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The Journal of Biological Chemistry Jun 1994Protein P0 in Saccharomyces cerevisiae is found only in the ribosomes and not free in a cytoplasmic pool like the structurally related acidic P1 and P2 proteins.... (Comparative Study)
Comparative Study
Protein P0 in Saccharomyces cerevisiae is found only in the ribosomes and not free in a cytoplasmic pool like the structurally related acidic P1 and P2 proteins. Analogously, P0 stays bound to the particles in conditions that release the other P proteins. Attempts to obtain yeast strains carrying an interrupted P0 gene by direct gene disruption techniques of different yeast strains always resulted in haploid cells carrying one disrupted and one intact copy of the gene. Disruption of the unique P0 genomic copy seems to induce a duplication and occasionally a chromosomal transposition of the gene. Conditional null mutants of P0 were then constructed carrying the P0 gene under the control of the inducible GAL1 promoter. A 2-3-fold excess of P0 mRNA is found in the conditional mutant when grown in galactose; however, only a small increase of the P0 protein is detected in total cell extracts. No P0 protein is detected in the cell supernatant, indicating that, like the standard ribosomal proteins and opposite to the other P proteins, the protein not bound to the ribosomes is degraded. Transfer of the mutants to the restrictive conditions causes, after some generations, a growth stop that finally leads to cell death. The growth decline is paralleled by a reduction in the polysome number and the appearance of half-mer particles as well as by an accumulation of 60 S particles deficient in P0 and in the acidic proteins P1 and P2. These results indicate that P0 is required for the interaction of the acidic P1 and P2 proteins with the ribosomes, and in its absence, deficient 60 S ribosomes are assembled which are inactive in protein synthesis resulting in cell lethality.
Topics: Blotting, Southern; DNA, Fungal; Escherichia coli; Genes, Fungal; Immunoblotting; Kinetics; Mutation; Phosphoproteins; Plasmids; Restriction Mapping; Ribosomal Proteins; Ribosomes; Saccharomyces cerevisiae; Transformation, Genetic
PubMed: 8195220
DOI: No ID Found -
Viruses Dec 2023Australian bat lyssavirus (ABLV) shows similar clinical symptoms as rabies, but there are currently no protein structures available for ABLV proteins. In lyssaviruses,...
Australian bat lyssavirus (ABLV) shows similar clinical symptoms as rabies, but there are currently no protein structures available for ABLV proteins. In lyssaviruses, the interaction between nucleoprotein (N) and phosphoprotein (N) in the absence of RNA generates a complex (NP) that is crucial for viral assembly, and understanding the interface between these two proteins has the potential to provide insight into a key feature: the viral lifecycle. In this study, we used recombinant chimeric protein expression and X-ray crystallography to determine the structure of ABLV nucleoprotein bound to residues 1-40 of its phosphoprotein chaperone. Comparison of our results with the recently generated structure of RABV CVS-11 NP demonstrated a highly conserved interface in this complex. Because the NP interface is conserved in the lyssaviruses of phylogroup I, it is an attractive therapeutic target for multiple rabies-causing viral species.
Topics: Animals; Lyssavirus; Rabies; Nucleoproteins; Chiroptera; Australia; Phosphoproteins; Rhabdoviridae Infections
PubMed: 38229694
DOI: 10.3390/v16010033 -
RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation.Journal of Virology Feb 2017The Flavivirus genus contains several arthropod-borne viruses that pose global health threats, including dengue viruses (DENV), yellow fever virus (YFV), and Zika virus...
UNLABELLED
The Flavivirus genus contains several arthropod-borne viruses that pose global health threats, including dengue viruses (DENV), yellow fever virus (YFV), and Zika virus (ZIKV). In order to understand how these viruses replicate in human cells, we previously conducted genome-scale RNA interference screens to identify candidate host factors. In these screens, we identified ribosomal proteins RPLP1 and RPLP2 (RPLP1/2) to be among the most crucial putative host factors required for DENV and YFV infection. RPLP1/2 are phosphoproteins that bind the ribosome through interaction with another ribosomal protein, RPLP0, to form a structure termed the ribosomal stalk. RPLP1/2 were validated as essential host factors for DENV, YFV, and ZIKV infection in two human cell lines: A549 lung adenocarcinoma and HuH-7 hepatoma cells, and for productive DENV infection of Aedes aegypti mosquitoes. Depletion of RPLP1/2 caused moderate cell-line-specific effects on global protein synthesis, as determined by metabolic labeling. In A549 cells, global translation was increased, while in HuH-7 cells it was reduced, albeit both of these effects were modest. In contrast, RPLP1/2 knockdown strongly reduced early DENV protein accumulation, suggesting a requirement for RPLP1/2 in viral translation. Furthermore, knockdown of RPLP1/2 reduced levels of DENV structural proteins expressed from an exogenous transgene. We postulate that these ribosomal proteins are required for efficient translation elongation through the viral open reading frame. In summary, this work identifies RPLP1/2 as critical flaviviral host factors required for translation.
IMPORTANCE
Flaviviruses cause important diseases in humans. Examples of mosquito-transmitted flaviviruses include dengue, yellow fever and Zika viruses. Viruses require a plethora of cellular factors to infect cells, and the ribosome plays an essential role in all viral infections. The ribosome is a complex macromolecular machine composed of RNA and proteins and it is responsible for protein synthesis. We identified two specific ribosomal proteins that are strictly required for flavivirus infection of human cells and mosquitoes: RPLP1 and RPLP2 (RPLP1/2). These proteins are part of a structure known as the ribosomal stalk and help orchestrate the elongation phase of translation. We show that flaviviruses are particularly dependent on the function of RPLP1/2. Our findings suggest that ribosome composition is an important factor for virus translation and may represent a regulatory layer for translation of specific cellular mRNAs.
Topics: Aedes; Animals; Cell Line; Dengue Virus; Flavivirus; Flavivirus Infections; Gene Expression; Gene Knockdown Techniques; Host-Pathogen Interactions; Humans; Phosphoproteins; Protein Binding; Protein Multimerization; Ribosomal Proteins; Viral Proteins; Virus Replication; Yellow fever virus
PubMed: 27974556
DOI: 10.1128/JVI.01706-16 -
Virologica Sinica Jun 2021Human parainfluenza virus type 3 (HPIV3), a member of the Paramyxoviridae family, can cause lower respiratory disease in infants and young children. The phosphoprotein...
Human parainfluenza virus type 3 (HPIV3), a member of the Paramyxoviridae family, can cause lower respiratory disease in infants and young children. The phosphoprotein (P) of HPIV3 is an essential cofactor of the viral RNA-dependent RNA polymerase large protein (L). P connects nucleocapsid protein (N) with L to initiate genome transcription and replication. Sumoylation influences many important pathways of the target proteins, and many viral proteins are also themselves sumoylated. In this study, we found that the P of HPIV3 could be sumoylated, and mutation of K492 and K532 to arginine (P) failed to be sumoylated within P, which enhances HPIV3 minigenome activity. Biochemical studies showed that P had no effect on its interactions with N, formation of homo-tetramers and formation of inclusion bodies. Finally, we found that incorporation of K492R/K532R into a recombinant HPIV3 (rHPIV3-P) increased viral production in culture cells, suggesting that sumoylation attenuates functions of P and down-regulates viral replication.
Topics: Child, Preschool; HEK293 Cells; HeLa Cells; Humans; Parainfluenza Virus 3, Human; Phosphoproteins; Sumoylation; Virus Replication
PubMed: 33197004
DOI: 10.1007/s12250-020-00314-2 -
PloS One 2013Perilipin-1 (Plin1), a prominent cytoplasmic lipid droplet (CLD) binding phosphoprotein and key physiological regulator of triglyceride storage and lipolysis in...
Perilipin-1 (Plin1), a prominent cytoplasmic lipid droplet (CLD) binding phosphoprotein and key physiological regulator of triglyceride storage and lipolysis in adipocytes, is thought to regulate the fragmentation and dispersion of CLD that occurs in response to β-adrenergic activation of adenylate cyclase. Here we investigate the dynamics and molecular determinants of these processes using cell lines stably expressing recombinant forms of Plin1 and/or other members of the perilipin family. Plin1 and a C-terminal CLD-binding fragment of Plin1 (Plin1CT) induced formation of single dense CLD clusters near the microtubule organizing center, whereas neither an N-terminal CLD-binding fragment of Plin1, nor Plin2 or Plin3 induced clustering. Clustered CLD coated by Plin1, or Plin1CT, dispersed in response to isoproterenol, or other agents that activate adenylate cyclase, in a process inhibited by the protein kinase A inhibitor, H89, and blocked by microtubule disruption. Isoproterenol-stimulated phosphorylation of CLD-associated Plin1 on serine 492 preceded their dispersion, and live cell imaging showed that cluster dispersion involved initial fragmentation of tight clusters into multiple smaller clusters, which then fragmented into well-dispersed individual CLD. siRNA knockdown of the cortical actin binding protein, moesin, induced disaggregation of tight clusters into multiple smaller clusters, and inhibited the reaggregation of dispersed CLD into tight clusters. Together these data suggest that the clustering and dispersion processes involve a complex orchestration of phosphorylation-dependent, microtubule-dependent and independent, and microfilament dependent steps.
Topics: Animals; Cytoplasm; HEK293 Cells; Humans; Isoproterenol; Lipid Metabolism; Mice; Microfilament Proteins; Microtubules; Mutation; Organelles; Phosphoproteins; Phosphorylation
PubMed: 23825572
DOI: 10.1371/journal.pone.0066837 -
The Journal of Biological Chemistry Mar 2002FAP52, a focal adhesion-associated phosphoprotein, is a member of a FAP52/PACSIN/syndapin family of proteins. They share a multidomain structure and are implicated in...
FAP52, a focal adhesion-associated phosphoprotein, is a member of a FAP52/PACSIN/syndapin family of proteins. They share a multidomain structure and are implicated in actin-based and endocytotic functions. We show, by using both native and recombinant proteins, that FAP52 selectively binds to the actin cross-linking protein filamin (ABP-280). This was based on an affinity purification followed by a sequence determination by mass spectrometry, co-immunoprecipitation, overlay binding, and surface plasmon resonance analysis. Binding studies with deletion mutants showed that the sites of the interaction map to the highly alpha-helical N-terminal part of FAP52 and to the C-terminal region of filamin, which also contains binding sites to some transmembrane signaling proteins. In immunofluorescence and immunoelectron microscopy of cultured fibroblasts, a different overall subcellular distribution was seen for filamin and FAP52 except for a stress fiber-focal adhesion junction where they showed a notable overlap. Overexpression of the full-length and mutant forms of FAP52 led to an extensive reorganization of actin and filamin in cultured fibroblasts. Thus, the results show that FAP52 interacts with filamin, and we propose that this interaction is important in linking and coordinating the events between focal adhesions and the actin cytoskeleton.
Topics: Actins; Animals; Cells, Cultured; Chick Embryo; Chromatography, Affinity; Contractile Proteins; Filamins; Focal Adhesions; Microfilament Proteins; Microscopy, Fluorescence; Microscopy, Immunoelectron; Mutagenesis; Phosphoproteins; Protein Binding; Recombinant Proteins; Spectrometry, Mass, Electrospray Ionization; Surface Plasmon Resonance
PubMed: 11790794
DOI: 10.1074/jbc.M111753200 -
Microbiology Spectrum Feb 2023Avian metapneumovirus subgroup C (aMPV/C) is an important pathogen that causes upper respiratory symptoms and egg production decline in turkeys and chickens. aMPV/C...
Avian metapneumovirus subgroup C (aMPV/C) is an important pathogen that causes upper respiratory symptoms and egg production decline in turkeys and chickens. aMPV/C infection leads to inhibition of the host antiviral immune response. However, our understanding of the molecular mechanisms underlying host immune response antagonized by aMPV/C infection is limited. In this study, we demonstrated that the aMPV/C phosphoprotein (P) inhibits the IFN antiviral signaling pathway triggered by melanoma differentiation gene 5 (MDA5) and reduces interferon β (IFN-β) production and IFN-stimulated genes (ISGs) by targeting IFN regulatory factor 7 (IRF7) but not nuclear factor κB (NF-κB) in DF-1 cells. Moreover, we found that aMPV/C P protein only blocks the nuclear translocation of IRF3 by interacting with IRF3 in HEK-293T cells, instead of affecting IRF3 phosphorylation and inducing IRF3 degradation, which suppresses IRF3 signaling activation and results in a decrease in IFN-β production. Collectively, these results reveal a novel mechanism by which aMPV/C infection disrupts IFN-β production in the host. The innate immune response is the first defense line of host cells and organisms against viral infections. When RNA viruses infect cells, viral RNA induces activation of retinoic acid-induced gene I and melanoma differentiation gene 5, which initiates downstream molecules and finally produces type I interferon (IFN-I) to regulate antiviral immune responses. The mechanism for avian metapneumovirus (aMPV) modulating IFN-I production to benefit its replication remains unknown. Here, we demonstrate that phosphoprotein of aMPV subgroup C (aMPV/C) selectively inhibits the nuclear translocation of interferon regulatory 3 (IRF3), instead of affecting the expression and phosphorylation of IRF3, which finally downregulates IFN-I production. This study showed a novel mechanism for aMPV/C infection antagonizing the host IFN response.
Topics: Animals; Chickens; Host-Pathogen Interactions; Interferon Regulatory Factor-3; Interferon Type I; Interferon-beta; Metapneumovirus; Phosphoproteins; Viral Proteins
PubMed: 36537793
DOI: 10.1128/spectrum.03413-22 -
PloS One Jul 2010Henipaviruses are newly emerged viruses within the Paramyxoviridae family. Their negative-strand RNA genome is packaged by the nucleoprotein (N) within alpha-helical...
Henipaviruses are newly emerged viruses within the Paramyxoviridae family. Their negative-strand RNA genome is packaged by the nucleoprotein (N) within alpha-helical nucleocapsid that recruits the polymerase complex made of the L protein and the phosphoprotein (P). To date structural data on Henipaviruses are scarce, and their N and P proteins have never been characterized so far. Using both computational and experimental approaches we herein show that Henipaviruses N and P proteins possess large intrinsically disordered regions. By combining several disorder prediction methods, we show that the N-terminal domain of P (PNT) and the C-terminal domain of N (NTAIL) are both mostly disordered, although they contain short order-prone segments. We then report the cloning, the bacterial expression, purification and characterization of Henipavirus PNT and NTAIL domains. By combining gel filtration, dynamic light scattering, circular dichroism and nuclear magnetic resonance, we show that both NTAIL and PNT belong to the premolten globule sub-family within the class of intrinsically disordered proteins. This study is the first reported experimental characterization of Henipavirus P and N proteins. The evidence that their respective N-terminal and C-terminal domains are highly disordered under native conditions is expected to be invaluable for future structural studies by helping to delineate N and P protein domains amenable to crystallization. In addition, following previous hints establishing a relationship between structural disorder and protein interactivity, the present results suggest that Henipavirus PNT and NTAIL domains could be involved in manifold protein-protein interactions.
Topics: Chromatography, Gel; Circular Dichroism; Henipavirus; Magnetic Resonance Spectroscopy; Nucleocapsid Proteins; Phosphoproteins; Viral Proteins
PubMed: 20657787
DOI: 10.1371/journal.pone.0011684 -
Bio-medical Materials and Engineering 2012The biochemical mechanism behind the strong binding between titanium and living bone has not been fully elucidated, in spite of worldwide clinical application of this...
The biochemical mechanism behind the strong binding between titanium and living bone has not been fully elucidated, in spite of worldwide clinical application of this phenomenon. We hypothesized that one of the core mechanisms may reside in the interaction between certain proteins in the host tissues and the implanted titanium. To verify the interaction between titanium and proteins, we chose the technique of chromatography in that titanium spherical beads (45 μm) were packed into a column to obtain a bed volume of 16×50 mm, which was eluted with phosphate buffered saline (PBS) and a straight gradient system made by using PBS and 25 mM NaOH. Fetal calf serum, albumin, lysozyme, casein, phosvitin and dentin phosphoprotein (phosphophoryn) were applied to the column. Most part of albumin and lysozyme eluted with the breakthrough peak, indicating practically no affinity to titanium. Fetal bovine serum also eluted mostly as the breakthrough peak, but distinct retained peak was observed. On the other hand, α-casein, phosvitin and phosphophoryn exhibited a distinct retained peak separated from the breakthrough peak. We proposed that phosphate groups (phosphoserines) in the major phosphoproteins, α-casein, phosvitin and phosphophoryn may be involved in the binding of these proteins with titanium.
Topics: Animals; Caseins; Cattle; Chromatography; Molecular Weight; Muramidase; Phosphates; Phosphoproteins; Phosvitin; Protein Binding; Serum Albumin; Titanium
PubMed: 23023145
DOI: 10.3233/BME-2012-0718 -
PloS One 2014The phosphoprotein (P) gene of most Paramyxovirinae encodes several proteins in overlapping frames: P and V, which share a common N-terminus (PNT), and C, which overlaps...
The phosphoprotein (P) gene of most Paramyxovirinae encodes several proteins in overlapping frames: P and V, which share a common N-terminus (PNT), and C, which overlaps PNT. Overlapping genes are of particular interest because they encode proteins originated de novo, some of which have unknown structural folds, challenging the notion that nature utilizes only a limited, well-mapped area of fold space. The C proteins cluster in three groups, comprising measles, Nipah, and Sendai virus. We predicted that all C proteins have a similar organization: a variable, disordered N-terminus and a conserved, α-helical C-terminus. We confirmed this predicted organization by biophysically characterizing recombinant C proteins from Tupaia paramyxovirus (measles group) and human parainfluenza virus 1 (Sendai group). We also found that the C of the measles and Nipah groups have statistically significant sequence similarity, indicating a common origin. Although the C of the Sendai group lack sequence similarity with them, we speculate that they also have a common origin, given their similar genomic location and structural organization. Since C is dispensable for viral replication, unlike PNT, we hypothesize that C may have originated de novo by overprinting PNT in the ancestor of Paramyxovirinae. Intriguingly, in measles virus and Nipah virus, PNT encodes STAT1-binding sites that overlap different regions of the C-terminus of C, indicating they have probably originated independently. This arrangement, in which the same genetic region encodes simultaneously a crucial functional motif (a STAT1-binding site) and a highly constrained region (the C-terminus of C), seems paradoxical, since it should severely reduce the ability of the virus to adapt. The fact that it originated twice suggests that it must be balanced by an evolutionary advantage, perhaps from reducing the size of the genetic region vulnerable to mutations.
Topics: Amino Acid Sequence; Binding Sites; Evolution, Molecular; Molecular Sequence Data; Paramyxovirinae; Phosphoproteins; STAT1 Transcription Factor; Sequence Alignment; Sequence Analysis; Sequence Homology, Nucleic Acid; Species Specificity; Viral Proteins
PubMed: 24587180
DOI: 10.1371/journal.pone.0090003