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MicrobiologyOpen Aug 2021The enormous complexity of the eukaryotic ribosome has been a real challenge in unlocking the mechanistic aspects of its amazing molecular function during mRNA...
The enormous complexity of the eukaryotic ribosome has been a real challenge in unlocking the mechanistic aspects of its amazing molecular function during mRNA translation and many non-canonical activities of ribosomal proteins in eukaryotic cells. While exploring the uncanny nature of ribosomal P proteins in malaria parasites Plasmodium falciparum, the 60S stalk ribosomal P2 protein has been shown to get exported to the infected erythrocyte (IE) surface as an SDS-resistant oligomer during the early to the mid-trophozoite stage. Inhibiting IE surface P2 either by monoclonal antibody or through genetic knockdown resulted in nuclear division arrest of the parasite. This strange and serendipitous finding has led us to explore more about un-canonical cell biology and the structural involvement of P2 protein in Plasmodium in the search for a novel biochemical role during parasite propagation in the human host.
Topics: Cell Division; Erythrocytes; Humans; Malaria, Falciparum; Membrane Proteins; Phosphoproteins; Plasmodium falciparum; Protein Transport; Ribosomal Proteins; Ribosomes
PubMed: 34459544
DOI: 10.1002/mbo3.1188 -
Bone Feb 2017Dentin phosphoprotein (DPP) is the most acidic protein in vertebrates and structurally is classified as an intrinsically disordered protein. Functionally, DPP is related...
Dentin phosphoprotein (DPP) is the most acidic protein in vertebrates and structurally is classified as an intrinsically disordered protein. Functionally, DPP is related to dentin and bone formation, however the specifics of such association remain unknown. Here, we used atomistic molecular dynamics simulations to screen selected binding domains of DPP onto hydroxyapatite (HA), which is one of its important interacting partners. From these results, we selected a functionally relevant peptide, Ace-SSDSSDSSDSSDSSD-NH2 (named P5) and its phosphorylated form (named P5P), for experimental characterization. SAXS experiments indicated that in solution P5 was disordered, possibly in an extended conformation while P5P displayed more compact globular conformations. Circular dichroism and FTIR confirmed that, either in the presence or absence of Ca/HA, P5 adopts a random coil structure, whereas its phosphorylated counterpart, P5P, has a more compact arrangement associated with conformations that display β-sheet and α-helix motifs when bound to HA. In solution, P5 inhibited HA crystal growth, whereas at similar concentrations, P5P stimulated it. These findings suggest that phosphorylation controls the transient formation of secondary and tertiary structure of DPP peptides, and, most likely of DPP itself, which in turn controls HA growth in solution and possibly HA growth in mineralized tissues.
Topics: Amino Acid Sequence; Calcification, Physiologic; Circular Dichroism; Durapatite; Extracellular Matrix Proteins; Molecular Dynamics Simulation; Peptides; Phosphoproteins; Phosphorylation; Protein Structure, Secondary; Scattering, Small Angle; Sialoglycoproteins; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction
PubMed: 27810285
DOI: 10.1016/j.bone.2016.10.028 -
PLoS Pathogens Dec 2020Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition.
Topics: COVID-19; Humans; Magnetic Resonance Spectroscopy; Molecular Docking Simulation; Nucleocapsid Proteins; Phosphoproteins; RNA, Viral; SARS-CoV-2
PubMed: 33264373
DOI: 10.1371/journal.ppat.1009100 -
Nature Jul 2009Reversible phosphorylation on serine, threonine and tyrosine is the most widely studied posttranslational modification of proteins. The number of phosphorylated sites on...
Reversible phosphorylation on serine, threonine and tyrosine is the most widely studied posttranslational modification of proteins. The number of phosphorylated sites on a protein (n) shows a significant increase from prokaryotes, with n = 7 sites, to eukaryotes, with examples having n >/= 150 sites. Multisite phosphorylation has many roles and site conservation indicates that increasing numbers of sites cannot be due merely to promiscuous phosphorylation. A substrate with n sites has an exponential number (2(n)) of phospho-forms and individual phospho-forms may have distinct biological effects. The distribution of these phospho-forms and how this distribution is regulated have remained unknown. Here we show that, when kinase and phosphatase act in opposition on a multisite substrate, the system can exhibit distinct stable phospho-form distributions at steady state and that the maximum number of such distributions increases with n. Whereas some stable distributions are focused on a single phospho-form, others are more diffuse, giving the phospho-proteome the potential to behave as a fluid regulatory network able to encode information and flexibly respond to varying demands. Such plasticity may underlie complex information processing in eukaryotic cells and suggests a functional advantage in having many sites. Our results follow from the unusual geometry of the steady-state phospho-form concentrations, which we show to constitute a rational algebraic curve, irrespective of n. We thereby reduce the complexity of calculating steady states from simulating 3 x 2(n) differential equations to solving two algebraic equations, while treating parameters symbolically. We anticipate that these methods can be extended to systems with multiple substrates and multiple enzymes catalysing different modifications, as found in posttranslational modification 'codes' such as the histone code. Whereas simulations struggle with exponentially increasing molecular complexity, mathematical methods of the kind developed here can provide a new language in which to articulate the principles of cellular information processing.
Topics: Eukaryotic Cells; Kinetics; Mathematics; Models, Biological; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Kinases
PubMed: 19536158
DOI: 10.1038/nature08102 -
Scientific Reports Nov 2017The phosphoprotein (P) is the main and essential cofactor of the RNA polymerase (L) of non-segmented, negative-strand RNA viruses. P positions the viral polymerase onto...
The phosphoprotein (P) is the main and essential cofactor of the RNA polymerase (L) of non-segmented, negative-strand RNA viruses. P positions the viral polymerase onto its nucleoprotein-RNA template and acts as a chaperone of the nucleoprotein (N), thereby preventing nonspecific encapsidation of cellular RNAs. The phosphoprotein of human metapneumovirus (HMPV) forms homotetramers composed of a stable oligomerization domain (P) flanked by large intrinsically disordered regions (IDRs). Here we combined x-ray crystallography of P with small angle x-ray scattering (SAXS)-based ensemble modeling of the full-length P protein and several of its fragments to provide a structural description of P that captures its dynamic character, and highlights the presence of varyingly stable structural elements within the IDRs. We discuss the implications of the structural properties of HMPV P for the assembly and functioning of the viral transcription/replication machinery.
Topics: DNA-Directed RNA Polymerases; Humans; Metapneumovirus; Nucleoproteins; Phosphoproteins; Protein Stability; Scattering, Small Angle; Viral Proteins; Virus Replication; X-Ray Diffraction
PubMed: 29093501
DOI: 10.1038/s41598-017-14448-z -
Neuroreport Jul 2012MicroRNAs are important in the development, functioning, and pathophysiology of the central nervous system. Here, we show that increasing the levels of microRNA-320...
MicroRNAs are important in the development, functioning, and pathophysiology of the central nervous system. Here, we show that increasing the levels of microRNA-320 (miR-320) for 3 days markedly increases neurite length, and at 4 days, reduces the total cell number in Neuro-2A cells. In-silico analysis of possible miR-320 targets identified cAMP-regulated phosphoprotein-19 kDa (ARPP-19) and semaphorin 3a as potential targets that could be involved. ARPP-19 was validated by showing reduced mRNA and protein levels when miR-320 was overexpressed, whereas miR-320 had no effect on semaphorin 3a expression. ARPP-19 is known to inhibit protein phosphatase-2A activity, which inhibits mitosis and induces neurite outgrowth, making this the likely mechanism. Thus, increased levels of miR-320 lead to decreased levels of ARPP-19, increased neurite length, and fewer total cells. These data suggest that miR-320 could play a role in neuronal development and might be a target to enhance neuronal regeneration following injury.
Topics: Animals; Cell Count; Cell Death; Cell Line, Tumor; Gene Targeting; Mice; MicroRNAs; Neurites; Phosphoproteins
PubMed: 22617447
DOI: 10.1097/WNR.0b013e3283540394 -
Virology Aug 1996Autographa californica nuclear polyhedrosis virus (AcMNPV) replicates in the nucleus and produces a viral-modified form of the nuclear matrix called the virogenic...
Autographa californica nuclear polyhedrosis virus (AcMNPV) replicates in the nucleus and produces a viral-modified form of the nuclear matrix called the virogenic stroma. The virogenic stroma is the site of viral DNA packaging and nucleocapsid assembly and is thought to be the site of viral DNA replication and RNA transcription. AcMNPV encodes a phosphoprotein, pp31, which localizes to the nucleus of uninfected insect cells and to the virogenic stroma of infected insect cells. pp31 has DNA binding activity and has been identified as a late expression factor. Thus, the intracellular location of pp31, its DNA binding activity, and its identification as a late transcription factor suggest that it participates in replicative events that occur in the virogenic stroma during AcMNPV infection. The purpose of this study was to map the pp31 domains needed for nuclear localization, virogenic stroma localization, and DNA binding. We focused on four basic amino acid regions (BRs 1-4) and used site-directed mutagenesis and gene fusion techniques to probe their functions. The amino-terminal basic region (BR1) was most important for nuclear localization of pp31 in uninfected cells. Three of the four BRs were needed to efficiently localize pp31 to the nucleus and virogenic stroma in infected cells. BR3 was identified as the DNA binding domain of pp31. These data indicated that BR1, BR3, and BR4 are important functional or multifunctional domains within the AcMNPV pp31 protein.
Topics: Animals; Binding Sites; Biological Transport; Cell Line; Cell Nucleus; DNA; DNA-Binding Proteins; Nucleopolyhedroviruses; Phosphoproteins; Protein Binding; Recombinant Fusion Proteins; Spodoptera
PubMed: 8806516
DOI: 10.1006/viro.1996.0429 -
International Journal of Molecular... Dec 2014The cAMP-regulated phosphoprotein 19 (ARPP-19) plays a key role in cell mitotic G2/M transition. Expression of ARPP-19 was increased in human hepatocellular carcinoma...
The cAMP-regulated phosphoprotein 19 (ARPP-19) plays a key role in cell mitotic G2/M transition. Expression of ARPP-19 was increased in human hepatocellular carcinoma (HCC) compared to adjacent non-tumorous liver tissues in 36 paired liver samples, and the level of ARPP-19 in HCC tissues was positively correlated with the tumor size. To determine the interrelationship between ARPP-19 expression and HCC, we silenced ARPP-19 expression in the human hepatocarcinoma HepG2 and SMMC-7721 cells using lentivirus encoding ARPP-19 siRNA. HepG2 and SMMC-7721 cells with ARPP-19 knockdown displayed lowered cell growth rate, retarded colony formation and increased arrest at the G2/M phase transition. Silencing ARPP-19 in HCC cells resulted in decreased protein levels of phospho-(Ser) CDKs substrates and increased levels of inactivated cyclin division cycle 2 (Cdc2). Therefore, ARPP-19 may play a role in HCC pathogenesis through regulating cell proliferation.
Topics: Adult; Aged; Aged, 80 and over; CDC2 Protein Kinase; Carcinoma, Hepatocellular; Case-Control Studies; Cell Proliferation; Cyclin-Dependent Kinases; Female; Hep G2 Cells; Humans; Liver Neoplasms; Male; Middle Aged; Phosphoproteins; Up-Regulation
PubMed: 25547487
DOI: 10.3390/ijms16010178 -
Analytical Chemistry Apr 2018Protein phosphorylation is a ubiquitous and critical post-translational modification (PTM) involved in numerous cellular processes. Mass spectrometry (MS)-based...
Protein phosphorylation is a ubiquitous and critical post-translational modification (PTM) involved in numerous cellular processes. Mass spectrometry (MS)-based proteomics has emerged as the preferred technology for protein identification, characterization, and quantification. Whereas ionization/detection efficiency of peptides in electrospray ionization (ESI)-MS are markedly influenced by the presence of phosphorylation, the physicochemical properties of intact proteins are assumed not to vary significantly due to the relatively smaller modification on large intact proteins. Thus, the ionization/detection efficiency of intact phosphoprotein is hypothesized not to alter appreciably for subsequent MS quantification. However, this hypothesis has never been rigorously tested. Herein, we systematically investigated the impact of phosphorylation on ESI-MS quantification of mono- and multiply phosphorylated proteins. We verified that a single phosphorylation did not appreciably affect the ESI-MS quantification of phosphoproteins as demonstrated in the enigma homolog isoform 2 (28 kDa) with monophosphorylation. Moreover, different ionization and desolvation parameters did not impact phosphoprotein quantification. In contrast to monophosphorylation, multiphosphorylation noticeably affected ESI-MS quantification of phosphoproteins likely due to differential ionization/detection efficiency between unphosphorylated and phosphorylated proteoforms as shown in the pentakis-phosphorylated β-casein (24 kDa).
Topics: Adaptor Proteins, Signal Transducing; Caseins; Chromatography, High Pressure Liquid; Humans; LIM Domain Proteins; Phosphopeptides; Phosphoproteins; Phosphorylation; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 29565561
DOI: 10.1021/acs.analchem.7b05246 -
Cell Jun 2000
Review
Topics: Animals; Cell Adhesion Molecules; Cell Movement; DNA-Binding Proteins; Humans; Microfilament Proteins; Phosphoproteins
PubMed: 10892738
DOI: 10.1016/s0092-8674(00)80879-x