Novel FGF9 variant contributes to multiple synostoses syndrome 3.

Multiple synostoses syndromes (SYNS) are autosomal dominant syndromes characterized by multiple joint fusions commonly involving the carpal-tarsal, interphalangeal, humeroradial, and cervical spine joints. They display genetic heterogeneity with pathogenic variants reported in four separate genes (NOG, GDF5, FGF9, and GDF6) defining four different SYNS forms. FGF9 variants have been reported in SYNS3, a SYNS with multiple synostoses, normal cognition, normal hearing, and craniosynostosis. Here, we report a novel FGF9 c.569G > C p.(Arg190Thr) variant identified by whole-exome sequencing in a patient with multiple bony abnormalities. The patient initially presented with elbow instability and decreased range of motion. Imaging revealed bilateral radial head deformities, carpal-tarsal fusions, brachydactyly, and osteoarthritis of the sacroiliac joints. In silico protein modeling of the identified FGF9 variant predicts decreased stability of ligand-receptor binding supporting the pathogenicity of this finding. This finding expands the repertoire of FGF9 variants and phenotypic information reported for SYNS3 and suggest that genotype phenotype correlations due to localization seem less likely and more so due to the consequence of the pathogenic variant on the receptor. This is useful in the counseling in families as more de novo variants emerge.

FGF9 mutation causes craniosynostosis along with multiple synostoses.

Craniosynostosis is commonly caused by mutations in fibroblast growth factor receptors (FGFRs), highlighting the essential role of FGF-mediated signaling in skeletal development. We set out to identify the molecular defect in a family referred for craniosynostosis and in whom no mutation was previously detected. Using next-generation sequencing, we identified a novel missense mutation in FGF9. Modeling based upon the crystal structure and functional studies confirmed its pathogenicity showing that it impaired homodimerization and FGFR3 binding. Only one FGF9 mutation has been previously reported in a multigeneration family with multiple synostoses (SYNS3) but no signs of craniosynostosis. In contrast, our family has a greater phenotypic resemblance to that observed in the Fgf9 spontaneous mouse mutant, elbow-knee-synostosis, Eks, with both multiple synostoses and craniosynostosis. We have demonstrated for the first time that mutations in FGF9 cause craniosynostosis in humans and confirm that FGF9 mutations cause multiple synostoses.

EP24.15 as a Potential Regulator of Kisspeptin Within the Neuroendocrine Hypothalamus.

The neuropeptide kisspeptin (Kiss1) is integral to the advent of puberty and the generation of cyclical LH surges. Although many complex actions of Kiss1 are known, the mechanisms governing the processing/regulation of this peptide have not been unveiled. The metallo enzyme, endopeptidase 24.15 (thimet oligopeptidase), has been demonstrated to play a key role in the processing and thus the duration of action of the reproductive neuropeptide, GnRH, which signals downstream of Kiss1. Initial in silico modeling implied that Kiss1 could also be a putative substrate for EP24.15. Coincubation of Kiss1 and EP24.15 demonstrated multiple cleavages of the peptide predominantly between Arg29-Gly30 and Ser47-Phe48 (corresponding to Ser5-Phe6 in Kiss-10; Kiss-10 as a substrate had an additional cleavage between Phe6-Gly7) as determined by mass spectrometry. Vmax for the reaction was 2.37±0.09 pmol/min · ng with a Km of 19.68 ± 2.53µM, which is comparable with other known substrates of EP24.15. EP24.15 immunoreactivity, as previously demonstrated, is distributed in cell bodies, nuclei, and processes throughout the hypothalamus. Kiss1 immunoreactivity is localized primarily to cell bodies and fibers within the mediobasal and anteroventral-periventricular hypothalamus. Double-label immunohistochemistry indicated coexpression of EP24.15 and Kiss1, implicating that the regulation of Kiss1 by EP24.15 could occur in vivo. Further studies will be directed at determining the precise temporal sequence of EP24.15 effects on Kiss1 as it relates to the control of reproductive hormone secretion and treatment of fertility issues.

Age-dependent Effects of 17ß-estradiol on the dynamics of estrogen receptor ß (ERß) protein-protein interactions in the ventral hippocampus.

Recent clinical evidence suggests that the neuroprotective and beneficial effects of hormone therapy may be limited by factors related to age and reproductive status. The patient's age and length of time without circulating ovarian hormones are likely to be key factors in the specific neurological outcomes of hormone therapy. However, the mechanisms underlying age-related changes in hormone efficacy have not been determined. We hypothesized that there are intrinsic changes in estrogen receptor ß (ERß) function that determine its ability to mediate the actions of 17ß-estradiol (E2) in brain regions such as the ventral hippocampus. In this study, we identified and quantified a subset of ERß protein interactions in the ventral hippocampus that were significantly altered by E2 replacement in young and aged animals, using two-dimensional differential gel electrophoresis coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry. This study demonstrates quantitative changes in ERß protein-protein interactions with E2 replacement that are dependent upon age in the ventral hippocampus and how these changes could alter processes such as transcriptional regulation. Thus, our data provide evidence that changes in ERß protein interactions are a potential mechanism for age-related changes in E2 responsiveness in the brain after menopause.

Primate genome gain and loss: a bone dysplasia, muscular dystrophy, and bone cancer syndrome resulting from mutated retroviral-derived MTAP transcripts.

Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal-dominant syndrome characterized by bone dysplasia, myopathy, and bone cancer. We previously mapped the DMS-MFH tumor-suppressing-gene locus to chromosomal region 9p21-22 but failed to identify mutations in known genes in this region. We now demonstrate that DMS-MFH results from mutations in the most proximal of three previously uncharacterized terminal exons of the gene encoding methylthioadenosine phosphorylase, MTAP. Intriguingly, two of these MTAP exons arose from early and independent retroviral-integration events in primate genomes at least 40 million years ago, and since then, their genomic integration has gained a functional role. MTAP is a ubiquitously expressed homotrimeric-subunit enzyme critical to polyamine metabolism and adenine and methionine salvage pathways and was believed to be encoded as a single transcript from the eight previously described exons. Six distinct retroviral-sequence-containing MTAP isoforms, each of which can physically interact with archetype MTAP, have been identified. The disease-causing mutations occur within one of these retroviral-derived exons and result in exon skipping and dysregulated alternative splicing of all MTAP isoforms. Our results identify a gene involved in the development of bone sarcoma, provide evidence of the primate-specific evolution of certain parts of an existing gene, and demonstrate that mutations in parts of this gene can result in human disease despite its relatively recent origin.

Mild isolated craniosynostosis due to a novel FGFR3 mutation, p.Ala334Thr.

Craniosynostosis is the premature fusion of one or more sutures of the skull, which can be syndromic or isolated. Mutations in FGFR1, FGFR2, or FGFR3, among others, are often responsible for these syndromic cases. The associated of FGFR3 mutations with craniosynostosis has been restricted to three mutations, the common p.Pro250Arg in Muenke syndrome, p.Ala391Glu in Crouzon syndrome with acanthosis nigricans, and p.Pro250Leu identified in a family with isolated craniosynostosis. Other FGFR3 mutations result in various skeletal dysplasias: achondroplasia, hypochondroplasia, and thanatophoric dysplasia. Here, we report a novel mutation in exon 8 (IIIc) of FGFR3, p.Ala334Thr, in a young boy with mild craniosynostosis. The mutation segregated with mild craniosynostosis in the family and was absent in 188 normal controls. Alanine 334 is evolutionarily conserved in vertebrates and is located at the amino terminus of the ßF loop in the FGFR3c isoform. The mutation is predicted to alter the protein tertiary structure which may impair its binding to its ligand, FGF1. The identification of a mutation in these clinically heterogeneous disorders can aid recurrence risk assessments. Although the implementation of a stepwise screening strategy is useful in diagnostics, mutations in unscreened regions of genes associated with craniosynostosis may explain a small proportion of craniosynostosis cases.

Signaling pathway adaptations and novel protein kinase A substrates related to behavioral sensitization to cocaine.

Behavioral sensitization is an animal model for aspects of cocaine addiction. Cocaine-sensitized rats exhibit increased AMPA receptor (AMPAR) surface expression in the nucleus accumbens (NAc) which may in turn enhance drug seeking. To identify signaling pathways contributing to AMPAR up-regulation, we measured AMPAR surface expression and signaling pathway activation in the NAc of cocaine-sensitized rats, cocaine-exposed rats that failed to sensitize and saline controls on withdrawal days (WD) 1, 7, and 21. We focused on calcium/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated protein kinase (ERK), and protein kinase A (PKA). In sensitized rats, AMPAR surface expression was elevated on WD7 and WD21 but not WD1. ERK2 activation followed a parallel time-course, suggesting a role in AMPAR up-regulation. Both sensitized and non-sensitized rats exhibited CaMKII activation on WD7, suggesting that CaMKII activation is not sufficient for AMPAR up-regulation. PKA phosphorylation, measured using an antibody recognizing phosphorylated PKA substrates, increased gradually over withdrawal in sensitized rats, from below control levels on WD1 to significantly greater than controls on WD21. Using proteomics, novel sensitization-related PKA substrates were identified, including two structural proteins (CRMP-2 and alpha-tubulin) that we speculate may link PKA signaling to previously reported dendritic remodeling in NAc neurons of cocaine-sensitized rats.

LHRH-(1-5): a bioactive peptide regulating reproduction.

Luteinizing hormone-releasing hormone-I (LHRH-I) was isolated from the mammalian hypothalamus and shown to be the primary regulator of reproduction through its initiation of pituitary gonadotropin release. Subsequently, it has also been shown to have non-pituitary actions. Although the regulation of LHRH-I synthesis and release has been extensively studied, there is additional evidence to suggest that processing of the peptide represents another layer of regulation. The focus of this review will be on evidence for the action of LHRH-(1-5), the pentapeptide metabolite of LHRH-I, in regulating LHRH-I synthesis, secretion and reproductive behavior. The involvement of LHRH-(1-5) in the control of aspects of reproduction might represent yet another level of regulatory complexity through neuropeptide processing.

Mapping protease substrates by using a biotinylated phage substrate library.

We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobic is the scissile bond.

Modulation of bradykinin signaling by EP24.15 and EP24.16 in cultured trigeminal ganglia.

Metalloendopeptidases expressed in neural tissue are characterized in terms of their neuropeptide substrates. One such neuropeptide, bradykinin (BK), is an important inflammatory mediator that activates the type-2 BK receptor (B2R) on the terminal endings of specialized pain-sensing neurons known as nociceptors. Among several metalloendopeptidases that metabolize and inactivate BK, EP24.15 and EP24.16 are known to associate with the plasma membrane in several immortalized cell lines. Potentially, the colocalization of EP24.15/16 and B2R at plasma membrane microdomains known as lipid rafts in a physiologically relevant nociceptive system would allow for discrete, peptidase regulation of BK signaling. Western blot analysis of crude subcellular fractions and lipid raft preparations of cultured rat trigeminal ganglia demonstrate similar expression profiles between EP24.15/16 and B2R on a subcellular level. Furthermore, the treatment of primary cultures of trigeminal ganglia with inhibitors of EP24.15/16 led to the potentiation of several bradykinin-induced events that occur downstream of B2R activation. EP24.15/16 inhibition by N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-AlalTyr-p-aminobenzoate (cFP) resulted in a 1000-fold increase in B2R sensitivity to BK as measured by inositol phosphate accumulation. In addition, cFP treatment resulted in a 31.1+/-5.0% potentiation of the ability of BK to inhibit protein kinase B (Akt) activity. Taken together, these data demonstrate that EP24.15/16 modulate intracellular, peptidergic signaling cascades through B2R in a physiologically relevant nociceptive system.

Facilitation of lordosis in rats by a metabolite of luteinizing hormone releasing hormone.

In the female rat, ovulation is preceded by a marked increase in the release of the decapeptide, LHRH, culminating in a preovulatory LH surge, which coincides with a period of sexual receptivity. The decapeptide, LHRH, is processed by a zinc metalloendopeptidase EC 3.4.24.15 (EP24.15) that cleaves the hormone at the Tyr(5)-Gly(6) bond. We have previously reported that the autoregulation of LHRH gene expression can also be mediated by its metabolite, LHRH-(1-5). Given the central function of LHRH in reproduction and reproductive behavior, we examined the role of the metabolite, LHRH-(1-5), in mediation of LHRH-facilitated reproductive behavior. Intracerebroventricular administration of LHRH-(1-5) facilitated sexual behavior responses, similar to those facilitated by the decapeptide LHRH, in ovariectomized estradiol-primed female rats. Furthermore, immunoneutralization of EP24.15 resulted in the inhibition of the LHRH-facilitated lordosis but had no inhibitory effects on LHRH-(1-5)-facilitated lordosis. The LHRH antagonist, Antide, was capable of inhibiting LHRH-facilitated lordosis, without affecting LHRH-(1-5)-facilitated lordosis. Collectively, these results suggest a role for LHRH metabolites in the facilitation of female receptive behavior in rats.

A prohormone convertase cleavage site within a predicted alpha-helix mediates sorting of the neuronal and endocrine polypeptide VGF into the regulated secretory pathway.

Distinct intracellular pathways are involved in regulated and constitutive protein secretion from neuronal and endocrine cells, yet the peptide signals and molecular mechanisms responsible for targeting and retention of soluble proteins in secretory granules are incompletely understood. By using confocal microscopy and subcellular fractionation, we examined trafficking of the neuronal and endocrine peptide precursor VGF that is stored in large dense core vesicles and undergoes regulated secretion. VGF cofractionated with secretory vesicle membranes but was not detected in detergent-resistant lipid rafts. Deletional analysis using epitope-tagged VGF suggested that the C-terminal 73-amino acid fragment of VGF, containing two predicted alpha-helical loops and four potential prohormone convertase (PC) cleavage sites, was necessary and sufficient with an N-terminal signal peptide-containing domain, for large dense core vesicle sorting and regulated secretion from PC12 and INS-1 cells. Further transfection analysis identified the sorting sequence as a compact C-terminal alpha-helix and embedded 564RRR566 PC cleavage site; mutation of the 564RRR566 PC site in VGF-(1-65): GFP:VGF-(545-617) blocked regulated secretion, whereas disruption of the alpha-helix had no effect. Mutation of the adjacent 567HFHH570 motif, a charged region that might enhance PC cleavage in acidic environments, also blocked regulated release. Finally, inhibition of PC cleavage in PC12 cells using the membrane-permeable synthetic peptide chloromethyl ketone (decanoyl-RVKR-CMK) blocked regulated secretion of VGF. Our studies define a critical RRR-containing C-terminal domain that targets VGF into the regulated pathway in neuronal PC12 and endocrine INS-1 cells, providing additional support for the proposed role that PCs and their cleavage sites play in regulated peptide secretion.

Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity.

The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). The constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. In the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function.

Stimulation of luteinizing hormone-releasing hormone (LHRH) gene expression in GT1-7 cells by its metabolite, LHRH-(1-5).

Given the central role of the decapeptide LHRH in reproduction and reproductive behavior, it is important to focus on delineating the possible effects of this gene and its products in the regulation of hormone-dependent reproductive processes. In the female, ovulation is preceded by a marked increase in LHRH release; the increase in LHRH release culminates in a preovulatory LH surge, which coincides with a period of sexual receptivity. In contrast to the belief that the proteolytic metabolism of LHRH serves only as a degradative process that removes excess LHRH and attenuates signal transduction through the LHRH receptor, we hypothesized that a metabolite of the decapeptide, LHRH-(1-5), can directly regulate LHRH neuronal function. This study demonstrates the ability of LHRH-(1-5) peptide to regulate LHRH gene expression in the LHRH neuronal cell line, the GT(1-7) cell. The results show that LHRH-(1-5) stimulated LHRH gene expression at the posttranscriptional level. In contrast to the LHRH suppression of its own gene expression, the coadministration of LHRH with the metalloendopeptidase, EC 3.4.24.15, an endopeptidase known to cleave LHRH to form LHRH(1-5), shows a reversal of effect, a stimulation of LHRH gene expression. Finally, the effect of LHRH-(1-5) on LHRH gene expression appears to be mediated by the calcium/calmodulin-dependent protein kinase. The present study supports the hypothesis that the physiological metabolite of LHRH, LHRH-(1-5), is functionally capable of regulating the reproductive neuroendocrine system.

The role of neuropeptide processing enzymes in endocrine (prostate) cancer: EC 3.4.24.15 (EP24.15).

The zinc metalloendopeptidase EC3.4.24.15 [EP24.15, thimet oligopeptidase], a neuropeptide processing enzyme, is central to the formation and degradation of many bioactive peptides in the neural proteome, and is highly expressed in normal prostate. EP24.15 actions are increased in androgen-dependent prostate cancer compared to androgen-independent; augmented by androgen treatment, and inhibited by clinical GnRH analogs. The "neural" prostate includes: neuropeptides, cognate receptors and processing enzymes regulating signaling of peptide-mediated neural inputs.

Regulation of endopeptidases EC3.4.24.15 and EC3.4.24.16 in vascular endothelial cells by cyclic strain: role of Gi protein signaling.

OBJECTIVE: Endopeptidase EC3.4.24.15 (EP24.15)- and EC3.4.24.16 (EP24.16)-specific peptide hydrolysis plays an important role in endothelium-mediated vasoregulation. Given the significant influence of hemodynamic forces on vascular homeostasis and pathology, we postulated that these related peptidases may be mechanosensitive. The objective of this study, therefore, was to investigate the putative role of cyclic strain in regulating the expression and enzymatic activity of EP24.15 and EP24.16 in bovine aortic endothelial cells (BAECs). METHODS AND RESULTS: BAECs were cultured under conditions of defined cyclic strain (0% to 10% stretch, 60 cycles/min, 0 to 24 hours). Strain significantly increased EP24.15 and EP24.16 soluble activity in a force- and time-dependent manner, with elevations of 2.3+/-0.4- and 1.9+/-0.3-fold for EP24.15 and EP24.16, respectively, after 24 hours at 10% strain. Pharmacological agents and dominant-negative G protein mutants used to selectively disrupt Gi(alpha)- and Gbetagamma-mediated signaling pathways attenuated strain-dependent (24 hours, 5%) increases for both enzymes. Differences in the inhibitory profile for both enzymes were also noted, with EP24.15 displaying greater sensitivity to Gi(alpha2/3) inhibition and EP24.16 exhibiting greater sensitivity to Gi(alpha1/2) and Gbetagamma inhibition. Cyclic strain also increased levels of secreted EP24.15 and EP24.16 activity by 2.6+/-0.02- and 3.6+/-0.2-fold, respectively, in addition to mRNA levels for both enzymes (EP24.15 +42%, EP24.16 +56%). CONCLUSIONS: Our findings suggest that cyclic strain putatively regulates both the mRNA expression and enzymatic function of EP24.15 and EP24.16 in BAECs via alternate Gi protein signaling pathways.

EP24.15 is associated with lipid rafts.

Metalloendopeptidase EC 3.4.24.15 (EP24.15, thimet oligopeptidase) is a neuropeptide-metabolizing peptidase expressed throughout the body, but primarily in the brain, gonads, and pituitary. For EP24.15 to have its greatest effect upon peptides in the periphery, it must be targeted and released into the extracellular space. Western blot analysis of fractions taken from discontinuous sucrose density gradients carried out on crude plasma membrane fractions from AtT-20 cells reveals colocalization of EP24.15 and flotillin-1, a known lipid raft marker. Further analysis revealed that an intracellular membrane marker and non-lipid raft, plasma membrane marker, failed to colocalize, supporting EP24.15/lipid raft association. Furthermore, EP24.15 immunoreactivity in lipid raft fractions generated from cells treated with methyl beta-cyclodextrin (MbetaCD) was greatly reduced. Finally, treatment with MbetaCD resulted in the accumulation of EP24.15 in the media of drug-treated cells over vehicle-treated cells, suggesting that a large percentage of EP24.15 associating with lipid rafts resides on the extracellular surface of the plasma membrane. With this exofacial localization, EP24.15 could have ample access to neuropeptides not only in the immediate microenvironment, but the ability to degrade or modify peptides bound for receptor interaction.

Mutations in capillary morphogenesis gene-2 result in the allelic disorders juvenile hyaline fibromatosis and infantile systemic hyalinosis.

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are autosomal recessive syndromes of unknown etiology characterized by multiple, recurring subcutaneous tumors, gingival hypertrophy, joint contractures, osteolysis, and osteoporosis. Both are believed to be allelic disorders; ISH is distinguished from JHF by its more severe phenotype, which includes hyaline deposits in multiple organs, recurrent infections, and death within the first 2 years of life. Using the previously reported chromosome 4q21 JHF disease locus as a guide for candidate-gene identification, we identified and characterized JHF and ISH disease-causing mutations in the capillary morphogenesis factor-2 gene (CMG2). Although CMG2 encodes a protein upregulated in endothelial cells during capillary formation and was recently shown to function as an anthrax-toxin receptor, its physiologic role is unclear. Two ISH family-specific truncating mutations, E220X and the 1-bp insertion P357insC that results in translation of an out-of-frame stop codon, were generated by site-directed mutagenesis and were shown to delete the CMG-2 transmembrane and/or cytosolic domains, respectively. An ISH compound mutation, I189T, is predicted to create a novel and destabilizing internal cavity within the protein. The JHF family-specific homoallelic missense mutation G105D destabilizes a von Willebrand factor A extracellular domain alpha-helix, whereas the other mutation, L329R, occurs within the transmembrane domain of the protein. Finally, and possibly providing insight into the pathophysiology of these diseases, analysis of fibroblasts derived from patients with JHF or ISH suggests that CMG2 mutations abrogate normal cell interactions with the extracellular matrix.