Selective binding of RGMc/hemojuvelin, a key protein in systemic iron metabolism, to BMP-2 and neogenin

2008 ◽  
Vol 294 (4) ◽  
pp. C994-C1003 ◽  
Author(s):  
Robin Kuns-Hashimoto ◽  
David Kuninger ◽  
Mahta Nili ◽  
Peter Rotwein
Keyword(s):  
Signaling Proteins ◽  
Wild Type ◽  
Single Chain ◽  
Gene Encoding ◽  
Morphogenetic Protein ◽  
Biochemical Assays ◽  
Juvenile Hemochromatosis ◽  
Repulsive Guidance Molecule ◽  
Membrane Spanning ◽  
Guidance Molecule

Juvenile hemochromatosis is a severe and rapidly progressing hereditary disorder of iron overload, and it is caused primarily by defects in the gene encoding repulsive guidance molecule c/hemojuvelin (RGMc/HJV), a recently identified protein that undergoes a complicated biosynthetic pathway in muscle and liver, leading to cell membrane-linked single-chain and heterodimeric species, and two secreted single-chain isoforms. RGMc modulates expression of the hepatic iron regulatory factor, hepcidin, potentially through effects on signaling by the bone morphogenetic protein (BMP) family of soluble growth factors. To date, little is known about specific pathogenic defects in disease-causing RGMc/HJV proteins. Here we identify functional abnormalities in three juvenile hemochromatosis-linked mutants. Using a combination of approaches, we first show that BMP-2 could interact in biochemical assays with single-chain RGMc species, and also could bind to cell-associated RGMc. Two mouse RGMc amino acid substitution mutants, D165E and G313V (corresponding to human D172E and G320V), also could bind BMP-2, but less effectively than wild-type RGMc, while G92V (human G99V) could not. In contrast, the membrane-spanning protein, neogenin, a receptor for the related molecule, RGMa, preferentially bound membrane-associated heterodimeric RGMc and was able to interact on cells only with wild-type RGMc and G92V. Our results show that different isoforms of RGMc/HJV may play unique physiological roles through defined interactions with distinct signaling proteins and demonstrate that, in some disease-linked RGMc mutants, these interactions are defective.

scholarly journals Proteomic analysis and molecular modelling characterize the iron-regulatory protein haemojuvelin/repulsive guidance molecule c

2013 ◽  
Vol 452 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Mahta Nili ◽  
Larry David ◽  
Johannes Elferich ◽  
Ujwal Shinde ◽  
Peter Rotwein
Keyword(s):  
Ab Initio ◽  
Molecular Modelling ◽  
Electron Transfer Dissociation ◽  
Structural Information ◽  
Regulatory Protein ◽  
Structural Features ◽  
Single Chain ◽  
Disulfide Linkages ◽  
Repulsive Guidance Molecule ◽  
Guidance Molecule

HJV (haemojuvelin) plays a key role in iron metabolism in mammals by regulating expression of the liver-derived hormone hepcidin, which controls systemic iron uptake and release. Mutations in HJV cause juvenile haemochromatosis, a rapidly progressing iron overload disorder in humans. HJV, also known as RGMc (repulsive guidance molecule c), is a member of the three-protein RGM family. RGMs are GPI (glycosylphosphatidylinositol)-linked glycoproteins that share ~50% amino acid identity and several structural motifs, including the presence of 14 cysteine residues in analogous locations. Unlike RGMa and RGMb, HJV/RGMc is composed of both single-chain and two-chain isoforms. To date there is no structural information for any member of the RGM family. In the present study we have mapped the disulfide bonds in mouse HJV/RGMc using a proteomics strategy combining sequential MS steps composed of ETD (electron transfer dissociation) and CID (collision-induced dissociation), in which ETD induces cleavage of disulfide linkages, and CID establishes disulfide bond assignments between liberated peptides. The results of the present study identified an HJV/RGMc molecular species containing four disulfide linkages. We predict using ab initio modelling that this molecule is a single-chain HJV/RGMc isoform. Our observations outline a general approach using tandem MS and ab initio molecular modelling to define unknown structural features in proteins.

scholarly journals Repulsive Guidance Molecule RGMa Alters Utilization of Bone Morphogenetic Protein (BMP) Type II Receptors by BMP2 and BMP4

2007 ◽  
Vol 282 (25) ◽  
pp. 18129-18140 ◽  
Author(s):  
Yin Xia ◽  
Paul B. Yu ◽  
Yisrael Sidis ◽  
Hideyuki Beppu ◽  
Kenneth D. Bloch ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Type Ii ◽  
Morphogenetic Protein ◽  
Repulsive Guidance Molecule ◽  
Guidance Molecule

scholarly journals Cooperation among c-subunits of FoF1-ATP synthase in rotation-coupled proton translocation

2021 ◽  
Author(s):  
Noriyo Mitome ◽  
Shintaroh Kubo ◽  
Sumie Ohta ◽  
Hikaru Takashima ◽  
Yuto Shigefuji ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Proton Pump ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Wild Type ◽  
Single Chain ◽  
Proton Release ◽  
Biochemical Assays ◽  
Proton Uptake ◽  
Fof1 Atp Synthase

In FoF1-ATP synthase, proton translocation through Fo drives rotation of the c-subunit oligomeric ring relative to the a-subunit. Recent studies suggest that in each step of the rotation, key glutamic acid residues in different c-subunits contribute to proton release to and proton uptake from the a-subunit. However, no studies have demonstrated cooperativity among c-subunits toward FoF1-ATP synthase activity. Here, we addressed this using Bacillus PS3 ATP synthase harboring c-ring with various combinations of wild-type and cE56D, enabled by genetically fused single-chain c-ring. ATP synthesis and proton pump activities were significantly decreased by a single cE56D mutation and further decreased by double cE56D mutations. Moreover, activity further decreased as the two mutation sites were separated, indicating cooperation among c-subunits. Similar results were obtained for proton transfer-coupled molecular simulations. Simulations revealed that prolonged proton uptake in mutated c-subunits is shared between two c-subunits, explaining the cooperation observed in biochemical assays.

scholarly journals Repulsive Guidance Molecule (RGMa), a DRAGON Homologue, Is a Bone Morphogenetic Protein Co-receptor

2005 ◽  
Vol 280 (33) ◽  
pp. 29820-29827 ◽  
Author(s):  
Jodie L. Babitt ◽  
Ying Zhang ◽  
Tarek A. Samad ◽  
Yin Xia ◽  
Jie Tang ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Morphogenetic Protein ◽  
Repulsive Guidance Molecule ◽  
Guidance Molecule

scholarly journals Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-PTS Show Enhanced Post-Exponential Phase Fitness

2021 ◽  
Author(s):  
Lin Zeng ◽  
Alejandro R Walker ◽  
Kyulim Lee ◽  
Zachary A Taylor ◽  
Robert A Burne
Keyword(s):  
Central Carbon Metabolism ◽  
Arginine Deiminase ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Gene Encoding ◽  
Streptococcus Sanguinis ◽  
Biochemical Assays ◽  
Production Of Hydrogen ◽  
Spontaneous Mutants ◽  
Production Of Ammonia

Genetic truncations in a gene encoding a putative glucose-PTS protein (manL, EIIABMan) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. Using an engineered mutant of manL and its complemented derivative, we showed that the ManL-deficient strain had improved bacterial viability in stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H2O2) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small, but significant percentage (~10%), of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. Significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e. excess carbohydrates and low pH, are those associated with caries development, we propose that the glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H2O2.

scholarly journals BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep

Reproduction ◽  
2017 ◽  
Vol 153 (4) ◽  
pp. 395-404 ◽  
Author(s):  
Anthony Estienne ◽  
Belén Lahoz ◽  
Peggy Jarrier ◽  
Loys Bodin ◽  
José Folch ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Rate Control ◽  
Activin A ◽  
Ovulation Rate ◽  
Loss Of Function ◽  
Wild Type ◽  
Homozygous State ◽  
Gene Encoding ◽  
Morphogenetic Protein ◽  
Ovarian Inhibin

Polymorphisms in the gene encoding bone morphogenetic protein 15 (BMP15) have been associated with multiple ovulations in sheep. As BMP15 regulates inhibin expression in rodents, we assumed that the ovarian inhibin/activin system could mediate part of the effect of BMP15 mutations in the regulation of ovulation rate in sheep. To answer this question, we have studied the effects of two natural loss-of-function mutations of BMP15 on the expression of components of this system. The FecXR and the FecXGr mutations, when present respectively in Rasa Aragonesa ewes at the heterozygous state and in Grivette ewes at the homozygous state, were associated with a twofold increase in ovulation rate. There were only small differences between mutant and wild-type ewes for mRNA expression of INHA, INHBA, ACVR1B, ACVR2A, FST or TGFBR3 in granulosa cells and inhibin A or activin A concentrations in follicular fluid. Moreover, the effects of mutations differed between breeds. In cultures of granulosa cells from wild-type ewes, BMP15, acting alone or in synergy with GDF9, stimulated INHA, INHBA and FST expression, but inhibited the expression of TGFBR3. Activin A did not affect INHBA expression, but inhibited the expression of ACVR2A also. The complexity of the inhibin/activin system, including positive and antagonistic elements, and the differential regulation of these elements by BMP15 and activin can explain that the effects of BMP15 mutations differ when present in different genetic backgrounds. In conclusion, the ovarian inhibin/activin system is unlikely to participate in the increase of ovulation rate associated with BMP15 mutations in sheep.

scholarly journals Repulsive guidance molecule is a structural bridge between neogenin and bone morphogenetic protein

2015 ◽  
Vol 22 (6) ◽  
pp. 458-465 ◽  
Author(s):  
Eleanor G Healey ◽  
Benjamin Bishop ◽  
Jonathan Elegheert ◽  
Christian H Bell ◽  
Sergi Padilla-Parra ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Morphogenetic Protein ◽  
Repulsive Guidance Molecule ◽  
Guidance Molecule

scholarly journals Inactivation of lmpA, Encoding a LIMPII-related Endosomal Protein, Suppresses the Internalization and Endosomal Trafficking Defects in Profilin-null Mutants

2000 ◽  
Vol 11 (6) ◽  
pp. 2019-2031 ◽  
Author(s):  
Lesly Temesvari ◽  
Linyi Zhang ◽  
Brent Fodera ◽  
Klaus-Peter Janssen ◽  
Michael Schleicher ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Fluid Phase ◽  
Actin Binding ◽  
Endosomal Trafficking ◽  
Wild Type ◽  
Gene Encoding ◽  
Biochemical Assays ◽  
Endosomal Pathway ◽  
Trafficking Defects

Profilin is a key phosphoinositide and actin-binding protein connecting and coordinating changes in signal transduction pathways with alterations in the actin cytoskeleton. Using biochemical assays and microscopic approaches, we demonstrate that profilin-null cells are defective in macropinocytosis, fluid phase efflux, and secretion of lysosomal enzymes but are unexpectedly more efficient in phagocytosis than wild-type cells. Disruption of the lmpA gene encoding a protein (DdLIMP) belonging to the CD36/LIMPII family suppressed, to different degrees, most of the profilin-minus defects, including the increase in F-actin, but did not rescue the secretion defect. Immunofluorescence microscopy indicated that DdLIMP, which is also capable of binding phosphoinositides, was associated with macropinosomes but was not detected in the plasma membrane. Also, inactivation of the lmpA gene in wild-type strains resulted in defects in macropinocytosis and fluid phase efflux but not in phagocytosis. These results suggest an important role for profilin in regulating the internalization of fluid and particles and the movement of material along the endosomal pathway; they also demonstrate a functional interaction between profilin and DdLIMP that may connect phosphoinositide-based signaling through the actin cytoskeleton with endolysosomal membrane trafficking events.

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