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Molecular Biology Reports Nov 2021Ribonucleotide reductases (RNR) catalyze the rate-limiting step in DNA synthesis during the S-phase of the cell cycle. Its constant activity in order to maintain dNTP...
Ribonucleotide reductases (RNR) catalyze the rate-limiting step in DNA synthesis during the S-phase of the cell cycle. Its constant activity in order to maintain dNTP homeostasis is a fascinating area of research and an attractive candidate for cancer research and antiviral drugs. Redox modification such as S-glutathionylation of the R1 subunit of mammalian RNR protein has been presumed to regulate the activity of RNR during catalytic cycles. Herein, we report S-glutathionylation of the R2 subunit. We have also shown Grx1 system can efficiently deglutathionylate the S-glutathionylated R2 subunit. Additionally, our data also showed for the very first time S-glutathionylation of mammalian p53R2 subunit that regulates DNA synthesis outside S-phase during DNA damage and repair. Taken together, these data will open new avenues for future research relating to exact physiological significance, target thiols, and/or overall RNR activity due to S-glutathionylation of R2 and p53R2 subunits and provide valuable insights for effective treatment regimes.
Topics: Animals; Cell Cycle Proteins; DNA Replication; Glutathione; Mice; Protein Subunits; Ribonucleotide Reductases; S Phase
PubMed: 34599703
DOI: 10.1007/s11033-021-06721-2 -
Computational Biology and Chemistry Dec 2013The structural dynamics of the cAMP-dependent protein kinase catalytic subunit were modeled using molecular dynamics computational methods. It was shown that the...
The structural dynamics of the cAMP-dependent protein kinase catalytic subunit were modeled using molecular dynamics computational methods. It was shown that the structure of this protein as well as its complexes with ATP and peptide ligand PKI(5-24) consisted of a large number of rapidly inter-converting conformations which could be grouped into subsets proceeding from their similarity. This cluster analysis revealed that conformations which correspond to the "opened" and "closed" structures of the protein were already present in the free enzyme, and most surprisingly co-existed in enzyme-ATP and enzyme-PKI(5-24) complexes as well as in the ternary complex, which included both of these ligands. The results also demonstrated that the most mobile structure segments of the protein were located in the regions of substrate binding sites and that their dynamics were most significantly affected by the binding of the ATP and peptide ligand.
Topics: Cyclic AMP-Dependent Protein Kinases; Models, Molecular; Molecular Dynamics Simulation; Protein Conformation; Protein Subunits
PubMed: 23938955
DOI: 10.1016/j.compbiolchem.2013.06.004 -
Journal of Molecular Biology Feb 1961
Topics: Protein Subunits; Proteins; Tymovirus; Viruses
PubMed: 13711747
DOI: 10.1016/s0022-2836(61)80014-4 -
Annual Review of Biochemistry 2007The fact that ions of macromolecular complexes produced by electrospray ionization can be maintained intact in a mass spectrometer has stimulated exciting new lines of... (Review)
Review
The fact that ions of macromolecular complexes produced by electrospray ionization can be maintained intact in a mass spectrometer has stimulated exciting new lines of research. In this review we chart the progress of this research from the observation of simple homo-oligomers to complex heterogeneous macromolecular assemblies of mega-Dalton proportions. The applications described herein not only confirm the status of mass spectrometry (MS) as a structural biology approach to complement X-ray analysis or electron microscopy, but also highlight unique attributes of the methodology. This is exemplified in studies of the biogenesis of macromolecular complexes and in the exchange of subunits between macromolecular complexes. Moreover, recent successes in revealing the overall subunit architecture of complexes are set to promote MS from a complementary approach to a structural biology tool in its own right.
Topics: Mass Spectrometry; Models, Molecular; Molecular Weight; Multiprotein Complexes; Protein Conformation; Protein Subunits; Solutions
PubMed: 17328674
DOI: 10.1146/annurev.biochem.76.061005.090816 -
The Journal of Physiology Oct 2014At depolarized membrane potentials, the conductance of some voltage-gated K(+) channels is reduced by C-type inactivation. This gating process is voltage independent in...
At depolarized membrane potentials, the conductance of some voltage-gated K(+) channels is reduced by C-type inactivation. This gating process is voltage independent in Kv1 and involves a conformational change in the selectivity filter that is mediated by cooperative subunit interactions. C-type inactivation in hERG1 K(+) channels is voltage-dependent, much faster in onset and greatly attenuates currents at positive potentials. Here we investigate the potential role of subunit interactions in C-type inactivation of hERG1 channels. Point mutations in hERG1 known to eliminate (G628C/S631C), inhibit (S620T or S631A) or enhance (T618A or M645C) C-type inactivation were introduced into subunits that were combined with wild-type subunits to form concatenated tetrameric channels with defined subunit composition and stoichiometry. Channels were heterologously expressed in Xenopus oocytes and the two-microelectrode voltage clamp was used to measure the kinetics and steady-state properties of inactivation of whole cell currents. The effect of S631A or T618A mutations on inactivation was a graded function of the number of mutant subunits within a concatenated tetramer as predicted by a sequential model of cooperative subunit interactions, whereas M645C subunits increased the rate of inactivation of concatemers, as predicted for subunits that act independently of one another. For mutations located within the inactivation gate proper (S620T or G628C/S631C), the presence of a single subunit in a concatenated hERG1 tetramer disrupted gating to the same extent as that observed for mutant homotetramers. Together, our findings indicate that the final step of C-type inactivation of hERG1 channels involves a concerted, all-or-none cooperative interaction between all four subunits, and that probing the mechanisms of channel gating with concatenated heterotypic channels should be interpreted with care, as conclusions regarding the nature of subunit interactions may depend on the specific mutation used to probe the gating process.
Topics: Animals; Ether-A-Go-Go Potassium Channels; Humans; Ion Channel Gating; Point Mutation; Protein Binding; Protein Subunits; Xenopus
PubMed: 25063820
DOI: 10.1113/jphysiol.2014.277483 -
Protein Science : a Publication of the... Jun 2019The multi-subunit Ca /calmodulin-dependent protein kinase II (CaMKII) holoenzyme plays a critical role in animal learning and memory. The kinase domain of CaMKII is...
The multi-subunit Ca /calmodulin-dependent protein kinase II (CaMKII) holoenzyme plays a critical role in animal learning and memory. The kinase domain of CaMKII is connected by a flexible linker to a C-terminal hub domain that assembles into a 12- or 14-subunit scaffold that displays the kinase domains around it. Studies on CaMKII suggest that the stoichiometry and dynamic assembly/disassembly of hub oligomers may be important for CaMKII regulation. Although CaMKII is a metazoan protein, genes encoding predicted CaMKII-like hub domains, without associated kinase domains, are found in the genomes of some green plants and bacteria. We show that the hub domains encoded by three related green algae, Chlamydomonas reinhardtii, Volvox carteri f. nagarensis, and Gonium pectoral, assemble into 16-, 18-, and 20-subunit oligomers, as assayed by native protein mass spectrometry. These are the largest known CaMKII hub domain assemblies. A crystal structure of the hub domain from C. reinhardtii reveals an 18-subunit organization. We identified four intra-subunit hydrogen bonds in the core of the fold that are present in the Chlamydomonas hub domain, but not in metazoan hubs. When six point mutations designed to recapitulate these hydrogen bonds were introduced into the human CaMKII-α hub domain, the mutant protein formed assemblies with 14 and 16 subunits, instead of the normal 12- and 14-subunit assemblies. Our results show that the stoichiometric balance of CaMKII hub assemblies can be shifted readily by small changes in sequence.
Topics: Amino Acid Sequence; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Crystallography, X-Ray; Humans; Models, Molecular; Protein Domains; Protein Subunits; Sequence Alignment
PubMed: 30942928
DOI: 10.1002/pro.3614 -
Proceedings of the National Academy of... Feb 2021Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from four different, but evolutionarily related, subunits. These subunits...
Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from four different, but evolutionarily related, subunits. These subunits assemble with a precise stoichiometry and arrangement such that two chemically distinct agonist-binding sites are formed between specific subunit pairs. How this subunit complexity evolved and became entrenched is unclear. Here we show that a single historical amino acid substitution is able to constrain the subunit stoichiometry of functional acetylcholine receptors. Using a combination of ancestral sequence reconstruction, single-channel electrophysiology, and concatenated subunits, we reveal that an ancestral β-subunit can not only replace the extant β-subunit but can also supplant the neighboring δ-subunit. By forward evolving the ancestral β-subunit with a single amino acid substitution, we restore the requirement for a δ-subunit for functional channels. These findings reveal that a single historical substitution necessitates an increase in acetylcholine receptor complexity and, more generally, that simple stepwise mutations can drive subunit entrenchment in this model heteromeric protein.
Topics: Amino Acid Substitution; Cell Line; Evolution, Molecular; Humans; Protein Binding; Protein Domains; Protein Multimerization; Protein Subunits; Receptors, Nicotinic
PubMed: 33579823
DOI: 10.1073/pnas.2018731118 -
International Journal of Biological... Jun 2016Xanthine oxidase (XOD) is the members of the molybdenum hydroxylase flavoprotein family and it plays a vital role in the body's purine catabolism. In this study, we...
Xanthine oxidase (XOD) is the members of the molybdenum hydroxylase flavoprotein family and it plays a vital role in the body's purine catabolism. In this study, we cloned the XOD 37kDa subunit protein by using RT-PCR and pMD-18-T clone vector based on the total RNA extracted from chicken liver. The cloning XOD subunit protein gene was ligated into the pET-32a to construct the recombinant plasmid pET-XOD. After the pET-XOD expression vector was transformed into host cells Rosetta (DE3), the recombinant XOD subunit proteins (54.8kDa) were successfully induced by isopropy1 β-d-thiogalactoside (IPTG). Rabbit antiserums were produced by using the purification of the recombinant XOD subunit protein as antigen. The titer of the antiserum was more than 1:102,400 determined by using ELISA. The result of Western blot demonstrated that the antiserum could specifically recognize the chicken liver XOD. Immunohistochemistry and immunofluorescence showed that the XOD mainly presented in the cytoplasm of chicken hepatocytes and proximal tubular epithelial cells. Our results indicated that the XOD subunit protein polyclonal antibody prepared by this method could be used for the further researches of the biological function of the XOD in the chicken.
Topics: Amino Acid Sequence; Animals; Base Sequence; Chickens; Epitopes; Escherichia coli; Gene Expression; Genetic Engineering; Genetic Vectors; Kidney; Liver; Protein Sorting Signals; Protein Subunits; Xanthine Oxidase
PubMed: 26949113
DOI: 10.1016/j.ijbiomac.2016.03.001 -
Hereditas Dec 2014Wheat bread-making quality is mainly determined by glutenin proteins in the grain, which exist in a wide range of variable alleles with differential influence on...
Wheat bread-making quality is mainly determined by glutenin proteins in the grain, which exist in a wide range of variable alleles with differential influence on processing attributes. A recently identified allele, Bx7 over-expression (Bx7(oe) ), has been showing highly significant positive effects on wheat dough strength over the normally expressed Bx7 allele. SDS-PAGE and normal RP-HPLC procedures failed to separate the two alleles. In the current study, an extensively optimised MALDI-TOF based procedure and a refined DNA based marker for efficiently differentiating Bx7(oe) from normal Bx7 allele were established. Results indicated that the MALDI-TOF procedure is cost effective, high throughput, and proven reliable, while the refined PCR marker only amplifies Bx7(oe) allele, a clear advantage over the previously developed codominant marker.
Topics: Alleles; DNA, Plant; Genetic Markers; Glutens; Protein Subunits; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Triticum
PubMed: 25588305
DOI: 10.1111/hrd2.00069 -
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