-
Molecules (Basel, Switzerland) Apr 2019Members of the polo-like kinase (Plk) family of serine/threonine protein kinases play crucial roles in cell cycle regulation and proliferation. Of the five Plks...
Members of the polo-like kinase (Plk) family of serine/threonine protein kinases play crucial roles in cell cycle regulation and proliferation. Of the five Plks (Plk1-5), Plk1 is recognized as an anticancer drug target. Plk1 contains multiple structural components that are important for its proper biological function. These include an N-terminal catalytic domain and a C-terminal non-catalytic polo-box domain (PBD). The PBD binds to phosphothreonine (pT) and phosphoserine-containing sequences. Blocking PBD-dependent interactions offers a potential means of down-regulating Plk1 function that is distinct from targeting its ATP-binding site. Previously, we demonstrated by tethering alkylphenyl chains from the (π)-position of the His residue in the 5-mer PLHSpT, that we were able to access a hydrophobic "cryptic" binding pocket on the surface of the PBD, and in so doing enhance binding affinities by approximately 1000-fold. More recently, we optimized these PBD-ligand interactions using an oxime ligation-based strategy. Herein, using azide-alkyne cycloaddition reactions, we explore new triazole-containing PBD-binding antagonists. Some of these ligands retain the high PBD-binding affinity of the parent peptide, while showing desirable enhanced selectivity for the PBD of Plk1 relative to the PBDs of Plk2 and Plk3.
Topics: Cell Cycle Proteins; Peptides; Phosphoserine; Phosphothreonine; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Triazoles; Polo-Like Kinase 1
PubMed: 31014020
DOI: 10.3390/molecules24081488 -
Nature Structural & Molecular Biology May 2008Cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) is required for translation and is typically synthesized by cysteinyl-tRNA synthetase (CysRS). However, Methanocaldococcus jannaschii...
Cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) is required for translation and is typically synthesized by cysteinyl-tRNA synthetase (CysRS). However, Methanocaldococcus jannaschii synthesizes Cys-tRNA(Cys) by an indirect pathway, whereby O-phosphoseryl-tRNA synthetase (SepRS) acylates tRNA(Cys) with phosphoserine (Sep), and Sep-tRNA-Cys-tRNA synthase (SepCysS) converts the tRNA-bound phosphoserine to cysteine. We show here that M. jannaschii SepRS differs from CysRS by recruiting the m1G37 modification as a determinant for aminoacylation, and in showing limited discrimination against mutations of conserved nucleotides. Kinetic and binding measurements show that both SepRS and SepCysS bind the reaction intermediate Sep-tRNA(Cys) tightly, and these two enzymes form a stable binary complex that promotes conversion of the intermediate to the product and sequesters the intermediate from binding to elongation factor EF-1alpha or infiltrating into the ribosome. These results highlight the importance of the protein binary complex for efficient synthesis of Cys-tRNA(Cys).
Topics: Aminoacylation; Base Sequence; DNA Mutational Analysis; Kinetics; Methanococcaceae; Molecular Sequence Data; Multiprotein Complexes; Nucleic Acid Conformation; Phosphoserine; RNA, Transfer, Amino Acyl
PubMed: 18425141
DOI: 10.1038/nsmb.1423 -
Nucleic Acids Research Jul 2003The CRP (Cleavage of Radiolabeled Phosphoproteins) program guides the design and interpretation of experiments to identify protein phosphorylation sites by Edman...
The CRP (Cleavage of Radiolabeled Phosphoproteins) program guides the design and interpretation of experiments to identify protein phosphorylation sites by Edman sequencing of unseparated peptides. Traditionally, phosphorylation sites are determined by cleaving the phosphoprotein and separating the peptides for Edman 32P-phosphate release sequencing. CRP analysis of a phosphoprotein's sequence accelerates this process by omitting the separation step: given a protein sequence of interest, the CRP program performs an in silico proteolytic cleavage of the sequence and reports the predicted Edman cycles in which radioactivity would be observed if a given serine, threonine or tyrosine were phosphorylated. Experimentally observed cycles containing 32P can be compared with CRP predictions to confirm candidate sites and/or explore the ability of additional cleavage experiments to resolve remaining ambiguities. To reduce ambiguity, the phosphorylated residue (P-Tyr, P-Ser or P-Thr) can be determined experimentally, and CRP will ignore sites with alternative residues. CRP also provides simple predictions of likely phosphorylation sites using known kinase recognition motifs. The CRP interface is available at http://fasta.bioch.virginia.edu/crp.
Topics: Humans; Internet; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphothreonine; Phosphotyrosine; Radioactive Tracers; Sequence Analysis, Protein; Software
PubMed: 12824437
DOI: 10.1093/nar/gkg513 -
Journal of Bone and Mineral Research :... Sep 1991Aqueous tissue processing and demineralization procedures may adversely affect the inorganic mineral phase of a calcified sample and, where mineral and organic...
Effects of fixation and demineralization on the retention of bone phosphoprotein and other matrix components as evaluated by biochemical analyses and quantitative immunocytochemistry.
Aqueous tissue processing and demineralization procedures may adversely affect the inorganic mineral phase of a calcified sample and, where mineral and organic constituents interact, may consequently also indirectly alter organic matrix ultrastructure and distribution. In the present work, the effects of demineralization have been investigated on the retention in chicken bone of two phosphoamino acids, O-phosphoserine and O-phosphothreonine, found in bone phosphoproteins proposed to be important in vertebrate mineralization and, more specifically, on the retention and distribution of a 66 kD bone phosphoprotein (66 kD BPP, osteopontin) also implicated in the calcification process. In tibiae fixed initially with 1% glutaraldehyde and then demineralized in 0.5 N HCl, 0.5 N acetic acid, or 0.1 M EDTA (all containing 1% glutaraldehyde), amino acid analyses and quantitative immunocytochemistry revealed that the phosphoamino acid content and the distribution of the 66 kD BPP were essentially the same as in fixed undemineralized controls. However, demineralization slightly altered the ultrastructural appearance of immunolabeled, electron-dense patches of organic material in the bone matrix. In unfixed bone demineralized with any of these acids, there was a substantial loss of phosphoamino acids and the 66 kD BPP from the bone matrix. The relative ability of these acids to extract phosphoproteins from unfixed bone was found to decrease in the order EDTA greater than HCl greater than acetic acid. These results emphasize the differential effects on structural components of various demineralization and extraction procedures for biochemical and immunocytochemical studies of biologic tissues. Furthermore, they demonstrate that initial fixation with glutaraldehyde retains phosphoproteins in bone, with or without demineralization, while being adequate for immunocytochemical localization of certain bone matrix proteins and that an understanding of the action of specimen preparation on organic constituents (as well as inorganic components) is essential for accurately describing ultrastructural matrix-mineral relationships.
Topics: Amino Acids; Animals; Bone Matrix; Bone and Bones; Chick Embryo; Collagen; Glutaral; Immunohistochemistry; Phosphoproteins; Phosphoserine; Phosphothreonine; Tissue Fixation
PubMed: 1789141
DOI: 10.1002/jbmr.5650060907 -
Microbiology (Reading, England) May 2015L-serine is one of the proteinogenic amino acids and participates in several essential processes in all organisms. In plants, the light-dependent photorespiratory and...
L-serine is one of the proteinogenic amino acids and participates in several essential processes in all organisms. In plants, the light-dependent photorespiratory and the light-independent phosphoserine pathways contribute to serine biosynthesis. In cyanobacteria, the light-dependent photorespiratory pathway for serine synthesis is well characterized, but the phosphoserine pathway has not been identified. Here, we investigated three candidate genes for enzymes of the phosphoserine pathway in Synechocystis sp. PCC 6803. Only the gene for the D-3-phosphoglycerate dehydrogenase is correctly annotated in the genome database, whereas the 3-phosphoserine transaminase and 3-phosphoserine phosphatase (PSP) proteins are incorrectly annotated and were identified here. All enzymes were obtained as recombinant proteins and showed the activities necessary to catalyse the three-step phosphoserine pathway. The genes coding for the phosphoserine pathway were found in most cyanobacterial genomes listed in CyanoBase. The pathway seems to be essential for cyanobacteria, because it was impossible to mutate the gene coding for PSP in Synechocystis sp. PCC 6803 or in Synechococcus elongatus PCC 7942. A model approach indicates a 30-60% contribution of the phosphoserine pathway to the overall serine pool. Hence, this study verified that cyanobacteria, similar to plants, use the phosphoserine pathway in addition to photorespiration for serine biosynthesis.
Topics: Amino Acid Sequence; Enzyme Activation; Gene Expression Regulation, Enzymologic; Light; Metabolic Networks and Pathways; Molecular Sequence Data; Phosphoglycerate Dehydrogenase; Phosphoserine; Sequence Alignment; Serine; Substrate Specificity; Synechocystis
PubMed: 25701735
DOI: 10.1099/mic.0.000055 -
Chemistry & Biology Jun 2012Many protein-protein interactions in cells are mediated by functional domains that recognize and bind to motifs containing phosphorylated serine and threonine residues....
Many protein-protein interactions in cells are mediated by functional domains that recognize and bind to motifs containing phosphorylated serine and threonine residues. To create small molecules that inhibit such interactions, we developed methodology for the synthesis of a prodrug that generates a phosphoserine peptidomimetic in cells. For this study, we synthesized a small molecule inhibitor of 14-3-3 proteins that incorporates a nonhydrolyzable difluoromethylenephosphoserine prodrug moiety. The prodrug is cytotoxic at low micromolar concentrations when applied to cancer cells and induces caspase activation resulting in apoptosis. The prodrug reverses the 14-3-3-mediated inhibition of FOXO3a resulting from its phosphorylation by Akt1 in a concentration-dependent manner that correlates well with its ability to inhibit cell growth. This methodology can be applied to target a variety of proteins containing phosphoserine and other phosphoamino acid binding domains.
Topics: 14-3-3 Proteins; Antineoplastic Agents; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Ligands; Molecular Mimicry; Molecular Structure; Phosphoserine; Prodrugs; Structure-Activity Relationship; Tumor Cells, Cultured
PubMed: 22726690
DOI: 10.1016/j.chembiol.2012.05.011 -
Cell Reports Jul 2019In budding yeast, a single DNA double-strand break (DSB) triggers the activation of Mec1-dependent DNA damage checkpoint. After about 12 h, cells turn off the checkpoint...
In budding yeast, a single DNA double-strand break (DSB) triggers the activation of Mec1-dependent DNA damage checkpoint. After about 12 h, cells turn off the checkpoint signaling and adapt despite the persistence of the DSB. We report that the adaptation involves the autophosphorylation of Mec1 at site S1964. A non-phosphorylatable mec1-S1964A mutant causes cells to arrest permanently in response to a single DSB without affecting the initial kinase activity of Mec1. Autophosphorylation of S1964 is dependent on Ddc1 and Dpb11, and it correlates with the timing of adaptation. We also report that Mec1's binding partner, Ddc2, is an inherently stable protein that is degraded specifically upon DNA damage. Ddc2 is regulated extensively through phosphorylation, which, in turn, regulates the localization of the Mec1-Ddc2 complex to DNA lesions. Taken together, these results suggest that checkpoint response is regulated through the autophosphorylation of Mec1 kinase and through the changes in Ddc2 abundance and phosphorylation.
Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Cell Cycle Proteins; DNA Damage; DNA Mutational Analysis; DNA Repair; Intracellular Signaling Peptides and Proteins; Mutation; Phosphorylation; Phosphoserine; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction
PubMed: 31340146
DOI: 10.1016/j.celrep.2019.06.068 -
Autophagy 2015Autophagy is a potentially inimical pathway and together with apoptosis, may be activated by similar stress stimuli that can lead to cell death. The molecular cues that...
Autophagy is a potentially inimical pathway and together with apoptosis, may be activated by similar stress stimuli that can lead to cell death. The molecular cues that dictate the cell fate choice between autophagy and apoptosis remain largely unknown. Here we report that the proapoptotic protein BBC3/PUMA (BCL2 binding component 3) is a bona fide substrate of chaperone-mediated autophagy (CMA). BBC3 associates with HSPA8/HSC70 (heat shock 70kDa protein 8), leading to its lysosome translocation and uptake. Inhibition of CMA results in stabilization of BBC3, which in turn sensitizes tumor cells to undergo apoptosis. We further demonstrate that upon TNF (tumor necrosis factor) treatment, IKBKB/IKKβ (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase β)-mediated BBC3 Ser10 phosphorylation is crucial for BBC3 stabilization via blocking its degradation by CMA. Mechanistically, Ser10 phosphorylation facilitates BBC3 translocation from the cytosol to mitochondria. BBC3 stabilization resulting from either Ser10 phosphorylation or CMA inhibition potentiates TNF-induced apoptotic cell death. Our findings thus reveal that the selective degradation of BBC3 underlies the prosurvival role of CMA and define a previously unappreciated proapoptotic role of IKBKB that acts through phosphorylation-mediated stabilization of BBC3, thereby promoting TNF-triggered apoptosis.
Topics: Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Cell Line, Tumor; Humans; I-kappa B Kinase; Models, Biological; Molecular Chaperones; Phosphorylation; Phosphoserine; Protein Stability; Proteolysis; Proto-Oncogene Proteins; Serine; Tumor Necrosis Factor-alpha
PubMed: 26212789
DOI: 10.1080/15548627.2015.1075688 -
Open Biology Jun 2016The underwater silk of an aquatic casemaking caddisfly larvae (Hesperophylax occidentalis) is viscoelastic, and displays distinct yield behaviour, large strain cycle...
The underwater silk of an aquatic casemaking caddisfly larvae (Hesperophylax occidentalis) is viscoelastic, and displays distinct yield behaviour, large strain cycle hysteresis and near complete recovery of its initial strength and stiffness when unloaded. Yield followed by a stress plateau has been attributed to sequential rupture of serial Ca(2+)-cross-linked phosphoserine (pS) β-domains. Spontaneous recovery has been attributed to refolding of the Ca(2+)/pS domains powered by an elastic network. In this study, native Ca(2+) ions were exchanged with other metal ions, followed by combined mechanical and FTIR analysis to probe the contribution of pS/metal ion complexes to silk mechanical properties. After exchange of Ca(2+) with Na(+), the fibres are soft elastomers and the infrared spectra are consistent with Cv3 symmetry of the -[Formula: see text] groups. Multivalent metal ions decreased the -[Formula: see text] symmetry and the symmetric stretching modes (vs) split in a manner characteristic of ordered phosphate compounds, such as phosphate minerals and lamellar bilayers of phosphatidic acid lipids. Integrated intensities of the vs bands, indicative of the metal ion's effect on transition dipole moment of the P-O bonds, and thereby the strength of the phosphate metal complex, increased in the order: Na(+) < Mg(2+) < Sr(2+) < Ba(2+) < Ca(2+) < Eu(3+) < La(3+) < Zn(2+) < Fe(2+) With a subset of the metal ion series, the initial stiffness and yield stress of metal ion-exchanged fibres increased in the same order: [Formula: see text] [Formula: see text] establishing the link between phosphate transition dipole moments and silk fibre strength.
Topics: Animals; Calcium; Elasticity; Holometabola; Insect Proteins; Molecular Structure; Phosphoserine; Protein Structure, Secondary; Silk; Sodium; Spectroscopy, Fourier Transform Infrared
PubMed: 27278649
DOI: 10.1098/rsob.160067 -
ACS Chemical Biology Jul 2019Genetic Code Expansion (GCE) can use TAG stop codons to guide site-specific incorporation of phosphoserine (pSer) into proteins. To eliminate prematurely truncated...
Genetic Code Expansion (GCE) can use TAG stop codons to guide site-specific incorporation of phosphoserine (pSer) into proteins. To eliminate prematurely truncated peptides, improve yields, and enhance the production of multiphosphorylated proteins, Release Factor 1 (RF1)-deficient expression hosts were developed, yet these grew slowly and their use was associated with extensive misincorporation of natural amino acids instead of pSer. Here, we merge a healthy RF1-deficient cell line with a high-efficiency pSer GCE translation system to produce a versatile pSer GCE platform in which only trace misincorporation of natural amino acids is detected even when five phosphoserines were introduced into one protein. Approximately 400 and 200 mg of singly and doubly phosphorylated GFP per liter of culture were obtained. Importantly, the lack of truncated protein permits expression of oligomeric proteins and the use of N-terminal solubility-enhancing proteins to aid phospho-protein expression and purification. To illustrate the enhanced utility of this system, we produce doubly phosphorylated STING (Stimulator of Interferon Genes), as well as triply phosphorylated BAD (Bcl2-associated agonist of cell death) complexed with 14-3-3, in quantity, purity, and homogeneity sufficient for structural biology applications. We anticipate that the facile access to phosphoproteins enabled by this system, which we call pSer-3.1G, will expand studies of the phospho-proteome.
Topics: Escherichia coli; Escherichia coli Proteins; Gene Deletion; Genetic Code; Green Fluorescent Proteins; Humans; Models, Molecular; Peptide Termination Factors; Phosphorylation; Phosphoserine; Protein Biosynthesis; bcl-Associated Death Protein
PubMed: 31243963
DOI: 10.1021/acschembio.9b00307