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Applied Biochemistry and Biotechnology Jun 2012Protein phosphatase 2A is the major enzyme that dephosphorylates the serine/threonine residues of proteins in the cytoplasm of animal cells. This phosphatase is most...
Protein phosphatase 2A is the major enzyme that dephosphorylates the serine/threonine residues of proteins in the cytoplasm of animal cells. This phosphatase is most strongly inhibited by okadaic acid. Besides okadaic acid, several other toxins and antibiotics have been shown to inhibit protein phosphatase 2A, including microsystin-LR, calyculin-A, tautomycib, nodularin, cantharidine, and fostriecin. This makes protein phosphatase 2A a valuable tool for detecting and assaying these toxins. High-scale production of active protein phosphatase 2A requires processing kilograms of animal tissue and involves several chromatographic steps. To avoid this, in this work we report the recombinant expression and characterization of the active catalytic subunit α of the protein phosphatase 2A in Trichoplusia ni insect larvae. Larvae were infected with baculovirus carrying the coding sequence for the catalytic subunit α of protein phosphatase 2A under the control of the polyhedrin promoter and containing a poly-His tag in the carboxyl end. The catalytic subunit was identified in the infected larvae extracts, and it was calculated to be present at 250 μg per gram of infected larvae, by western blot. Affinity chromatography was used for protein purification. Protein purity was determined by western blot. The activity of the enzyme, determined by the p-nitrophenyl phosphate method, was 94 μmol/min/mg of purified protein. The catalytic subunit was further characterized by inhibition with okadaic acid and dinophysis toxin 2. The results presented in this work show that this method allows the production of large quantities of the active enzyme cost-effectively. Also, the enzyme activity was stable up to 2 months at -20 °C.
Topics: Animals; Enzyme Assays; Enzyme Inhibitors; Gene Expression; Humans; Isoenzymes; Larva; Moths; Protein Phosphatase 2; Protein Subunits
PubMed: 22639363
DOI: 10.1007/s12010-012-9737-1 -
Molecular & Cellular Proteomics : MCP May 2012Molybdopterin (MPT) synthase is an essential enzyme involved in the synthesis of the molybdenum cofactor precursor molybdopterin. The molybdenum cofactor biosynthetic...
Molybdopterin (MPT) synthase is an essential enzyme involved in the synthesis of the molybdenum cofactor precursor molybdopterin. The molybdenum cofactor biosynthetic pathway is conserved from prokaryotes to Metazoa. CG10238 is the Drosophila homolog of the MoaE protein, a subunit of MPT synthase, and is found in a fusion with the mitogen-activated protein kinase (MAPK)-upstream protein kinase-binding inhibitory protein (MBIP). This fused protein inhibits the activation of c-Jun N-terminal kinase (JNK). dMoaE (CG10238) carries out this function as a subunit of the ATAC histone acetyltransferase complex. In this study, we demonstrate that Drosophila MoaE (CG10238) also interacts with Drosophila MoaD and with itself to form a complex with stoichiometry identical to the MPT synthase holoenzyme in addition to its function in ATAC. We also show that sequence determinants that regulate MAPK signaling are located within the MoaE region of dMoaE (CG10238). Analysis of other metazoan MBIPs reveals that MBIP protein sequences have an N-terminal region that appears to have been derived from the MoaE protein, although it has lost residues responsible for catalytic activity. Thus, intact and modified copies of the MoaE protein may have been conscripted to play a new, noncatalytic role in MAPK signaling in Metazoa as part of the ATAC complex.
Topics: Algorithms; Animals; Cell Line; Conserved Sequence; Drosophila melanogaster; Enzyme Activation; Evolution, Molecular; Immunoprecipitation; MAP Kinase Signaling System; Molecular Sequence Data; Phylogeny; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Quaternary; Protein Subunits; Sequence Analysis, Protein; Sulfurtransferases
PubMed: 22345504
DOI: 10.1074/mcp.M111.015818 -
Proceedings of the National Academy of... Dec 2003We have determined the solution structure of Mth11 (Mth Rpp29), an essential subunit of the RNase P enzyme from the archaebacterium Methanothermobacter...
We have determined the solution structure of Mth11 (Mth Rpp29), an essential subunit of the RNase P enzyme from the archaebacterium Methanothermobacter thermoautotrophicus (Mth). RNase P is a ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving the 5' leader sequence during maturation of tRNAs in all three domains of life. In eubacteria, this enzyme is made up of two subunits: a large RNA ( approximately 120 kDa) responsible for mediating catalysis, and a small protein cofactor ( approximately 15 kDa) that modulates substrate recognition and is required for efficient in vivo catalysis. In contrast, multiple proteins are associated with eukaryotic and archaeal RNase P, and these proteins exhibit no recognizable homology to the conserved bacterial protein subunit. In reconstitution experiments with recombinantly expressed and purified protein subunits, we found that Mth Rpp29, a homolog of the Rpp29 protein subunit from eukaryotic RNase P, is an essential protein component of the archaeal holoenzyme. Consistent with its role in mediating protein-RNA interactions, we report that Mth Rpp29 is a member of the oligonucleotide/oligosaccharide binding fold family. In addition to a structured beta-barrel core, it possesses unstructured N- and C-terminal extensions bearing several highly conserved amino acid residues. To identify possible RNA contacts in the protein-RNA complex, we examined the interaction of the 11-kDa protein with the full 100-kDa Mth RNA subunit by using NMR chemical shift perturbation. Our findings represent a critical step toward a structural model of the RNase P holoenzyme from archaebacteria and higher organisms.
Topics: Amino Acid Sequence; Archaeal Proteins; Base Sequence; Cloning, Molecular; Escherichia coli; Magnetic Resonance Spectroscopy; Methanobacteriaceae; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Protein Conformation; Protein Subunits; Ribonuclease P; Sequence Alignment; Sequence Homology, Amino Acid; Thermodynamics; Transcription, Genetic
PubMed: 14673079
DOI: 10.1073/pnas.2535887100 -
Molecular and Cellular Biology Jan 2008Protein kinase CK2 (formerly casein kinase II) is a highly conserved and ubiquitous serine/threonine kinase that is composed of two catalytic subunits (CK2alpha and/or...
Protein kinase CK2 (formerly casein kinase II) is a highly conserved and ubiquitous serine/threonine kinase that is composed of two catalytic subunits (CK2alpha and/or CK2alpha') and two CK2beta regulatory subunits. CK2 has many substrates in cells, and key roles in yeast cell physiology have been uncovered by introducing subunit mutations. Gene-targeting experiments have demonstrated that in mice, the CK2beta gene is required for early embryonic development, while the CK2alpha' subunit appears to be essential only for normal spermatogenesis. We have used homologous recombination to disrupt the CK2alpha gene in the mouse germ line. Embryos lacking CK2alpha have a marked reduction in CK2 activity in spite of the presence of the CK2alpha' subunit. CK2alpha(-/-) embryos die in mid-gestation, with abnormalities including open neural tubes and reductions in the branchial arches. Defects in the formation of the heart lead to hydrops fetalis and are likely the cause of embryonic lethality. Thus, CK2alpha appears to play an essential and uncompensated role in mammalian development.
Topics: Animals; Biomarkers; Casein Kinase II; Catalytic Domain; Cell Differentiation; Cells, Cultured; Embryo, Mammalian; Gene Expression Regulation, Developmental; Gene Targeting; Heart; Mice; Mice, Knockout; Myocardium; Protein Subunits; RNA, Messenger; Transcription, Genetic
PubMed: 17954558
DOI: 10.1128/MCB.01119-07 -
Biomolecular NMR Assignments Oct 2012Guanine-nucleotide binding proteins (G proteins) act as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at...
Guanine-nucleotide binding proteins (G proteins) act as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of GDP-bound form of the G protein α subunit (Gα(GDP)) and G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα(GTP)) and Gβγ. Then, Gα(GTP) and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Each family transduces the signaling from different GPCRs to the specific effectors. Here, we established the backbone resonance assignments of human Gα(i3), a member of the i/o family, with a molecular weight of 41 K in complex with a GTP analogue, GTPγS.
Topics: GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine Triphosphate; Humans; Nuclear Magnetic Resonance, Biomolecular; Protein Subunits
PubMed: 22274999
DOI: 10.1007/s12104-012-9361-6 -
Biochimica Et Biophysica Acta 2013Early structures of the cytochrome bc1 complex revealed heterogeneity in the position of the soluble portion of the Rieske iron sulfur protein subunit, implicating a... (Review)
Review
Early structures of the cytochrome bc1 complex revealed heterogeneity in the position of the soluble portion of the Rieske iron sulfur protein subunit, implicating a movement of this domain during function. Subsequent biochemical and biophysical works have firmly established that the motion of this subunit acts in the capacity of a conformationally assisted electron transfer step during the already complicated catalytic mechanism described within the modified version of Peter Mitchells Q cycle. How the movement of this subunit is initiated or how the frequency of its motion is controlled as a function of other steps during the catalysis remain topics of debate within the active research communities. This review addresses the historical aspects of the discovery and description of this movement, while attempting to provide a context for the involvement of conformational motion in the catalysis and efficiency of the enzyme. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.
Topics: Biocatalysis; Electron Transport; Electron Transport Complex III; Heme; Iron-Sulfur Proteins; Models, Chemical; Models, Molecular; Protein Conformation; Protein Subunits
PubMed: 23876289
DOI: 10.1016/j.bbabio.2013.07.007 -
Current Opinion in Structural Biology Oct 2016With the convergence of breakthroughs in structural biology, specifically breaking the resolution barriers in cryo-electron microscopy and with continuing developments... (Review)
Review
With the convergence of breakthroughs in structural biology, specifically breaking the resolution barriers in cryo-electron microscopy and with continuing developments in crystallography, novel interfaces with other biophysical methods are emerging. Here we consider how mass spectrometry can inform these techniques by providing unambiguous definition of subunit stoichiometry. Moreover recent developments that increase mass spectral resolution enable molecular details to be ascribed to unassigned density within high-resolution maps of membrane and soluble protein complexes. Importantly we also show how developments in mass spectrometry can define optimal solution conditions to guide downstream structure determination, particularly of challenging biomolecules that refuse to crystallise.
Topics: Biology; Crystallography; DNA; Humans; Mass Spectrometry; Protein Subunits; RNA
PubMed: 27721169
DOI: 10.1016/j.sbi.2016.09.008 -
Biophysical Journal Oct 2011We developed a novel, to our knowledge, technique for real-time monitoring of subunit exchange in homooligomeric proteins, using deuteration-assisted small-angle neutron...
We developed a novel, to our knowledge, technique for real-time monitoring of subunit exchange in homooligomeric proteins, using deuteration-assisted small-angle neutron scattering (SANS), and applied it to the tetradecamer of the proteasome α7 subunit. Isotopically normal and deuterated tetradecamers exhibited identical SANS profiles in 81% D(2)O solution. After mixing these solutions, the isotope sensitive SANS intensity in the low-q region gradually decreased, indicating subunit exchange, whereas the small-angle x-ray scattering profile remained unchanged confirming the structural integrity of the tetradecamer particles during the exchange. Kinetic analysis of zero-angle scattering intensity indicated that 1), only two of the 14 subunits were exchanged in each tetradecamer and 2), the exchange process involves at least two steps. This study underscores the usefulness of deuteration-assisted SANS, which can provide quantitative information not only on the molecular sizes and shapes of homooligomeric proteins, but also on their kinetic properties.
Topics: Deuterium; Humans; Kinetics; Models, Molecular; Neutron Diffraction; Proteasome Endopeptidase Complex; Protein Multimerization; Protein Structure, Quaternary; Protein Subunits; Scattering, Small Angle
PubMed: 22004758
DOI: 10.1016/j.bpj.2011.09.004 -
ACS Synthetic Biology Oct 2018Exploiting the ability of proteins to self-assemble into architectural templates may provide novel routes for the positioning of functional molecules in nanotechnology....
Exploiting the ability of proteins to self-assemble into architectural templates may provide novel routes for the positioning of functional molecules in nanotechnology. Here we report the engineering of multicomponent protein templates composed of distinct monomers that assemble in repeating orders into a dynamic functional structure. This was achieved by redesigning the protein-protein interfaces of a molecular chaperone with helical sequences to create unique subunits that assemble through orthogonal coiled-coils into filaments up to several hundred nanometers in length. Subsequently, it was demonstrated that functional proteins could be fused to the subunits to achieve ordered alignment along filaments. Importantly, the multicomponent filaments had molecular chaperone activity and could prevent other proteins from thermal-induced aggregation, a potentially useful property for the scaffolding of enzymes. The design in this work is presented as proof-of-concept for the creation of modular templates that could potentially be used to position functional molecules, stabilize other proteins such as enzymes, and enable controlled assembly of nanostructures with unique topologies.
Topics: Circular Dichroism; Cytoskeleton; Models, Molecular; Molecular Chaperones; Protein Conformation, beta-Strand; Protein Engineering; Protein Refolding; Protein Subunits; Proteins
PubMed: 30234970
DOI: 10.1021/acssynbio.8b00241 -
Endocrine Reviews Apr 2016The TSH receptor (TSHR) on the surface of thyrocytes is unique among the glycoprotein hormone receptors in comprising two subunits: an extracellular A-subunit, and a... (Review)
Review
The TSH receptor (TSHR) on the surface of thyrocytes is unique among the glycoprotein hormone receptors in comprising two subunits: an extracellular A-subunit, and a largely transmembrane and cytosolic B-subunit. Unlike its ligand TSH, whose subunits are encoded by two genes, the TSHR is expressed as a single polypeptide that subsequently undergoes intramolecular cleavage into disulfide-linked subunits. Cleavage is associated with removal of a C-peptide region, a mechanism similar in some respects to insulin cleavage into disulfide linked A- and B-subunits with loss of a C-peptide region. The potential pathophysiological importance of TSHR cleavage into A- and B-subunits is that some A-subunits are shed from the cell surface. Considerable experimental evidence supports the concept that A-subunit shedding in genetically susceptible individuals is a factor contributing to the induction and/or affinity maturation of pathogenic thyroid-stimulating autoantibodies, the direct cause of Graves' disease. The noncleaving gonadotropin receptors are not associated with autoantibodies that induce a "Graves' disease of the gonads." We also review herein current information on the location of the cleavage sites, the enzyme(s) responsible for cleavage, the mechanism by which A-subunits are shed, and the effects of cleavage on receptor signaling.
Topics: Animals; Humans; Protein Multimerization; Protein Subunits; Protein Transport; Proteolysis; Receptors, Thyrotropin; Thyroid Epithelial Cells
PubMed: 26799472
DOI: 10.1210/er.2015-1098