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Molecular Biology Reports Jun 2011The clathrin-associated adaptor protein (AP) complexes are the primary clathrin adaptors that contribute to the formation of clathrin-coated vesicles (CCVs). The GhAPm...
Molecular cloning and characterization of GhAPm, a gene encoding the μ subunit of the clathrin-associated adaptor protein complex that is associated with cotton (Gossypium hirsutum) fiber development.
The clathrin-associated adaptor protein (AP) complexes are the primary clathrin adaptors that contribute to the formation of clathrin-coated vesicles (CCVs). The GhAPm gene (GenBank accession number: GU359054), which encodes the medium subunit of the AP complexes, was cloned from cotton by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA was 1590 bp in size and encoded an open reading frame (ORF) of 416 amino acids with a molecular weight of 46 kDa. The GhAPm protein shared 81-85% identity at the amino acid level with the AP complex μ subunits isolated from Vitis vinifera, Glycine max, Populus trichocarpa, Ricinus communis and Arabidopsis thaliana, respectively. The corresponding genomic DNA, containing eight exons and seven introns, was isolated and analyzed. Also, a 5'-flanking region was analyzed, and a group of putative cis-acting elements were identified. DNA gel blot analysis showed that there is only one GhAPm gene in the cotton genome. Real-time RT-PCR analysis revealed that GhAPm is expressed in the root, stem, leaf, petal, ovule, and fiber. However, the interesting finding is that GhAPm expression level was shown to increase steadily as the cotton fiber develops. In 30 DPA fibers, expression increases sharply and arrives at a peak then the expression levels decrease rapidly. Based on these data, we propose that GhAPm has a critical role in cotton membrane trafficking and fiber development.
Topics: Adaptor Proteins, Vesicular Transport; Amino Acid Sequence; Base Sequence; Cloning, Molecular; Gene Expression Regulation, Plant; Gossypium; Models, Molecular; Molecular Sequence Data; Plant Proteins; Protein Structure, Tertiary; Protein Subunits; Sequence Alignment; Sequence Analysis, DNA
PubMed: 21225463
DOI: 10.1007/s11033-010-0436-0 -
Quarterly Reviews of Biophysics Aug 2011The rotary ATPase family of membrane protein complexes may have only three members, but each one plays a fundamental role in biological energy conversion. The... (Review)
Review
The rotary ATPase family of membrane protein complexes may have only three members, but each one plays a fundamental role in biological energy conversion. The F₁F(o)-ATPase (F-ATPase) couples ATP synthesis to the electrochemical membrane potential in bacteria, mitochondria and chloroplasts, while the vacuolar H⁺-ATPase (V-ATPase) operates as an ATP-driven proton pump in eukaryotic membranes. In different species of archaea and bacteria, the A₁A(o)-ATPase (A-ATPase) can function as either an ATP synthase or an ion pump. All three of these multi-subunit complexes are rotary molecular motors, sharing a fundamentally similar mechanism in which rotational movement drives the energy conversion process. By analogy to macroscopic systems, individual subunits can be assigned to rotor, axle or stator functions. Recently, three-dimensional reconstructions from electron microscopy and single particle image processing have led to a significant step forward in understanding of the overall architecture of all three forms of these complexes and have allowed the organisation of subunits within the rotor and stator parts of the motors to be more clearly mapped out. This review describes the emerging consensus regarding the organisation of the rotor and stator components of V-, A- and F-ATPases, examining core similarities that point to a common evolutionary origin, and highlighting key differences. In particular, it discusses how newly revealed variation in the complexity of the inter-domain connections may impact on the mechanics and regulation of these molecular machines.
Topics: Adenosine Triphosphatases; Animals; Humans; Microscopy, Electron; Protein Structure, Tertiary; Protein Subunits; Rotation
PubMed: 21426606
DOI: 10.1017/S0033583510000338 -
MBio Jun 2010The Escherichia coli signal recognition particle (SRP) system plays an important role in membrane protein biogenesis. Previous studies have suggested indirectly that in...
The Escherichia coli signal recognition particle (SRP) system plays an important role in membrane protein biogenesis. Previous studies have suggested indirectly that in addition to its role during the targeting of ribosomes translating membrane proteins to translocons, the SRP might also have a quality control role in preventing premature synthesis of membrane proteins in the cytoplasm. This proposal was studied here using cells simultaneously overexpressing various membrane proteins and either SRP, the SRP protein Ffh, its 4.5S RNA, or the Ffh M domain. The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins. We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes. Since E. coli SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed.
Topics: Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Membrane Proteins; Protein Stability; Protein Structure, Tertiary; Protein Subunits; Signal Recognition Particle
PubMed: 20714446
DOI: 10.1128/mBio.00020-10 -
Biochemical Society Transactions Jan 2011We review recent results on the complete structure of the archaeal RNAP (RNA polymerase) enzyme of Sulfolobus shibatae. We compare the three crystal forms in which this... (Review)
Review
We review recent results on the complete structure of the archaeal RNAP (RNA polymerase) enzyme of Sulfolobus shibatae. We compare the three crystal forms in which this RNAP packs (space groups P2₁2₁2₁, P2₁2₁2 and P2₁) and provide a preliminary biophysical characterization of the newly identified 13-subunit Rpo13. The availability of different crystal forms for this RNAP allows the analysis of the packing degeneracy and the intermolecular interactions that determine this degeneracy. We observe the pivotal role played by the protruding stalk composed of subunits Rpo4 and Rpo7 in the lattice contacts. Aided by MALLS (multi-angle laser light scattering), we have initiated the biophysical characterization of the recombinantly expressed and purified subunit Rpo13, a necessary step towards the understanding of Rpo13's role in archaeal transcription.
Topics: Amino Acid Sequence; Archaeal Proteins; DNA-Directed RNA Polymerases; Models, Molecular; Molecular Sequence Data; Protein Conformation; Protein Subunits; RNA, Archaeal; Sulfolobus; Transcription, Genetic
PubMed: 21265742
DOI: 10.1042/BST0390025 -
Frontiers in Bioscience : a Journal and... Jan 2007The last few years have seen an explosion in the number of voltage-dependent ion channel sequences detected in sperm and testes. The complex structural paradigm of these... (Review)
Review
The last few years have seen an explosion in the number of voltage-dependent ion channel sequences detected in sperm and testes. The complex structural paradigm of these channels is now known to include a pore-forming alpha1 subunit(s) whose electrophysiological properties are modulated by an intracellular beta subunit, a disulfide-linked complex of a membrane-spanning delta subunit with an extracellular alpha2 subunit, and a transmembrane gamma subunit. Many of these are alternatively spliced. Furthermore, the known number of genes coding each subtype has expanded significantly (10 alpha1, 4 beta, 4 alpha2delta, 8 gamma). Recently, the CatSper gene family has been characterized based on similarity to the voltage-dependent calcium channel alpha1 subunit. From among this multiplicity, a wide cross-section is active in sperm, including many splice variants. For example, expression of the various alpha1 subunits appears strictly localized in discrete domains of mature sperm, and seems to control distinct physiological roles such as cellular signaling pathways. These include alpha1 alternative splicing variants that are regulated by ions passed by channels in developing sperm. Various combinations of ion channel sequence variants have been studies in research models and in a variety of human diseases, including male infertility. For example, rats that are genetically resistant to testes damage by lead seem to respond to lead ions by increasing alpha1 alternative splicing. In contrast, in varicocele-associated male infertility, the outcome from surgical correction correlates with suppression of alpha1 alternative splicing, Ion channel blockers remain attractive model contraceptive drugs because of their ability to modulate cholesterol levels. However, the large number of sperm ion channel variants shared with other cell types make ion channels less attractive targets for male contraceptive development than a few years ago. In this review, the genetics, structure and function of voltage-dependent calcium channels and related CatSper molecules will be discussed, and several practical clinical applications associated with these channels will be reported.
Topics: Alternative Splicing; Animals; Calcium Channels; Contraception; Humans; Male; Mice; Protein Subunits; Rats; Spermatozoa; Testis
PubMed: 17127392
DOI: 10.2741/2158 -
Nucleic Acids Research Jan 2019BbvCI, a Type IIT restriction endonuclease, recognizes and cleaves the seven base pair sequence 5'-CCTCAGC-3', generating 3-base, 5'-overhangs. BbvCI is composed of two...
BbvCI, a Type IIT restriction endonuclease, recognizes and cleaves the seven base pair sequence 5'-CCTCAGC-3', generating 3-base, 5'-overhangs. BbvCI is composed of two protein subunits, each containing one catalytic site. Either site can be inactivated by mutation resulting in enzyme variants that nick DNA in a strand-specific manner. Here we demonstrate that the holoenzyme is labile, with the R1 subunit dissociating at low pH. Crystallization of the R2 subunit under such conditions revealed an elongated dimer with the two catalytic sites located on opposite sides. Subsequent crystallization at physiological pH revealed a tetramer comprising two copies of each subunit, with a pair of deep clefts each containing two catalytic sites appropriately positioned and oriented for DNA cleavage. This domain organization was further validated with single-chain protein constructs in which the two enzyme subunits were tethered via peptide linkers of variable length. We were unable to crystallize a DNA-bound complex; however, structural similarity to previously crystallized restriction endonucleases facilitated creation of an energy-minimized model bound to DNA, and identification of candidate residues responsible for target recognition. Mutation of residues predicted to recognize the central C:G base pair resulted in an altered enzyme that recognizes and cleaves CCTNAGC (N = any base).
Topics: Amino Acid Sequence; Base Sequence; Binding Sites; Catalytic Domain; DNA Cleavage; DNA Restriction Enzymes; Escherichia coli; Holoenzymes; Mutation; Peptides; Protein Multimerization; Protein Subunits
PubMed: 30395313
DOI: 10.1093/nar/gky1059 -
Science in China. Series C, Life... May 2009The influenza virus RNA-dependent RNA polymerase is a heterotrimeric complex (PA, PB1 and PB2) with multiple enzymatic activities for catalyzing viral RNA transcription... (Review)
Review
The influenza virus RNA-dependent RNA polymerase is a heterotrimeric complex (PA, PB1 and PB2) with multiple enzymatic activities for catalyzing viral RNA transcription and replication. The roles of PB1 and PB2 have been clearly defined, but PA is less well understood. The critical role of the polymerase complex in the influenza virus life cycle and high sequence conservation suggest it should be a major target for therapeutic intervention. However, until very recently, functional studies and drug discovery targeting the influenza polymerase have been hampered by the lack of three-dimensional structural information. We will review the recent progress in the structure and function of the PA subunit of influenza polymerase, and discuss prospects for the development of anti-influenza therapeutics based on available structures.
Topics: DNA-Directed RNA Polymerases; Models, Molecular; Orthomyxoviridae; Protein Multimerization; Protein Structure, Quaternary; Protein Structure, Tertiary; Protein Subunits; Structure-Activity Relationship; Viral Proteins
PubMed: 19471867
DOI: 10.1007/s11427-009-0060-1 -
PloS One 2016Type 2 asparaginases, a subfamily of N-terminal nucleophile (Ntn) hydrolases, are activated by limited proteolysis. This activation yields a heterodimer and a loop...
Type 2 asparaginases, a subfamily of N-terminal nucleophile (Ntn) hydrolases, are activated by limited proteolysis. This activation yields a heterodimer and a loop region at the C-terminus of the α-subunit is released. Since this region is unresolved in all type 2 asparaginase crystal structures but is close to the active site residues, we explored this loop region in six members of the type 2 asparaginase family using homology modeling. As the loop model for the childhood cancer-relevant protease Taspase1 differed from the other members, Taspase1 activation as well as the conformation and dynamics of the 56 amino acids loop were investigated by CD and NMR spectroscopy. We propose a helix-turn-helix motif, which can be exploited as novel anticancer target to inhibit Taspase1 proteolytic activity.
Topics: Endopeptidases; Humans; Molecular Dynamics Simulation; Myeloid-Lymphoid Leukemia Protein; Protein Structure, Secondary; Protein Subunits; Proton Magnetic Resonance Spectroscopy; Structure-Activity Relationship
PubMed: 26974973
DOI: 10.1371/journal.pone.0151431 -
Biochimica Et Biophysica Acta Feb 2009Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from...
Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from Leishmania amazonensis (LaRPA-1) has previously been affinity-purified on a column containing a G-rich telomeric DNA. LaRPA-1 binds and co-localizes with parasite telomeres in vivo. Here we describe the purification and characterization of native recombinant LaRPA-1 (rLaRPA-1). The protein was initially re-solubilized from inclusion bodies by using urea. After dialysis, rLaRPA-1 was soluble but contaminated with DNA, which was removed by an anion-exchange chromatography of the protein solubilized in urea. However, rLaRPA-1 precipitated after dialysis to remove urea. To investigate whether the contaminating DNA was involved in chaperoning the refolding of rLaRPA-1, salmon sperm DNA or heparin was added to the solution before dialysis. The addition of either of these substances prevented the precipitation of rLaRPA-1. The resulting rLaRPA-1 was soluble, correctly folded, and able to bind telomeric DNA. This is the first report showing the characterization of rLaRPA1 and of the importance of additives in chaperoning the refolding of this protein. The availability of rLaRPA-1 should be helpful in assessing the importance of this protein as a potential drug target.
Topics: Animals; DNA; Heparin; Leishmania; Molecular Chaperones; Protein Binding; Protein Folding; Protein Subunits; Recombinant Proteins; Replication Protein A; Solubility
PubMed: 19056467
DOI: 10.1016/j.bbagen.2008.10.011 -
The Journal of Physiology Jan 2015NMDA receptors (NMDARs) are a class of ionotropic glutamate receptors (iGluRs) that are essential for neuronal development, synaptic plasticity, learning and cell... (Review)
Review
NMDA receptors (NMDARs) are a class of ionotropic glutamate receptors (iGluRs) that are essential for neuronal development, synaptic plasticity, learning and cell survival. Several features distinguish NMDARs from other iGluRs and underlie the crucial roles NMDARs play in nervous system physiology. NMDARs display slow deactivation kinetics, are highly Ca(2+) permeable, and require depolarization to relieve channel block by external Mg(2+) ions, thereby making them effective coincidence detectors. These properties and others differ among NMDAR subtypes, which are defined by the subunits that compose the receptor. NMDARs, which are heterotetrameric, commonly are composed of two GluN1 subunits and two GluN2 subunits, of which there are four types, GluN2A-D. 'Diheteromeric' NMDARs contain two identical GluN2 subunits. Gating and ligand-binding properties (e.g. deactivation kinetics) and channel properties (e.g. channel block by Mg(2+)) depend strongly on the GluN2 subunit contained in diheteromeric NMDARs. Recent work shows that two distinct regions of GluN2 subunits control most diheteromeric NMDAR subtype-dependent properties: the N-terminal domain is responsible for most subtype dependence of gating and ligand-binding properties; a single residue difference between GluN2 subunits at a site termed the GluN2 S/L site is responsible for most subtype dependence of channel properties. Thus, two structurally and functionally distinct regions underlie the majority of subtype dependence of NMDAR properties. This topical review highlights recent studies of recombinant diheteromeric NMDARs that uncovered the involvement of the N-terminal domain and of the GluN2 S/L site in the subtype dependence of NMDAR properties.
Topics: Protein Subunits; Receptors, N-Methyl-D-Aspartate
PubMed: 25556790
DOI: 10.1113/jphysiol.2014.273763