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International Journal of Molecular... Jan 2018Intrinsically disordered proteins (IDPs) represent approximately 30% of the human genome and play key roles in cell proliferation and cellular signaling by modulating... (Review)
Review
Intrinsically disordered proteins (IDPs) represent approximately 30% of the human genome and play key roles in cell proliferation and cellular signaling by modulating the function of target proteins via protein-protein interactions. In addition, IDPs are involved in various human disorders, such as cancer, neurodegenerative diseases, and amyloidosis. To understand the underlying molecular mechanism of IDPs, it is important to study their structural features during their interactions with target proteins. However, conventional biochemical and biophysical methods for analyzing proteins, such as X-ray crystallography, have difficulty in characterizing the features of IDPs because they lack an ordered three-dimensional structure. Here, we present biochemical and biophysical studies on nucleolar phosphoprotein 140 (Nopp140), which mostly consists of disordered regions, during its interaction with casein kinase 2 (CK2), which plays a central role in cell growth. Surface plasmon resonance and electron paramagnetic resonance studies were performed to characterize the interaction between Nopp140 and CK2. A single-molecule fluorescence resonance energy transfer study revealed conformational change in Nopp140 during its interaction with CK2. These studies on Nopp140 can provide a good model system for understanding the molecular function of IDPs.
Topics: Animals; Casein Kinase II; Fluorescence Resonance Energy Transfer; Humans; Intrinsically Disordered Proteins; Magnetic Resonance Spectroscopy; Molecular Dynamics Simulation; Nuclear Proteins; Phosphoproteins; Protein Binding
PubMed: 29382046
DOI: 10.3390/ijms19020381 -
International Journal of Molecular... Feb 2021phosphoproteins (P) are essential co-factors of the viral polymerase by serving as a linchpin between the catalytic subunit and the ribonucleoprotein template. They... (Review)
Review
phosphoproteins (P) are essential co-factors of the viral polymerase by serving as a linchpin between the catalytic subunit and the ribonucleoprotein template. They have highly diverged, but their overall architecture is conserved. They are multidomain proteins, which all possess an oligomerization domain that separates - and -terminal domains. Large intrinsically disordered regions constitute their hallmark. Here, we exemplify their structural features and interaction potential, based on the P proteins. These P proteins are rather small, and their oligomerization domain is the only part with a defined 3D structure, owing to a quaternary arrangement. All other parts are either flexible or form short-lived secondary structure elements that transiently associate with the rest of the protein. P proteins interact with several viral and cellular proteins that are essential for viral transcription and replication. The combination of intrinsic disorder and tetrameric organization enables them to structurally adapt to different partners and to act as adaptor-like platforms to bring the latter close in space. Transient structures are stabilized in complex with protein partners. This class of proteins gives an insight into the structural versatility of non-globular intrinsically disordered protein domains.
Topics: Amino Acid Sequence; Animals; Binding Sites; Gene Expression Regulation, Viral; Humans; Intrinsically Disordered Proteins; Models, Molecular; Mononegavirales; Phosphoproteins; Pneumovirus; Protein Binding; Protein Conformation; Protein Folding; Protein Interaction Domains and Motifs; Respiratory Syncytial Virus, Human; Structure-Activity Relationship; Viral Proteins
PubMed: 33546457
DOI: 10.3390/ijms22041537 -
Pharmacology & Therapeutics Sep 2014Phosphoprotein enriched in astrocytes-15 (PEA-15) is a cytoplasmic protein that sits at an important junction in intracellular signalling and can regulate diverse... (Review)
Review
Phosphoprotein enriched in astrocytes-15 (PEA-15) is a cytoplasmic protein that sits at an important junction in intracellular signalling and can regulate diverse cellular processes, such as proliferation and apoptosis, dependent upon stimulation. Regulation of these processes occurs by virtue of the unique interaction of PEA-15 with other signalling proteins. PEA-15 acts as a cytoplasmic tether for the mitogen-activated protein kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) preventing nuclear localisation. In order to release ERK1/2, PEA-15 requires to be phosphorylated via several potential pathways. PEA-15 (and its phosphorylation state) therefore regulates many ERK1/2-dependent processes, including proliferation, via regulating ERK1/2 nuclear translocation. In addition, PEA-15 contains a death effector domain (DED) which allows interaction with other DED-containing proteins. PEA-15 can bind the DED-containing apoptotic adaptor molecule, Fas-associated death domain protein (FADD) which is also dependent on the phosphorylation status of PEA-15. PEA-15 binding of FADD can inhibit apoptosis as bound FADD cannot participate in the assembly of apoptotic signalling complexes. Through these protein-protein interactions, PEA-15-regulated cellular effects have now been investigated in a number of disease-related studies. Changes in PEA-15 expression and regulation have been observed in diabetes mellitus, cancer, neurological disorders and the cardiovascular system. These changes have been suggested to contribute to the pathology related to each of these disease states. As such, new therapeutic targets based around PEA-15 and its associated interactions are now being uncovered and could provide novel avenues for treatment strategies in multiple diseases.
Topics: Animals; Apoptosis Regulatory Proteins; Brain Diseases; Cardiovascular Diseases; Endocrine System Diseases; Humans; Intracellular Signaling Peptides and Proteins; Neoplasms; Phosphoproteins; Protein Conformation
PubMed: 24657708
DOI: 10.1016/j.pharmthera.2014.03.006 -
Protein Science : a Publication of the... Aug 2021Extant fold-switching proteins remodel their secondary structures and change their functions in response to environmental stimuli. These shapeshifting proteins regulate...
Extant fold-switching proteins remodel their secondary structures and change their functions in response to environmental stimuli. These shapeshifting proteins regulate biological processes and are associated with a number of diseases, including tuberculosis, cancer, Alzheimer's, and autoimmune disorders. Thus, predictive methods are needed to identify more fold-switching proteins, especially since all naturally occurring instances have been discovered by chance. In response to this need, two high-throughput predictive methods have recently been developed. Here we test them on ORF9b, a newly discovered fold switcher and potential therapeutic target from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Promisingly, both methods correctly indicate that ORF9b switches folds. We then tested the same two methods on ORF9b1, the ORF9b homolog from SARS-CoV-1. Again, both methods predict that ORF9b1 switches folds, a finding consistent with experimental binding studies. Together, these results (a) demonstrate that protein fold switching can be predicted using high-throughput computational approaches and (b) suggest that fold switching might be a general characteristic of ORF9b homologs.
Topics: Coronavirus Nucleocapsid Proteins; Humans; Models, Molecular; Phosphoproteins; Protein Folding; Protein Structure, Secondary; SARS-CoV-2
PubMed: 33934422
DOI: 10.1002/pro.4097 -
Journal of Virology Jan 2022Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein (N), forming an RNA-N...
Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein (N), forming an RNA-N complex (N), which serves as the template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping, and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to N and L, allowing the attachment of the L polymerase to the N template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between HMPV P and N, which has not been demonstrated yet. Here, we finely characterized the binding P-N interaction domains by using recombinant proteins, combined with a functional assay for the polymerase complex activity, and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to N and that P binds to the N-terminal domain of N (N), and we identified conserved N residues critical for the interaction. Our results allowed us to propose a structural model for the HMPV P-N interaction. Human metapneumovirus (HMPV) is a leading cause of severe respiratory infections in children but also affects human populations of all ages worldwide. Currently, no vaccine or efficient antiviral treatments are available for this pneumovirus. A better understanding of the molecular mechanisms involved in viral replication could help the design or discovery of specific antiviral compounds. In this work, we have investigated the interaction between two major viral proteins involved in HMPV RNA synthesis, the N and P proteins. We finely characterized their domains of interaction and identified a pocket on the surface of the N protein, a potential target of choice for the design of compounds interfering with N-P complexes and inhibiting viral replication.
Topics: Animals; Binding Sites; Cell Line; Cricetinae; Inclusion Bodies; Metapneumovirus; Models, Molecular; Mutation; Nucleocapsid Proteins; Phosphoproteins; Protein Binding; Protein Interaction Domains and Motifs; RNA, Viral; RNA-Dependent RNA Polymerase; Virus Replication
PubMed: 34730389
DOI: 10.1128/JVI.00909-21 -
Frontiers in Bioscience (Landmark... Jan 2009Enabled/VASP Homology-1 (EVH1) domains function primarily as interaction modules that link signaling proteins by binding to proline-rich sequences. EVH1 domains are ~115... (Review)
Review
Enabled/VASP Homology-1 (EVH1) domains function primarily as interaction modules that link signaling proteins by binding to proline-rich sequences. EVH1 domains are ~115 residues in length and adopt the pleckstrin homology (PH) fold. Four different protein families contain EVH1 domains: Ena/VASP, Homer, WASP and SPRED. Except for the SPRED domains, for which no binding partners are known, EVH1 domains use a conserved hydrophobic cleft to bind a four-residue motif containing 2-4 prolines. Conserved aromatic residues, including an invariant tryptophan, create a wedge-shaped groove on the EVH1 surface that matches the triangular profile of a polyproline type II helix. Hydrophobic residues adjacent to the polyproline motif dock into complementary sites on the EVH1 domain to enhance ligand binding specificity. Pseudosymmetry in the polyproline type II helix allows peptide ligands to bind in either of two N-to-C terminal orientations, depending on interactions between sequences flanking the prolines and the EVH1 domain. EVH1 domains also recognize non-proline motifs, as illustrated by the structure of an EVH1:LIM3 complex and the extended EVH1 ligands of the verprolin family.
Topics: Cell Adhesion Molecules; Microfilament Proteins; Peptides; Phosphoproteins; Substrate Specificity
PubMed: 19273103
DOI: 10.2741/3281 -
Nature Communications May 2024Differences in gene-expression profiles between individual cells can give rise to distinct cell fate decisions. Yet how localisation on a micropattern impacts initial...
Differences in gene-expression profiles between individual cells can give rise to distinct cell fate decisions. Yet how localisation on a micropattern impacts initial changes in mRNA, protein, and phosphoprotein abundance remains unclear. To identify the effect of cellular position on gene expression, we developed a scalable antibody and mRNA targeting sequential fluorescence in situ hybridisation (ARTseq-FISH) method capable of simultaneously profiling mRNAs, proteins, and phosphoproteins in single cells. We studied 67 (phospho-)protein and mRNA targets in individual mouse embryonic stem cells (mESCs) cultured on circular micropatterns. ARTseq-FISH reveals relative changes in both abundance and localisation of mRNAs and (phospho-)proteins during the first 48 hours of exit from pluripotency. We confirm these changes by conventional immunofluorescence and time-lapse microscopy. Chemical labelling, immunofluorescence, and single-cell time-lapse microscopy further show that cells closer to the edge of the micropattern exhibit increased proliferation compared to cells at the centre. Together these data suggest that while gene expression is still highly heterogeneous position-dependent differences in mRNA and protein levels emerge as early as 12 hours after LIF withdrawal.
Topics: Animals; In Situ Hybridization, Fluorescence; Mice; Mouse Embryonic Stem Cells; RNA, Messenger; Phosphoproteins; Single-Cell Analysis; Time-Lapse Imaging; Gene Expression Profiling; Cell Differentiation
PubMed: 38724524
DOI: 10.1038/s41467-024-48107-5 -
Developmental Dynamics : An Official... Oct 2021Mutation in Odontogenesis-associated phosphoprotein (ODAPH) has been reported to cause recessive hypomineralized amelogenesis imperfecta (AI) in human. However, the...
BACKGROUND
Mutation in Odontogenesis-associated phosphoprotein (ODAPH) has been reported to cause recessive hypomineralized amelogenesis imperfecta (AI) in human. However, the exact role of ODAPH in amelogenesis is still unknown.
RESULTS
ODAPH was identified as a novel constituent of the atypical basal lamina located at the interface between maturation ameloblasts and the enamel by dual immunofluorescence staining of ODAPH and LAMC2. Odaph knockout mice were generated to explore the function of ODAPH in amelogenesis. Odaph mice teeth showed severely attrition and reduced enamel mineralization. Histological analysis showed from transition or early-maturation stage, ameloblasts were rapidly shortened, lost cell polarity, and exhibited cell pathology. Abundant enamel matrix marked by amelogenin was retained. Temporary cyst-like structures were formed between flattened epithelial cells and the enamel from maturation stage to eruption. The integrity of the atypical basal lamina was impaired indicated by the reduced diffuse expression of LAMC2 and AMTN. The expression of maturation stage related genes of Amtn, Klk4, Integrinβ6 and Slc24a4 were significantly decreased.
CONCLUSIONS
Our results suggested Odaph played vital roles during amelogenesis by maintaining the integrity of the atypical basal lamina in maturation stage, which may contribute to a better understanding of the pathophysiology of human AI.
Topics: Ameloblasts; Amelogenesis; Animals; Dental Enamel; Extracellular Matrix Proteins; Laminin; Mice; Mice, Knockout; Phosphoproteins
PubMed: 33772937
DOI: 10.1002/dvdy.336 -
RNA Biology 2010The phosphoprotein P of non-segmented negative-sense RNA viruses is an essential component of the replication and transcription complex and acts as a co-factor for the... (Review)
Review
The structure of the nucleoprotein binding domain of lyssavirus phosphoprotein reveals a structural relationship between the N-RNA binding domains of Rhabdoviridae and Paramyxoviridae.
The phosphoprotein P of non-segmented negative-sense RNA viruses is an essential component of the replication and transcription complex and acts as a co-factor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. We have obtained the structure of the C-terminal domain of P of Mokola virus (MOKV), a lyssavirus that belongs to the Rhabdoviridae family and mapped at the amino acid level the crucial positions involved in interaction with N and in the formation of the viral replication complex. Comparison of the N-RNA binding domains of P solved to date suggests that the N-RNA binding domains are structurally conserved among paramyxoviruses and rhabdoviruses in spite of low sequence conservation. We also review the numerous other functions of this domain and more generally of the phosphoprotein.
Topics: Humans; Lyssavirus; Models, Biological; Models, Molecular; Nucleoproteins; Paramyxoviridae; Phosphoproteins; Protein Structure, Tertiary; RNA; RNA-Binding Proteins; Rhabdoviridae; Structure-Activity Relationship
PubMed: 20458178
DOI: 10.4161/rna.7.3.11931 -
Substitution of S179P in the Lyssavirus Phosphoprotein Impairs Its Interferon Antagonistic Function.Journal of Virology Nov 2022Lyssaviruses cause rabies, which is an acute neurological disease responsible for more than 59,000 human deaths annually and has no available effective treatments. The...
Lyssaviruses cause rabies, which is an acute neurological disease responsible for more than 59,000 human deaths annually and has no available effective treatments. The phosphoprotein (P) of lyssaviruses (lyssavirus-P) plays multiple roles in virus replication and immune evasion. Lyssavirus-P has been identified as the major type I interferon (IFN-I) antagonist, while the precise site and precise molecular mechanism remain unclear. Herein, we found that substitution of site 179 of lyssavirus-P from serine (Ser) to proline (Pro) impairs its antagonism function of IFN-I by sequence alignment and site mutations. Subsequent studies demonstrated that lyssavirus-P containing S179 specifically interacted with I-kappa B kinase ε (IKKε). Specifically, lyssavirus-P containing S179 interacted simultaneously with the kinase domain (KD) and scaffold dimerization domain (SDD) of IKKε, competing with TNF receptor-associated factor 3 (TRAF3) and IFN regulatory factor 3 (IRF3) for binding with IKKε, leading to the inhibition of IFN production. Furthermore, S179 was involved in the viral pathogenicity of the typical lyssavirus rabies virus in a mouse model. Interestingly, we found that S179 is conserved among most lyssavirus-P and functional for IFN antagonism. Collectively, we identified S179 of lyssavirus-P is essential for IFN-I inhibition, which provides deep insight into the immune evasion strategies of lyssaviruses. Interferon (IFN) and the IFN-induced cellular antiviral response constitute the first line of defense against viral invasion. Evading host innate immunity, especially IFN signaling, is the key step required for lyssaviruses to establish infection. In this study, S179 of lyssavirus phosphoprotein (lyssavirus-P) was identified as the key site for antagonizing IFN-I production. Mechanistically, lyssavirus-P containing S179 specifically targets the key kinase IKKε and disrupts its interaction with TRAF3 and IRF3. S179P mutation in the P protein of the typical lyssavirus rabies virus (RABV) attenuated its pathogenicity in a mouse model. Our findings provide deep insight into the immune evasion strategies of lyssaviruses, which is helpful for the development of effective antiviral therapeutics.
Topics: Animals; Mice; Humans; Lyssavirus; I-kappa B Kinase; Phosphoproteins; TNF Receptor-Associated Factor 3; Rabies virus; Interferon Type I; Antiviral Agents
PubMed: 36326274
DOI: 10.1128/jvi.01125-22