Patients with high lncRNA GNAS-AS1 expression represented shorter overall survival and was an independent prognostic predictor of OS. LncRNA GNAS-AS1 knockdown inhibited cell proliferation, migration, and invasion by regulated miR-490-3p partly at least. CONCLUSIONS: LncRNA GNAS-AS1 can be used as a prognostic indicator and its inhibition suppress the development of OS, suggesting its value as novel therapeutic strategies in OS.

Peptidomic analysis of endogenous plasma peptides from patients with pancreatic neuroendocrine tumours.

RATIONALE: Diagnosis of pancreatic neuroendocrine tumours requires the study of patient plasma with multiple immunoassays, using multiple aliquots of plasma. The application of mass spectrometry based techniques could reduce the cost and amount of plasma required for diagnosis. METHODS: Plasma samples from two patients with pancreatic neuroendocrine tumours were extracted using an established acetonitrile-based plasma peptide enrichment strategy. The circulating peptidome was characterised using nano and high flow rate liquid chromatography/mass spectrometry (LC/MS) analyses. To assess the diagnostic potential of the analytical approach, a large sample batch (68 plasmas) from control subjects, and aliquots from subjects harbouring two different types of pancreatic neuroendocrine tumour (insulinoma and glucagonoma), were analysed using a 10-min LC/MS peptide screen. RESULTS: The untargeted plasma peptidomics approach identified peptides derived from the glucagon prohormone, chromogranin A, chromogranin B and other peptide hormones and proteins related to control of peptide secretion. The glucagon prohormone derived peptides that were detected were compared against putative peptides that were identified using multiple antibody pairs against glucagon peptides. Comparison of the plasma samples for relative levels of selected peptides showed clear separation between the glucagonoma and the insulinoma and control samples. CONCLUSIONS: The combination of the organic solvent extraction methodology with high flow rate analysis could potentially be used to aid diagnosis and monitor treatment of patients with functioning pancreatic neuroendocrine tumours. However, significant validation will be required before this approach can be clinically applied.

Study of adenylyl cyclase-GαS interactions and identification of novel AC ligands.

Adenylyl cyclases (ACs) are membrane bound enzymes that catalyze the production of cAMP from ATP in response to the activation by G-protein Gαs. Different isoforms of ACs are ubiquitously expressed in different tissues involved in regulatory mechanisms in response to specific stimulants. There are 9 AC isoforms present in humans, with AC5 and AC6 proposed to play a vital role in cardiac functions. The activity of AC6 is sensitive to nitric oxide, such that nitrosylation of the protein might regulate its function. However, the information on structural determinants of nitrosylation in ACs and how they interact with Gαs is limited. Here we used homology modeling to build a molecular model of human AC6 bound to Gαs. Based on this 3D model, we predict the nitrosylation amenable cysteines, and identify potential novel ligands of AC6 using virtual ligand screening. Our model suggests Cys1004 in AC6 (subunit C2) and Cys174 in Gαs present at the AC-Gαs interface as the possible residues that might undergo reversible nitrosylation. Docking analysis predicted novel ligands of AC6 that include forskolin-based compounds and its derivatives. Further work involving site-directed mutagenesis of the predicted residues will allow manipulation of AC activity using novel ligands, and crucial insights on the role of nitrosylation of these proteins in pathophysiological conditions.

Chromogranins: from discovery to current times.

The discovery in 1953 of the chromaffin granules as co-storage of catecholamines and ATP was soon followed by identification of a range of uniquely acidic proteins making up the isotonic vesicular storage complex within elements of the diffuse sympathoadrenal system. In the mid-1960s, the enzymatically inactive, major core protein, chromogranin A was shown to be exocytotically discharged from the stimulated adrenal gland in parallel with the co-stored catecholamines and ATP. A prohormone concept was introduced when one of the main storage proteins collectively named granins was identified as the insulin release inhibitory polypeptide pancreastatin. A wide range of granin-derived biologically active peptides have subsequently been identified. Both chromogranin A and chromogranin B give rise to antimicrobial peptides of relevance for combat of pathogens. While two of the chromogranin A-derived peptides, vasostatin-I and pancreastatin, are involved in modulation of calcium and glucose homeostasis, respectively, vasostatin-I and catestatin are important modulators of endothelial permeability, angiogenesis, myocardial contractility, and innate immunity. A physiological role is now evident for the full-length chromogranin A and vasostatin-I as circulating stabilizers of endothelial integrity and in protection against myocardial injury. The high circulating levels of chromogranin A and its fragments in patients suffering from various inflammatory diseases have emerged as challenges for future research and clinical applications.

Glycobiomarker, Fucosylated Short-Form Secretogranin III Levels Are Increased in Serum of Patients with Small Cell Lung Carcinoma.

Secretogranin III (SgIII) is a member of the chromogranin/secretogranin family of neuroendocrine secretory proteins. Granins are expressed in endocrine and neuroendocrine cells and subsequently processed into bioactive hormones. Although granin-derived peptide expression is correlated with neuroendocrine carcinomas, little is known about SgIII. We previously identified SgIII by a comparative glycoproteomics approach for elucidation of glycobiomarker candidates in lung carcinoma. Here, we examined the expression, secretion, and glycosylation of SgIII to identify novel biomarkers of small cell lung carcinoma (SCLC). In comparative immunohistochemical analysis and secretion profiling, SgIII was observed in all types of lung cancer. However, low-molecular-weight SgIII (short-form SgIII) was specifically found in SCLC culture medium. Glycoproteomics analysis showed that a fucosylated glycan was attached to the first of three potential N-glycosylation sites and an unfucosylated glycan was detected on the second site; however, the third site was not glycosylated. Next, we performed lectin capture with a fucose-binding lectin and detected short-form SgIII specifically in the sera of patients with SCLC. The results suggested an association between the fucosylated glycoform of short-form SgIII and SCLC. Thus, fucosylated short-form SgIII may be a valuable biomarker for SCLC and could be used to monitor development of the disease. All MS data are available via ProteomeXchange and jPOST with identifiers PXD007626 and JPST000313, respectively.

Rare Variant Analysis of Human and Rodent Obesity Genes in Individuals with Severe Childhood Obesity.

Obesity is a genetically heterogeneous disorder. Using targeted and whole-exome sequencing, we studied 32 human and 87 rodent obesity genes in 2,548 severely obese children and 1,117 controls. We identified 52 variants contributing to obesity in 2% of cases including multiple novel variants in GNAS, which were sometimes found with accelerated growth rather than short stature as described previously. Nominally significant associations were found for rare functional variants in BBS1, BBS9, GNAS, MKKS, CLOCK and ANGPTL6. The p.S284X variant in ANGPTL6 drives the association signal (rs201622589, MAF~0.1%, odds ratio = 10.13, p-value = 0.042) and results in complete loss of secretion in cells. Further analysis including additional case-control studies and population controls (N = 260,642) did not support association of this variant with obesity (odds ratio = 2.34, p-value = 2.59 × 10-3), highlighting the challenges of testing rare variant associations and the need for very large sample sizes. Further validation in cohorts with severe obesity and engineering the variants in model organisms will be needed to explore whether human variants in ANGPTL6 and other genes that lead to obesity when deleted in mice, do contribute to obesity. Such studies may yield druggable targets for weight loss therapies.

An aromatic amino acid within intracellular loop 2 of the prostaglandin EP2 receptor is a prerequisite for selective association and activation of Gαs.

We previously demonstrated that the aromatic moiety of Tyr143 within the intracellular loop 2 (ICL2) region of the prostaglandin EP2 receptor plays a crucial role in Gs coupling. Here we investigated whether the ICL2 of the EP2 receptor directly binds to Gαs and whether an aromatic moiety affects this interaction. In Chinese hamster ovary cells, mutations of Tyr143 reduced the ability of the EP2 receptor to interact with G proteins as demonstrated by GTPγS sensitivity, as well as the ability of agonist-induced cAMP formation, with the rank order of Phe>Tyr (wild-type)=Trp>Leu>Ala (=0). We found that the wild-type ICL2 peptide (i2Y) and its mutant with Phe at Tyr143 (i2F) inhibited receptor-G protein complex formation of wild-type EP2 in membranes, whereas the Ala-substituted mutant (i2A) did not. Specific interactions between these peptides and the Gαs protein were detected by surface plasmon resonance, but Gαs showed different association rates, with a rank order of i2F>i2Y≫i2A, with similar dissociation rates. Moreover, i2F and i2Y, but not i2A activated membrane adenylyl cyclase. These results indicate that the ICL2 region of the EP2 receptor is its potential interaction site with Gαs, and that the aromatic side chain moiety at position 143 is a determinant for the accessibility of the ICL2 to the Gαs protein.

Internalized Receptor for Glucose-dependent Insulinotropic Peptide stimulates adenylyl cyclase on early endosomes.

Until very recently, G-protein dependent signal of GPCRs was thought to originate exclusively from the plasma membrane and internalized GPCRs were considered silent. Here, we demonstrated that, once internalized and located in the membrane of early endosomes, glucose-dependent Insulinotropic receptor (GIPR) continues to trigger production of cAMP and PKA activation. Direct evidence is based on identification of the active form of Gαs in early endosomes containing GIPR using a genetically encoded GFP tagged nanobody, and on detection of a distinct FRET signal accounting for cAMP production at the surface of endosomes containing GIP, compared to endosomes without GIP. Furthermore, decrease of the sustained phase of cAMP production and PKA activation kinetics as well as reversibility of cAMP production and PKA activity following GIP washout in cells treated with a pharmacological inhibitor of GIPR internalization, and continuous increase of cAMP level over time in the presence of dominant-negative Rab7, which causes accumulation of early endosomes in cells, were noticed. Hence the GIPR joins the few GPCRs which signal through G-proteins both at plasma membrane and on endosomes.

Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling.

Although the importance of the C terminus of the α subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the Gα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the ß2-adrenergic receptor (ß2-AR)-Gαs and V1A receptor (V1AR)-Gαq The Gαs and Gαq peptides adopt different orientations in ß2-AR and V1AR, respectively. The ß2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in Gαq prevents this peptide from interacting strongly with ß2-AR. Mutagenesis to the Gαs counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions.

Malignant cell-derived extracellular vesicles express different chromogranin epitopes compared to prostasomes.

BACKGROUND: Prostasomes are nanosized extracellular vesicles exocytosed by prostate epithelial cells. They have been assigned many roles propitious to sperm in favor of fertilization. Prostatic cancer cells can also produce and secrete extracellular vesicles. METHODS: We assessed using ELISA, the surface expression of chromogranin proproteins on prostasomes and malignant extracellular vesicles of four different prostate cancer cell-lines, two hormone sensitive and two hormone refractory. We used a panel of chromogranin A and chromogranin B antibodies against peptides in-between hypothetical cleavage sites along the proproteins. RESULTS: A diverging pattern of chromogranin peptides was apparent when comparing prostasomes and malignant extracellular vesicles indicating a phenotypical change. We also compared western blot patterns (prostasomes and malignant extracellular vesicles) for selected antibodies that displayed high absorbances in the ELISA. Western blot analyses revealed various cleavage patterns of those proproteins that were analyzed in prostasomes and extracellular vesicles. CONCLUSION: Chromogranins are constituents of not only prostasomes but also of malignant prostate cell-derived extracellular vesicles with different amino acid sequences exposed at the membrane surface giving rise to a mosaic pattern. These findings may be of relevance for designing new assays for detection or even possible treatment of prostate cancers.

Activation of neutrophils by the two-component leukotoxin LukE/D from Staphylococcus aureus: proteomic analysis of the secretions.

Staphylococcus aureus is responsible for severe bacterial infections in hospitals and healthcare facilities. It produces single and bicomponent toxins (leukotoxins and hemolysins) that hinder innate immune function. Leukotoxin subunits bind to leukocyte cell membrane thus inducing transmembrane pores and subsequently, cell lysis. Leukotoxin LukE/D is a member of the bicomponent toxin family, but to date, no study concerning its involvement in host-pathogen interactions has been reported. In the present study, we performed the proteomic analysis of the secretions recovered after activation of human neutrophils by leukotoxin LukE/D. The neutrophil secretions were purified by RP-HPLC and different fractions were analyzed by Edman sequencing, LC-MS/MS, immunoblotted for chromogranin-derived peptides and further analyzed for antimicrobial properties. Proteomic analysis revealed that neutrophil secretions constitute a large number of proteins related with immune boosting mechanisms, proteolytic degradation, inflammatory process and antioxidant reactions.

The extended granin family: structure, function, and biomedical implications.

The chromogranins (chromogranin A and chromogranin B), secretogranins (secretogranin II and secretogranin III), and additional related proteins (7B2, NESP55, proSAAS, and VGF) that together comprise the granin family subserve essential roles in the regulated secretory pathway that is responsible for controlled delivery of peptides, hormones, neurotransmitters, and growth factors. Here we review the structure and function of granins and granin-derived peptides and expansive new genetic evidence, including recent single-nucleotide polymorphism mapping, genomic sequence comparisons, and analysis of transgenic and knockout mice, which together support an important and evolutionarily conserved role for these proteins in large dense-core vesicle biogenesis and regulated secretion. Recent data further indicate that their processed peptides function prominently in metabolic and glucose homeostasis, emotional behavior, pain pathways, and blood pressure modulation, suggesting future utility of granins and granin-derived peptides as novel disease biomarkers.

The antimicrobial peptides derived from chromogranin/secretogranin family, new actors of innate immunity.

Chromogranins/secretogranins are members of the granin family present in secretory vesicles of nervous, endocrine and immune cells. In chromaffin cells, activation of nicotinic cholinergic receptors induces the release, with catecholamines, of bioactive peptides resulting from a natural processing. During the past decade, our laboratory has characterized new antimicrobial chromogranin-derived peptides in the secretions of stimulated bovine chromaffin cells. They act at the micromolar range against bacteria, fungi, yeasts, and are non-toxic for the mammalian cells. They are recovered in several biological fluids involved in defence mechanisms (human serum, neutrophil secretions and saliva). These new antimicrobial peptides demonstrate the major role of the adrenal medulla in innate immunity. In this review we focus on the antimicrobial peptides derived from human and bovine chromogranin A (CGA), chromogranin B (CGB) and secretogranin II (SGII) emphasizing their direct action against pathogens and their effects on immune cells.

New insights into granin-derived peptides: evolution and endocrine roles.

The granin protein family is composed of two chromogranin and five secretogranin members that are acidic, heat-stable proteins in secretory granules in cells of the nervous and endocrine systems. We report that there is little evidence for evolutionary relationships among the granins except for the chromogranin group. The main granin members, including chromogranin A and B, and secretogranin II are moderately conserved in the vertebrates. Several small bioactive peptides can be generated by proteolysis from those homologous domains existing within the granin precursors, reflecting the conservation of biological activities in different vertebrates. In this context, we focus on reviewing the distribution and function of the major granin-derived peptides, including vasostatin, bovine CgB(1-41) and secretoneurin in vertebrate endocrine systems, especially those associated with growth, glucose metabolism and reproduction.

A large form of secretogranin III functions as a sorting receptor for chromogranin A aggregates in PC12 cells.

Granin-family proteins, including chromogranin A and secretogranin III, are sorted to the secretory granules in neuroendocrine cells. We previously demonstrated that secretogranin III binds chromogranin A and targets it to the secretory granules in pituitary corticotrope-derived AtT-20 cells. However, secretogranin III has not been identified in adrenal chromaffin and PC12 cells, where chromogranin A is correctly sorted to the secretory granules. In this study, low levels of a large and noncleaved secretogranin III have been identified in PC12 cells and rat adrenal glands. Although the secretogranin III expression was limited in PC12 cells, when the FLAG-tagged secretogranin III lacking the secretory granule membrane-binding domain was expressed excessively, hemagglutinin-tagged chromogranin A was unable to target to the secretory granules at the tips and shifted to the constitutive secretory pathway. Secretogranin III was able to bind the aggregated form of chromogranin A, suggesting that a small quantity of secretogranin III is enough to carry a large quantity of chromogranin A. Furthermore, secretogranin III bound adrenomedullin, a major peptide hormone in chromaffin cells. Indeed, small interfering RNA-directed secretogranin III depletion impaired intracellular retention of chromogranin A and adrenomedullin, suggesting that they are constitutively released to the medium. We suggest that the sorting function of secretogranin III for chromogranin A is common in PC12 and chromaffin cells as well as in other endocrine cells, and a small amount of secretogranin III is able to sort chromogranin A aggregates together with adrenomedullin to secretory granules.

Characterization of natural vasostatin-containing peptides in rat heart.

Chromogranin A (CGA) is a protein that is stored and released together with neurotransmitters and hormones in the nervous, endocrine and diffuse neuroendocrine systems. As human vasostatins I and II [CGA(1-76) and CGA(1-113), respectively] have been reported to affect vessel motility and exert concentration-dependent cardiosuppressive effects on isolated whole heart preparations of eel, frog and rat (i.e. negative inotropism and antiadrenergic activity), we investigated the presence of vasostatin-containing peptides in rat heart. Rat heart extracts were purified by RP-HPLC, and the resulting fractions analyzed for the presence of CGA N-terminal fragments using dot-blot analysis. CGA-immunoreactive fractions were submitted to western blot and MS analysis using the TOF/TOF technique. Four endogenous N-terminal CGA-derived peptides [CGA(4-113), CGA(1-124), CGA(1-135) and CGA(1-199)] containing the vasostatin sequence were characterized. The following post-translational modifications of these fragments were identified: phosphorylation at Ser96, O-glycosylation (trisaccharide, NAcGal-Gal-NeuAc) at Thr126, and oxidation at three methionine residues. This first identification of CGA-derived peptides containing the vasostatin motif in rat heart supports their role in cardiac physiology by an autocrine/paracrine mechanism.

Differential expression and processing of chromogranin A and secretogranin II in relation to the secretory status of endocrine cells.

Chromogranin A (CgA) and secretogranin II (SgII) are neuroendocrine secretory proteins that participate in regulation of the secretory pathway and also serve as precursors of biologically active peptides. To investigate whether there is a relationship between the expression, distribution, and processing of CgA and SgII and the degree of secretory activity, we employed two melanotrope subpopulations of the pituitary intermediate lobe that exhibit opposite secretory phenotypes. Thus, although one of the melanotrope subtypes shows high secretory activity, the other exhibits characteristics of a hormone storage phenotype. Our data show that SgII expression levels were higher in secretory melanotropes, whereas CgA expression showed similar rates in both cell subsets. The use of various antibodies revealed the presence of the unprocessed proteins as well as three CgA-derived peptides (67, 45, and 30 kDa) and six SgII-derived peptides (81, 66, 55, 37, 32, and 30 kDa) in both subpopulations. However, the smallest molecular forms of both granins predominated in secretory melanotropes, whereas the largest SgII- and CgA-immunoreactive peptides were more abundant in storage melanotropes, which is suggestive of a more extensive processing of granins in the secretory subset. Confocal microscopy studies showed that CgA immunoreactivity was higher in storage cells, but SgII immunoreactivity was higher in secretory melanotropes. Taken together, our results indicate that SgII and CgA are differentially regulated in melanotrope subpopulations. Thus, SgII expression is strongly related to the secretory activity of melanotrope cells, whereas CgA expression may not be related to secretory rate, but, rather, to hormone storage in this endocrine cell type.

Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II.

MCoTI-I and MCoTI-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTI-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue #10 within the open-chain variant of MCoTI-II by the non-natural isosteric nucleo amino acid AlaG [beta-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude. While the cyclic inhibitors MCoTI-I and MCoTI-II were found to be very potent trypsin inhibitors, with picomolar inhibition constants, the open-chain variants displayed an approximately 10-fold lower affinity. These data suggest that the formation of a circular backbone in the MCoTI squash inhibitors results in enhanced affinity and therefore is a determinant of biological activity.