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Open Biology Jun 2021Herpes simplex virus type 1 (HSV-1) is one of the nine herpesviruses that infect humans. HSV-1 encodes seven proteins to replicate its genome in the hijacked human cell.... (Review)
Review
Herpes simplex virus type 1 (HSV-1) is one of the nine herpesviruses that infect humans. HSV-1 encodes seven proteins to replicate its genome in the hijacked human cell. Among these are the herpes virus DNA helicase and primase that are essential components of its replication machinery. In the HSV-1 replisome, the helicase-primase complex is composed of three components including UL5 (helicase), UL52 (primase) and UL8 (non-catalytic subunit). UL5 and UL52 subunits are functionally interdependent, and the UL8 component is required for the coordination of UL5 and UL52 activities proceeding in opposite directions with respect to the viral replication fork. Anti-viral compounds currently under development target the functions of UL5 and UL52. Here, we review the structural and functional properties of the UL5/UL8/UL52 complex and highlight the gaps in knowledge to be filled to facilitate molecular characterization of the structure and function of the helicase-primase complex for development of alternative anti-viral treatments.
Topics: Animals; Antiviral Agents; DNA Helicases; DNA Primase; Drug Development; Herpes Simplex; Herpesvirus 1, Human; Humans; Models, Molecular; Multienzyme Complexes; Protein Binding; Protein Interaction Domains and Motifs; Protein Subunits; Structure-Activity Relationship; Virus Replication
PubMed: 34102080
DOI: 10.1098/rsob.210011 -
Scientific Reports Mar 2021γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, exerts its effect through the activation of GABA receptors. GABA...
γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, exerts its effect through the activation of GABA receptors. GABA receptors are ligand-gated chloride channels composed of five subunit proteins. Mammals have 19 different GABA receptor subunits (α1-6, β1-3, γ1-3, δ, ε, π, θ, and ρ1-3), the physiological properties of which have been assayed by electrophysiology. However, the evolutionary conservation of the physiological characteristics of diverged GABA receptor subunits remains unclear. Zebrafish have 23 subunits (α1, α2a, α2b, α3-5, α6a, α6b, β1-4, γ1-3, δ, π, ζ, ρ1, ρ2a, ρ2b, ρ3a, and ρ3b), but the electrophysiological properties of these subunits have not been explored. In this study, we cloned the coding sequences for zebrafish GABA receptor subunits and investigated their expression patterns in larval zebrafish by whole-mount in situ hybridization. We also performed electrophysiological recordings of GABA-evoked currents from Xenopus oocytes injected with one or multiple zebrafish GABA receptor subunit cRNAs and calculated the half-maximal effective concentrations (EC50s) for each. Our results revealed the spatial expressions and electrophysiological GABA sensitivities of zebrafish GABA receptors, suggesting that the properties of GABA receptor subunits are conserved among vertebrates.
Topics: Animals; Cloning, Molecular; Conserved Sequence; Gene Expression; Genetic Vectors; In Situ Hybridization, Fluorescence; Kinetics; Larva; Membrane Potentials; Oocytes; Phylogeny; Protein Subunits; Receptors, GABA-A; Recombinant Proteins; Xenopus; Zebrafish; Zebrafish Proteins; gamma-Aminobutyric Acid
PubMed: 33737538
DOI: 10.1038/s41598-021-84646-3 -
International Journal of Molecular... Jul 2023Protein phosphatase 2A (PP2A) is a strongly conserved and major protein phosphatase in all eukaryotes. The canonical PP2A complex consists of a catalytic (C),... (Review)
Review
Protein phosphatase 2A (PP2A) is a strongly conserved and major protein phosphatase in all eukaryotes. The canonical PP2A complex consists of a catalytic (C), scaffolding (A), and regulatory (B) subunit. Plants have three groups of evolutionary distinct B subunits: B55, B' (B56), and B''. Here, the Arabidopsis B' group is reviewed and compared with other eukaryotes. Members of the B'α/B'β clade are especially important for chromatid cohesion, and dephosphorylation of transcription factors that mediate brassinosteroid (BR) signaling in the nucleus. Other B' subunits interact with proteins at the cell membrane to dampen BR signaling or harness immune responses. The transition from vegetative to reproductive phase is influenced differentially by distinct B' subunits; B'α and B'β being of little importance, whereas others (B'γ, B'ζ, B'η, B'θ, B'κ) promote transition to flowering. Interestingly, the latter B' subunits have three motifs in a conserved manner, i.e., two docking sites for protein phosphatase 1 (PP1), and a POLO consensus phosphorylation site between these motifs. This supports the view that a conserved PP1-PP2A dephosphorelay is important in a variety of signaling contexts throughout eukaryotes. A profound understanding of these regulators may help in designing future crops and understand environmental issues.
Topics: Arabidopsis; Arabidopsis Proteins; Phosphorylation; Physiological Phenomena; Protein Phosphatase 2; Protein Subunits; Transcription Factors
PubMed: 37569631
DOI: 10.3390/ijms241512255 -
Toxins Jul 2019Pertussis toxin (PT) is a multimeric complex of six proteins. The PTS1 subunit is an ADP-ribosyltransferase that inactivates the alpha subunit of heterotrimeric G/... (Review)
Review
Pertussis toxin (PT) is a multimeric complex of six proteins. The PTS1 subunit is an ADP-ribosyltransferase that inactivates the alpha subunit of heterotrimeric G/ proteins. The remaining PT subunits form a pentamer that positions PTS1 in and above the central cavity of the triangular structure. Adhesion of this pentamer to glycoprotein or glycolipid conjugates on the surface of a target cell leads to endocytosis of the PT holotoxin. Vesicle carriers then deliver the holotoxin to the endoplasmic reticulum (ER) where PTS1 dissociates from the rest of the toxin, unfolds, and exploits the ER-associated degradation pathway for export to the cytosol. Refolding of the cytosolic toxin allows it to regain an active conformation for the disruption of cAMP-dependent signaling events. This review will consider the intracellular trafficking of PT and the order-disorder-order transitions of PTS1 that are essential for its cellular activity.
Topics: Animals; Cytosol; Humans; Pertussis Toxin; Protein Subunits; Protein Transport
PubMed: 31349590
DOI: 10.3390/toxins11080437 -
Proceedings of the National Academy of... Aug 2023Cells of vertebrate and invertebrate organisms express proteins specialized in membrane channel-based cell-cell communication that are absent in unicellular organisms....
Cells of vertebrate and invertebrate organisms express proteins specialized in membrane channel-based cell-cell communication that are absent in unicellular organisms. We recently described the prediction of some members of the large-pore channel family in kinetoplastids, consisting of proteins called unnexins, which share several structural features with innexin and pannexin proteins. Here, we demonstrated that the unnexin1 protein (Unx1) is delivered to the cell membrane, displaying a topology consisting of four transmembrane domains with C and N termini on the cytoplasmic side and form large-pore channels that are permeable to small molecules. Low extracellular Ca/Mg levels or extracellular alkalinization, but not mechanical stretching, increases channel activity. The Unx1 channel mediates the influx of Ca and does not form intercellular dye coupling between HeLa Unx1 transfected cells. Unx1 channel function was further evidenced by its ability to mediate ionic currents when expressed in oocytes. Downregulation of Unx1 mRNA with morpholine contains invasion. Phylogenetic analysis revealed the presence of Unx1 homologs in other protozoan parasites, suggesting a conserved function for these channel parasites in other protists. Our data demonstrate that Unx1 forms large-pore membrane channels, which may serve as a diffusional pathway for ions and small molecules that are likely to be metabolic substrates or waste products, and signaling autocrine and paracrine molecules that could be involved in cell invasion. As morpholinos-induced downregulation of Unx1 reduces the infectivity of trypomastigotes, the Unx1 channels might be an attractive target for developing trypanocide drugs.
Topics: Protein Subunits; Phylogeny; Cell Membrane; Cytoplasm; Morpholinos
PubMed: 37487087
DOI: 10.1073/pnas.2307898120 -
Biochimica Et Biophysica Acta.... Feb 2020Methylation of proteins is emerging to be an important regulator of protein function. SET7/9, a protein lysine methyltransferase, catalyses methylation of several...
Methylation of proteins is emerging to be an important regulator of protein function. SET7/9, a protein lysine methyltransferase, catalyses methylation of several proteins involved in diverse biological processes. SET7/9-mediated methylation often regulates the stability, sub-cellular localization and protein-protein interactions of its substrate proteins. Here, we aimed to identify novel biological processes regulated by SET7/9 by identifying new interaction partners. For this we used yeast two-hybrid screening and identified the large subunit ribosomal protein, eL42 as a potential interactor of SET7/9. We confirmed the SET7/9-eL42 interaction by co-immunoprecipitation and GST pulldown studies. The N-terminal MORN domain of SET7/9 is essential for its interaction with eL42. Importantly, we identified that SET7/9 methylates eL42 at three different lysines - Lys53, Lys80 and Lys100 through site-directed mutagenesis. By puromycin incorporation assay, we find that SET7/9-mediated methylation of eL42 affects global translation. This study identifies a new role of the functionally versatile SET7/9 lysine methyltransferase in the regulation of global protein synthesis.
Topics: Amino Acid Sequence; HEK293 Cells; Histone-Lysine N-Methyltransferase; Humans; Lysine; Methylation; Protein Biosynthesis; Protein Domains; Protein Interaction Domains and Motifs; Protein Subunits; RNA Interference; RNA, Small Interfering; Ribosomal Proteins; Two-Hybrid System Techniques
PubMed: 31751593
DOI: 10.1016/j.bbamcr.2019.118611 -
PloS One 2023Most proteins form complexes consisting of two or more subunits, where complex assembly can proceed via two competing pathways: co-translational assembly of a mature and...
Most proteins form complexes consisting of two or more subunits, where complex assembly can proceed via two competing pathways: co-translational assembly of a mature and a nascent subunit, and post-translational assembly by two mature protein subunits. Assembly pathway dominance, i.e., which of the two pathways is predominant under which conditions, is poorly understood. Here, we introduce a reaction-diffusion system that describes protein complex formation via post- and co-translational assembly and use it to analyze the dominance of both pathways. Special features of this new system are (i) spatially inhomogeneous sources of reacting species, (ii) a combination of diffusing and immobile species, and (iii) an asymmetric binding competition between the species. We study assembly pathway dominance for the spatially homogeneous system and find that the ratio of production rates of the two protein subunits determines the long-term pathway dominance. This result is independent of the binding rate constants for post- and co-translational assembly and implies that a system with an initial post-translational assembly dominance can eventually exhibit co-translational assembly dominance and vice versa. For exactly balanced production of both subunits, the assembly pathway dominance is determined by the steady state concentration of the subunit that can bind both nascent and mature partners. The introduced system of equations can be applied to describe general dynamics of assembly processes involving both diffusing and immobile components.
Topics: Protein Subunits; Protein Biosynthesis
PubMed: 36827413
DOI: 10.1371/journal.pone.0281964 -
Cell Apr 2020Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class...
Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.
Topics: Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Enzyme Activators; G1 Phase; Humans; Multiprotein Complexes; Phenothiazines; Phosphorylation; Protein Phosphatase 2; Protein Subunits; Trans-Activators; Transcription Factors
PubMed: 32315619
DOI: 10.1016/j.cell.2020.03.051 -
Nature May 2020Most proteins associate into multimeric complexes with specific architectures, which often have functional properties such as cooperative ligand binding or allosteric...
Most proteins associate into multimeric complexes with specific architectures, which often have functional properties such as cooperative ligand binding or allosteric regulation. No detailed knowledge is available about how any multimer and its functions arose during evolution. Here we use ancestral protein reconstruction and biophysical assays to elucidate the origins of vertebrate haemoglobin, a heterotetramer of paralogous α- and β-subunits that mediates respiratory oxygen transport and exchange by cooperatively binding oxygen with moderate affinity. We show that modern haemoglobin evolved from an ancient monomer and characterize the historical 'missing link' through which the modern tetramer evolved-a noncooperative homodimer with high oxygen affinity that existed before the gene duplication that generated distinct α- and β-subunits. Reintroducing just two post-duplication historical substitutions into the ancestral protein is sufficient to cause strong tetramerization by creating favourable contacts with more ancient residues on the opposing subunit. These surface substitutions markedly reduce oxygen affinity and even confer cooperativity, because an ancient linkage between the oxygen binding site and the multimerization interface was already an intrinsic feature of the protein's structure. Our findings establish that evolution can produce new complex molecular structures and functions via simple genetic mechanisms that recruit existing biophysical features into higher-level architectures.
Topics: Allosteric Regulation; Binding Sites; Evolution, Molecular; Heme; Hemoglobins; Humans; Iron; Models, Molecular; Oxygen; Protein Multimerization; Protein Structure, Quaternary; Protein Subunits
PubMed: 32461643
DOI: 10.1038/s41586-020-2292-y -
International Journal of Molecular... Oct 2021As the central node between nutrition signaling input and the metabolic pathway, AMP-activated protein kinase (AMPK) is tightly regulated to maintain energy homeostasis.... (Review)
Review
As the central node between nutrition signaling input and the metabolic pathway, AMP-activated protein kinase (AMPK) is tightly regulated to maintain energy homeostasis. Subcellular compartmentalization of AMPK is one of the critical regulations that enables AMPK to access proper targets and generate appropriate responses to specific perturbations and different levels of stress. One of the characterized localization mechanisms is RanGTPase-driven CRM1 that recognizes the nuclear export sequence (NES) on the α subunit to translocate AMPK into the cytoplasm. Nuclear localization putatively employs RanGTPase-driven importin that might recognize the nuclear localization signal (NLS) present on the AMPKα2 kinase domain. Nucleo-cytoplasmic shuttling of AMPK is influenced by multiple factors, such as starvation, exercise, heat shock, oxidant, cell density, and circadian rhythm. Tissue-specific localization, which distributes AMPK trimers with different combinations, has also been shown to be vital in maintaining tissue-specific metabolism. Tissue-specific and subcellular distribution of AMPK might be attributed to differences in the expression of the subunit, the stabilization by protein regulators, tissue activity, and the localization of AMPK activators. Considering the importance of AMPK localization in coordinating signaling and metabolism, further research is due to fully elucidate the largely unknown complex mechanism underlying this regulation.
Topics: AMP-Activated Protein Kinases; Active Transport, Cell Nucleus; Cell Nucleus; Circadian Rhythm; Cytoplasm; Energy Metabolism; Heat-Shock Response; Humans; Karyopherins; Protein Subunits; Receptors, Cytoplasmic and Nuclear; Exportin 1 Protein
PubMed: 34681581
DOI: 10.3390/ijms222010921