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Cell Apr 2023Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases....
Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases. Here, we uncover how cellular stress disrupts the metastable host-virus equilibrium in persistent infection and induces viral replication in a culture model of mumps virus. Using a combination of cell biology, whole-cell proteomics, and cryo-electron tomography, we show that persistent viral replication factories are dynamic condensates and identify the largely disordered viral phosphoprotein as a driver of their assembly. Upon stress, increased phosphorylation of the phosphoprotein at its interaction interface with the viral polymerase coincides with the formation of a stable replication complex. By obtaining atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events constitute a stress-mediated switch within viral condensates that provide an environment to support upregulation of viral replication.
Topics: Humans; Mumps virus; Nucleocapsid; Persistent Infection; Phosphoproteins; Virus Replication
PubMed: 37116470
DOI: 10.1016/j.cell.2023.03.015 -
Journal of Proteome Research Jul 2008A significant consequence of protein phosphorylation is to alter protein-protein interactions, leading to dynamic regulation of the components of protein complexes that...
A significant consequence of protein phosphorylation is to alter protein-protein interactions, leading to dynamic regulation of the components of protein complexes that direct many core biological processes. Recent proteomic studies have populated databases with extensive compilations of cellular phosphoproteins and phosphorylation sites and a similarly deep coverage of the subunit compositions and interactions in multiprotein complexes. However, considerably less data are available on the dynamics of phosphorylation, composition of multiprotein complexes or that define their interdependence. We describe a method to identify candidate phosphoprotein complexes by combining phosphoprotein affinity chromatography, separation by size, denaturing gel electrophoresis, protein identification by tandem mass spectrometry, and informatics analysis. Toward developing phosphoproteome profiling, we have isolated native phosphoproteins using a phosphoprotein affinity matrix, Pro-Q Diamond resin (Molecular Probes-Invitrogen). This resin quantitatively retains phosphoproteins and associated proteins from cell extracts. Pro-Q Diamond purification of a yeast whole cell extract followed by 1-D PAGE separation, proteolysis and ESI LC-MS/MS, a method we term PA-GeLC-MS/MS, yielded 108 proteins, a majority of which were known phosphoproteins. To identify proteins that were purified as parts of phosphoprotein complexes, the Pro-Q eluate was separated into two fractions by size, <100 kDa and >100 kDa, before analysis by PAGE and ESI LC-MS/MS and the component proteins queried against databases to identify protein-protein interactions. The <100 kDa fraction was enriched in phosphoproteins indicating the presence of monomeric phosphoproteins. The >100 kDa fraction contained 171 proteins of 20-80 kDa, nearly all of which participate in known protein-protein interactions. Of these 171, few are known phosphoproteins, consistent with their purification by participation in protein complexes. By comparing the results of our phosphoprotein profiling with the informational databases on phosphoproteomics, protein-protein interactions and protein complexes, we have developed an approach to examining the correlation between protein interactions and protein phosphorylation.
Topics: Chromatography, Liquid; Databases, Factual; Fungal Proteins; Humans; K562 Cells; Multiprotein Complexes; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphothreonine; Phosphotyrosine; Proteome; Tandem Mass Spectrometry; Yeasts
PubMed: 18510356
DOI: 10.1021/pr700816k -
Cellular and Molecular Life Sciences :... Sep 2017In this review, we summarize computational and experimental data gathered so far showing that structural disorder is abundant within paramyxoviral nucleoproteins (N) and... (Review)
Review
In this review, we summarize computational and experimental data gathered so far showing that structural disorder is abundant within paramyxoviral nucleoproteins (N) and phosphoproteins (P). In particular, we focus on measles, Nipah, and Hendra viruses and highlight both commonalities and differences with respect to the closely related Sendai virus. The molecular mechanisms that control the disorder-to-order transition undergone by the intrinsically disordered C-terminal domain (N) of their N proteins upon binding to the C-terminal X domain (XD) of the homologous P proteins are described in detail. By having a significant residual disorder, N-XD complexes are illustrative examples of "fuzziness", whose possible functional significance is discussed. Finally, the relevance of N-P interactions as promising targets for innovative antiviral approaches is underscored, and the functional advantages of structural disorder for paramyxoviruses are pinpointed.
Topics: Antiviral Agents; Evolution, Molecular; Intrinsically Disordered Proteins; Molecular Chaperones; Nucleoproteins; Paramyxoviridae; Phosphoproteins; Protein Structure, Quaternary; RNA, Viral; Viral Proteins; Virus Replication
PubMed: 28600653
DOI: 10.1007/s00018-017-2556-3 -
Nature Jan 2024Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation. Mitotic entry is initiated by increased phosphorylation of mitotic...
Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation. Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases, whereas mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55. Although the role of kinases in mitotic entry is well established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited. Inhibition of PP2A:B55 is achieved by the intrinsically disordered proteins ARPP19 and FAM122A. Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the single-particle cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies, both intrinsically disordered proteins bind PP2A:B55, but do so in highly distinct manners, leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provide a molecular roadmap for the development of therapeutic interventions for PP2A:B55-related diseases.
Topics: Humans; Cryoelectron Microscopy; Intracellular Signaling Peptides and Proteins; Intrinsically Disordered Proteins; Mitosis; Nuclear Magnetic Resonance, Biomolecular; Phosphoproteins; Phosphorylation; Protein Phosphatase 2
PubMed: 38123684
DOI: 10.1038/s41586-023-06870-3 -
The Journal of Biological Chemistry Jul 2021More than half a century ago, reversible protein phosphorylation was linked to mitochondrial metabolism through the regulation of pyruvate dehydrogenase. Since this... (Review)
Review
More than half a century ago, reversible protein phosphorylation was linked to mitochondrial metabolism through the regulation of pyruvate dehydrogenase. Since this discovery, the number of identified mitochondrial protein phosphorylation sites has increased by orders of magnitude, driven largely by technological advances in mass spectrometry-based phosphoproteomics. However, the majority of these modifications remain uncharacterized, rendering their function and relevance unclear. Nonetheless, recent studies have shown that disruption of resident mitochondrial protein phosphatases causes substantial metabolic dysfunction across organisms, suggesting that proper management of mitochondrial phosphorylation is vital for organellar and organismal homeostasis. While these data suggest that phosphorylation within mitochondria is of critical importance, significant gaps remain in our knowledge of how these modifications influence organellar function. Here, we curate publicly available datasets to map the extent of protein phosphorylation within mammalian mitochondria and to highlight the known functions of mitochondrial-resident phosphatases. We further propose models by which phosphorylation may affect mitochondrial enzyme activities, protein import and processing, and overall organellar homeostasis.
Topics: Animals; Humans; Mitochondrial Proteins; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Kinases; Proteome
PubMed: 34144036
DOI: 10.1016/j.jbc.2021.100880 -
Cancer Detection and Prevention 2006Nucleophosmin (NPM) is a nucleolar phosphoprotein that shuttles between the nucleus and cytoplasm during the cell cycle. NPM has several interacting partners and diverse... (Review)
Review
Nucleophosmin (NPM) is a nucleolar phosphoprotein that shuttles between the nucleus and cytoplasm during the cell cycle. NPM has several interacting partners and diverse cellular functions, including the processing of ribosomal RNA, centrosome duplication and the control of cellular processes to ensure genomic stability. Subcellular localization of NPM appears to be strongly correlated with NPM functions and cell proliferation. NPM is phosphorylated mainly at its central acidic domain by several upstream kinases, and its phosphorylation appears to be involved in regulating its functions in ribosome biogenesis and centrosome duplication. Recent studies suggest that NPM may act as a licensing factor to maintain proper centrosome duplication and that the Ran/CRM1 nucleocytoplasmic complex regulates local trafficking of NPM to centrosomes by interacting through its nuclear export sequence motif. Here, we provide a brief overview of NPM functions and its roles in human carcinogenesis, and discuss our recent findings related to the potential mechanisms underlying its regulation of centrosome duplication.
Topics: Cell Cycle; Cell Transformation, Neoplastic; Centrosome; Humans; Neoplasms; Nuclear Proteins; Nucleophosmin; Phosphoproteins; Phosphorylation
PubMed: 17113241
DOI: 10.1016/j.cdp.2006.10.008 -
The Journal of Medical Investigation :... 2022Aging induces pathological cardiovascular changes such as cardiac dysfunction and arteriosclerosis. With aging, heart cells, especially, become more susceptible to...
Aging induces pathological cardiovascular changes such as cardiac dysfunction and arteriosclerosis. With aging, heart cells, especially, become more susceptible to lethal damage. In this report, we tried to understand the precise mechanism of myocardial change resulting from aging by examining the heart proteome in aging mice using two-dimensional gel electrophoresis (2DE). The proteins were stained with fluorescence dyes (SYPRO Ruby and Pro-Q Diamond) and identified by subsequent MALDI-TOF-MS / MS. As a result, markedly altered levels of 14 proteins and 7 phosphoproteins were detected in the hearts of 3-, 7-, 11-, and 20-month-old mice. The functions of these identified proteins and phosphoproteins were energy metabolism, muscle contraction, glycolysis, and cytoskeletal support. Additionally, the results of Western blotting confirmed changes in the expression of FTH, CPNE5, and SUCLA2. These findings showed that aging modified the expression of proteins and phosphoproteins in the heart. We suggest that changes in the expression of these proteins are critical to the development of cardiac dysfunction resulting from aging. J. Med. Invest. 69 : 217-223, August, 2022.
Topics: Aging; Animals; Diamond; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Heart Diseases; Mice; Phosphoproteins; Proteome; Proteomics; Tandem Mass Spectrometry
PubMed: 36244772
DOI: 10.2152/jmi.69.217 -
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 -
PLoS Pathogens Dec 2022Rabies virus (RABV) transcription and replication take place within viral factories having liquid properties, called Negri bodies (NBs), that are formed by liquid-liquid...
Rabies virus (RABV) transcription and replication take place within viral factories having liquid properties, called Negri bodies (NBs), that are formed by liquid-liquid phase separation (LLPS). The co-expression of RABV nucleoprotein (N) and phosphoprotein (P) in mammalian cells is sufficient to induce the formation of cytoplasmic biocondensates having properties that are like those of NBs. This cellular minimal system was previously used to identify P domains that are essential for biocondensates formation. Here, we constructed fluorescent versions of N and analyzed by FRAP their dynamics inside the biocondensates formed in this minimal system as well as in NBs of RABV-infected cells using FRAP. The behavior of N appears to be different of P as there was no fluorescence recovery of N proteins after photobleaching. We also identified arginine residues as well as two exposed loops of N involved in condensates formation. Corresponding N mutants exhibited distinct phenotypes in infected cells ranging from co-localization with NBs to exclusion from them associated with a dominant-negative effect on infection. We also demonstrated that in vitro, in crowded environments, purified P as well as purified N0-P complex (in which N is RNA-free) form liquid condensates. We identified P domains required for LLPS in this acellular system. P condensates were shown to associate with liposomes, concentrate RNA, and undergo a liquid-gel transition upon ageing. Conversely, N0-P droplets were disrupted upon incubation with RNA. Taken together, our data emphasize the central role of P in NBs formation and reveal some physicochemical features of P and N0-P droplets relevant for explaining NBs properties such as their envelopment by cellular membranes at late stages of infection and nucleocapsids ejections from the viral factories.
Topics: Animals; Rabies virus; Nucleoproteins; Rabies; Nucleocapsid; Phosphoproteins; Virus Replication; Mammals
PubMed: 36480574
DOI: 10.1371/journal.ppat.1011022 -
Expert Review of Proteomics Aug 2011The response to extracellular stimuli often alters the phosphorylation state of plasma membrane- associated proteins. In this regard, generation of a comprehensive... (Review)
Review
The response to extracellular stimuli often alters the phosphorylation state of plasma membrane- associated proteins. In this regard, generation of a comprehensive membrane phosphoproteome can significantly enhance signal transduction and drug mechanism studies. However, analysis of this subproteome is regarded as technically challenging, given the low abundance and insolubility of integral membrane proteins, combined with difficulties in isolating, ionizing and fragmenting phosphopeptides. In this article, we highlight recent advances in membrane and phosphoprotein enrichment techniques resulting in improved identification of these elusive peptides. We also describe the use of alternative fragmentation techniques, and assess their current and future value to the field of membrane phosphoproteomics.
Topics: Amino Acid Sequence; Animals; Cell Membrane; Humans; Molecular Sequence Data; Phosphopeptides; Phosphoproteins; Phosphorylation; Proteomics; Solubility
PubMed: 21819303
DOI: 10.1586/epr.11.40