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BMC Anesthesiology Jan 2014Propofol is a safe and effective intravenous anesthetic that is widely used for the induction and maintenance of anesthesia during surgery. However, the mechanism by...
BACKGROUND
Propofol is a safe and effective intravenous anesthetic that is widely used for the induction and maintenance of anesthesia during surgery. However, the mechanism by which propofol exerts its anesthetic effect remains unknown. The rapid onset of phosphorylation modifications coincides with that of propofol anesthesia.
METHODS
Propofol-anesthetized rat models were built and phosphorylated proteins in the thalamus, hippocampus and frontal lobe were enriched the to analyze the changes in these phosphoproteins after propofol anesthesia.
RESULTS
Sixteen of these phosphoprotein spots were successfully identified using MALDI-TOF MS and a subsequent comparative sequence search in the Mascot database. Of these proteins, keratin 18 and the tubulin 2c chain are cytoskeletal proteins; keratin 18 and gelsolin are relevant to alcohol drowsiness. Based on Western blot analysis, we also confirmed that the phosphorylation of these proteins is directly induced by propofol, indicating that propofol anesthesia may be relevant to cytoskeletal proteins and alcohol drowsiness.
CONCLUSIONS
These identified propofol-induced phosphorylations of proteins provide meaningful contributions for further studying the anesthetic mechanism of propofol.
Topics: Anesthetics, Intravenous; Animals; Frontal Lobe; Hippocampus; Male; Phosphoproteins; Propofol; Proteomics; Rats; Rats, Sprague-Dawley; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thalamus
PubMed: 24410762
DOI: 10.1186/1471-2253-14-3 -
Journal of Molecular Biology Sep 2013Here, we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously, we demonstrated that one of the immunoglobulin...
Here, we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously, we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct filamentous actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and cross-linking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin cross-linking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo.
Topics: Actins; Amino Acid Substitution; Animals; COS Cells; Chlorocebus aethiops; Cytoskeletal Proteins; Immunoglobulins; Mice; Models, Molecular; Mutagenesis, Site-Directed; Phosphoproteins; Protein Binding; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Protein Transport; Rabbits; Transfection
PubMed: 23806659
DOI: 10.1016/j.jmb.2013.06.016 -
Journal of Virology May 2023Human metapneumovirus (HMPV) is a negative-strand RNA virus that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. A...
Specific Residues in the C-Terminal Domain of the Human Metapneumovirus Phosphoprotein Are Indispensable for Formation of Viral Replication Centers and Regulation of the Function of the Viral Polymerase Complex.
Human metapneumovirus (HMPV) is a negative-strand RNA virus that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. A hallmark of HMPV infection is the formation of membraneless, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). The HMPV phosphoprotein (P) and nucleoprotein (N) are the minimal viral proteins necessary to form IB-like structures, and both proteins are required for the viral polymerase to synthesize RNA during infection. HMPV P is a homotetramer with regions of intrinsic disorder and has several known and predicted phosphorylation sites of unknown function. In this study, we found that the P C-terminal intrinsically disordered domain (CTD) must be present to facilitate IB formation with HMPV N, while either the N-terminal intrinsically disordered domain or the central oligomerization domain was dispensable. Alanine substitution at a single tyrosine residue within the CTD abrogated IB formation and reduced coimmunoprecipitation with HMPV N. Mutations to C-terminal phosphorylation sites revealed a potential role for phosphorylation in regulating RNA synthesis and P binding partners within IBs. Phosphorylation mutations which reduced RNA synthesis in a reporter assay produced comparable results in a recombinant viral rescue system, measured as an inability to produce infectious viral particles with genomes containing these single P mutations. This work highlights the critical role HMPV P plays in facilitating a key step of the viral life cycle and reveals the potential role for phosphorylation in regulating the function of this significant viral protein. Human metapneumovirus (HMPV) infects global populations, with severe respiratory tract infections occurring in infants, the elderly, and the immunocompromised. There are currently no FDA-approved therapeutics available to prevent or treat HMPV infection. Therefore, understanding how HMPV replicates is vital for the identification of novel targets for therapeutic development. During HMPV infection, viral RNA synthesis proteins localize to membraneless structures called inclusion bodies (IBs), which are sites of genome replication and transcription. The HMPV phosphoprotein (P) is necessary for IBs to form and for the virus to synthesize RNA, but it is not known how this protein contributes to IB formation or if it is capable of regulating viral replication. We show that the C-terminal domain of P is the location of a molecular interaction driving IB formation and contains potential phosphorylation sites where amino acid charge regulates the function of the viral polymerase complex.
Topics: Aged; Humans; Cell Line; Metapneumovirus; Nucleotidyltransferases; Paramyxoviridae Infections; Phosphoproteins; Respiratory Tract Infections; RNA; Viral Proteins; Viral Replication Compartments; Virus Replication; Inclusion Bodies, Viral
PubMed: 37092993
DOI: 10.1128/jvi.00030-23 -
Scientific Reports Jun 2016Cdc37, as a kinase-specific co-chaperone of the chaperone Hsp90AA1 (Hsp90), actively aids with the maturation, stabilization and activation of the cellular or viral...
Cdc37, as a kinase-specific co-chaperone of the chaperone Hsp90AA1 (Hsp90), actively aids with the maturation, stabilization and activation of the cellular or viral kinase/kinase-like targets. Phosphoprotein (P) of rabies virus (RABV) is a multifunctional, non-kinase protein involved in interferon antagonism, viral transcription and replication. Here, we demonstrated that the RABV non-kinase P is chaperoned by Cdc37 and Hsp90 during infection. We found that Cdc37 and Hsp90 affect the RABV life cycle directly. Activity inhibition and knockdown of Cdc37 and Hsp90 increased the instability of the viral P protein. Overexpression of Cdc37 and Hsp90 maintained P's stability but did not increase the yield of infectious RABV virions. We further demonstrated that the non-enzymatic polymerase cofactor P protein of all the genotypes of lyssaviruses is a target of the Cdc37/Hsp90 complex. Cdc37, phosphorylated or unphosphorylated on Ser13, aids the P protein to load onto the Hsp90 machinery, with or without Cdc37 binding to Hsp90. However, the interaction between Cdc37 and Hsp90 appears to have additional allosteric regulation of the conformational switch of Hsp90. Our study highlighted a novel mechanism in which Cdc37/Hsp90 chaperones a non-kinase target, which has significant implications for designing therapeutic targets against Rabies.
Topics: Allosteric Regulation; Animals; Cell Cycle Proteins; Cell Line; Gene Expression Regulation; HSP90 Heat-Shock Proteins; Mice; Molecular Chaperones; Phosphoproteins; Phosphorylation; Protein Binding; Protein Conformation; Protein Stability; Rabies; Rabies virus; Viral Structural Proteins
PubMed: 27251758
DOI: 10.1038/srep27123 -
Journal of Virology Jul 2013The phosphoprotein (P) is virally encoded by the Rhabdoviridae and Paramyxoviridae in the order Mononegavirales. P is a self-associated oligomer and forms complexes with...
The phosphoprotein (P) is virally encoded by the Rhabdoviridae and Paramyxoviridae in the order Mononegavirales. P is a self-associated oligomer and forms complexes with the large viral polymerase protein (L), the nucleocapsid protein (N), and the assembled nucleocapsid. P from different viruses has shown structural diversities even though their essential functions are the same. We systematically mapped the domains in mumps virus (MuV) P and investigated their interactions with nucleocapsid-like particles (NLPs). Similar to other P proteins, MuV P contains N-terminal, central, and C-terminal domains with flexible linkers between neighboring domains. By pulldown assays, we discovered that in addition to the previously proposed nucleocapsid binding domain (residues 343 to 391), the N-terminal region of MuV P (residues 1 to 194) could also bind NLPs. Further analysis of binding kinetics was conducted using surface plasmon resonance. This is the first observation that both the N- and C-terminal regions of a negative-strand RNA virus P are involved in binding the nucleocapsid. In addition, we defined the oligomerization domain (POD) of MuV P as residues 213 to 277 and determined its crystal structure. The tetrameric MuV POD is formed by one pair of long parallel α-helices with another pair in opposite orientation. Unlike the parallel orientation of each α-helix in the tetramer of Sendai virus POD, this represents a novel orientation of a POD where both the N- and the C-terminal domains are at either end of the tetramer. This is consistent with the observation that both the N- and the C-terminal domains are involved in binding the nucleocapsid.
Topics: Computational Biology; Crystallization; Escherichia coli; Kinetics; Models, Molecular; Mumps virus; Nucleocapsid Proteins; Phosphoproteins; Plasmids; Protein Conformation; Protein Structure, Tertiary; Surface Plasmon Resonance; X-Ray Diffraction
PubMed: 23637399
DOI: 10.1128/JVI.00653-13 -
Virology Mar 2014To the best of our knowledge, two phosphorylation sites have been reported previously, among 11 known Vaccinia virus phosphoproteins. Here, via phosphopeptide mass...
To the best of our knowledge, two phosphorylation sites have been reported previously, among 11 known Vaccinia virus phosphoproteins. Here, via phosphopeptide mass spectrometry, up to 189 phosphorylation sites were identified among 48 proteins in preparations of purified Vaccinia mature virus (MV). 8.5% of phospho-residues were pTyr. Viral phosphoproteins were found in diverse functional classes, including structural proteins, membrane proteins and RNA polymerase subunits. Among the nine identified membrane phosphoproteins, the sites in just one, namely A14L, were deduced to be internal with respect to the accompanying membrane. Examination of sites in known substrates of the Vaccinia-encoded protein kinase VPK2, indicated VPK2 to be a proline-dependent kinase. The MV phosphoproteome was enriched in potential substrates of cellular kinases belonging to the CDK2/CDK3, CK2, and p38 groups. Quantitative mass spectrometry identified several sites that became phosphorylated during intravirion kinase activation in vitro, each showing one of two distinct pH-dependency profiles.
Topics: Amino Acid Sequence; Humans; Mass Spectrometry; Molecular Sequence Data; Phosphoproteins; Phosphorylation; Vaccinia; Vaccinia virus; Viral Proteins; Virion
PubMed: 24606709
DOI: 10.1016/j.virol.2014.01.012 -
The Journal of Veterinary Medical... Aug 2017Attenuated derivative rabies virus Ni-CE replicates in muscle cells less efficiently than does the parental pathogenic strain Nishigahara. To examine the mechanism...
Attenuated derivative rabies virus Ni-CE replicates in muscle cells less efficiently than does the parental pathogenic strain Nishigahara. To examine the mechanism underlying the less efficient replication of Ni-CE, we compared the activities of Ni-CE and Nishigahara phosphoproteins, viral interferon (IFN) antagonists, to suppress IFN-β promoter activity in muscle cells and we demonstrated a defect of Ni-CE phosphoprotein in this ability. Treatment with an IFN-β-neutralizing antibody improved the replication efficiency of Ni-CE in muscle cells, indicating that produced IFN inhibits Ni-CE replication. The results indicate the importance of IFN antagonism of rabies virus phosphoprotein for viral replication in muscle cells.
Topics: Animals; Cell Line; Interferons; Mice; Muscle Cells; Phosphoproteins; Rabies; Rabies virus; Viral Proteins; Virulence; Virus Replication
PubMed: 28674326
DOI: 10.1292/jvms.17-0054 -
Microbiology (Reading, England) Aug 2015Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens...
Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB-RB transitions.
Topics: Animals; Bacterial Proteins; Chlamydia; Chlamydia Infections; Electrophoresis, Gel, Two-Dimensional; Gene Expression Regulation, Bacterial; Humans; Mice; Phosphoproteins; Tandem Mass Spectrometry
PubMed: 25998263
DOI: 10.1099/mic.0.000116 -
Journal of Neurochemistry Jul 2000The role of the dopamine- and cyclic AMP-regulated phosphoprotein of M(r) 32,000 (DARPP-32) in dopaminergic regulation of gene transcription in striatum and globus...
The role of the dopamine- and cyclic AMP-regulated phosphoprotein of M(r) 32,000 (DARPP-32) in dopaminergic regulation of gene transcription in striatum and globus pallidus was examined. Mice with targeted disruption of the gene encoding DARPP-32, its homologue, inhibitor-1, or both, were used. Pharmacological characterization showed that mutant mice had normal basal levels of dopamine D(1) and D(2) receptors and adenosine A(2A) receptors. Basal expression levels of the striatonigral-specific neuropeptides substance P and prodynorphin and the immediate early genes c-fos and NGFI-A were also unaltered in mutant mice. A full D(1) receptor agonist, SKF 82958, up-regulated the expression of these neuropeptides and immediate early genes significantly more in wild-type mice than in mice lacking DARPP-32. Moreover, the additive stimulation of SKF 82958 and quinelorane, a D(2) receptor agonist, on c-fos mRNA in globus pallidus was significantly decreased in DARPP-32 and DARPP-32/I-1 knockout mice. No changes in dopamine receptor-induced gene expression were found in I-1 knockout mice. These results demonstrate an important involvement of DARPP-32 in dopamine receptor-mediated regulation of gene expression both in striatal neurons, which are enriched in DARPP-32, and in pallidal neurons, which do not contain DARPP-32.
Topics: Animals; Benzazepines; Caudate Nucleus; Corpus Striatum; Dopamine Agonists; Dopamine Antagonists; Dopamine and cAMP-Regulated Phosphoprotein 32; Gene Expression Regulation; Genes, fos; Globus Pallidus; Mice; Mice, Knockout; Nerve Tissue Proteins; Nucleus Accumbens; Phosphoproteins; Quinolines; RNA, Messenger; Receptor, Adenosine A2A; Receptors, Dopamine D1; Receptors, Dopamine D2; Receptors, Purinergic P1; Transcription, Genetic
PubMed: 10854268
DOI: 10.1046/j.1471-4159.2000.0750248.x -
Molecular Plant Apr 2016While photosynthetic linear electron flow produces both ATP and NADPH, cyclic electron flow (CEF) around photosystem I (PSI) and cytochrome b6f generates only ATP. CEF...
While photosynthetic linear electron flow produces both ATP and NADPH, cyclic electron flow (CEF) around photosystem I (PSI) and cytochrome b6f generates only ATP. CEF is thus essential to balance the supply of ATP and NADPH for carbon fixation; however, it remains unclear how the system tunes the relative levels of linear and cyclic flow. Here, we show that PETO, a transmembrane thylakoid phosphoprotein specific of green algae, contributes to the stimulation of CEF when cells are placed in anoxia. In oxic conditions, PETO co-fractionates with other thylakoid proteins involved in CEF (ANR1, PGRL1, FNR). In PETO-knockdown strains, interactions between these CEF proteins are affected. Anoxia triggers a reorganization of the membrane, so that a subpopulation of PSI and cytochrome b6f now co-fractionates with the CEF effectors in sucrose gradients. The absence of PETO impairs this reorganization. Affinity purification identifies ANR1 as a major interactant of PETO. ANR1 contains two ANR domains, which are also found in the N-terminal region of NdhS, the ferredoxin-binding subunit of the plant ferredoxin-plastoquinone oxidoreductase (NDH). We propose that the ANR domain was co-opted by two unrelated CEF systems (PGR and NDH), possibly as a sensor of the redox state of the membrane.
Topics: Chlamydomonas; Electron Transport; Gene Knockdown Techniques; Oxygen; Phosphoproteins; Plant Proteins; Protein Binding; Thylakoids
PubMed: 26768121
DOI: 10.1016/j.molp.2015.12.017