Characterization Of Calcium Binding Research Articles

Identification and Characterization of Calcium Binding and Coiled Coil Domain 1 (Calcoco1) in Skeletal Muscle

Skeletal muscle atrophy results in loss of muscle mass and reduced strength and is caused by disparate physiological conditions including aging, cancer, corticosteroid use, and denervation. To better understand the molecular genetic events that lead to muscle atrophy, a previous study isolated skeletal muscle from mice following 3 and 14 days of denervation. Gene expression was then analyzed by microarray and compared to control muscle to identify novel, atrophy‐induced genes. The microarray revealed that Calcium Binding and Coiled Coil Domain 1 (Calcoco1) is expressed in skeletal muscle and induced in response to denervation. To confirm that Calcoco1 is expressed in skeletal muscle, the cDNA was successfully amplified and cloned from cultured C2C12 cells. Moreover, quantitative PCR was conducted using RNA isolated from proliferating and differentiated muscle cells to determine the expression profile of Calcoco1 at the transcriptional level. We observed moderate activation of Calcoco1 through myoblast proliferation and early differentiation, followed by a robust increase in activation by the later stages of myotube differentiation. These results were mirrored at the protein level where it was observed that Calcoco1 is expressed during early proliferation and increases as muscle cell differentiation progresses. To elucidate a functional role for Calcoco1 in muscle, we transfected cultured muscle cells with a Calcoco1 expression plasmid and then harvested the cells at timepoints ranging from proliferation through late differentiation. The cell lysates were probed by Western blot for markers of muscle cell differentiation, including Myosin Heavy Chain (MyHC) and myogenin, which both show significant repression in response to Calcoco1 overexpression. The Calcoco1 protein is predicted to contain both a calcium binding domain and a coiled‐coiled domain, suggesting that it may play a role in protein‐protein interactions or as a putative transcription factor. To investigate these possibilities, we sought to determine the sub‐cellular localization of Calcoco1 in muscle cells by fusing Calcoco1 cDNA to Green Fluorescent Protein (GFP) and expressing it in muscle cells. Visualization by confocal microscopy revealed clear nuclear‐exclusion of Calcoco1 protein in unchallenged myoblast cells, suggesting that it may not participate directly in gene regulation. The discovery that Calcoco1 is expressed in skeletal muscle and is induced in response to neurogenic atrophy helps further our understanding of the molecular and cellular events of muscle wasting and may eventually contribute to the identification of new therapeutic targets for the treatment and of muscle atrophy.

Characterization of Calcium Binding and Coiled Coil Domain 1 (Calcoco1) in Skeletal Muscle

Skeletal muscle atrophy results in loss of muscle mass and reduced strength and is caused by a range of physiological conditions including aging, cancer, corticosteroid use, and denervation. To further our understanding of the molecular genetic events associated with muscle atrophy, a previous study isolated skeletal muscle from mice following 3 days and 14 days of denervation and the gene expression profile was analyzed by microarray and compared to control muscle in order to identify novel, atrophy‐induced genes. The microarray revealed that Calcium Binding and Coiled Coil Domain 1 (Calcoco1) is expressed in skeletal muscle and induced in response to denervation. The cDNA of Calcoco1 was successfully amplified and cloned from cultured C2C12 cells, demonstrating that this gene is expressed in muscle cells. Quantitative PCR was subsequently conducted using RNA isolated from proliferating and differentiating muscle cells to determine the expression profile of Calcoco1 at the transcriptional level. We observed moderate activation of Calcoco1 through myoblast proliferation and early differentiation, followed by a robust increase in activation by the later stages of myotube differentiation. These results were mirrored at the protein level where it was observed that Calcoco1 is expressed in early proliferation and increases as differentiation progresses. To elucidate Calcoco1's functional role in muscle, we transfected cultured muscle cells with a Calcoco1 expression plasmid and then harvested the cells at timepoints ranging from proliferation through late differentiation. The cell lysates were probed by Western blot for markers of muscle cell differentiation, including Myosin Heavy Chain (MyHC) and myogenin, which both show significant repression in response to Calcoco1 over expression. The Calcoco1 protein is predicted to contain both a calcium binding domain and a coiled‐coiled domain, suggesting that it may play a role in protein‐protein interactions or as a putative transcription factor. To begin to investigate these possibilities, we sought to determine the sub‐cellular localization of Calcoco1 in muscle cells by fusing Calcoco1 cDNA to Green Fluorescent Protein (GFP) and expressing it in muscle cells. Visualization by confocal microscopy revealed clear nuclear‐exclusion of Calcoco1 protein in unchallenged myoblast cells, suggesting that it may not participate directly in gene regulation. The discovery that Calcoco1 is expressed in skeletal muscle and is induced in response to neurogenic atrophy, in combination with its apparent role in muscle cell differentiation, helps further our understanding of the molecular genetic and cellular events of muscle atrophy and may eventually contribute to the identification of new therapeutic targets for the treatment and prevention of muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification and Characterization of Calcium Binding and Coiled Coil Domain 1 (Calcoco1) in Skeletal Muscle

Skeletal muscle atrophy results in reduced muscle size and strength and is caused by a range of physiological conditions, including aging, cancer, corticosteroid use, and denervation. In order to better characterize the molecular genetic events of atrophy, skeletal muscle was isolated from mice following 3 days and 14 days of denervation. The gene expression profile of the denervated muscle tissue was analyzed by microarray and compared to control muscle in order to identify novel atrophy‐induced genes. The microarray revealed that Calcium Binding and Coiled Coil Domain 1 (Calcoco1) is expressed in skeletal muscle and is induced in response to denervation. To confirm that Calcoco1 is expressed in muscle, we successfully cloned the cDNA of Calcoco1 from cultured muscle cells, demonstrating that this gene is transcriptionally active. In addition, quantitative PCR (qPCR) was used to assess Calcoco1 expression in both proliferating and differentiated muscle cells and the results demonstrated that Calcoco1 expression levels are moderate in proliferating myoblasts, but show significant elevation in differentiated myotubes. Furthermore, Western Blot analysis of protein homogenates isolated from muscle cells confirmed that Calcoco1 is expressed in both proliferating myoblasts and differentiated myotubes. In order to better characterize the transcriptional regulation of Calcoco1, fragments of the proximal promoter located immediately upstream of the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells in combination with myogenic regulatory factor (MRF) expression plasmids, which resulted in significant activation of reporter gene activity. Furthermore, bioinformatic analysis of the promoter region of Calcoco1 revealed a conserved canonical E‐box element, which is a known myogenic regulatory factor (MRF) binding site, further suggesting that Calcoco1 may be regulated by muscle specific transcription factors. Finally, the Calcoco1 protein is predicted to have both a calcium binding domain and a coiled‐coil domain, suggesting that it may function in protein‐protein interactions and/or as a putative transcription factor. Therefore, in order to determine the sub‐cellular localization of Calcoco1 in muscle cells, the Calcoco1 cDNA was fused with the green fluorescent protein (GFP), expressed in muscle cells, and visualized by confocal microscopy revealing that Calcoco1 is localized exclusively to the cytoplasm in unchallenged myoblast cells. The discovery that Calcoco1 is expressed in skeletal muscle combined with the observation that this gene is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of muscle atrophy and may eventually lead to the identification of new therapeutic targets for the treatment and prevention of muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants to D.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of calcium binding to adipocyte plasma membranes.

Calcium binding to adipocyte plasma membranes has been assessed by equilibrium dialysis and by membrane filtration techniques. Calcium binding was specific and saturable, displaying two distinct classes of binding sites. The affinity constants and maximum binding capacities in the presence of 0.1 M KCl were 4.5 X 10(4) M-1 and 1.8 nmol/mg of protein and 2.0 X 10(3) M-1 and 13.7 nmol/mg for the high and low affinity sites, respectively. Bound calcium was totally dissociated in the presence of excess calcium within 11.0 min in two distinct phases corresponding to the two classes of sites. Association and dissociation rate constants for the high affinity sites were 7.7 X 10(2) M-1S-1 and 9.2 X 10(-3S-1 respectively. Free energy changes at 24 degrees were +6.4 kcal mol-1 for the high affinity sites and +4.5 kcal mol-1 for the low affinity sites. The high affinity sites demonstrated a pH optimum of 7.0 whereas the binding to the low affinity sites progressively increased between pH 6.0 and 9.0. Low concentrations of MgCl2 (less than 300 muM) enhanced calcium binding slightly, whereas high concentrations of KCl and MgCl2 were noncompetitive inhibitors of calcium binding. Procaine and ruthenium red had no effect on calcium binding and lanthanum was a poor inhibitor of calcium binding. This represents the first report of calcium binding to adipocyte plasma membranes and the first kinetic analysis of calcium binding to biological membranes. The specificity of this calcium-binding system in adipocyte plasma membranes suggests its importance in cellular bioregulation.