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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 -
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 -
Antiviral Research Mar 2014The nucleocapsid phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV N protein) packages the viral genome into a helical ribonucleocapsid (RNP)... (Review)
Review
The nucleocapsid phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV N protein) packages the viral genome into a helical ribonucleocapsid (RNP) and plays a fundamental role during viral self-assembly. It is a protein with multifarious activities. In this article we will review our current understanding of the N protein structure and its interaction with nucleic acid. Highlights of the progresses include uncovering the modular organization, determining the structures of the structural domains, realizing the roles of protein disorder in protein-protein and protein-nucleic acid interactions, and visualizing the ribonucleoprotein (RNP) structure inside the virions. It was also demonstrated that N-protein binds to nucleic acid at multiple sites with a coupled-allostery manner. We propose a SARS-CoV RNP model that conforms to existing data and bears resemblance to the existing RNP structures of RNA viruses. The model highlights the critical role of modular organization and intrinsic disorder of the N protein in the formation and functions of the dynamic RNP capsid in RNA viruses. This paper forms part of a symposium in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses."
Topics: Coronavirus Nucleocapsid Proteins; Humans; Macromolecular Substances; Models, Biological; Models, Molecular; Nucleocapsid Proteins; Phosphoproteins; Protein Binding; Protein Conformation; RNA, Viral; Severe acute respiratory syndrome-related coronavirus; Virus Assembly
PubMed: 24418573
DOI: 10.1016/j.antiviral.2013.12.009 -
Viruses Jan 2022Particles of many paramyxoviruses include small amounts of proteins with a molecular weight of about 20 kDa. These proteins, termed "C", are basic, have low amino acid... (Review)
Review
Particles of many paramyxoviruses include small amounts of proteins with a molecular weight of about 20 kDa. These proteins, termed "C", are basic, have low amino acid homology and some secondary structure conservation. C proteins are encoded in alternative reading frames of the phosphoprotein gene. Some viruses express nested sets of C proteins that exert their functions in different locations: In the nucleus, they interfere with cellular transcription factors that elicit innate immune responses; in the cytoplasm, they associate with viral ribonucleocapsids and control polymerase processivity and orderly replication, thereby minimizing the activation of innate immunity. In addition, certain C proteins can directly bind to, and interfere with the function of, several cytoplasmic proteins required for interferon induction, interferon signaling and inflammation. Some C proteins are also required for efficient virus particle assembly and budding. C-deficient viruses can be grown in certain transformed cell lines but are not pathogenic in natural hosts. C proteins affect the same host functions as other phosphoprotein gene-encoded proteins named V but use different strategies for this purpose. Multiple independent systems to counteract host defenses may ensure efficient immune evasion and facilitate virus adaptation to new hosts and tissue environments.
Topics: Animals; Defective Interfering Viruses; Genome, Viral; Humans; Immune Evasion; Immunity, Innate; Inflammasomes; Open Reading Frames; Paramyxoviridae Infections; Paramyxovirinae; Phosphoproteins; Phylogeny; Viral Proteins; Virus Assembly; Virus Replication
PubMed: 35062341
DOI: 10.3390/v14010137