Identification of a calcium binding protein highly localized to the cochlear nucleus

A protein, designated as protein 10 (MW = 29 kDa, pI = 5.3), was shown to be highly localized to the cochlear nuclei of guinea-pigs and rats by the use of two-dimensional gel electrophoresis. In gels from guinea-pig brain, this protein appeared greatest in amounts in the ventral cochlear nucleus and within the deep regions of the dorsal nucleus. Intermediate amounts of protein 10 were found in the lateral superior olivary nucleus and dorsal nucleus of the lateral lemniscus with lesser amounts in telencephalic brain regions. Finally, incubation of nitrocellulose blots in ⁴⁵Ca²⁺ revealed that protein 10 is a calcium binding protein.     

Identification of a novel hepatic calcium binding protein

Calcium binding activity in the 100,000 X g supernatant of bovine liver has been isolated by a procedure involving DEAE cellulose and Sephadex G-100 chromatography. In addition to calmodulin, two new high affinity calcium binding proteins have been identified. On gel filtration chromatography these proteins migrate with apparent molecular weights of 83,700 and 51,400; whereas by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the two proteins migrate identically with Mr 63,000. In the presence of millimolar Mg2+, both proteins bind up to one mol Ca2+/mol protein. Half-maximal binding occurs at approximately 0.1 microM Ca2+. Amino acid compositional analysis reveals that both proteins are acidic, and contain about 40% glx and asx. Peptide mapping procedures suggest that these proteins may be highly homologous or multiple forms of a single protein. The results show the existence of calcium binding protein(s) other than calmodulin in hepatic cytosol.     

Stoichiometry of calcium binding to a synthetic heterodimeric troponin-C domain.

In this work we describe calcium binding to two synthetic 34-residue peptides, determined by 1H-nmr spectroscopy. The peptides investigated, SCIII and SCIV, encompass the calcium-binding sites III and IV, respectively, of troponin-C. In the absence of calcium it has previously been shown that each of these peptides possesses little regular secondary structure. Further, the 1H-nmr spectra of an equimolar mixture of both of these apo-peptides (apo-SCIII/SCIV) shows that little interaction occurs between peptides. Upon calcium binding the spectral changes that occur to SCIII/SCIV are consistent with global conformational changes in both peptides. We have shown previously that these conformational changes are a product of calcium binding to SCIII and SCIV to form a two-site heterodimer Ca2-SCIII/SCIV. It is proposed that this calcium-induced folding proceeds via calcium binding to SCIII to form Ca-SCIII, peptide association with apo-SCIV to form the heterodimer Ca-SCIII/SCIV, and calcium binding to form Ca2-SCIII/SCIV. The dissociation constants involved in this pathway, K1, Kd, and K2, respectively, have been determined by stoichiometric calcium titration of SCIII/SCIV, monitored by 1H-nmr spectroscopy. Using this procedure it has been determined that K1 = 3 microM, Kd = 10 microM, and K2 = 2 microM.     

Identification of a novel calcium binding protein from bovine brain

A novel Ca²⁺ binding protein, named caligulin, was extracted from the heat-treated 100 000 × g supernatant of bovine brain and purified to electrophoretic homogeneity. The apparent M_r of caligulin determined on sodium dodecyl sulfate polyacrylamide gels was 24 000. Analysis by gel filtration chromatography indicated an apparent M_r of 33 000, suggesting a monomeric protein. Amino acid composition data demonstrated the presence of 25% acidic residues, 12% basic residues and 10% leucine. In the presence of 1 mM MgCl₂ and 0.15 M KCl, caligulin bound 1 mol Ca²⁺/mol protein with half-maximal binding at about 0.2 μM Ca²⁺.     

Identification of ‘cystic fibrosis protein’ as a complex of two calcium-binding proteins present in human cells of myeloid origin

Cystic fibrosis protein is a serum protein characterized by a pI close to 8.4 and present with a higher concentration in serum and plasma of cystic fibrosis carriers than in controls. This protein was found immunologically indistinguishable from the cystic fibrosis antigen isolated from granulocytes and presenting a sequence analogous to that of MRP-8, a calcium-binding protein expressed in the myeloid cell lineage. Using antibodies directed against MRP-8 and its closely associated calcium-binding protein, MRP-14, we demonstrate here that cystic fibrosis protein purified from serum is a complex of the two proteins MRP-8 and MRP-14.     

Formation and intracellular transport of a heterodimeric viral spike protein complex

We have analyzed the heterodimerization and intracellular transport from the ER to the Golgi complex (GC) of two membrane glycoproteins of a bunyavirus (Uukuniemi virus) that matures by a budding process in the GC. The glycoproteins G1 and G2, which form the viral spikes, are cotranslationally cleaved in the ER from a 110,000-D precursor. Newly synthesized G1 was transported to the GC and incorporated into virus particles about 30-45 min faster than newly synthesized G2. Analysis of the kinetics of intrachain disulfide bond formation showed that G1 acquired its mature form within 10 min, while completion of disulfide bond formation of G2 required a considerably longer time (up to 60 min). During the maturation process, G2 was transiently associated with the IgG heavy chain binding protein for a longer time than G1. Protein disulfide isomerase also coprecipitated with antibodies against G1 and G2. In virus particles, G1 and G2 were present exclusively as heterodimers. Immunoprecipitation with monoclonal antibodies showed that heterodimerization occurred rapidly, probably in the ER, between newly made G1 and mature, dimerization competent G2. Taken together, our results show that these two viral glycoproteins have different maturation kinetics in the ER. We conclude that the apparent different kinetics of ER to GC transport of G1 and G2 is due to the different rates by which these proteins fold and become competent to enter into heterodimeric complexes prior to exit from the ER.     

Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti

Background

Legumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca²⁺ concentration, which is a fundamental prerequisite for any Ca²⁺-based signalling system, is accomplished by complex mechanisms including Ca²⁺ binding proteins acting as Ca²⁺ buffers. In this work we investigated the occurrence of Ca²⁺ binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus.

Results

A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca²⁺-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca²⁺-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca²⁺ binding ability of the M. loti protein was demonstrated by ⁴⁵Ca²⁺-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca²⁺ adducts.

Conclusions

The present data indicate that ferredoxin II is a major Ca²⁺ binding protein in M. loti that may participate in Ca²⁺ homeostasis and suggest an evolutionarily ancient origin for protein-based Ca²⁺ regulatory systems.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0352-5) contains supplementary material, which is available to authorized users.     

Identification of AKAP79 as a protein phosphatase 1 catalytic binding protein

The ubiquitously expressed and highly promiscuous protein phosphatase 1 (PP1) regulates many cellular processes. Targeting PP1 to specific locations within the cell allows for the regulation of PP1 by conferring substrate specificity. In the present study, we identified AKAP79 as a novel PP1 regulatory subunit. Immunoprecipitaiton of the AKAP from rat brain extract found that the PP1 catalytic subunit copurified with the anchoring protein. This is a direct interaction, demonstrated by pulldown experiments using purified proteins. Interestingly, the addition of AKAP79 to purified PP1 catalytic subunit decreased phosphatase activity with an IC(50) of 811 ± 0.56 nM of the anchoring protein. Analysis of AKAP79 identified a PP1 binding site that conformed to a consensus PP1 binding motif (FxxR/KxR/K) in the first 44 amino acids of the anchoring protein. This was confirmed when a peptide mimicking this region of AKAP79 was able to bind PP1 by both pulldown assay and surface plasmon resonance. However, PP1 was still able to bind to AKAP79 upon deletion of this region, suggesting additional sites of contact between the anchoring protein and the phosphatase. Importantly, this consensus PP1 binding motif was found not to be responsible for PP1 inhibition, but rather enhanced phosphatase activity, as deletion of this domain resulted in an increased inhibition of PP1 activity. Instead, a second interaction domain localized to residues 150-250 of AKAP79 was required for the inhibition of PP1. However, the inhibitory actions of AKAP79 on PP1 are substrate dependent, as the anchoring protein did not inhibit PP1 dephosphorylation of phospho-PSD-95, a substrate found in AKAP79 complexes in the brain. These combined observations suggest that AKAP79 acts as a PP1 regulatory subunit that can direct PP1 activity toward specific targets in the AKAP79 complex.     

Identification of caveolin-1 as a fatty acid binding protein

In an attempt to identify high affinity, fatty acid binding proteins present in 3T3-L1 adipocytes plasma membranes, we labeled proteins in purified plasma membranes with the photoreactive fatty acid analogue, 11-m-diazirinophenoxy[11-3H]undecanoate. A single membrane protein of 22 kDa was covalently labeled after photolysis. This protein fractionated with caveolin-1 containing caveolae and was immunoprecipitated by an anti-caveolin-1 monoclonal antibody. Furthermore, 2D-PAGE analysis revealed that both the alpha and beta isoforms of caveolin-1 could be labeled by the photoreactive fatty acid upon photolysis, indicating that both bind fatty acids. The saturable binding of the photoreactive fatty acid suggests caveolin-1 has a lipid binding site that may either operate during intracellular lipid traffic or regulate caveolin-1 function.     

Identification of 'cystic fibrosis protein' as a complex of two calcium-binding proteins present in human cells of myeloid origin

Cystic fibrosis protein is a serum protein characterized by a pI close to 8.4 and present with a higher concentration in serum and plasma of cystic fibrosis carriers than in controls. This protein was found immunologically indistinguishable from the cystic fibrosis antigen isolated from granulocytes and presenting a sequence analogous to that of MRP-8, a calcium-binding protein expressed in the myeloid cell lineage. Using antibodies directed against MRP-8 and its closely associated calcium-binding protein, MRP-14, we demonstrate here that cystic fibrosis protein purified from serum is a complex of the two proteins MRP-8 and MRP-14.     

The Kar3p kinesin-related protein forms a novel heterodimeric structure with its associated protein Cik1p

Proteins that physically associate with members of the kinesin superfamily are critical for the functional diversity observed for these microtubule motor proteins. However, quaternary structures of complexes between kinesins and kinesin-associated proteins are poorly defined. We have analyzed the nature of the interaction between the Kar3 motor protein, a minus-end-directed kinesin from yeast, and its associated protein Cik1. Extraction experiments demonstrate that Kar3p and Cik1p are tightly associated. Mapping of the interaction domains of the two proteins by two-hybrid analyses indicates that Kar3p and Cik1p associate in a highly specific manner along the lengths of their respective coiled-coil domains. Sucrose gradient velocity centrifugation and gel filtration experiments were used to determine the size of the Kar3-Cik1 complex from both mating pheromone-treated cells and vegetatively growing cells. These experiments predict a size for this complex that is consistent with that of a heterodimer containing one Kar3p subunit and one Cik1p subunit. Finally, immunoprecipitation of epitope-tagged and untagged proteins confirms that only one subunit of Kar3p and Cik1p are present in the Kar3-Cik1 complex. These findings demonstrate that the Kar3-Cik1 complex has a novel heterodimeric structure not observed previously for kinesin complexes.     

钙结合蛋白calreticulin

钙结合蛋白ｃａｌｒｅｔｉｃｕｌｉｎ由Ｎ、Ｐ和Ｃ三个区域的氨在酸序列组成,具有很主的钙结合容量,主要存在于内质网上;其蛋白和基因在生物进化过程中具有极高的保守性,提示它在许多细胞功能的调节中发挥重要作用;它是一种独特的内质网膜分子伴侣,参与胞内钙信号、细胞粘际及基因表达的调控。     

Identification of protein kinase A catalytic subunit beta as a novel binding partner of p73 and regulation of p73 function

Post-translational modifications play a crucial role in regulation of the protein stability and pro-apoptotic function of p53 as well as its close relative p73. Using a yeast two-hybrid screening based on the Sos recruitment system, we identified protein kinase A catalytic subunit beta (PKA-Cbeta) as a novel binding partner of p73. Co-immunoprecipitation and glutathione S-transferase pull-down assays revealed that p73alpha associated with PKA-Cbeta in mammalian cells and that their interaction was mediated by both the N- and C-terminal regions of p73alpha. In contrast, p53 failed to bind to PKA-Cbeta. In vitro phosphorylation assay demonstrated that glutathione S-transferase-p73alpha-(1-130), which has one putative PKA phosphorylation site, was phosphorylated by PKA. Enforced expression of PKA-Cbeta resulted in significant inhibition of the transactivation function and pro-apoptotic activity of p73alpha, whereas a kinase-deficient mutant of PKA-Cbeta had no detectable effect. Consistent with this notion, treatment with H-89 (an ATP analog that functions as a PKA inhibitor) reversed the dibutyryl cAMP-mediated inhibition of p73alpha. Of particular interest, PKA-Cbeta facilitated the intramolecular interaction of p73alpha, thereby masking the N-terminal transactivation domain with the C-terminal inhibitory domain. Thus, our findings indicate a PKA-Cbeta-mediated inhibitory mechanism of p73 function.     

Identification of TBC7 having TBC domain as a novel binding protein to TSC1-TSC2 complex

TBC7, a TBC (Tre-2/Bub2/Cdc16) 1 domain protein, was identified as a novel binding protein to the TSC1-TSC2 tumor suppressor complex by peptide mass fingerprinting analysis of the proteins immunoprecipitated with FLAG-epitope tagged TSC1 and TSC2 from the transfected mammalian cells. The in vivo and in vitro association of TBC7 and the TSC1-TSC2 complex was confirmed by the co-immunoprecipitation and pull-down analysis, respectively, and TBC7 was revealed to bind to the C-terminal half region of TSC1, which is distinct from the binding site with TSC2. The immunofluorescence microscopy and subcellular fractionation showed that TBC7 co-localizes with the tumor suppressor complex in the endomembrane. Overexpression of TBC7 enhanced ubiquitination of TSC1 and increased phosphorylation of S6 protein by S6 kinase, that is located in the mTOR-signaling pathway. These results indicate TBC7 could take a part in the negative regulation of the tumor suppressor complex through facilitating the downregulation of TSC1.     

Identification of a clathrin binding subunit in the HA2 adaptor protein complex

The HA2 adaptor complex, comprising alpha-, beta-, 50-kDa, and 16-kDa subunits, was partially dissociated into its constituents with 3 M urea, and the beta-subunit was purified from the mixture by hydroxylapatite and affinity chromatography. The renatured beta-subunit behaves hydrodynamically as a single polypeptide of Mr approximately 128,000. In a sedimentation assay the purified beta-polypeptide co-sediments with pre-formed clathrin cages. The beta-polypeptide, however, will not induce assembly of clathrin triskelia. Our results support the conjecture that the beta-type subunits (beta and beta') of the HA2 and HA2 adaptor complexes serve to attach the HA-2 adaptor complex to clathrin (Ahle, S., Mann, A., Eichelsbacher, U., and Ungewickell, E. (1988) EMBO J. 7, 919-929), while the other subunits may determine the specificity of binding to docking proteins and receptors on cytoplasmic membrane surfaces.     

Identification of calnexin as a binding protein for Amadori-modified glycated albumin

Albumin modified by Amadori glucose adducts (glycated albumin) selectively binds to glomerular mesangial cells and triggers signal transduction processes that modulate cellular function. To identify glycated albumin binding proteins, we applied membrane extracts prepared from murine mesangial cells to a column of lysine-Sepharose followed by application to an affinity column of fructosyllysine-Sepharose. This procedure yielded an approximately 90 kDa polypeptide that immunoreacted with Amadori-modified but not carbohydrate-free albumin. MALDI mass fingerprinting matched 9 out of 25 peptides with calnexin, and amino acid analysis showed homology with this transmembrane calcium-binding protein of the calreticulin family. These results indicate that one of the mesangial cell receptors for glycated albumin is a calnexin-like protein.     

Visinin: a novel calcium binding protein expressed in retinal cone cells

Visinin is a retinal cone cell-specific protein (molecular weight 24,000, pI 5.1). To investigate its function, visinin cDNA was isolated from a chick retinal lambda gt11 cDNA library, using anti-visinin serum. The beta-galactosidase-visinin fusion protein was used for purifying epitope-selected antibody. The purified visinin antibody reacted only with a 24 kd protein in retinal cone cells. Visinin mRNA was expressed only in the retinal photoreceptor layer. The nucleotide sequence of the cDNA revealed that visinin has three E-F hand structures and is a Ca2+ binding protein. Visinin protein expressed in E. coli exhibited Ca2+ binding activity. These results suggest that visinin is a photoreceptor-specific Ca2+ binding protein and may be involved in phototransduction in the cone cells.     

Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein

Background  

The p49/STRAP (or SRFBP1) protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart.