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Current Molecular Pharmacology 2015Voltage gated calcium channels (Cav) are composed of up to five proteins: The ion conducting pore subunit α1 and the auxiliary subunits α2, δ, β, and γ. Recent... (Review)
Review
Voltage gated calcium channels (Cav) are composed of up to five proteins: The ion conducting pore subunit α1 and the auxiliary subunits α2, δ, β, and γ. Recent reports show that Cavα1 and Cavβ comprise the calcium channel core complex and that β, α2 δ and γ may serve additional roles that are independent of the Cavα1 subunit. This short review will summarize these emerging functions.
Topics: Animals; Calcium; Calcium Channels; Humans; Ion Channel Gating; Models, Biological; Muscle, Skeletal; Myocardium; Protein Binding; Protein Subunits
PubMed: 25966689
DOI: 10.2174/1874467208666150507110202 -
Current Protein & Peptide Science 2016Among three major replicative DNA polymerases of the B-family, Pol α, Pol δ and Pol ε, Pol δ plays an essential role in chromosomal DNA replication and is also... (Review)
Review
Among three major replicative DNA polymerases of the B-family, Pol α, Pol δ and Pol ε, Pol δ plays an essential role in chromosomal DNA replication and is also involved in various DNA repair processes in eukaryotes. Human Pol δ is commonly viewed as a heterotetrameric complex, consisting of the catalytic subunit p125 and second subunit p50, together with two additional accessory subunits, p68 and p12. A growing body of research has shown that the latter subunits play a critical role in the regulation of Pol δ functions. The formation of a new form of Pol δ, heterotrimer Pol δ3, is found by virtue of the depletion of p12 through the ubiquitin-proteasome pathway in response to DNA damages that are trigged by UV irradiation, alkylating agents, oxidative and replication stresses. Pol δ3 exhibits significant differences in properties to its progenitor with a major impact on cellular processes in genomic surveillance, DNA replication and DNA repair. Our recent studies indicate that there exists an alternative pathway for Pol δ3 formation by calpain-mediated proteolysis of p12 in a calcium-triggered apoptosis in living cells. In this article, we review and discuss the recent advances from our group and others in the studies of human Pol δ with an emphasis on the generation of its multiple forms by reconstitution and subsequent alternations in enzymatic properties, the multiple pathways of the Pol δ3 formation in living cells, and the phylogenetic analysis of the evolutionary history on POLD4 gene that is for the p12 subunit.
Topics: DNA; DNA Damage; DNA Polymerase III; DNA Repair; DNA Replication; Humans; Models, Genetic; Phylogeny; Protein Subunits; Signal Transduction
PubMed: 26916162
DOI: 10.2174/1389203717666160226145006 -
Journal of Labelled Compounds &... Jun 2019N-methyl-D-aspartate (NMDA) receptors play key roles in physiology by regulating the synaptic plasticity and the cellular mechanism involved in learning and memory. The... (Review)
Review
N-methyl-D-aspartate (NMDA) receptors play key roles in physiology by regulating the synaptic plasticity and the cellular mechanism involved in learning and memory. The GluN2A subunit is the most abundant expression of NMDA receptors in mature brain, and its dysfunction has been implicated in various neurological disorders. However, the function of GluN2A subunit in physiological and pathological conditions is not yet completely unveil due to the lack of subunit-selective ligands, including specific positron emission tomography (PET)/single photon emission computed tomography (SPECT) imaging probes. In this review, recent progresses in understanding its pathophysiological role, the structure-activity relationship, and the postulated mechanisms of novel GluN2A ligands as well as status of molecular imaging probes for PET are summarized.
Topics: Allosteric Regulation; Animals; Humans; Molecular Imaging; Nuclear Medicine; Protein Subunits; Receptors, N-Methyl-D-Aspartate
PubMed: 31037756
DOI: 10.1002/jlcr.3744 -
Science (New York, N.Y.) Nov 2015Assembly of protein complexes is considered a posttranslational process involving random collision of subunits. We show that within the Escherichia coli cytosol,...
Assembly of protein complexes is considered a posttranslational process involving random collision of subunits. We show that within the Escherichia coli cytosol, bacterial luciferase subunits LuxA and LuxB assemble into complexes close to the site of subunit synthesis. Assembly efficiency decreases markedly if subunits are synthesized on separate messenger RNAs from genes integrated at distant chromosomal sites. Subunit assembly initiates cotranslationally on nascent LuxB in vivo. The ribosome-associated chaperone trigger factor delays the onset of cotranslational interactions until the LuxB dimer interface is fully exposed. Protein assembly is thus directly coupled to the translation process and involves spatially confined, actively chaperoned cotranslational subunit interactions. Bacterial gene organization into operons therefore reflects a fundamental cotranslational mechanism for spatial and temporal regulation that is vital to effective assembly of protein complexes.
Topics: Bacteria; Bacterial Proteins; Escherichia coli; Gene Order; Genes, Bacterial; Green Fluorescent Proteins; Luciferases, Bacterial; Luminescent Proteins; Molecular Chaperones; Operon; Protein Biosynthesis; Protein Structure, Secondary; Protein Subunits; RNA, Messenger; Recombinant Fusion Proteins; Ribosomes; Vibrio
PubMed: 26405228
DOI: 10.1126/science.aac8171 -
The Biochemical Journal Jul 2016Calcium plays a key role in cell signalling by its intervention in a wide range of physiological processes. Its entry into cells occurs mainly via voltage-gated calcium... (Review)
Review
Calcium plays a key role in cell signalling by its intervention in a wide range of physiological processes. Its entry into cells occurs mainly via voltage-gated calcium channels (VGCC), which are found not only in the plasma membrane of excitable cells but also in cells insensitive to electrical signals. VGCC are composed of different subunits, α1, β, α2δ and γ, among which the cytosolic β subunit (Cavβ) controls the trafficking of the channel to the plasma membrane, its regulation and its gating properties. For many years, these were the main functions associated with Cavβ. However, a growing number of proteins have been found to interact with Cavβ, emphasizing the multifunctional role of this versatile protein. Interestingly, some of the newly assigned functions of Cavβ are independent of its role in the regulation of VGCC, and thus further increase its functional roles. Based on the identity of Cavβ protein partners, this review emphasizes the diverse cellular functions of Cavβ and summarizes both past findings as well as recent progress in the understanding of VGCC.
Topics: Animals; Calcium; Calcium Channels; Cell Membrane; Humans; Protein Binding; Protein Isoforms; Protein Subunits
PubMed: 27354560
DOI: 10.1042/BCJ20160125 -
International Journal of Developmental... May 2017The β1 nicotinic acetylcholine receptor (nAChR) subunit is a muscle type subunit of this family and as such, is found predominantly in muscle. Recent reports document...
The β1 nicotinic acetylcholine receptor (nAChR) subunit is a muscle type subunit of this family and as such, is found predominantly in muscle. Recent reports document its expression in other tissues and cell lines including adrenal glands, carcinomas, lung and brain. However, the majority of studies were of tissue lysates, thus the cellular distribution was not determined. This study aimed to determine the cellular distribution of the β1 nAChR subunit in the brain, at both the mRNA and protein levels, using non-radioactive in situ hybridization (ISH) and immunohistochemistry (IHC), respectively, and to compare it to two muscle tissue types, skeletal and placenta. Tissue was formalin fixed and paraffin embedded (all tissue types) and frozen (placenta) from humans. Additional control tissue from the piglet and mouse brain were also studied, as was mRNA for the α3 nAChR and N-methyl-d-aspartate receptor 1 (NR1) subunit. We found no β1 nAChR subunit mRNA expression in the human and piglet brain despite strong protein expression. Some signal was seen in the mouse brain but considered inconclusive given the probes designed were not of 100% homology to the mouse. In the skeletal muscle and placenta tissues, β1 nAChR subunit mRNA expression was prominent and mirrored protein expression. No α3 nAChR or NR1 mRNA was seen in the skeletal muscle, as expected, although both subunit mRNAs were present in the placenta. This study concludes that further experiments are required to conclusively state that the β1 nAChR subunit is expressed in the human, piglet and mouse brain.
Topics: Animals; Brain; Female; Humans; Mice; Muscle, Skeletal; Placenta; Pregnancy; Protein Subunits; RNA, Messenger; Receptors, Nicotinic; Swine
PubMed: 28153524
DOI: 10.1016/j.ijdevneu.2017.01.011 -
Methods in Molecular Biology (Clifton,... 2018The reconstitution of recombinant protein complexes is facilitated by methods that allow coexpression of their subunits from a single vector. Here we describe a detailed...
The reconstitution of recombinant protein complexes is facilitated by methods that allow coexpression of their subunits from a single vector. Here we describe a detailed step-by-step protocol for the biGBac cloning method which can be used to generate baculoviral transfer vectors coding for up to 25 subunits of a protein complex (Weissmann et al., Proc Natl Acad Sci U S A 113(19):E2564-E2569, 2016). biGBac is based on Gibson assembly reactions, optimized DNA linker sequences, and uses a hierarchical two-step assembly procedure. In the first assembly step, up to five expression cassettes are combined to generate a polygene cassette. In the second step, up to five polygene cassettes can then be combined to generate transfer vectors coding for up to 25 subunits.
Topics: Baculoviridae; Cloning, Molecular; Genetic Vectors; Humans; Multigene Family; Multiprotein Complexes; Protein Engineering; Protein Subunits; Recombinant Proteins
PubMed: 29605925
DOI: 10.1007/978-1-4939-7759-8_21 -
PloS One 2014Protein phosphatase 2A (PP2A) is a ubiquitous phospho-serine/threonine phosphatase that controls many diverse cellular functions. The predominant form of PP2A is a...
Protein phosphatase 2A (PP2A) is a ubiquitous phospho-serine/threonine phosphatase that controls many diverse cellular functions. The predominant form of PP2A is a heterotrimeric holoenzyme consisting of a scaffolding A subunit, a variable regulatory B subunit, and a catalytic C subunit. The C subunit also associates with other interacting partners, such as α4, to form non-canonical PP2A complexes. We report visualization of PP2A complexes in mammalian cells. Bimolecular fluorescence complementation (BiFC) analysis of PP2A subunit interactions demonstrates that the B subunit plays a key role in directing the subcellular localization of PP2A, and confirms that the A subunit functions as a scaffold in recruiting the B and C subunits to form a heterotrimeric holoenzyme. BiFC analysis also reveals that α4 promotes formation of the AC core dimer. Furthermore, we demonstrate visualization of specific ABC holoenzymes in cells by combining BiFC and fluorescence resonance energy transfer (BiFC-FRET). Our studies not only provide direct imaging data to support previous biochemical observations on PP2A complexes, but also offer a promising approach for studying the spatiotemporal distribution of individual PP2A complexes in cells.
Topics: Animals; Fluorescent Antibody Technique; Mice; NIH 3T3 Cells; Protein Multimerization; Protein Phosphatase 2; Protein Subunits
PubMed: 25536081
DOI: 10.1371/journal.pone.0116074 -
International Review of Neurobiology 2016The large-conductance, Ca(2+)- and voltage-activated K(+) (BK) channel is ubiquitously expressed in mammalian tissues and displays diverse biophysical or pharmacological... (Review)
Review
The large-conductance, Ca(2+)- and voltage-activated K(+) (BK) channel is ubiquitously expressed in mammalian tissues and displays diverse biophysical or pharmacological characteristics. This diversity is in part conferred by channel modulation with different regulatory auxiliary subunits. To date, two distinct classes of BK channel auxiliary subunits have been identified: β subunits and γ subunits. Modulation of BK channels by the four auxiliary β (β1-β4) subunits has been well established and intensively investigated over the past two decades. The auxiliary γ subunits, however, were identified only very recently, which adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. This chapter will review the current understanding of BK channel modulation by auxiliary β and γ subunits, especially the latest findings.
Topics: Animals; Cell Membrane; Humans; Ion Channel Gating; Large-Conductance Calcium-Activated Potassium Channels; Models, Molecular; Protein Subunits
PubMed: 27238261
DOI: 10.1016/bs.irn.2016.03.015 -
Journal of Structural Biology Jun 2021Sulfite reductase (SiR), a dodecameric complex of flavoprotein reductase subunits (SiRFP) and hemoprotein oxidase subunits (SiRHP), reduces sulfur for biomass...
Sulfite reductase (SiR), a dodecameric complex of flavoprotein reductase subunits (SiRFP) and hemoprotein oxidase subunits (SiRHP), reduces sulfur for biomass incorporation. Electron transfer within SiR requires intra- and inter-subunit interactions that are mediated by the relative position of each protein, governed by flexible domain movements. Using small-angle neutron scattering, we report the first solution structures of SiR heterodimers containing a single copy of each subunit. These structures show how the subunits bind and how both subunit binding and oxidation state impact SiRFP's conformation. Neutron contrast matching experiments on selectively deuterated heterodimers allow us to define the contribution of each subunit to the solution scattering. SiRHP binding induces a change in the position of SiRFP's flavodoxin-like domain relative to its ferredoxin-NADP reductase domain while compacting SiRHP's N-terminus. Reduction of SiRFP leads to a more open structure relative to its oxidized state, re-positioning SiRFP's N-terminal flavodoxin-like domain towards the SiRHP binding position. These structures show, for the first time, how both SiRHP binding to, and reduction of, SiRFP positions SiRFP for electron transfer between the subunits.
Topics: Ferredoxins; Models, Molecular; Neutron Diffraction; Oxidation-Reduction; Protein Domains; Protein Multimerization; Protein Subunits; Scattering, Small Angle; Solutions; Solvents; Sulfite Reductase (NADPH); Ultracentrifugation
PubMed: 33722582
DOI: 10.1016/j.jsb.2021.107724