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Connective Tissue Research 2014Biomineralization is the process by which living organisms deposit mineral in the extracellular matrix. In nature, almost 50% of biominerals are calcium-bearing... (Review)
Review
Biomineralization is the process by which living organisms deposit mineral in the extracellular matrix. In nature, almost 50% of biominerals are calcium-bearing minerals. In addition to calcium, we find biominerals formed from silica and magnetite. Calcium-containing biominerals could be either calcium phosphate as in apatite found in vertebrates or calcium carbonate as in calcite and aragonite found in many invertebrates. Since all biomineralization is matrix mediated, an understanding of the nature of the proteins involved is essential in elucidating its mechanism. This review will discuss some of the proteins involved in the process of biomineralization involving calcium. Two proteins, dentin matrix protein 1 and dentin phosphoprotein (Phosphophoryn) will serve as models for the vertebrate system, and two others - P16 and phosphodontin will serve as models for the invertebrate system.
Topics: Acids; Amino Acid Sequence; Animals; Calcification, Physiologic; Humans; Invertebrates; Molecular Sequence Data; Phosphoproteins; Vertebrates
PubMed: 24437603
DOI: 10.3109/03008207.2013.867336 -
European Journal of Oral Sciences Jun 2023This study aimed to evaluate the effects of phosphoproteins on bacterial mineralization. Dental calculus formation is attributed to bacterial mineralization in the oral...
This study aimed to evaluate the effects of phosphoproteins on bacterial mineralization. Dental calculus formation is attributed to bacterial mineralization in the oral cavity; however, the influence of phosphoproteins (which are abundant in saliva) is not clear. The model bacterium Escherichia coli was suspended in a calcification solution containing casein as a model phosphoprotein. To evaluate mineralization independent of bacterial metabolism, bacteria killed by heat treatment at 70°C were compared with viable bacteria. After incubation at 37°C for 24 h, the mode of calcification was observed using electron microscopy and energy dispersive x-ray spectroscopy. Solutions without casein produced precipitation in solution, which was identical to that in experiments without bacteria. In contrast, calcification solutions with 200 ppm casein only produced calcium phosphate deposition intracellularly. Without heat treatment, intracellular calcification rarely occurred, even when casein was added. Thus, phosphoproteins promoted intracellular calcification of dead bacteria; this is similar to the calcification of insoluble matrices, such as collagen fibrils, promoted by acidic polymers. We concluded that intracellular calcification is caused by the collagen fibril-like behavior of dead bacteria. The promotion of intracellular calcification of dead bacteria by phosphoproteins suggested a basic principle of dental calculus formation.
Topics: Humans; Phosphoproteins; Caseins; Dental Calculus; Bacteria; Collagen; Calcification, Physiologic
PubMed: 36929523
DOI: 10.1111/eos.12929 -
Molecular Neurobiology 1987This article summarizes some of our knowledge concerning intracellular protein phosphorylation pathways in nerve cells. It also summarizes, very briefly, recent direct... (Review)
Review
This article summarizes some of our knowledge concerning intracellular protein phosphorylation pathways in nerve cells. It also summarizes, very briefly, recent direct experimental evidence involving intracellular injection of protein kinases, protein kinase inhibitors, and substrates, indicating that protein phosphorylation mediates the actions of a variety of neurotransmitters on their target cells. Finally, it summarizes in somewhat greater detail the results of studies of three different types of substrate proteins that appear to regulate different types of biological responses in nerve cells: synapsin I, a substrate protein present in virtually all nerve terminals, which appears to regulate neurotransmitter release from those nerve terminals; the acetylcholine receptor, the phosphorylation of which regulates its rate of desensitization in the presence of acetylcholine; and DARPP-32, the phosphorylation of which converts it into a very potent phosphoprotein phosphatase inhibitor that may be involved in the regulation by the neuromodulator dopamine of the effects of the neurotransmitter glutamate. The identification and characterization of additional neuronal phosphoproteins can be expected to lead to the clarification of numerous additional molecular mechanisms by which signal transduction is carried out in nerve cells.
Topics: Animals; Nerve Tissue Proteins; Neurons; Neurotransmitter Agents; Phosphoproteins; Phosphorylation; Receptors, Neurotransmitter; Signal Transduction; Synapsins
PubMed: 2908293
DOI: 10.1007/BF02935265 -
Journal of the American Chemical Society Jan 2005We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid...
We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the alpha-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported.
Topics: Cyclic AMP-Dependent Protein Kinases; Phosphoproteins; RNA, Transfer, Amino Acid-Specific; Serine; Threonine
PubMed: 15656617
DOI: 10.1021/ja043875c -
Cell Communication & Adhesion 2006Posttranslational regulation of proteins via protein phosphorylation is one of the major means of protein regulation. Phosphorylation is a very rapid and reversible... (Review)
Review
Posttranslational regulation of proteins via protein phosphorylation is one of the major means of protein regulation. Phosphorylation is a very rapid and reversible method of changing the function of proteins. Detection of phosphorylated proteins and the identification of phosphorylation sites are necessary to molecularly link specific phosphorylated events with change in phosphoprotein function. Mass Spectrometry (MS) has become the methodology of choice for phosphosite identification. Here we review current approaches including sample separation and enrichment techniques (SDS-PAGE, immunoprecipitation, metal-assisted enrichment, strong cation exchange, dendrimer capture), quantitative MS analysis methods (SILAC, iTRAQ, AQUA), and the application of recently developed methods including electron transfer dissociation ionization and "top-down" proteomics to phosphoprotein analysis.
Topics: Mass Spectrometry; Phosphoproteins; Phosphorylation
PubMed: 17162667
DOI: 10.1080/15419060601077917 -
Methods in Molecular Biology (Clifton,... 2009Protein phosphorylation is a reversible and frequently occurring posttranslational modification regulating a large number of biological functions. Understanding the role... (Review)
Review
Protein phosphorylation is a reversible and frequently occurring posttranslational modification regulating a large number of biological functions. Understanding the role phosphorylation events play in biochemical pathways requires the detection of phosphorylated proteins and their phosphorylated amino acids. Mass spectrometry has developed as the premier method for characterizing phosphoproteins as it is sensitive to detecting phosphosites within a single protein or a complex protein mixture (phosphoproteomics). Here an overview is provided of the sample separation and mass spectrometry techniques commonly used for qualitative phosphoprotein analysis.
Topics: Amino Acid Sequence; Animals; Catalytic Domain; Electrophoresis, Polyacrylamide Gel; Humans; Mass Spectrometry; Models, Biological; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Proteomics
PubMed: 19241012
DOI: 10.1007/978-1-60327-834-8_12 -
Journal of Separation Science Apr 2022Selective separation and enrichment of phosphoproteins are essential for understanding their important functions in almost all cellular processes. Here, taking advantage...
Selective separation and enrichment of phosphoproteins are essential for understanding their important functions in almost all cellular processes. Here, taking advantage of the feature that cadmium ion (Cd ) has an overwhelming preference for phosphates, we developed a robust and simple Cd co-precipitation strategy for the selective isolation of intact phosphoproteins. After evaluating the feasibility of Cd in phosphoprotein precipitation, we compared the washing protocols for the removal of non-specific binding proteins and then used the best-performing protocol for the isolation of phosphoproteins from different complex samples. It was found that phosphoproteins can be specifically enriched from artificial protein mixtures containing α-casein, β-casein, and bovine serum albumin or plasma, in which bovine serum albumin or plasma were served as interferences with very high molar ratios. Applying this method to enrich phosphoproteins from complex cell lysates, a high specificity was confirmed by western blotting analysis with a phosphoprotein-specific kit. Finally, we successfully applied this method to the purification of caseins from drinking milk, highlighting its potential application in the studies where purified phosphoproteins were required. In a word, this Cd co-precipitation method enables universal and effective capture, enrichment, and detection of intact phosphoproteins, making it a powerful tool for the comprehensive analysis of the phosphoproteome.
Topics: Cadmium; Caseins; Phosphates; Phosphoproteins; Serum Albumin, Bovine
PubMed: 35108751
DOI: 10.1002/jssc.202100892 -
Calcified Tissue International May 1992Osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 are three acidic phosphoproteins that are isolated from the mineralized phase of bone matrix, are... (Review)
Review
Osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 are three acidic phosphoproteins that are isolated from the mineralized phase of bone matrix, are synthesized by osteoblastic cells, and are generally restricted in their distribution to calcified tissues. Although each is a distinct gene product, these proteins share aspartic/glutamic acid contents of 30-36% and each contains multiple phosphoryl and sialyl groups. These properties, plus a strict relationship of acidic macromolecules with cell-controlled mineralization throughout nature, suggest functions in calcium binding and nucleation of calcium hydroxyapatite crystal formation. However, direct proof for such roles is still largely indirect in nature. The purpose of this review is to present two speculative hypotheses regarding acidic phosphoprotein function. The goal was to use new sequence information along with database comparisons to develop a structural rationalization of how these proteins may function in calcium handling by bone. For example, our analysis has identified a conserved polyacidic stretch in all three phosphoproteins which we propose mediates metal binding. Also, conserved motifs were identified that are analogous with those for casein kinase II phosphorylation sites and whose number correlates well with that of phosphoryl groups/protein. A two-state conformational model of calcium binding by bone matrix acidic phosphoproteins is described which incorporates these findings.
Topics: Amino Acid Sequence; Animals; Bone Density; Bone Matrix; Calcification, Physiologic; Humans; Molecular Sequence Data; Phosphoproteins
PubMed: 1596774
DOI: 10.1007/BF00296767 -
Journal of the American Chemical Society Feb 2015Analysis of protein phosphorylation remains a significant challenge due to the low abundance of phosphoproteins and the low stoichiometry of phosphorylation, which...
Analysis of protein phosphorylation remains a significant challenge due to the low abundance of phosphoproteins and the low stoichiometry of phosphorylation, which requires effective enrichment of phosphoproteins. Here we have developed superparamagnetic nanoparticles (NPs) whose surface is functionalized by multivalent ligand molecules that specifically bind to the phosphate groups on any phosphoproteins. These NPs enrich phosphoproteins from complex cell and tissue lysates with high specificity as confirmed by SDS-PAGE analysis with a phosphoprotein-specific stain and mass spectrometry analysis of the enriched phosphoproteins. This method enables universal and effective capture, enrichment, and detection of intact phosphoproteins toward a comprehensive analysis of the phosphoproteome.
Topics: Ligands; Magnetite Nanoparticles; Phosphoproteins
PubMed: 25655481
DOI: 10.1021/ja511833y -
Methods in Molecular Biology (Clifton,... 2011Redox-dependent thylakoid protein phosphorylation regulates both the short- and long-term acclimation of the photosynthetic apparatus to changes in environmental...
Redox-dependent thylakoid protein phosphorylation regulates both the short- and long-term acclimation of the photosynthetic apparatus to changes in environmental conditions. The major thylakoid phosphoproteins belong to photosystem II (D1, D2, CP43, PsbH) and its light-harvesting antenna (Lhcb1, Lhcb2, CP29), but a number of minor phosphoproteins have also been identified. The detection methods traditionally include the radiolabeling techniques, electrophoretic separation of the phosphorylated and unphosphorylated forms of the protein, and the use of phosphoamino acid antibodies or phosphoprotein-specific dyes. The recent progress in mass spectrometry techniques and methods of proteomics allow for the successful identification and analyses of protein phosphorylation. In mass spectrometry approaches no exogenous tracer is needed and natural phosphorylation of proteins can be characterized with high sensitivity yielding the mapping of exact phosphorylation sites in the proteins as well. Various methods for the detection of thylakoid phosphoproteins, including the preparation of phosphopeptides for mass spectrometric analyses and techniques for phosphopeptide identification by electrospray ionization mass spectrometry (ESI-MS) are described. The experimental protocols for simultaneous identification of multiple phosphopeptides in complex peptide mixtures, enrichment of phosphopeptides by immobilized metal affinity chromatography (IMAC), and for their sequencing by tandem spectrometry are outlined.
Topics: Adenosine Triphosphate; Amino Acid Sequence; Antibodies; Antibody Specificity; Binding Sites; Chromatography, Affinity; Coloring Agents; Electrophoresis, Polyacrylamide Gel; Membranes, Artificial; Peptide Fragments; Phosphoproteins; Phosphorylation; Plant Proteins; Polyvinyls; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Thylakoids
PubMed: 20960130
DOI: 10.1007/978-1-60761-925-3_15