Chemotactic Signaling by Single-Chain Chemoreceptors

PubMed Central

Mowery, Patricia; Ames, Peter; Reiser, Rebecca H.; Parkinson, John S.

2015-01-01

Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit swapping interactions that will need to be taken into account in experimental applications of single-chain chemoreceptors. PMID:26709829

Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone.

PubMed

Ismaya, Wangsa T; Rozeboom, Henriëtte J; Weijn, Amrah; Mes, Jurriaan J; Fusetti, Fabrizia; Wichers, Harry J; Dijkstra, Bauke W

2011-06-21

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 Å resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ∼392 residues and two L subunits of ∼150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ∼100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 Å away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.

Bioinspired synthesis of pentalene-based chromophores from an oligoketone chain.

PubMed

Saito, Yuki; Higuchi, Masayuki; Yoshioka, Shota; Senboku, Hisanori; Inokuma, Yasuhide

2018-04-24

We report a bioinspired synthesis of 2,5-dihydropentalene-based chromophores from an aliphatic oligoketone bearing 1,3- and 1,4-diketone subunits. Unlike the natural polyketone sequence, fused five-membered rings were formed via an intramolecular aldol condensation. A subsequent Knoevenagel condensation reaction with malononitrile furnished a multiply cross-conjugated π-system with low-lying LUMO levels. Furthermore, pentalenes obtained from a non-conjugated aliphatic chain exhibited visible absorption and solid-state fluorescence.

Subunit composition and structure of subcomponent C1q of the first component of human complement.

PubMed

Reid, K B; Porter, R R

1976-04-01

1. Unreduced human subcomponent C1q was shown by electrophoresis on polyacrylamide gels run in the presence of sodium dodecyl sulphate to be composed of two types of non-covalently linked subunits of apparent mol.wts. 69 000 and 54 000. The ratio of the two subunits was markedly affected by the ionic strength of the applied sample. At a low ionic strength of applied sample, which gave the optimum value for the 54 000-apparent mol.wt. subunit, a ratio of 1.99:1.00 was obtained for the ratio of the 69 000-apparent mol.wt. subunit to the 5400-apparent-mol.wt. subunit. The amount of the 54 000-apparent-mol.wt. subunit detected in the expected position on the gel was found to be inversely proportional to increases in the ionic strength of the applled sample. 2. Human subcomponent C1q on reduction and alkylation, or oxidation, yields equimolar amounts of three chains designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. The results obtained by Yonemasu & Stroud [(1972) Immunochemistry 9, 545-554], which showed that the 69 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the A and B chains and that the 54 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the C chain, were confirmed. 3. Gel filtration on Sephadex G-200 in 6.0M-guanidinium chloride showed that both types of unreduced subunit were eluted together as a single symmetrical peak of apparent mol.wt. 49 000-50 000 when globular proteins were used as markers. The molecular weights of the oxidized or reduced A, B and C chains have been shown previously to be very similar all being in the range 23 000-24 000 [Reid et al. (1972) Biochem. J. 130, 749-763; Reid (1974) Biochem. J. 141, 189-203]. 4. It is proposed that subcomponent C1q (mol.wt. 410000) is composed of nine non-covalently linked subunits, i.e. six A-B dimers and three C-C dimers. 5. A structure for subcomponent C1q is proposed and is based on the assumption that the collagen-like regions of 78 residues in each of the A, B and C chains are combined to form a triple-helical structure of the same type as is found in collagens.

The Small Subunit of Snapdragon Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Tobacco Geranylgeranyl Diphosphate Synthase in Planta[W

PubMed Central

Orlova, Irina; Nagegowda, Dinesh A.; Kish, Christine M.; Gutensohn, Michael; Maeda, Hiroshi; Varbanova, Marina; Fridman, Eyal; Yamaguchi, Shinjiro; Hanada, Atsushi; Kamiya, Yuji; Krichevsky, Alexander; Citovsky, Vitaly; Pichersky, Eran; Dudareva, Natalia

2009-01-01

Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta. PMID:20028839

Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L.

PubMed

Zhu, Shaotong; Canales, Alejandra; Bedair, Mai; Vik, Steven B

2016-06-01

Complex I is a multi-subunit enzyme of the respiratory chain with seven core subunits in its membrane arm (A, H, J, K, L, M, and N). In the enzyme from Escherichia coli the C-terminal ten amino acids of subunit K lie along the lateral helix of subunit L, and contribute to a junction of subunits K, L and N on the cytoplasmic surface. Using double cysteine mutagenesis, the cross-linking of subunit K (R99C) to either subunit L (K581C) or subunit N (T292C) was attempted. A partial yield of cross-linked product had no effect on the activity of the enzyme, or on proton translocation, suggesting that the C-terminus of subunit K has no dynamic role in function. To further elucidate the role of subunit K genetic deletions were constructed at the C-terminus. Upon the serial deletion of the last 4 residues of the C-terminus of subunit K, various results were obtained. Deletion of one amino acid had little effect on the activity of Complex I, but deletions of 2 or more amino acids led to total loss of enzyme activity and diminished levels of subunits L, M, and N in preparations of membrane vesicles. Together these results suggest that while the C-terminus of subunit K has no dynamic role in energy transduction by Complex I, it is vital for the correct assembly of the enzyme.

Mitochondrial DNA Depletion in Respiratory Chain-Deficient Parkinson Disease Neurons.

PubMed

Grünewald, Anne; Rygiel, Karolina A; Hepplewhite, Philippa D; Morris, Christopher M; Picard, Martin; Turnbull, Doug M

2016-03-01

To determine the extent of respiratory chain abnormalities and investigate the contribution of mtDNA to the loss of respiratory chain complexes (CI-IV) in the substantia nigra (SN) of idiopathic Parkinson disease (IPD) patients at the single-neuron level. Multiple-label immunofluorescence was applied to postmortem sections of 10 IPD patients and 10 controls to quantify the abundance of CI-IV subunits (NDUFB8 or NDUFA13, SDHA, UQCRC2, and COXI) and mitochondrial transcription factors (TFAM and TFB2M) relative to mitochondrial mass (porin and GRP75) in dopaminergic neurons. To assess the involvement of mtDNA in respiratory chain deficiency in IPD, SN neurons, isolated with laser-capture microdissection, were assayed for mtDNA deletions, copy number, and presence of transcription/replication-associated 7S DNA employing a triplex real-time polymerase chain reaction (PCR) assay. Whereas mitochondrial mass was unchanged in single SN neurons from IPD patients, we observed a significant reduction in the abundances of CI and II subunits. At the single-cell level, CI and II deficiencies were correlated in patients. The CI deficiency concomitantly occurred with low abundances of the mtDNA transcription factors TFAM and TFB2M, which also initiate transcription-primed mtDNA replication. Consistent with this, real-time PCR analysis revealed fewer transcription/replication-associated mtDNA molecules and an overall reduction in mtDNA copy number in patients. This effect was more pronounced in single IPD neurons with severe CI deficiency. Respiratory chain dysfunction in IPD neurons not only involves CI, but also extends to CII. These deficiencies are possibly a consequence of the interplay between nDNA and mtDNA-encoded factors mechanistically connected via TFAM. © 2016 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.

PubMed

Cheng, C; Prince, L S; Snyder, P M; Welsh, M J

1998-08-28

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

PubMed Central

Shen, Peter S.; Park, Joseph; Qin, Yidan; Li, Xueming; Parsawar, Krishna; Larson, Matthew H.; Cox, James; Cheng, Yifan; Lambowitz, Alan M.; Weissman, Jonathan S.; Brandman, Onn; Frost, Adam

2015-01-01

In Eukarya, stalled translation induces 40S dissociation and recruitment of the Ribosome Quality control Complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here, we report cryoEM structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S at sites exposed after 40S dissociation, placing the Ltn1p RING domain near the exit channel and Rqc2p over the P-site tRNA. We further demonstrate that Rqc2p recruits alanine and threonine charged tRNA to the A-site and directs elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with Carboxy-terminal Ala and Thr extensions (“CAT tails”). PMID:25554787

Biogenesis of mitochondria in cauliflower (Brassica oleracea var. botrytis) curds subjected to temperature stress and recovery involves regulation of the complexome, respiratory chain activity, organellar translation and ultrastructure.

PubMed

Rurek, Michal; Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

2015-01-01

The biogenesis of the cauliflower curd mitochondrial proteome was investigated under cold, heat and the recovery. For the first time, two dimensional fluorescence difference gel electrophoresis was used to study the plant mitochondrial complexome in heat and heat recovery. Particularly, changes in the complex I and complex III subunits and import proteins, and the partial disintegration of matrix complexes were observed. The presence of unassembled subunits of ATP synthase was accompanied by impairment in mitochondrial translation of its subunit. In cold and heat, the transcription profiles of mitochondrial genes were uncorrelated. The in-gel activities of respiratory complexes were particularly affected after stress recovery. Despite a general stability of respiratory chain complexes in heat, functional studies showed that their activity and the ATP synthesis yield were affected. Contrary to cold stress, heat stress resulted in a reduced efficiency of oxidative phosphorylation likely due to changes in alternative oxidase (AOX) activity. Stress and stress recovery differently modulated the protein level and activity of AOX. Heat stress induced an increase in AOX activity and protein level, and AOX1a and AOX1d transcript level, while heat recovery reversed the AOX protein and activity changes. Conversely, cold stress led to a decrease in AOX activity (and protein level), which was reversed after cold recovery. Thus, cauliflower AOX is only induced by heat stress. In heat, contrary to the AOX activity, the activity of rotenone-insensitive internal NADH dehydrogenase was diminished. The relevance of various steps of plant mitochondrial biogenesis to temperature stress response and recovery is discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Production and characterization of murine models of classic and intermediate maple syrup urine disease

PubMed Central

Homanics, Gregg E; Skvorak, Kristen; Ferguson, Carolyn; Watkins, Simon; Paul, Harbhajan S

2006-01-01

Background Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model. Methods To create a murine model of classic MSUD, we used gene targeting and embryonic stem cell technologies to create a mouse line that lacked a functional E2 subunit gene of branched-chain keto acid dehydrogenase. To create a murine model of intermediate MSUD, we used transgenic technology to express a human E2 cDNA on the knockout background. Mice of both models were characterized at the molecular, biochemical, and whole animal levels. Results By disrupting the E2 subunit gene of branched-chain keto acid dehydrogenase, we created a gene knockout mouse model of classic MSUD. The homozygous knockout mice lacked branched-chain keto acid dehydrogenase activity, E2 immunoreactivity, and had a 3-fold increase in circulating branched-chain amino acids. These metabolic derangements resulted in neonatal lethality. Transgenic expression of a human E2 cDNA in the liver of the E2 knockout animals produced a model of intermediate MSUD. Branched-chain keto acid dehydrogenase activity was 5–6% of normal and was sufficient to allow survival, but was insufficient to normalize circulating branched-chain amino acids levels, which were intermediate between wildtype and the classic MSUD mouse model. Conclusion These mice represent important animal models that closely approximate the phenotype of humans with the classic and intermediate forms of MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease. PMID:16579849

Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.

PubMed

Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Gago, Gabriela; Spina, Lucie; Bardou, Fabienne; Lemassu, Anne; Quémard, Annaïk; Gramajo, Hugo

2017-04-01

Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two β subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C 24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen. © 2017 Federation of European Biochemical Societies.

Mitochondrial DNA Depletion in Respiratory Chain–Deficient Parkinson Disease Neurons

PubMed Central

Rygiel, Karolina A.; Hepplewhite, Philippa D.; Morris, Christopher M.; Picard, Martin; Turnbull, Doug M.

2016-01-01

Objective To determine the extent of respiratory chain abnormalities and investigate the contribution of mtDNA to the loss of respiratory chain complexes (CI–IV) in the substantia nigra (SN) of idiopathic Parkinson disease (IPD) patients at the single‐neuron level. Methods Multiple‐label immunofluorescence was applied to postmortem sections of 10 IPD patients and 10 controls to quantify the abundance of CI–IV subunits (NDUFB8 or NDUFA13, SDHA, UQCRC2, and COXI) and mitochondrial transcription factors (TFAM and TFB2M) relative to mitochondrial mass (porin and GRP75) in dopaminergic neurons. To assess the involvement of mtDNA in respiratory chain deficiency in IPD, SN neurons, isolated with laser‐capture microdissection, were assayed for mtDNA deletions, copy number, and presence of transcription/replication‐associated 7S DNA employing a triplex real‐time polymerase chain reaction (PCR) assay. Results Whereas mitochondrial mass was unchanged in single SN neurons from IPD patients, we observed a significant reduction in the abundances of CI and II subunits. At the single‐cell level, CI and II deficiencies were correlated in patients. The CI deficiency concomitantly occurred with low abundances of the mtDNA transcription factors TFAM and TFB2M, which also initiate transcription‐primed mtDNA replication. Consistent with this, real‐time PCR analysis revealed fewer transcription/replication‐associated mtDNA molecules and an overall reduction in mtDNA copy number in patients. This effect was more pronounced in single IPD neurons with severe CI deficiency. Interpretation Respiratory chain dysfunction in IPD neurons not only involves CI, but also extends to CII. These deficiencies are possibly a consequence of the interplay between nDNA and mtDNA‐encoded factors mechanistically connected via TFAM. ANN NEUROL 2016;79:366–378 PMID:26605748

Characterization of durum wheat high molecular weight glutenin subunits Bx20 and By20 sequences by a molecular and proteomic approach.

PubMed

Santagati, Vito Davide; Sestili, Francesco; Lafiandra, Domenico; D'Ovidio, Renato; Rogniaux, Helene; Masci, Stefania

2016-07-01

Wheat high molecular weight glutenin subunit variation is important because of its great influence on glutenin polymer structure, that is related to dough technological properties. Among the different subunits, the pair Bx20 and By20 is known to have a negative effect on quality, but the reasons are not clear: Bx20 has two cysteines, which theoretically make this subunit a chain extender of the glutenin polymer, just like the other Bx subunits, showing four cysteines, two of which should be involved in intra-molecular disulfide bonds. By20 has never been characterized so far at molecular level. Here we report the nucleotide sequences of Bx20 and By20 genes isolated from the durum wheat cultivar 'Lira 45' and the validation of the corresponding deduced amino acid sequences by using MALDI-TOF and LC-MS/MS. Four nucleotide differences were identified in the Bx20 gene with respect to the deduced sequence present in NCBI, causing two amino acid substitutions. For the By20 subunit, nucleotide and amino acid sequences revealed a great similarity to By15, both at gene and protein levels, showing five nucleotide changes generating two amino acid differences. No evidence of post-translational modifications has been found. Hypotheses are formulated in regard to relationships with technological quality. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Folding and trimerization of clathrin subunits at the triskelion hub.

PubMed

Näthke, I S; Heuser, J; Lupas, A; Stock, J; Turck, C W; Brodsky, F M

1992-03-06

The triskelion shape of the clathrin molecule enables it to form the polyhedral protein network that covers clathrin-coated pits and vesicles. Domains within the clathrin heavy chain that are responsible for maintaining triskelion shape and function were identified and localized. Sequences that mediate trimerization are distal to the carboxyl terminus and are adjacent to a domain that mediates both light chain binding and clathrin assembly. Structural modeling predicts that within this domain, the region of heavy chain-light chain interaction is a bundle of three or four alpha helices. These studies establish a low resolution model of clathrin subunit folding in the central portion (hub) of the triskelion, thus providing a basis for future mutagenesis experiments.

Molecular oxygen migration through the xenon docking sites of human hemoglobin in the R-state.

PubMed

Lepeshkevich, Sergei V; Gilevich, Syargey N; Parkhats, Marina V; Dzhagarov, Boris M

2016-09-01

A nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O2) rebinding to tetrameric human hemoglobin and its isolated α and β chains in buffer solutions equilibrated with 1atm of air and up to 25atm of xenon. Xenon binding to the isolated α chains and to the α subunits within tetrameric hemoglobin was found to cause a decrease in the efficiency of O2 escape by a factor of ~1.30 and 3.3, respectively. A kinetic model for O2 dissociation, rebinding, and migration through two alternative pathways in the hemoglobin subunits was introduced and discussed. It was shown that, in the isolated α chains and α subunits within tetrameric hemoglobin, nearly one- and two-third escaping molecules of O2 leave the protein via xenon docking sites, respectively. The present experimental data support the idea that O2 molecule escapes from the β subunits mainly through the His(E7) gate, and show unambiguously that, in the α subunits, in addition to the direct E7 channel, there is at least one alternative escape route leading to the exterior via the xenon docking sites. Copyright © 2016 Elsevier B.V. All rights reserved.

Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans

PubMed Central

MJ, Falk; Z, Zhang; Rosenjack; Nissim; E, Daikhin; Nissim; MM, Sedensky; M, Yudkoff; PG, Morgan

2008-01-01

C. elegans affords a model of primary mitochondrial dysfunction that provides insight into cellular adaptations which accompany mutations in nuclear gene that encode mitochondrial proteins. To this end, we characterized genome-wide expression profiles of C. elegans strains with mutations in nuclear-encoded subunits of respiratory chain complexes. Our goal was to detect concordant changes among clusters of genes that comprise defined metabolic pathways. Results indicate that respiratory chain mutants significantly upregulate a variety of basic cellular metabolic pathways involved in carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defense pathways such as the metabolism of P450 and glutathione. To further confirm and extend expression analysis findings, quantitation of whole worm free amino acid levels was performed in C. elegans mitochondrial mutants for subunits of complexes I, II, and III. Significant differences were seen for 13 of 16 amino acid levels in complex I mutants compared with controls, as well as overarching similarities among profiles of complex I, II, and III mutants compared with controls. The specific pattern of amino acid alterations observed provides novel evidence to suggest that an increase in glutamate-linked transamination reactions caused by the failure of NAD+ dependent oxidation of ketoacids occurs in primary mitochondrial respiratory chain mutants. Recognition of consistent alterations among patterns of nuclear gene expression for multiple biochemical pathways and in quantitative amino acid profiles in a translational genetic model of mitochondrial dysfunction allows insight into the complex pathogenesis underlying primary mitochondrial disease. Such knowledge may enable the development of a metabolomic profiling diagnostic tool applicable to human mitochondrial disease. PMID:18178500

Low-intensity laser irradiation at 660 nm stimulates transcription of genes involved in the electron transport chain.

PubMed

Masha, Roland T; Houreld, Nicolette N; Abrahamse, Heidi

2013-02-01

Low-intensity laser irradiation (LILI) has been shown to stimulate cellular functions leading to increased adenosine triphosphate (ATP) synthesis. This study was undertaken to evaluate the effect of LILI on genes involved in the mitochondrial electron transport chain (ETC, complexes I-IV) and oxidative phosphorylation (ATP synthase). Four human skin fibroblast cell models were used in this study: normal non-irradiated cells were used as controls while wounded, diabetic wounded, and ischemic cells were irradiated. Cells were irradiated with a 660 nm diode laser with a fluence of 5 J/cm(2) and gene expression determined by quantitative real-time reverse transcription (RT) polymerase chain reaction (PCR). LILI upregulated cytochrome c oxidase subunit VIb polypeptide 2 (COX6B2), cytochrome c oxidase subunit VIc (COX6C), and pyrophosphatase (inorganic) 1 (PPA1) in diabetic wounded cells; COX6C, ATP synthase, H+transporting, mitochondrial Fo complex, subunit B1 (ATP5F1), nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 alpha subcomplex, 11 (NDUFA11), and NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) in wounded cells; and ATPase, H+/K+ exchanging, beta polypeptide (ATP4B), and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C2 (subunit 9) (ATP5G2) in ischemic cells. LILI at 660 nm stimulates the upregulation of genes coding for subunits of enzymes involved in complexes I and IV and ATP synthase.

Small Subunits of Serine Palmitoyltransferase (ssSPTs) and Their Physiological Roles

DTIC Science & Technology

2014-02-12

showing that organisms also have unique sphingoid base chain lengths. In insects, such as Drosophila melanogaster , the predominant chain lengths of the ... Drosophila melanogaster mutant defective in male meiotic cytokinesis (‘Ghiberti’) has a mutation in a gene with low homology to the ssSPT subunits of...INTRODUCTION: Sphingolipid metabolism in Drosophila melanogaster (fly) is an active area of research. It is a good model system to study the roles of

The Assembly Pathway of Mitochondrial Respiratory Chain Complex I.

PubMed

Guerrero-Castillo, Sergio; Baertling, Fabian; Kownatzki, Daniel; Wessels, Hans J; Arnold, Susanne; Brandt, Ulrich; Nijtmans, Leo

2017-01-10

Mitochondrial complex I is the largest integral membrane enzyme of the respiratory chain and consists of 44 different subunits encoded in the mitochondrial and nuclear genome. Its biosynthesis is a highly complicated and multifaceted process involving at least 14 additional assembly factors. How these subunits assemble into a functional complex I and where the assembly factors come into play is largely unknown. Here, we applied a dynamic complexome profiling approach to elucidate the assembly of human mitochondrial complex I and its further incorporation into respiratory chain supercomplexes. We delineate the stepwise incorporation of all but one subunit into a series of distinct assembly intermediates and their association with known and putative assembly factors, which had not been implicated in this process before. The resulting detailed and comprehensive model of complex I assembly is fully consistent with recent structural data and the remarkable modular architecture of this multiprotein complex. Copyright © 2017 Elsevier Inc. All rights reserved.

Conformational coupling between the active site and residues within the K(C)-channel of the Vibrio cholerae cbb3-type (C-family) oxygen reductase.

PubMed

Ahn, Young O; Mahinthichaichan, Paween; Lee, Hyun Ju; Ouyang, Hanlin; Kaluka, Daniel; Yeh, Syun-Ru; Arjona, Davinia; Rousseau, Denis L; Tajkhorshid, Emad; Adelroth, Pia; Gennis, Robert B

2014-10-21

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K(C)-channel is a conserved glutamate in subunit III. However, the majority of the K(C)-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K(C)-channel, was found to depend on the conformation of Y241(Vc), located in subunit I at the interface with subunit III. Mutations of Y241(Vc) (to A/F/H/S) in the Vibrio cholerae cbb3 eliminate catalytic activity, but also cause perturbations that propagate over a 28-Å distance to the active site heme b3. The data suggest a linkage between residues lining the K(C)-channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241(Vc) and the active site cross-linked Y255(Vc), as well as two CuB histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.

Three-dimensional structure of human electron transfer flavoprotein to 2.1-Å resolution

PubMed Central

Roberts, David L.; Frerman, Frank E.; Kim, Jung-Ja P.

1996-01-01

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-Å resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the α subunit and the third domain is made up entirely by the β subunit. The N-terminal portion of the α subunit and the majority of the β subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the α subunit. Alignment of all the known sequences for the ETF α subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of αT266, suggesting why the pathogenic mutation, αT266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4′-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between αH286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins. PMID:8962055

Russell body inducing threshold depends on the variable domain sequences of individual human IgG clones and the cellular protein homeostasis.

PubMed

Stoops, Janelle; Byrd, Samantha; Hasegawa, Haruki

2012-10-01

Russell bodies are intracellular aggregates of immunoglobulins. Although the mechanism of Russell body biogenesis has been extensively studied by using truncated mutant heavy chains, the importance of the variable domain sequences in this process and in immunoglobulin biosynthesis remains largely unknown. Using a panel of structurally and functionally normal human immunoglobulin Gs, we show that individual immunoglobulin G clones possess distinctive Russell body inducing propensities that can surface differently under normal and abnormal cellular conditions. Russell body inducing predisposition unique to each immunoglobulin G clone was corroborated by the intrinsic physicochemical properties encoded in the heavy chain variable domain/light chain variable domain sequence combinations that define each immunoglobulin G clone. While the sequence based intrinsic factors predispose certain immunoglobulin G clones to be more prone to induce Russell bodies, extrinsic factors such as stressful cell culture conditions also play roles in unmasking Russell body propensity from immunoglobulin G clones that are normally refractory to developing Russell bodies. By taking advantage of heterologous expression systems, we dissected the roles of individual subunit chains in Russell body formation and examined the effect of non-cognate subunit chain pair co-expression on Russell body forming propensity. The results suggest that the properties embedded in the variable domain of individual light chain clones and their compatibility with the partnering heavy chain variable domain sequences underscore the efficiency of immunoglobulin G biosynthesis, the threshold for Russell body induction, and the level of immunoglobulin G secretion. We propose that an interplay between the unique properties encoded in variable domain sequences and the state of protein homeostasis determines whether an immunoglobulin G expressing cell will develop the Russell body phenotype in a dynamic cellular setting. Copyright © 2012 Elsevier B.V. All rights reserved.

Crotoxin: Structural Studies, Mechanism of Action and Cloning of Its Gene

DTIC Science & Technology

1987-03-01

other venoms and examine their toxin neutral- izing ability. The amino acid sequences of both crotoxin subunits were determined Is a prelude to cloning...be examined for their potential as anti-idiotype vaccines The complete amino acid sequence of the basic subunit and two of the three dic subunit chains...of crotoxin from the venom of C.d. terrificus has been de rmined. Sequence comparison data suggest that the non-toxic, acidic subunit was derived

(PS)2: protein structure prediction server version 3.0.

PubMed

Huang, Tsun-Tsao; Hwang, Jenn-Kang; Chen, Chu-Huang; Chu, Chih-Sheng; Lee, Chi-Wen; Chen, Chih-Chieh

2015-07-01

Protein complexes are involved in many biological processes. Examining coupling between subunits of a complex would be useful to understand the molecular basis of protein function. Here, our updated (PS)(2) web server predicts the three-dimensional structures of protein complexes based on comparative modeling; furthermore, this server examines the coupling between subunits of the predicted complex by combining structural and evolutionary considerations. The predicted complex structure could be indicated and visualized by Java-based 3D graphics viewers and the structural and evolutionary profiles are shown and compared chain-by-chain. For each subunit, considerations with or without the packing contribution of other subunits cause the differences in similarities between structural and evolutionary profiles, and these differences imply which form, complex or monomeric, is preferred in the biological condition for the subunit. We believe that the (PS)(2) server would be a useful tool for biologists who are interested not only in the structures of protein complexes but also in the coupling between subunits of the complexes. The (PS)(2) is freely available at http://ps2v3.life.nctu.edu.tw/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

PubMed

Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

1996-07-01

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2

PubMed Central

De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

2016-01-01

Integrins are heterodimeric cell-surface adhesion molecules comprising one of possible 18 α-chains and one of possible 8 β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalised by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalisation by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with AP2 C-µ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions. PMID:26779610

Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2.

PubMed

De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

2016-02-01

Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-μ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions.

Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

PubMed Central

Ghosh, D; Weeks, C M; Grochulski, P; Duax, W L; Erman, M; Rimsay, R L; Orr, J C

1991-01-01

The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3 alpha,20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the alpha/beta fold characteristic of the dinucleotide binding region. The fold of the rest of the subunit, the quaternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3 alpha- and 20 beta-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site. Images PMID:1946424

The biochemistry of the protein crystal toxin of Bacillus thuringiensis

Treesearch

Paul G. Fast

1985-01-01

The crystal consists of dimeric protein subunits. The monomer peptide chains are held together in the subunit and the subunit in the crystal by disulfide and non-covalent bonds. The monomer peptide has a molecular weight of about 130 kdaltons which, in the presence of proteases, is hydrolyzed to a protease-resistant-protein of 65 kda that is toxic both to larvae by...

The delta-subunit of murine guanine nucleotide exchange factor eIF-2B. Characterization of cDNAs predicts isoforms differing at the amino-terminal end.

PubMed

Henderson, R A; Krissansen, G W; Yong, R Y; Leung, E; Watson, J D; Dholakia, J N

1994-12-02

Protein synthesis in mammalian cells is regulated at the level of the guanine nucleotide exchange factor, eIF-2B, which catalyzes the exchange of eukaryotic initiation factor 2-bound GDP for GTP. We have isolated and sequenced cDNA clones encoding the delta-subunit of murine eIF-2B. The cDNA sequence encodes a polypeptide of 544 amino acids with molecular mass of 60 kDa. Antibodies against a synthetic polypeptide of 30 amino acids deduced from the cDNA sequence specifically react with the delta-subunit of mammalian eIF-2B. The cDNA-derived amino acid sequence shows significant homology with the yeast translational regulator Gcd2, supporting the hypothesis that Gcd2 may be the yeast homolog of the delta-subunit of mammalian eIF-2B. Primer extension studies and anchor polymerase chain reaction analysis were performed to determine the 5'-end of the transcript for the delta-subunit of eIF-2B. Results of these experiments demonstrate two different mRNAs for the delta-subunit of eIF-2B in murine cells. The isolation and characterization of two different full-length cDNAs also predicts the presence of two alternate forms of the delta-subunit of eIF-2B in murine cells. These differ at their amino-terminal end but have identical nucleotide sequences coding for amino acids 31-544.

Effect of single-point mutations on the stability and immunogenicity of a recombinant ricin A chain subunit vaccine antigen.

PubMed

Thomas, Justin C; O'Hara, Joanne M; Hu, Lei; Gao, Fei P; Joshi, Sangeeta B; Volkin, David B; Brey, Robert N; Fang, Jianwen; Karanicolas, John; Mantis, Nicholas J; Middaugh, C Russell

2013-04-01

There is great interest in the design and development of highly thermostable and immunogenic protein subunit vaccines for biodefense. In this study, we used two orthogonal and complementary computational protein design approaches to generate a series of single-point mutants of RiVax, an attenuated recombinant ricin A chain (RTA) protein subunit vaccine antigen. As assessed by differential scanning calorimetry, the conformational stabilities of the designed mutants ranged from 4°C less stable to 4.5°C more stable than RiVax, depending on solution pH. Two more thermostable (V18P, C171L) and two less thermostable (T13V, S89T) mutants that displayed native-like secondary and tertiary structures (as determined by circular dichroism and fluorescence spectral analysis, respectively) were tested for their capacity to elicit RTA-specific antibodies and toxin-neutralizing activity. Following a prime-boost regimen, we found qualitative differences with respect to specific antibody titers and toxin neutralizing antibody levels induced by the different mutants. Upon a second boost with the more thermostable mutant C171L, a statistically significant increase in RTA-specific antibody titers was observed when compared with RiVax-immunized mice. Notably, the results indicate that single residue changes can be made to the RiVax antigen that increase its thermal stability without adversely impacting the efficacy of the vaccine.

Molecular basis of maple syrup urine disease: Novel mutations at the E1[alpha] locus that impair E1([alpha][sub 2][beta][sub 2]) assembly or decrease steady-state E1[alpha] mRNA levels of branched-chain [alpha]-keto acid dehydrogenase complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, J.L.; Fisher, C.R.; Chuang, D.T.

1994-08-01

The authors report the occurrence of three novel mutations in the E1[alpha] (BCKDHA) locus of the branched-chain [alpha]-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1[alpha] gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1[alpha] subunit. Both the 8-bp deletion and the single C insertionmore » generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1[alpha] mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1[alpha] subunit impairs its proper assembly with the normal E1[beta]. Unassembled as well as misassembled E1[alpha] and E1[beta] subunits are degraded in the cell. 32 refs., 8 figs.« less

Metallothionein 2A affects the cell respiration by suppressing the expression of mitochondrial protein cytochrome c oxidase subunit II.

PubMed

Bragina, Olga; Gurjanova, Karina; Krishtal, Jekaterina; Kulp, Maria; Karro, Niina; Tõugu, Vello; Palumaa, Peep

2015-06-01

Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.

Cardiac metabolic pathways affected in the mouse model of barth syndrome.

PubMed

Huang, Yan; Powers, Corey; Madala, Satish K; Greis, Kenneth D; Haffey, Wendy D; Towbin, Jeffrey A; Purevjav, Enkhsaikhan; Javadov, Sabzali; Strauss, Arnold W; Khuchua, Zaza

2015-01-01

Cardiolipin (CL) is a mitochondrial phospholipid essential for electron transport chain (ETC) integrity. CL-deficiency in humans is caused by mutations in the tafazzin (Taz) gene and results in a multisystem pediatric disorder, Barth syndrome (BTHS). It has been reported that tafazzin deficiency destabilizes mitochondrial respiratory chain complexes and affects supercomplex assembly. The aim of this study was to investigate the impact of Taz-knockdown on the mitochondrial proteomic landscape and metabolic processes, such as stability of respiratory chain supercomplexes and their interactions with fatty acid oxidation enzymes in cardiac muscle. Proteomic analysis demonstrated reduction of several polypeptides of the mitochondrial respiratory chain, including Rieske and cytochrome c1 subunits of complex III, NADH dehydrogenase alpha subunit 5 of complex I and the catalytic core-forming subunit of F0F1-ATP synthase. Taz gene knockdown resulted in upregulation of enzymes of folate and amino acid metabolic pathways in heart mitochondria, demonstrating that Taz-deficiency causes substantive metabolic remodeling in cardiac muscle. Mitochondrial respiratory chain supercomplexes are destabilized in CL-depleted mitochondria from Taz knockdown hearts resulting in disruption of the interactions between ETC and the fatty acid oxidation enzymes, very long-chain acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase, potentially affecting the metabolic channeling of reducing equivalents between these two metabolic pathways. Mitochondria-bound myoglobin was significantly reduced in Taz-knockdown hearts, potentially disrupting intracellular oxygen delivery to the oxidative phosphorylation system. Our results identify the critical pathways affected by the Taz-deficiency in mitochondria and establish a future framework for development of therapeutic options for BTHS.

Decreased enzyme activity and contents of hepatic branched-chain alpha-keto acid dehydrogenase complex subunits in a rat model for type 2 diabetes mellitus.

PubMed

Bajotto, Gustavo; Murakami, Taro; Nagasaki, Masaru; Sato, Yuzo; Shimomura, Yoshiharu

2009-10-01

The mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC) is responsible for the committed step in branched-chain amino acid catabolism. In the present study, we examined BCKDC regulation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats both before (8 weeks of age) and after (25 weeks of age) the onset of type 2 diabetes mellitus. Long-Evans Tokushima Otsuka (LETO) rats were used as controls. Plasma branched-chain amino acid and branched-chain alpha-keto acid concentrations were significantly increased in young and middle-aged OLETF rats. Although the hepatic complex was nearly 100% active in all animals, total BCKDC activity and protein abundance of E1alpha, E1beta, and E2 subunits were markedly lower in OLETF than in LETO rats at 8 and 25 weeks of age. In addition, hepatic BCKDC activity and protein amounts were significantly decreased in LETO rats aged 25 weeks than in LETO rats aged 8 weeks. In skeletal muscle, E1beta and E2 proteins were significantly reduced, whereas E1alpha tended to increase in OLETF rats. Taken together, these results suggest that (1) whole-body branched-chain alpha-keto acid oxidation capacity is extremely reduced in OLETF rats independently of diabetes development, (2) the aging process decreases BCKDC activity and protein abundance in the liver of normal rats, and (3) differential posttranscriptional regulation for the subunits of BCKDC may exist in skeletal muscle.

Crotoxin: Structural Studies, Mechanism of Action and Cloning of Its gene

DTIC Science & Technology

1989-12-01

B-chain. Sequencing of the three peptides present in the acidic subunit, two of which are blocked by pyroglutamate , represents a significant...We have completed the sequence determination of both the basic and acidic subunits of crotoxin. The acidic subunit peptides were difficult, since two...of the three peptides were blocked at the amino-terminus by pyroglutamate . Earlier structural studies on crotoxin and related crotalid dimeric

Rattlesnake Neurotoxin Structure, Mechanism of Action, Immunology and Molecular Biology

DTIC Science & Technology

1992-09-10

and Kaiser, 1990). Sequencing of the three peptides present in the acidic subunit, two of which are blocked by pyroglutamate , represents a significant...deblock with pyroglutamate aminopeptidase were unsuccessful. The B-chain contained 35 amino acids and showed 91% amino acid identity witn the...similarities of all rattlesnake neurotoxins, showed that the acidic subunit plays more than a chaperone role for the basic subunit and is clearly

Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues.

PubMed

Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia; Calzia, Daniela; Beitia, Maider; Morelli, Alessandro; Rosano, Camillo

2017-01-22

F 1 F o -ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H + flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H + within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F 1 and F O domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo. Copyright © 2016 Elsevier Inc. All rights reserved.

Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia

F{sub 1}F{sub o}-ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H{sup +} flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H{sup +} within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F{sub 1} andmore » F{sub O} domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo.« less

URF6, Last Unidentified Reading Frame of Human mtDNA, Codes for an NADH Dehydrogenase Subunit

NASA Astrophysics Data System (ADS)

Chomyn, Anne; Cleeter, Michael W. J.; Ragan, C. Ian; Riley, Marcia; Doolittle, Russell F.; Attardi, Giuseppe

1986-10-01

The polypeptide encoded in URF6, the last unassigned reading frame of human mitochondrial DNA, has been identified with antibodies to peptides predicted from the DNA sequence. Antibodies prepared against highly purified respiratory chain NADH dehydrogenase from beef heart or against the cytoplasmically synthesized 49-kilodalton iron-sulfur subunit isolated from this enzyme complex, when added to a deoxycholate or a Triton X-100 mitochondrial lysate of HeLa cells, specifically precipitated the URF6 product together with the six other URF products previously identified as subunits of NADH dehydrogenase. These results strongly point to the URF6 product as being another subunit of this enzyme complex. Thus, almost 60% of the protein coding capacity of mammalian mitochondrial DNA is utilized for the assembly of the first enzyme complex of the respiratory chain. The absence of such information in yeast mitochondrial DNA dramatizes the variability in gene content of different mitochondrial genomes.

Secondary reduction of alpha7B integrin in laminin alpha2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle.

PubMed

Cohn, R D; Mayer, U; Saher, G; Herrmann, R; van der Flier, A; Sonnenberg, A; Sorokin, L; Voit, T

1999-03-01

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The alpha7beta1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the alpha7 integrin are laminin-1 (alpha1-beta1-gamma1), laminin-2 (alpha2-beta1-gamma1) and laminin-4 (alpha2-beta2-gamma1). Targeted deletion of the gene for the alpha7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the alpha7B and beta1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the alpha7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the beta1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of alpha7B integrin was significantly reduced at the sarcolemma in six patients with laminin alpha2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin alpha2 chain expressed. In contrast, the expression of the laminin alpha2 chain was not altered in the skeletal muscle of the alpha7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the alpha7 integrin subunit and that of the laminin alpha2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of alpha7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even in young boys (age <2 years). The expression of the beta1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of beta1D integrin was significantly reduced in the alpha7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of alpha7B in laminin alpha2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in alpha7 deficient mice, implies that the alpha7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

The Val192Leu mutation in the alpha-subunit of beta-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease.

PubMed Central

Hou, Y.; Vavougios, G.; Hinek, A.; Wu, K. K.; Hechtman, P.; Kaplan, F.; Mahuran, D. J.

1996-01-01

Substitution mutations adversely affecting the alpha-subunit of beta-hexosaminidase A (alphabeta) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-alpha chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an "active-site" residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all alpha-specific activity. This biochemical phenotype is referred to as the "B1-variant form" of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, alpha-Val192Leu. Chinese hamster ovary cells were permanently cotransfected with an alpha-cDNA-construct encoding the substitution and a mutant beta-cDNA (beta-Arg211Lys), encoding a beta-subunit that is inactive but normal in all other respects. We were surprised to find that the Val192Leu substitution, produced a pro-alpha chain that did not form alpha-beta dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val192Leu substitution does not specifically affect the alpha-active site. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8659543

The Val192Leu mutation in the alpha-subunit of beta-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease.

PubMed

Hou, Y; Vavougios, G; Hinek, A; Wu, K K; Hechtman, P; Kaplan, F; Mahuran, D J

1996-07-01

Substitution mutations adversely affecting the alpha-subunit of beta-hexosaminidase A (alphabeta) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-alpha chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an "active-site" residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all alpha-specific activity. This biochemical phenotype is referred to as the "B1-variant form" of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, alpha-Val192Leu. Chinese hamster ovary cells were permanently cotransfected with an alpha-cDNA-construct encoding the substitution and a mutant beta-cDNA (beta-Arg211Lys), encoding a beta-subunit that is inactive but normal in all other respects. We were surprised to find that the Val192Leu substitution, produced a pro-alpha chain that did not form alpha-beta dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val192Leu substitution does not specifically affect the alpha-active site.

Subunit assembly of hemoglobin: an important determinant of hematologic phenotype.

PubMed

Bunn, H F

1987-01-01

Hemoglobin's physiologic properties depend on the orderly assembly of its subunits in erythropoietic cells. The biosynthesis of alpha- and beta-globin polypeptide chains is normally balanced. Heme rapidly binds to the globin subunit, either during translation or shortly thereafter. The formation of the alpha beta-dimer is facilitated by electrostatic attraction of a positively charged alpha-subunit to a negatively charged beta-subunit. The alpha beta-dimer dissociates extremely slowly. The difference between the rate of dissociation of alpha beta- and alpha gamma-dimers with increasing pH explains the well-known alkaline resistance of Hb F. Two dimers combine to form the functioning alpha 2 beta 2-tetramer. This model of hemoglobin assembly explains the different levels of positively charged and negatively charged mutant hemoglobins that are encountered in heterozygotes and the effect of alpha-thalassemia and heme deficiency states in modifying the level of the variant hemoglobin as well as Hb A2. Electrostatic interactions also affect the binding of hemoglobin to the cytoplasmic surface of the red cell membrane and may underlie the formation of target cells. Enhanced binding of positively charged variants such as S and C trigger a normally dormant pathway for potassium and water loss. Thus, the positive charge on beta c is responsible for the two major contributors to the pathogenesis of Hb SC disease: increased proportion of Hb S and increased intracellular hemoglobin concentration. It is likely that electrostatic interactions play an important role in the assembly of a number of other multisubunit macromolecules, including membrane receptors, cytoskeletal proteins, and DNA binding proteins.

Isolation of amino acid activating subunit-pantetheine protein complexes: Their role in chain elongation in tyrocidine synthesis

PubMed Central

Lee, Sung G.; Lipmann, Fritz

1977-01-01

Dissociation of the multienzymes of tyrocidine synthesis by prolonged incubation of crude extracts of Bacillus brevis (Dubos strain, ATCC 8185) has yielded, on Sephadex G-100 chromatography, two fractions of amino acid activating subunits, a larger one of 70,000 daltons and a smaller one of 90,000 daltons; the latter was a complex consisting of the 70,000 dalton subunit and the pantetheine-carrying protein of about 20,000 daltons. When it dissociated, the intermediate enzyme, which activates three amino acids, contained two-thirds of the subunits in the 70,000 dalton and one-third in the 90,000 dalton fraction; the heavy enzyme, which activates six amino acids, contained five-sixths of the subunits in the former fraction and one-sixth in the latter. Both fractions showed ATP-PPi exchange with all amino acids that are activated by the respective polyenzymes. With proline as an example, the 70,000 dalton subunit exhibited a single low-affinity binding site, which should correspond to the peripheral thiol acceptor site, whereas the 90,000 dalton subunit showed both a low-affinity binding site and an additional high-affinity site for proline; the high-affinity site is attributed to the pantetheine present on the pantetheine-carrying protein, and suggests that amino acids are translocated from the peripheral SH to the pantetheine-carrying moiety during chain elongation. This was confirmed by the observation that the 90,000 dalton complex, when incubated with the light enzyme in the presence of phenylalanine and proline, produced DPhe-Pro dipeptide that cyclized into DPhe-Pro diketopiperazine, but the 70,000 dalton activating subunit, when similarly incubated, did not. After subunit dissociation, however, no further elongation occurred after the transfer from phenylalanine to proline. Images PMID:196286

Biogenesis of the yeast cytochrome bc1 complex.

PubMed

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-01-01

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.

Short communication: molecular characterization of dog and cat p65 subunits of NF-kappaB.

PubMed

Ishikawa, Shingo; Takemitsu, Hiroshi; Li, Gebin; Mori, Nobuko; Yamamoto, Ichiro; Arai, Toshiro

2015-04-01

Nuclear factor kappa B (NF-κB) plays an important role in the immune system. The p65 subunit is an important part of NF-κB unit, and studies of dog and cat p65 subunits of NF-κB (dp65 and cp65) are important in understanding their immune function. In this study, we described the molecular characterization of dp65 and cp65. The dp65 and cp65 complementary DNA encoded 542 and 555 amino acids, respectively, showing a high sequence homology with the mammalian p65 subunit (>87.5%). Quantitative polymerase chain reaction revealed that the p65 messenger RNA is highly expressed in the dog stomach and cat heart and adipose tissue. Functional NF-κB promoter-luciferase reporter vectors revealed that our isolated dp65 and cp65 cDNA encodes a functionally active protein. Transiently expressed dp65 and cp65 up-regulated pro-inflammatory cytokine expression levels in dog and cat, respectively. These findings suggest that dp65 and cp65 play important roles in regulating immune function. Copyright © 2015 Elsevier Ltd. All rights reserved.

The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain.

PubMed Central

Braun, H P; Emmermann, M; Kruft, V; Schmitz, U K

1992-01-01

The major mitochondrial processing activity removing presequences from nuclear encoded precursor proteins is present in the soluble fraction of fungal and mammalian mitochondria. We found that in potato, this activity resides in the inner mitochondrial membrane. Surprisingly, the proteolytic activity co-purifies with cytochrome c reductase, a protein complex of the respiratory chain. The purified complex is bifunctional, as it has the ability to transfer electrons from ubiquinol to cytochrome c and to cleave off the presequences of mitochondrial precursor proteins. In contrast to the nine subunit fungal complex, cytochrome c reductase from potato comprises 10 polypeptides. Protein sequencing of peptides from individual subunits and analysis of corresponding cDNA clones reveals that subunit III of cytochrome c reductase (51 kDa) represents the general mitochondrial processing peptidase. Images PMID:1324169

Motor Skills Training Enhances α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor Subunit mRNA Expression in the Ipsilateral Sensorimotor Cortex and Striatum of Rats Following Intracerebral Hemorrhage.

PubMed

Tamakoshi, Keigo; Ishida, Kazuto; Kawanaka, Kentaro; Takamatsu, Yasuyuki; Tamaki, Hiroyuki

2017-10-01

We investigated the effects of acrobatic training (AT) on expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits in the sensorimotor cortex and striatum after intracerebral hemorrhage (ICH). Male Wistar rats were divided into 4 groups: ICH without AT (ICH), ICH with AT (ICH + AT), sham operation without AT (SHAM), and sham operation with AT (SHAM + AT). ICH was induced by collagenase injection into the left striatum. The ICH + AT group performed 5 acrobatic tasks daily on days 4-28 post ICH. Forelimb sensorimotor function was evaluated using the forelimb placing test. On days 14 and 29, mRNA expression levels of AMPAR subunits GluR1-4 were measured by real-time reverse transcription-polymerase chain reaction. Forelimb placing test scores were significantly higher in the ICH + AT group than in the ICH group. Expression levels of all AMPAR subunit mRNAs were significantly higher in the ipsilateral sensorimotor cortex of rats in the ICH + AT group than in that of rats in the ICH group on day 29. GluR3 and GluR4 expression levels were reduced in the ipsilateral striatum of rats in the ICH group compared with that of rats in the SHAM group on day 14. These changes may play a critical role in motor skills training-induced recovery after ICH. Copyright © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Cytochrome b in human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of the components in liver mitochondria and chromosome assignment of the genes for the large (SDHC) and small (SDHD) subunits to 1q21 and 11q23.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Kojima, S; Kita, K

1997-01-01

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex in both the tricarboxylic acid cycle and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms. In this study, the amino acid sequences of the large (cybL) and small (cybS) subunits of cytochrome b in human liver complex II were deduced from cDNAs isolated by homology probing with mixed primers for the polymerase chain reaction. The mature cybL and cybS contain 140 and 103 amino acids, respectively, and show little similarity to the amino acid sequences of the subunits from other species in contrast to the highly conserved features of the flavoprotein (Fp) subunit and iron-sulfur protein (Ip) subunit. From hydrophobicity analysis, both cybL and cybS appear to have three transmembrane segments, indicating their role as membrane-anchors for the enzyme complex. Histidine residues, which are possible heme axial ligands in cytochrome b of complex II, were found in the second transmembrane segment of each subunit. The genes for cybL (SDHC) and cybS (SDHD) were mapped to chromosome 1q21 and 11q23, respectively by fluorescent in situ hybridization (FISH).

Differences in the phenotypic effects of mutations in homologous MrpA and MrpD subunits of the multi-subunit Mrp-type Na+/H+ antiporter.

PubMed

Morino, Masato; Ogoda, Shinichiro; Krulwich, Terry Ann; Ito, Masahiro

2017-01-01

Mrp antiporters are the sole antiporters in the Cation/Proton Antiporter 3 family of transporter databases because of their unusual structural complexity, 6-7 hydrophobic proteins that function as a hetero-oligomeric complex. The two largest and homologous subunits, MrpA and MrpD, are essential for antiport activity and have direct roles in ion transport. They also show striking homology with proton-conducting, membrane-embedded Nuo subunits of respiratory chain complex I of bacteria, e.g., Escherichia coli. MrpA has the closest homology to the complex I NuoL subunit and MrpD has the closest homology to the complex I NuoM and N subunits. Here, introduction of mutations in MrpD, in residues that are also present in MrpA, led to defects in antiport function and/or complex formation. No significant phenotypes were detected in strains with mutations in corresponding residues of MrpA, but site-directed changes in the C-terminal region of MrpA had profound effects, showing that the MrpA C-terminal region has indispensable roles in antiport function. The results are consistent with a divergence in adaptations that support the roles of MrpA and MrpD in secondary antiport, as compared to later adaptations supporting homologs in primary proton pumping by the respiratory chain complex I.

The Non-catalytic B Subunit of Coagulation Factor XIII Accelerates Fibrin Cross-linking*

PubMed Central

Souri, Masayoshi; Osaki, Tsukasa; Ichinose, Akitada

2015-01-01

Covalent cross-linking of fibrin chains is required for stable blood clot formation, which is catalyzed by coagulation factor XIII (FXIII), a proenzyme of plasma transglutaminase consisting of catalytic A (FXIII-A) and non-catalytic B subunits (FXIII-B). Herein, we demonstrate that FXIII-B accelerates fibrin cross-linking. Depletion of FXIII-B from normal plasma supplemented with a physiological level of recombinant FXIII-A resulted in delayed fibrin cross-linking, reduced incorporation of FXIII-A into fibrin clots, and impaired activation peptide cleavage by thrombin; the addition of recombinant FXIII-B restored normal fibrin cross-linking, FXIII-A incorporation into fibrin clots, and activation peptide cleavage by thrombin. Immunoprecipitation with an anti-fibrinogen antibody revealed an interaction between the FXIII heterotetramer and fibrinogen mediated by FXIII-B and not FXIII-A. FXIII-B probably binds the γ-chain of fibrinogen with its D-domain, which is near the fibrin polymerization pockets, and dissociates from fibrin during or after cross-linking between γ-chains. Thus, FXIII-B plays important roles in the formation of a ternary complex between proenzyme FXIII, prosubstrate fibrinogen, and activator thrombin. Accordingly, congenital or acquired FXIII-B deficiency may result in increased bleeding tendency through impaired fibrin stabilization due to decreased FXIII-A activation by thrombin and secondary FXIII-A deficiency arising from enhanced circulatory clearance. PMID:25809477

Revealing Ligand Binding Sites and Quantifying Subunit Variants of Noncovalent Protein Complexes in a Single Native Top-Down FTICR MS Experiment

NASA Astrophysics Data System (ADS)

Li, Huilin; Wongkongkathep, Piriya; Van Orden, Steve L.; Ogorzalek Loo, Rachel R.; Loo, Joseph A.

2014-12-01

"Native" mass spectrometry (MS) has been proven to be increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode, and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD of the hADH dimer shows that each subunit (E and S chains) binds not only to two zinc atoms, but also the NAD/NADH ligand, with a higher NAD/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.

Human L-ferritin deficiency is characterized by idiopathic generalized seizures and atypical restless leg syndrome

PubMed Central

Cozzi, Anna; Santambrogio, Paolo; Privitera, Daniela; Broccoli, Vania; Rotundo, Luisa Ida; Garavaglia, Barbara; Benz, Rudolf; Altamura, Sandro; Goede, Jeroen S.; Muckenthaler, Martina U.

2013-01-01

The ubiquitously expressed iron storage protein ferritin plays a central role in maintaining cellular iron homeostasis. Cytosolic ferritins are composed of heavy (H) and light (L) subunits that co-assemble into a hollow spherical shell with an internal cavity where iron is stored. The ferroxidase activity of the ferritin H chain is critical to store iron in its Fe3+ oxidation state, while the L chain shows iron nucleation properties. We describe a unique case of a 23-yr-old female patient affected by a homozygous loss of function mutation in the L-ferritin gene, idiopathic generalized seizures, and atypical restless leg syndrome (RLS). We show that L chain ferritin is undetectable in primary fibroblasts from the patient, and thus ferritin consists only of H chains. Increased iron incorporation into the FtH homopolymer leads to reduced cellular iron availability, diminished levels of cytosolic catalase, SOD1 protein levels, enhanced ROS production and higher levels of oxidized proteins. Importantly, key phenotypic features observed in fibroblasts are also mirrored in reprogrammed neurons from the patient’s fibroblasts. Our results demonstrate for the first time the pathophysiological consequences of L-ferritin deficiency in a human and help to define the concept for a new disease entity hallmarked by idiopathic generalized seizure and atypical RLS. PMID:23940258

Cytoplasmic Dynein Regulation by Subunit Heterogeneity and Its Role in Apical Transport

PubMed Central

Tai, Andrew W.; Chuang, Jen-Zen; Sung, Ching-Hwa

2001-01-01

Despite the existence of multiple subunit isoforms for the microtubule motor cytoplasmic dynein, it has not yet been directly shown that dynein complexes with different compositions exhibit different properties. The 14-kD dynein light chain Tctex-1, but not its homologue RP3, binds directly to rhodopsin's cytoplasmic COOH-terminal tail, which encodes an apical targeting determinant in polarized epithelial Madin-Darby canine kidney (MDCK) cells. We demonstrate that Tctex-1 and RP3 compete for binding to dynein intermediate chain and that overexpressed RP3 displaces endogenous Tctex-1 from dynein complexes in MDCK cells. Furthermore, replacement of Tctex-1 by RP3 selectively disrupts the translocation of rhodopsin to the MDCK apical surface. These results directly show that cytoplasmic dynein function can be regulated by its subunit composition and that cytoplasmic dynein is essential for at least one mode of apical transport in polarized epithelia. PMID:11425878

Hyperexcitability of bladder afferent neurons associated with reduction of Kv1.4 α-subunit in rats with spinal cord injury.

PubMed

Takahashi, Ryosuke; Yoshizawa, Tsuyoshi; Yunoki, Takakazu; Tyagi, Pradeep; Naito, Seiji; de Groat, William C; Yoshimura, Naoki

2013-12-01

To clarify the functional and molecular mechanisms inducing hyperexcitability of C-fiber bladder afferent pathways after spinal cord injury we examined changes in the electrophysiological properties of bladder afferent neurons, focusing especially on voltage-gated K channels. Freshly dissociated L6-S1 dorsal root ganglion neurons were prepared from female spinal intact and spinal transected (T9-T10 transection) Sprague Dawley® rats. Whole cell patch clamp recordings were performed on individual bladder afferent neurons. Kv1.2 and Kv1.4 α-subunit expression levels were also evaluated by immunohistochemical and real-time polymerase chain reaction methods. Capsaicin sensitive bladder afferent neurons from spinal transected rats showed increased cell excitability, as evidenced by lower spike activation thresholds and a tonic firing pattern. The peak density of transient A-type K+ currents in capsaicin sensitive bladder afferent neurons from spinal transected rats was significantly less than that from spinal intact rats. Also, the KA current inactivation curve was displaced to more hyperpolarized levels after spinal transection. The protein and mRNA expression of Kv1.4 α-subunits, which can form transient A-type K+ channels, was decreased in bladder afferent neurons after spinal transection. Results indicate that the excitability of capsaicin sensitive C-fiber bladder afferent neurons is increased in association with reductions in transient A-type K+ current density and Kv1.4 α-subunit expression in injured rats. Thus, the Kv1.4 α-subunit could be a molecular target for treating overactive bladder due to neurogenic detrusor overactivity. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension.

PubMed

Afolayan, Adeleye J; Eis, Annie; Alexander, Maxwell; Michalkiewicz, Teresa; Teng, Ru-Jeng; Lakshminrusimha, Satyan; Konduri, Girija G

2016-01-01

Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that a decreased endothelial nitric oxide synthase level leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAECs) from normal and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels, and ATP levels in PAECs and lung tissue of PPHN fetal lambs at baseline compared with gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) and sirtuin-1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O2 was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared with unventilated PPHN lambs. iNO administration, which facilitated weaning of FiO2 , partly restored mtDNA copy number, ETC subunit levels, and ATP levels. DetaNONOate increased eNOS phosphorylation and its interaction with heat shock protein 90 (HSP90); increased levels of superoxide dismutase 2 (SOD2) mRNA, protein, and activity; and decreased the mitochondrial superoxide levels in PPHN-PAECs. Knockdown of eNOS decreased ETC protein levels in control PAECs. We conclude that ventilation with 100% O2 amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by iNO and weaning of oxygen. Copyright © 2016 the American Physiological Society.

Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis.

PubMed Central

Tyler, S D; Johnson, W M; Lior, H; Wang, G; Rozee, K R

1991-01-01

A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively. An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes. Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms. Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure. Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b. For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b. Two strains of E. coli O91:H21 contained both VT2v-a and VT2v-b B subunit genes. The VT2 reference strain of E. coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E. coli, pEB1, possessed only that of the VT2 gene. The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per microgram of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per microgram of protein in strains carrying only the VT2 variant target (TCD50 is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype. Images PMID:1679436

Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phillips, J.; Warby, C; Whitby, F

2009-01-01

Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connectedmore » by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.« less

Insect Ferritins: typical or atypical?

PubMed Central

Pham, Daphne Q. D.; Winzerling, Joy J.

2010-01-01

Insects transmit millions of cases of disease each year, and cost millions of dollars in agricultural losses. The control of insect-borne diseases is vital for numerous developing countries, and the management of agricultural insect pests is a very serious business for developed countries. Control methods should target insect-specific traits in order to avoid non-target effects, especially in mammals. Since insect cells have had a billion years of evolutionary divergence from those of vertebrates, they differ in many ways that might be promising for the insect control field—especially, in iron metabolism because current studies have indicated that significant differences exist between insect and mammalian systems. Insect iron metabolism differs from that of vertebrates in the following respects. Insect ferritins have a heavier mass than mammalian ferritins. Unlike their mammalian counterparts, the insect ferritin subunits are often glycosylated and are synthesized with a signal peptide. The crystal structure of insect ferritin also shows a tetrahedral symmetry consisting of 12 heavy chain and 12 light chain subunits in contrast to that of mammalian ferritin that exhibits an octahedral symmetry made of 24 heavy chain and 24 light chain subunits. Insect ferritins associate primarily with the vacuolar system and serve as iron transporters—quite the opposite of the mammalian ferritins, which are mainly cytoplasmic and serve as iron storage proteins. This review will discuss these differences. PMID:20230873

Polysialic acid in human milk. CD36 is a new member of mammalian polysialic acid-containing glycoprotein.

PubMed

Yabe, Uichiro; Sato, Chihiro; Matsuda, Tsukasa; Kitajima, Ken

2003-04-18

The neural cell adhesion molecule and the voltage-sensitive sodium channel alpha-subunit are the only two molecules in mammals known to be modified by alpha-2,8-linked polysialic acid (polySia). We found a new polySia-containing glycoprotein in human milk and identified it as CD36, a member of the B class of the scavenger receptor superfamily. The polySia-containing glycan chain(s) were removed by alkaline treatment but not by peptide:N-glycanase F digestion, indicating that milk CD36 contained polySia on O-linked glycan chain(s). Polysialylation of CD36 occurs not only in human milk but also in mouse milk. However, CD36 in human platelets is not polysialylated. PolySia CD36 is secreted in milk at any lactation stage and reaches peak level at 1 month after parturition. Thus, it is suggested that polySia of milk CD36 is significant for neonatal development in terms of protection and nutrition.

Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits.

PubMed

McElduff, A; Watkinson, A; Hedo, J A; Gorden, P

1986-11-01

The insulin receptor is synthesized as a 190,000-Mr single-chain precursor that contains exclusively asparagine-N-linked high-mannose-type carbohydrate chains. In this study we have characterized the structure of the pro-receptor oligosaccharides. IM-9 lymphocytes were pulse-chase-labelled with [3H]mannose, and the insulin pro-receptor was isolated by immunoprecipitation and SDS/polyacrylamide-gel electrophoresis. The pro-receptor oligosaccharides were removed from the protein backbone with endoglycosidase H and analysed by h.p.l.c. Immediately after a [3H]mannose pulse the largest oligosaccharide found in the pro-receptor was Glc1Man9GlcNAc2; this structure represented only a small fraction (3%) of the total. The predominant oligosaccharides present in the pro-receptor were Man9GlcNAc2 (25%) and Man8GlcNAc2 (48%). Smaller oligosaccharides were also detected: Man7GlcNAc2 (18%), Man6GlcNAc2 (3%) and Man5GlcNAc2 (3%). The relative distribution of the different oligosaccharides did not change at 1, 2 or 3 h after the pulse with the exception of the rapid disappearance of the Glc1Man9GlcNAc2 component. The mature alpha- and beta-subunits of the insulin receptor are known to contain both high-mannose-type and complex-type oligosaccharides. We have also examined here the structure of the high-mannose chains of these subunits. The predominant species in the alpha-subunit was Man8GlcNAc2 whereas in the beta-subunit it was Man7GlcNAc2. These results demonstrate that most (approx. 75%) oligosaccharides of the insulin pro-receptor are chains of the type Man8GlcNAc2 or Man9GlcNAc2. Thus, assuming that a Glc3Man9GlcNAc2 species is transferred co-translationally, carbohydrate processing of the pro-receptor appears to be very rapid and limited to the removal of the three glucose residues and one mannose residue. Further mannose removal does not occur until the pro-receptor has been proteolytically cleaved. In addition, the degree of mannose trimming appears to be different in the alpha- and beta-subunits.

Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits.

PubMed Central

McElduff, A; Watkinson, A; Hedo, J A; Gorden, P

1986-01-01

The insulin receptor is synthesized as a 190,000-Mr single-chain precursor that contains exclusively asparagine-N-linked high-mannose-type carbohydrate chains. In this study we have characterized the structure of the pro-receptor oligosaccharides. IM-9 lymphocytes were pulse-chase-labelled with [3H]mannose, and the insulin pro-receptor was isolated by immunoprecipitation and SDS/polyacrylamide-gel electrophoresis. The pro-receptor oligosaccharides were removed from the protein backbone with endoglycosidase H and analysed by h.p.l.c. Immediately after a [3H]mannose pulse the largest oligosaccharide found in the pro-receptor was Glc1Man9GlcNAc2; this structure represented only a small fraction (3%) of the total. The predominant oligosaccharides present in the pro-receptor were Man9GlcNAc2 (25%) and Man8GlcNAc2 (48%). Smaller oligosaccharides were also detected: Man7GlcNAc2 (18%), Man6GlcNAc2 (3%) and Man5GlcNAc2 (3%). The relative distribution of the different oligosaccharides did not change at 1, 2 or 3 h after the pulse with the exception of the rapid disappearance of the Glc1Man9GlcNAc2 component. The mature alpha- and beta-subunits of the insulin receptor are known to contain both high-mannose-type and complex-type oligosaccharides. We have also examined here the structure of the high-mannose chains of these subunits. The predominant species in the alpha-subunit was Man8GlcNAc2 whereas in the beta-subunit it was Man7GlcNAc2. These results demonstrate that most (approx. 75%) oligosaccharides of the insulin pro-receptor are chains of the type Man8GlcNAc2 or Man9GlcNAc2. Thus, assuming that a Glc3Man9GlcNAc2 species is transferred co-translationally, carbohydrate processing of the pro-receptor appears to be very rapid and limited to the removal of the three glucose residues and one mannose residue. Further mannose removal does not occur until the pro-receptor has been proteolytically cleaved. In addition, the degree of mannose trimming appears to be different in the alpha- and beta-subunits. Images Fig. 1. PMID:3827820

Two new γ chain variants: Hb F-Augusta GA [(G)γ59(E3)Lys → Arg; HBG2: c.179A > G] and Hb F-Port Royal-II [(A)γ125(H3)Glu → Ala; HBG1: c.377A > C].

PubMed

Kutlar, Ferdane; Ameri, Afshin; Patel, Niren H; Zhuang, Lina; Johnson, Lee E; Cheng, Michael L; Kutlar, Abdullah

2014-01-01

The total number of hemoglobin (Hb) variants so far reported to the HbVar database is 1598 (April 9 2014) and 130 of them are fetal Hb variants. Fetal Hb are categorized as two different subunits, (G)γ- and (A)γ-globin chains, and γ chain variants can be observed in both subunits. There are 72 (G)γ- and 58 (A)γ-globin chain variants. Most of them are clinically silent and detected during newborn screening programs in the USA and outside the USA. In this report, we discuss the molecular characteristics and diagnostic difficulties of two new γ-globin chain variants found in an African American baby with no clinical symptoms. One is a new (G)γ-globin chain variant, Hb F-Augusta GA [(G)γ59(E3)Lys → Arg; HBG2: c.179A > G] and the other one is Hb F-Port Royal-II [(A)γ125(H3)Glu → Ala; HBG1: c.377A > C].

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression.

PubMed

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Kandimalla, Ramesh J L; Bal, Amanjit; Gill, Kiran Dip

2013-12-01

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10mg/kgb.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) and Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits-NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. © 2013.

Inferring High-Confidence Human Protein-Protein Interactions

DTIC Science & Technology

2012-01-01

comprised proteins that had the same specific func- tion or were subunits of the same protein complex, such as branched chain keto acid E1 alpha (BCKDHA...and branched chain keto acid E1 beta (BCKDHB) [3,29], and dynein cytoplasmic 2 intermediate chain 1 (D2LIC) and dynein cytoplasmic 2 heavy chain 1...474.3 28.0 1337.0 BCKDHA 5 Branched chain keto acid dehydro. E1, alpha BCKDHB 4 Branched chain keto acid dehydro. E1, beta 4 471.4 29.0 1337.5 ARTN 2

Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement.

PubMed

Kublik, Anja; Deobald, Darja; Hartwig, Stefanie; Schiffmann, Christian L; Andrades, Adarelys; von Bergen, Martin; Sawers, R Gary; Adrian, Lorenz

2016-09-01

Dehalococcoides mccartyi strain CBDB1 is an obligate organohalide-respiring bacterium using only hydrogen as electron donor and halogenated organics as electron acceptor. Here, we studied proteins involved in the respiratory chain under non-denaturing conditions. Using blue native gel electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250-270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) colocalised with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster containing subunit (CbdbA131) of the hydrogen uptake hydrogenase (Hup). No colocalisation between the catalytically active subunits of hydrogenase and reductive dehalogenase was found. By two-dimensional BN/SDS-PAGE the stability of the complex towards detergents was assessed, demonstrating stepwise disintegration with increasing detergent concentrations. Chemical cross-linking confirmed the presence of a higher molecular mass reductive dehalogenase protein complex composed of RdhA, CISM I and Hup hydrogenase and proved to be a potential tool for stabilising protein-protein interactions of the dehalogenating complex prior to membrane solubilisation. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

O-mannosylation of the Mycobacterium tuberculosis Adhesin Apa Is Crucial for T Cell Antigenicity during Infection but Is Expendable for Protection

PubMed Central

Dobos, Karen M.; Lucas, Megan; Spencer, John S.; Fang, Sunan; McDonald, Melissa A.; Pohl, Jan; Birkness, Kristin; Chamcha, Venkateswarlu; Ramirez, Melissa V.; Plikaytis, Bonnie B.; Posey, James E.; Amara, Rama Rao

2013-01-01

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis. PMID:24130497

O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

PubMed

Nandakumar, Subhadra; Kannanganat, Sunil; Dobos, Karen M; Lucas, Megan; Spencer, John S; Fang, Sunan; McDonald, Melissa A; Pohl, Jan; Birkness, Kristin; Chamcha, Venkateswarlu; Ramirez, Melissa V; Plikaytis, Bonnie B; Posey, James E; Amara, Rama Rao; Sable, Suraj B

2013-01-01

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

Regulation of succinate-ubiquinone reductase and fumarate reductase activities in human complex II by phosphorylation of its flavoprotein subunit.

PubMed

Tomitsuka, Eriko; Kita, Kiyoshi; Esumi, Hiroyasu

2009-01-01

Complex II (succinate-ubiquinone reductase; SQR) is a mitochondrial respiratory chain enzyme that is directly involved in the TCA cycle. Complex II exerts a reverse reaction, fumarate reductase (FRD) activity, in various species such as bacteria, parasitic helminths and shellfish, but the existence of FRD activity in humans has not been previously reported. Here, we describe the detection of FRD activity in human cancer cells. The activity level was low, but distinct, and it increased significantly when the cells were cultured under hypoxic and glucose-deprived conditions. Treatment with phosphatase caused the dephosphorylation of flavoprotein subunit (Fp) with a concomitant increase in SQR activity, whereas FRD activity decreased. On the other hand, treatment with protein kinase caused an increase in FRD activity and a decrease in SQR activity. These data suggest that modification of the Fp subunit regulates both the SQR and FRD activities of complex II and that the phosphorylation of Fp might be important for maintaining mitochondrial energy metabolism within the tumor microenvironment.

Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.

PubMed

Roloff, Gabrielle A; Henry, Michael F

2015-08-15

Three mitochondrial DNA-encoded proteins, Cox1, Cox2, and Cox3, comprise the core of the cytochrome c oxidase complex. Gene-specific translational activators ensure that these respiratory chain subunits are synthesized at the correct location and in stoichiometric ratios to prevent unassembled protein products from generating free oxygen radicals. In the yeast Saccharomyces cerevisiae, the nuclear-encoded proteins Mss51 and Pet309 specifically activate mitochondrial translation of the largest subunit, Cox1. Here we report that Mam33 is a third COX1 translational activator in yeast mitochondria. Mam33 is required for cells to adapt efficiently from fermentation to respiration. In the absence of Mam33, Cox1 translation is impaired, and cells poorly adapt to respiratory conditions because they lack basal fermentative levels of Cox1. © 2015 Roloff and Henry. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Reverse Transcription Quantitative Polymerase Chain Reaction for Detection of and Differentiation Between RNA and DNA of HIV-1-Based Lentiviral Vectors.

PubMed

Pavlovic, Melanie; Koehler, Nina; Anton, Martina; Dinkelmeier, Anna; Haase, Maren; Stellberger, Thorsten; Busch, Ulrich; Baiker, Armin E

2017-08-01

The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described. Numerous controls are included in order to monitor the technical procedure.

Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria.

PubMed

Formosa, Luke E; Hofer, Annette; Tischner, Christin; Wenz, Tina; Ryan, Michael T

2016-01-01

In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10 mg/kg b.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) andmore » Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits–NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. - Highlights: • Aluminium decreases the mRNA levels of mitochondrial and nuclear encoded subunits. • It decreases the mtDNA copy number and mitochondrial content in rat brain. • It down-regulates the mRNA and protein levels of PGC-1α, NRF-1, NRF-2 and Tfam. • It also disturbs the mitochondrial or nuclear architecture of neurons. • Finally it also decreases mitochondrial number in HC and CS regions of rat brain.« less

Crystal structures of ricin toxin's enzymatic subunit (RTA) in complex with neutralizing and non-neutralizing single-chain antibodies.

PubMed

Rudolph, Michael J; Vance, David J; Cheung, Jonah; Franklin, Matthew C; Burshteyn, Fiana; Cassidy, Michael S; Gary, Ebony N; Herrera, Cristina; Shoemaker, Charles B; Mantis, Nicholas J

2014-08-26

Ricin is a select agent toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins. In this study, we determined X-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA's RNA N-glycosidase activity or induced notable allosteric changes in the toxin's subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 (CDR, complementarity-determining region) elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ferritin Assembly in Enterocytes of Drosophila melanogaster

PubMed Central

Rosas-Arellano, Abraham; Vásquez-Procopio, Johana; Gambis, Alexis; Blowes, Liisa M.; Steller, Hermann; Mollereau, Bertrand; Missirlis, Fanis

2016-01-01

Ferritins are protein nanocages that accumulate inside their cavity thousands of oxidized iron atoms bound to oxygen and phosphates. Both characteristic types of eukaryotic ferritin subunits are present in secreted ferritins from insects, but here dimers between Ferritin 1 Heavy Chain Homolog (Fer1HCH) and Ferritin 2 Light Chain Homolog (Fer2LCH) are further stabilized by disulfide-bridge in the 24-subunit complex. We addressed ferritin assembly and iron loading in vivo using novel transgenic strains of Drosophila melanogaster. We concentrated on the intestine, where the ferritin induction process can be controlled experimentally by dietary iron manipulation. We showed that the expression pattern of Fer2LCH-Gal4 lines recapitulated iron-dependent endogenous expression of the ferritin subunits and used these lines to drive expression from UAS-mCherry-Fer2LCH transgenes. We found that the Gal4-mediated induction of mCherry-Fer2LCH subunits was too slow to effectively introduce them into newly formed ferritin complexes. Endogenous Fer2LCH and Fer1HCH assembled and stored excess dietary iron, instead. In contrast, when flies were genetically manipulated to co-express Fer2LCH and mCherry-Fer2LCH simultaneously, both subunits were incorporated with Fer1HCH in iron-loaded ferritin complexes. Our study provides fresh evidence that, in insects, ferritin assembly and iron loading in vivo are tightly regulated. PMID:26861293

CHANGES IN FERRITIN H- AND L-CHAINS IN CANINE LENSES WITH AGE-RELATED NUCLEAR CATARACT

PubMed Central

Goralska, Małgorzata; Nagar, Steven; Colitz, Carmen M.H.; Fleisher, Lloyd N.; McGahan, M. Christine

2014-01-01

PURPOSE To determine potential differences in the characteristics of the iron storage protein, ferritin and its heavy (H) and light (L) subunits in fiber cells from cataractous and normal lenses of older dogs. METHODS Lens fiber cell homogenates were analyzed by SDS-PAGE and ferritin chains were immunodetected with ferritin chain-specific antibodies. Ferritin concentration was measured by ELISA. Immunohistochemistry was used to localize ferritin chains in lens sections. RESULTS The concentration of assembled ferritin was comparable in normal and cataractous lenses of similarly aged dogs. The ferritin L-chain detected in both lens types was modified and was about 11 kDa larger (30 kDa) than standard L-chain (19 kDa) purified from canine liver. The H-chain identified in cataractous fiber cells (29 kDa) differed from 21 kDa standard canine H-chain and from 12 kDa modified H-chain present in fiber cells of normal lenses. Histologic analysis revealed that the H-chain was distributed differently throughout cataractous lenses when compared to normal lenses. There was also a difference in subunit makeup of assembled ferritin between the two lens types. Ferritin from cataractous lenses contained more H-chain and bound 11-fold more iron than ferritin from normal lenses. CONCLUSIONS There are significant differences in the characteristics of ferritin H-chain and its distribution in canine cataractous lenses as compared to normal lenses. The higher content of H-chain in assembled ferritin allows this molecule to sequester more iron. In addition the accumulation of H-chain in deeper fiber layers of the lens may be part of a defense mechanism by which the cataractous lens limits iron-catalyzed oxidative damage. PMID:18708625

The Val{sup 192}Leu mutation in the {alpha}-subunit of {beta}-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Y.; Vavougios, G.; Hinek, A.

1996-07-01

Substitution mutations adversely affecting the {alpha}-subunit of {beta}-hexosaminidase A ({alpha}{beta}) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-{alpha} chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an {open_quotes}active-site{close_quotes} residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all {alpha}-specific activity. This biochemical phenotype is referred to as the {open_quotes}B1-variant form{close_quotes} of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and bothmore » subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, {alpha}-Val{sup 192}Leu. Chinese hamster ovary cells were permanently cotransfected with an {alpha}-cDNA-construct encoding the substitution and a mutant {beta}-cDNA ({beta}-Arg{sup 211}Lys), encoding a {beta}-subunit that is inactive but normal in all other respects. We were surprised to find that the Val{sup 192}Leu substitution produced a pro-{alpha} chain that did not form {alpha}-{beta} dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val{sup 192}Leu substitution does not specifically affect the {alpha}-active site. 23 refs., 4 figs., 2 tabs.« less

Detection of trypanosomatid Phytomonas parasitic in plants by polymerase chain reaction amplification of small subunit ribosomal DNA.

PubMed

Teixeira, M M; Campaner, M; Camargo, E P

1994-01-01

To improve the diagnosis of Phytomonas infections in plants, we developed a polymerase chain reaction (PCR) assay using synthetic oligonucleotides complementary to conserved sequences of the 18S small subunit ribosomal (SSU) gene. From 10 ng upward of DNA of cultures of Phytomonas isolated from plants, fruits, and insects, PCR amplified an 800-bp DNA band that, after restriction analysis and probe hybridization, proved to be of 18S rDNA Phytomonas origin. PCR was also done with sap samples of tomatoes experimentally infected with Phytomonas, yielding amplified 800-bp ribosomal DNA bands before any flagellate could be detected by microscopic examination of the fruit sap.

Mrp Antiporters Have Important Roles in Diverse Bacteria and Archaea.

PubMed

Ito, Masahiro; Morino, Masato; Krulwich, Terry A

2017-01-01

Mrp (Multiple resistance and pH) antiporter was identified as a gene complementing an alkaline-sensitive mutant strain of alkaliphilic Bacillus halodurans C-125 in 1990. At that time, there was no example of a multi-subunit type Na + /H + antiporter comprising six or seven hydrophobic proteins, and it was newly designated as the monovalent cation: proton antiporter-3 (CPA3) family in the classification of transporters. The Mrp antiporter is broadly distributed among bacteria and archaea, not only in alkaliphiles. Generally, all Mrp subunits, mrpA-G , are required for enzymatic activity. Two exceptions are Mrp from the archaea Methanosarcina acetivorans and the eubacteria Natranaerobius thermophilus , which are reported to sustain Na + /H + antiport activity with the MrpA subunit alone. Two large subunits of the Mrp antiporter, MrpA and MrpD, are homologous to membrane-embedded subunits of the respiratory chain complex I, NuoL, NuoM, and NuoN, and the small subunit MrpC has homology with NuoK. The functions of the Mrp antiporter include sodium tolerance and pH homeostasis in an alkaline environment, nitrogen fixation in Schizolobium meliloti , bile salt tolerance in Bacillus subtilis and Vibrio cholerae , arsenic oxidation in Agrobacterium tumefaciens , pathogenesis in Pseudomonas aeruginosa and Staphylococcus aureus , and the conversion of energy involved in metabolism and hydrogen production in archaea. In addition, some Mrp antiporters transport K + and Ca 2+ instead of Na + , depending on the environmental conditions. Recently, the molecular structure of the respiratory chain complex I has been elucidated by others, and details of the mechanism by which it transports protons are being clarified. Based on this, several hypotheses concerning the substrate transport mechanism in the Mrp antiporter have been proposed. The MrpA and MrpD subunits, which are homologous to the proton transport subunit of complex I, are involved in the transport of protons and their coupling cations. Herein, we outline other recent findings on the Mrp antiporter.

Mrp Antiporters Have Important Roles in Diverse Bacteria and Archaea

PubMed Central

Ito, Masahiro; Morino, Masato; Krulwich, Terry A.

2017-01-01

Mrp (Multiple resistance and pH) antiporter was identified as a gene complementing an alkaline-sensitive mutant strain of alkaliphilic Bacillus halodurans C-125 in 1990. At that time, there was no example of a multi-subunit type Na+/H+ antiporter comprising six or seven hydrophobic proteins, and it was newly designated as the monovalent cation: proton antiporter-3 (CPA3) family in the classification of transporters. The Mrp antiporter is broadly distributed among bacteria and archaea, not only in alkaliphiles. Generally, all Mrp subunits, mrpA–G, are required for enzymatic activity. Two exceptions are Mrp from the archaea Methanosarcina acetivorans and the eubacteria Natranaerobius thermophilus, which are reported to sustain Na+/H+ antiport activity with the MrpA subunit alone. Two large subunits of the Mrp antiporter, MrpA and MrpD, are homologous to membrane-embedded subunits of the respiratory chain complex I, NuoL, NuoM, and NuoN, and the small subunit MrpC has homology with NuoK. The functions of the Mrp antiporter include sodium tolerance and pH homeostasis in an alkaline environment, nitrogen fixation in Schizolobium meliloti, bile salt tolerance in Bacillus subtilis and Vibrio cholerae, arsenic oxidation in Agrobacterium tumefaciens, pathogenesis in Pseudomonas aeruginosa and Staphylococcus aureus, and the conversion of energy involved in metabolism and hydrogen production in archaea. In addition, some Mrp antiporters transport K+ and Ca2+ instead of Na+, depending on the environmental conditions. Recently, the molecular structure of the respiratory chain complex I has been elucidated by others, and details of the mechanism by which it transports protons are being clarified. Based on this, several hypotheses concerning the substrate transport mechanism in the Mrp antiporter have been proposed. The MrpA and MrpD subunits, which are homologous to the proton transport subunit of complex I, are involved in the transport of protons and their coupling cations. Herein, we outline other recent findings on the Mrp antiporter. PMID:29218041

Three-dimensional structure of homodimeric cholesterol esterase-ligand complex at 1.4 Å resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pletnev, V.; Addlagatta, A.; Wawrzak, Z.

2010-03-08

The three-dimensional structure of a Candida cylindracea cholesterol esterase (ChE) homodimer (534 x 2 amino acids) in complex with a ligand of proposed formula C{sub 23}H{sub 48}O{sub 2} has been determined at 1.4 {angstrom} resolution in space group P1 using synchrotron low-temperature data. The structure refined to R = 0.136 and R{sub free} = 0.169 and has revealed new stereochemical details in addition to those detected for the apo- and holo-forms at 1.9 and 2.0 {angstrom} resolution, respectively [Ghosh et al. (1995), Structure, 3, 279-288]. The cholesterol esterase structure is a dimer with four spatially separated interfacial contact areas andmore » two symmetry-related pairs of openings to an internal intradimer cavity. Hydrophobic active-site gorges in each subunit face each other across a central interfacial cavity. The ChE subunits have carbohydrate chains attached to their Asn314 and Asn351 residues, with two ordered N-acetyl-D-glucosoamine moieties visible at each site. The side chains of 14 residues have two alternative conformations with occupancy values of 0.5 {+-} 0.2. For each subunit the electron density in the enzyme active-site gorge is well modeled by a C{sub 23}-chain fatty acid.« less

Biology of Symbioses between Marine Invertebrates and Intracellular Bacteria

DTIC Science & Technology

1990-01-30

bisphosphate carboxylase We designed from published sequence information oligonucleotide primers which are complementary to conserved regions on RubisCO ...large and small subunit genes. These primers were used successfully to amplify using polymerase chain reaction (PCR) specific regions of RubisCO ...for the large subunit of ribulose bisphosphate carboxylase/oxygenase ( RubisCO ) to symbiont DNA shows that the symbionts from both deep-sea and shallow

RNA Polymerase Structure, Function, Regulation, Dynamics, Fidelity, and Roles in GENE EXPRESSION | Center for Cancer Research

Cancer.gov

Multi-subunit RNA polymerases (RNAP) are ornate molecular machines that translocate on a DNA template as they generate a complementary RNA chain. RNAPs are highly conserved in evolution among eukarya, eubacteria, archaea, and some viruses. As such, multi-subunit RNAPs appear to be an irreplaceable advance in the evolution of complex life on earth. Because of their stepwise

Subunit mass fingerprinting of mitochondrial complex I.

PubMed

Morgner, Nina; Zickermann, Volker; Kerscher, Stefan; Wittig, Ilka; Abdrakhmanova, Albina; Barth, Hans-Dieter; Brutschy, Bernhard; Brandt, Ulrich

2008-10-01

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica. Using 5-10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis1[w

PubMed Central

Czechowski, Tomasz; Stitt, Mark; Altmann, Thomas; Udvardi, Michael K.; Scheible, Wolf-Rüdiger

2005-01-01

Gene transcripts with invariant abundance during development and in the face of environmental stimuli are essential reference points for accurate gene expression analyses, such as RNA gel-blot analysis or quantitative reverse transcription-polymerase chain reaction (PCR). An exceptionally large set of data from Affymetrix ATH1 whole-genome GeneChip studies provided the means to identify a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis (Arabidopsis thaliana). Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions. Most of these were expressed at much lower levels than traditional reference genes, making them very suitable for normalization of gene expression over a wide range of transcript levels. Specific and efficient primers were developed for 22 genes and tested on a diverse set of 20 cDNA samples. Quantitative reverse transcription-PCR confirmed superior expression stability and lower absolute expression levels for many of these genes, including genes encoding a protein phosphatase 2A subunit, a coatomer subunit, and an ubiquitin-conjugating enzyme. The developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels in Arabidopsis in the future. PMID:16166256

Myosin light chains: Teaching old dogs new tricks

PubMed Central

Heissler, Sarah M; Sellers, James R

2014-01-01

The myosin holoenzyme is a multimeric protein complex consisting of heavy chains and light chains. Myosin light chains are calmodulin family members which are crucially involved in the mechanoenzymatic function of the myosin holoenzyme. This review examines the diversity of light chains within the myosin superfamily, discusses interactions between the light chain and the myosin heavy chain as well as regulatory and structural functions of the light chain as a subunit of the myosin holoenzyme. It covers aspects of the myosin light chain in the localization of the myosin holoenzyme, protein-protein interactions and light chain binding to non-myosin binding partners. Finally, this review challenges the dogma that myosin regulatory and essential light chain exclusively associate with conventional myosin heavy chains while unconventional myosin heavy chains usually associate with calmodulin. PMID:26155737

[Co5(mu3-OH)2(btec)2(bpp)]n: a three-dimensional homometallic molecular metamagnet built from the mixed hydroxide/carboxylate-bridged ferrimagnetic-like chains.

PubMed

Jia, Hong-Peng; Li, Wei; Ju, Zhan-Feng; Zhang, Jie

2007-09-07

A three-dimensional homometallic complex [Co(5)(mu(3)-OH)(2)(btec)(2)(bpp)](n) is built from the mixed hydroxide/carboxylate bridged cobalt(ii) chains linked by the 1,2,4,5-benzenetetracarboxylate (btec(4-)) anion and 1,3-bis(4-pyridyl)-propane molecule (bpp). Within each chain, two mu(3)-OH-bridged metal triangles connect to each other by sharing a common vertex to give rise to a bow-tie type Co(5)(mu(3)-OH)(2) subunit, which is joined to adjacent subunits by four mu(1,1)-carboxylate bridges to form a step-like metal-oxygen backbone. The magnetic studies revealed that the coexistence of ferromagnetic and antiferrimagnetic interactions resulted in a ferrimagnetic-like behavior of the homometallic chains. Below a critical temperature (T(N) = 12.5 K), bulk antiferromagnetic ordering was observed at low field due to the weak interchain antiferromagnetic interactions. A metamagnetic transition occurred at a magnetic field of ca. 5 kOe at 2 K.

Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

PubMed Central

Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

2011-01-01

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

Cloning of the chaperonin t-complex polypeptide 1 gene from Schistosoma mansoni and studies of its expression levels under heat shock and oxidative stress.

PubMed

Campos, E G; Hamdan, F F

2000-03-01

The protein TCP-1 (t-complex polypeptide 1) is a subunit of the hetero-oligomeric complex CCT (chaperonin containing TCP- 1) present in the eukaryotic cytosol. Chaperone function may be critical for the development and survival of the different life stages of Schistosoma mansoni, a parasite that is exposed to drastic environmental changes during its development. We isolated a full-length S. mansoni TCP-1 cDNA (SmTCP-1A) encoding a protein highly homologous with TCP-1. The deduced SmTCP-1A amino-acid sequence shows up to 65% identity with other eukaryotic CCT family members. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the mRNA expression levels of SmTCP-1A in adult S. mansoni were down-regulated in worms subjected to heat shock and oxidative stress conditions. This down-regulation of SmTCP-1A mRNA may reflect a switch in CCT subunits as an adaptive response to heat shock and oxidative stress conditions.

Functional and composition differences between mitochondrial complex II in Arabidopsis and rice are correlated with the complex genetic history of the enzyme.

PubMed

Huang, Shaobai; Taylor, Nicolas L; Narsai, Reena; Eubel, Holger; Whelan, James; Millar, A Harvey

2010-02-01

Complex II plays a central role in mitochondrial metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. However, the composition and function of the plant enzyme has been elusive and differs from the well-characterised enzymes in mammals and bacteria. Herewith, we demonstrate that mitochondrial Complex II from Arabidopsis and rice differ significantly in several aspects: (1) Stability-Rice complex II in contrast to Arabidopsis is not stable when resolved by native electrophoresis and activity staining. (2) Composition-Arabidopsis complex II contains 8 subunits, only 7 of which have homologs in the rice genome. SDH 1 and 2 subunits display high levels of amino acid identity between two species, while the remainder of the subunits are not well conserved at a sequence level, indicating significant divergence. (3) Gene expression-the pairs of orthologous SDH1 and SDH2 subunits were universally expressed in both Arabidopsis and rice. The very divergent genes for SDH3 and SDH4 were co-expressed in both species, consistent with their functional co-ordination to form the membrane anchor. The plant-specific SDH5, 6 and 7 subunits with unknown functions appeared to be differentially expressed in both species. (4) Biochemical regulation -succinate-dependent O(2) consumption and SDH activity of isolated Arabidopsis mitochondria were substantially stimulated by ATP, but a much more minor effect of ATP was observed for the rice enzyme. The ATP activation of succinate-dependent reduction of DCPIP in frozen-thawed and digitonin-solubilised mitochondrial samples, and with or without the uncoupler CCCP, indicate that the differential ATP effect on SDH is not via the protonmotive force but likely due to an allosteric effect on the plant SDH enzyme itself, in contrast to the enzyme in other organisms.

Cell cholesterol modulates metalloproteinase-dependent shedding of low-density lipoprotein receptor-related protein-1 (LRP-1) and clearance function

PubMed Central

Selvais, Charlotte; D'Auria, Ludovic; Tyteca, Donatienne; Perrot, Gwenn; Lemoine, Pascale; Troeberg, Linda; Dedieu, Stéphane; Noël, Agnès; Nagase, Hideaki; Henriet, Patrick; Courtoy, Pierre J.; Marbaix, Etienne; Emonard, Hervé

2011-01-01

Low-density lipoprotein receptor-related protein-1 (LRP-1) is a plasma membrane scavenger and signaling receptor, composed of a large ligand-binding subunit (515-kDa α-chain) linked to a shorter transmembrane subunit (85-kDa β-chain). LRP-1 cell-surface level and function are controlled by proteolytic shedding of its ectodomain. Here, we identified ectodomain sheddases in human HT1080 cells and demonstrated regulation of the cleavage by cholesterol by comparing the classical fibroblastoid type with a spontaneous epithelioid variant, enriched ∼2-fold in cholesterol. Two membrane-associated metalloproteinases were involved in LRP-1 shedding: a disintegrin and metalloproteinase-12 (ADAM-12) and membrane-type 1 matrix metalloproteinase (MT1-MMP). Although both variants expressed similar levels of LRP-1, ADAM-12, MT1-MMP, and specific tissue inhibitor of metalloproteinases-2 (TIMP-2), LRP-1 shedding from epithelioid cells was ∼4-fold lower than from fibroblastoid cells. Release of the ectodomain was triggered by cholesterol depletion in epithelioid cells and impaired by cholesterol overload in fibroblastoid cells. Modulation of LRP-1 shedding on clearance was reflected by accumulation of gelatinases (MMP-2 and MMP-9) in the medium. We conclude that cholesterol exerts an important control on LRP-1 levels and function at the plasma membrane by modulating shedding of its ectodomain, and therefore represents a novel regulator of extracellular proteolytic activities.—Selvais, C., D'Auria, L., Tyteca, D., Perrot, G, Lemoine, P., Troeberg, L., Dedieu, S., Noël, A., Nagase, H., Henriet, P., Courtoy, P. J., Marbaix, E., Emonard, H. Cell cholesterol modulates metalloproteinase-dependent shedding of low-density lipoprotein receptor-related protein-1 (LRP-1) and clearance function. PMID:21518850

Molecular cloning and expression of heteromeric ACCase subunit genes from Jatropha curcas.

PubMed

Gu, Keyu; Chiam, Huihui; Tian, Dongsheng; Yin, Zhongchao

2011-04-01

Acetyl-CoA carboxylase (ACCase) catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, which is the essential first step in the biosynthesis of long-chain fatty acids. ACCase exists as a multi-subunit enzyme in most prokaryotes and the chloroplasts of most plants and algae, while it is present as a multi-domain enzyme in the endoplasmic reticulum of most eukaryotes. The heteromeric ACCase of higher plants consists of four subunits: an α-subunit of carboxyltransferase (α-CT, encoded by accA gene), a biotin carboxyl carrier protein (BCCP, encoded by accB gene), a biotin carboxylase (BC, encoded by accC gene) and a β-subunit of carboxyltransferase (β-CT, encoded by accD gene). In this study, we cloned and characterized the genes accA, accB1, accC and accD that encode the subunits of heteromeric ACCase in Jatropha (Jatropha curcas), a potential biofuel plant. The full-length cDNAs of the four subunit genes were isolated from a Jatropha cDNA library and by using 5' RACE, whereas the genomic clones were obtained from a Jatropha BAC library. They encode a 771 amino acid (aa) α-CT, a 286-aa BCCP1, a 537-aa BC and a 494-aa β-CT, respectively. The single-copy accA, accB1 and accC genes are nuclear genes, while the accD gene is located in chloroplast genome. Jatropha α-CT, BCCP1, BC and β-CT show high identity to their homologues in other higher plants at amino acid level and contain all conserved domains for ACCase activity. The accA, accB1, accC and accD genes are temporally and spatially expressed in the leaves and endosperm of Jatropha plants, which are regulated by plant development and environmental factors. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Identification of a Novel HADHB Gene Mutation in an Iranian Patient with Mitochondrial Trifunctional Protein Deficiency.

PubMed

Shahrokhi, Mahdiyeh; Shafiei, Mohammad; Galehdari, Hamid; Shariati, Gholamreza

2017-01-01

Mitochondrial trifunctional protein (MTP) is a hetero-octamer composed of eight parts (subunits): four α-subunits containing LCEH (long-chain 2,3-enoyl-CoA  hydratase) and LCHAD (long-chain 3-hydroxyacyl CoA dehydrogenase) activity, and four β-subunits that possess LCKT (long-chain  3-ketoacyl-CoA thiolase) activity which catalyzes three out of four steps in β-oxidation spiral of long-chain fatty acid. Its deficiency is an autosomal recessive disorder that causes a clinical spectrum of diseases. A blood spot was collected from the patient's original newborn screening card with parental informed consent. A newborn screening test and quantity plasma acylcarnitine profile analysis by MS/MS were performed. After isolation of DNA and Amplification of all exons of the HADHA and HADHB, directly Sequence analyses of all exons and the flanking introns both of genes were performed. Here, we report a novel mutation in a patient with MTP deficiency diagnosed with newborn screening test and quantity plasma acylcarnitine profile analysis by MS/MS and then confirmed by enzyme analysis in cultured fibroblasts and direct sequencing of the HADHA and HADHB genes. Molecular analysis of causative genes showed a missense mutation (p.Q385P) c.1154A > C in exon 14 of HADHB gene. Since this mutation was not found in 50 normal control cases; so it was concluded that c.1154A > C mutation was a causative mutation. Phenotype analysis of this mutation predicted pathogenesis which reduces the stability of the MTP protein complex.

Amino-acid sequence and predicted three-dimensional structure of pea seed (Pisum sativum) ferritin.

PubMed Central

Lobreaux, S; Yewdall, S J; Briat, J F; Harrison, P M

1992-01-01

The iron storage protein, ferritin, is widely distributed in the living kingdom. Here the complete cDNA and derived amino-acid sequence of pea seed ferritin are described, together with its predicted secondary structure, namely a four-helix-bundle fold similar to those of mammalian ferritins, with a fifth short helix at the C-terminus. An N-terminal extension of 71 residues contains a transit peptide (first 47 residues) responsible for plastid targetting as in other plant ferritins, and this is cleaved before assembly. The second part of the extension (24 residues) belongs to the mature subunit; it is cleaved during germination. The amino-acid sequence of pea seed ferritin is aligned with those of other ferritins (49% amino-acid identity with H-chains and 40% with L-chains of human liver ferritin in the aligned region). A three-dimensional model has been constructed by fitting the aligned sequence to the coordinates of human H-chains, with appropriate modifications. A folded conformation with an 11-residue helix is predicted for the N-terminal extension. As in mammalian ferritins, 24 subunits assemble into a hollow shell. In pea seed ferritin, its N-terminal extension is exposed on the outside surface of the shell. Within each pea subunit is a ferroxidase centre resembling those of human ferritin H-chains except for a replacement of Glu-62 by His. The channel at the 4-fold-symmetry axes defined by E-helices, is predicted to be hydrophilic in plant ferritins, whereas it is hydrophobic in mammalian ferritins. Images Fig. 3. Fig. 5. Fig. 6. PMID:1472006

Structural symmetry and protein function.

PubMed

Goodsell, D S; Olson, A J

2000-01-01

The majority of soluble and membrane-bound proteins in modern cells are symmetrical oligomeric complexes with two or more subunits. The evolutionary selection of symmetrical oligomeric complexes is driven by functional, genetic, and physicochemical needs. Large proteins are selected for specific morphological functions, such as formation of rings, containers, and filaments, and for cooperative functions, such as allosteric regulation and multivalent binding. Large proteins are also more stable against denaturation and have a reduced surface area exposed to solvent when compared with many individual, smaller proteins. Large proteins are constructed as oligomers for reasons of error control in synthesis, coding efficiency, and regulation of assembly. Symmetrical oligomers are favored because of stability and finite control of assembly. Several functions limit symmetry, such as interaction with DNA or membranes, and directional motion. Symmetry is broken or modified in many forms: quasisymmetry, in which identical subunits adopt similar but different conformations; pleomorphism, in which identical subunits form different complexes; pseudosymmetry, in which different molecules form approximately symmetrical complexes; and symmetry mismatch, in which oligomers of different symmetries interact along their respective symmetry axes. Asymmetry is also observed at several levels. Nearly all complexes show local asymmetry at the level of side chain conformation. Several complexes have reciprocating mechanisms in which the complex is asymmetric, but, over time, all subunits cycle through the same set of conformations. Global asymmetry is only rarely observed. Evolution of oligomeric complexes may favor the formation of dimers over complexes with higher cyclic symmetry, through a mechanism of prepositioned pairs of interacting residues. However, examples have been found for all of the crystallographic point groups, demonstrating that functional need can drive the evolution of any symmetry.

Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

NASA Astrophysics Data System (ADS)

Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

1995-02-01

The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

Amine-controlled assembly of metal-sulfite architecture from 1D chains to 3D framework.

PubMed

Austria, Cristina; Zhang, Jian; Valle, Henry; Zhang, Qichun; Chew, Emily; Nguyen, Dan-Tam; Gu, J Y; Feng, Pingyun; Bu, Xianhui

2007-08-06

Whereas open-framework materials have been made in a variety of chemical compositions, few are known in which 3-connected SO3(2)- anions serve as basic building units. Here, we report four new metal-sulfite polymeric structures, (ZnSO3)Py (1, py = pyridine), (ZnSO3)2(2,2'-bipy)H2O (2, 2,2'-bipy = 2,2'-bipyridine), (ZnSO3)2(TMDPy) (3, TMDPy = 4,4'-trimethylenedipyridine), and (MnSO3)2en (4, en = ethylenediamine) that have been synthesized hydrothermally and structurally characterized. In these compounds, low-dimensional 1D and 2D inorganic subunits are assembled into higher 2D or 3D covalent frameworks by organic ligands. In addition to the structure-directing effect of organic ligands, the flexible coordination chemistry of Zn2+ and SO3(2)- also contributes to the observed structural diversity. In compounds 1-3, Zn2+ sites alternate with trigonal pyramidal SO3(2)- anions to form three types of [ZnSO3]n chains, whereas in compound 4, a 2D-corrugated [MnSO3]n layer is present. Compound 1 features a rail-like chain with pendant pyridine rings. The pi-pi interaction between 2,2'-bipy ligands is found between adjacent chains in compound 2, resulting in 2D sheets that are further stacked through interlayer hydrogen bonds. Compound 3 exhibits a very interesting inorganic [(ZnSO3)2]n chain constructed from two chairlike subunits, and such chains are bridged by TMDPy ligands into a 2D sheet. In compound 4, side-by-side helical chains permeate through 2D-corrugated [MnSO3]n layers, which are pillared by neutral ethylenediamine molecules into a 3D framework that can be topologically represented as a (3,6)-connected net. The results presented here illustrate the rich structural chemistry of metal-sulfites and the potential of sulfite anions as a unique structural building block for the construction of novel open-framework materials, in particular, those containing polymeric inorganic subunits that may have interesting physical properties such as low-dimensional magnetism or electronic properties.

Synthesis of hemoglobin Gun Hill: increased synthesis of the heme-free βGH globin chain and subunit exchange with a free α-chain pool

PubMed Central

Rieder, Ronald F.

1971-01-01

Hemoglobin Gun Hill is an unstable mutant hemoglobin associated with mild compensated hemolysis. This abnormal protein has a deletion of five amino acids in the β-chains. The deletion includes the heme-binding proximal histidine at position 92. The β-chains of hemoglobin Gun Hill lack heme groups. Approximately 32% of the circulating hemoglobin in heterozygous subjects consists of the mutant hemoglobin. When reticulocytes were incubated with radioactive amino acid the specific activity of hemoglobin Gun Hill was three to six times that of hemoglobin A. Total incorporation of radioactivity into hemoglobin Gun Hill was two to three times that into hemoglobin A. There were 20-50% more total counts in β-Gun Hill (βGH) than in βA. These results indicate that in reticulocytes there was greater synthesis of the abnormal β-chains than βA-chains. The ratio of the specific activities of the α-chains of hemoglobin Gun Hill to the α-chains of hemoglobin A was 20: 1. There was evidence of a free pool of α-chains in the reticulocytes containing hemoglobin Gun Hill. After 10 min of incubation approximately 40% of the total α-chain radioactivity was in the free pool. When protein synthesis was blocked by incubation of reticulocytes with puromycin, the specific activity of the α-chains of hemoglobin Gun Hill continued to increase due to direct exchange of α-subunits between the free pool and preformed hemoglobin Gun Hill. Studies of the assembly of βA and βGH revealed that the rates of translation of the two polypeptide chains were equal and uniform. No evidence was obtained for the existence of “slow points” in the process of globin chain assembly. The studies also suggest that lack of strong heme-globin binding does not hinder the synthesis of globin chains. PMID:5540175

Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins.

PubMed

Li, Xiao-Ping; Tumer, Nilgun E

2017-04-11

Both ricin and Shiga holotoxins display no ribosomal activity in their native forms and need to be activated to inhibit translation in a cell-free translation inhibition assay. This is because the ribosome binding site of the ricin A chain (RTA) is blocked by the B subunit in ricin holotoxin. However, it is not clear why Shiga toxin 1 (Stx1) or Shiga toxin 2 (Stx2) holotoxin is not active in a cell-free system. Here, we compare the ribosome binding and depurination activity of Stx1 and Stx2 holotoxins with the A1 subunits of Stx1 and Stx2 using either the ribosome or a 10-mer RNA mimic of the sarcin/ricin loop as substrates. Our results demonstrate that the active sites of Stx1 and Stx2 holotoxins are blocked by the A2 chain and the B subunit, while the ribosome binding sites are exposed to the solvent. Unlike ricin, which is enzymatically active, but cannot interact with the ribosome, Stx1 and Stx2 holotoxins are enzymatically inactive but can interact with the ribosome.

Purification and characterization of a milk-clotting aspartic proteinase from globe artichoke (Cynara scolymus L.).

PubMed

Llorente, Berta E; Brutti, Cristina B; Caffini, Néstor O

2004-12-29

The study of proteinase expression in crude extracts from different organs of the globe artichoke (Cynara scolymus L.) disclosed that enzymes with proteolytic and milk-clotting activity are mainly located in mature flowers. Maximum proteolytic activity was recorded at pH 5.0, and inhibition studies showed that only pepstatin, specific for aspartic proteinases, presented a significant inhibitory effect. Such properties, in addition to easy enzyme inactivation by moderate heating, make this crude protease extract potentially useful for cheese production. Adsorption with activated carbon, together with anion exchange and affinity chromatography, led to the isolation of a heterodimeric milk-clotting proteinase consisting of 30- and 15-kDa subunits. MALDI-TOF MS of the 15-kDa chain determined a 15.358-Da mass, and the terminal amino sequence presented 96% homology with the smaller cardosin A subunit. The amino terminal sequence of the 30-kDa chain proved to be identical to the larger cardosin A subunit. Electrophoresis evidenced proteinase self-processing that was confirmed by immunoblots presenting 62-, 30-, and 15-kDa bands.

Characterization of the interaction between the heavy and light chains of bovine factor Va.

PubMed

Walker, F J

1992-10-05

Bovine factor Va has been previously been shown to consist of heavy (M(r) = 94,000) and light chains (M(r) = 81,000), that interact in a manner dependent upon the presence of either calcium or manganese ions. In an attempt to understand the mechanism of subunit interaction we have studied the effects of temperature and ions on factor Va stability. The rates of formation of factor Va from isolated chains and dissociation were temperature-dependent with an energy of activation of 6.2 and 1.3 kcal mol-1, respectively. The yield of factor Va from isolated chains was inversely related to the amount of time the chains were incubated at 4 degrees C. Incubation of individual chains revealed that the heavy chain is cold-labile, an effect that is reversible. Manganese ion was observed to prevent the conversion to the inactive form. High salt tends to stabilize the two-chain structure of factor Va, but is inhibitory to its formation from isolated chains. High concentrations of either manganese or calcium ions also inhibited reconstitution of activity. The light chain, in particular, was sensitive to the presence of manganese or calcium ion. Heavy chain that had been cleaved by activated protein C had a weakened interaction with the light chain, and the resulting complex had no procoagulant activity. Cooling of the heavy chain to 4 degrees C enhanced its intrinsic fluorescence. Manganese ion prevented some of this enhancement. The heavy chain fluorescence returned to the room temperature value with a half-life of approximately 10 min. In the presence of manganese ion relaxation was accelerated. The intrinsic fluorescence of activated protein C-cleaved heavy chain was not increased when the temperature was decreased. These data suggest that the heavy chain can exist in two forms. Elevated temperature converts it to a form that can bind ions and have a productive interaction with the light chain. However, conditions that prevent the heavy chain from combining with the light chain also stabilize the two subunit structure, suggesting that the high affinity of the complex is due to conformational changes that occur after chain interaction.

Definition of the mutation responsible for maple syrup urine disease in Poll Shorthorns and genotyping Poll Shorthorns and Poll Herefords for maple syrup urine disease alleles.

PubMed

Dennis, J A; Healy, P J

1999-08-01

The organisation of the E1alpha subunit of bovine branched-chain alpha-keto acid dehydrogenase gene was established. c DNA was cloned from Poll Shorthorn x Poll Hereford calves affected with Maple Syrup Urine Disease to identify the mutation responsible for the disease in Poll Shorthorns. Clones containing the c DNA sequences inherited from the Poll Shorthorn sire of the affected calves were identified. Paternal clones were sequenced and a cytidine to thymidine transition was found at nucleotide 1380. The mutation is predicted to substitute leucine in place of a highly conserved proline at codon 372. A polymerase chain reaction procedure was developed for detection of the 1380C-->T mutation in genomic DNA. Three Poll Shorthorn parents of affected calves and three affected Poll Shorthorn x Poll Hereford calves were heterozygous and an affected Poll Shorthorn calf was homozygous for this mutation. An improved polymerase chain reaction procedure was also devised to genotype Poll Herefords for the 248C-->T mutation. The procedures will facilitate disease prevention programs and assist in differential diagnosis of conditions in new-born calves that present with a rapid onset of progressive neurological disease and are characterised histologically by 'status spongiosus'. Maple Syrup Urine Disease (MSUD) is an autosomal recessive defect reported in humans (Danner and Elsas 1989), and in Poll Hereford (PH) and Poll Shorthorn (PS) calves (Harper et al 1986, Healy et al 1992). The clinical, biochemical and pathological manifestations of the disease are identical in the two breeds of cattle, and are characterised by the rapid onset of progressive neurological disease, leading to death within a few days of birth. The disease is caused by a deficiency of activity of the mitochondrial enzyme branched-chain alpha-keto acid dehydrogenase (BCKADH). This deficiency leads to elevated concentrations, in blood and tissues, of branched chain alpha-keto acids and their precursors, the branched chain amino acids, valine, leucine and isoleucine. BCKADH consists of four subunits E1alpha, E1beta, E2 and E3 that are encoded by separate genes, and MSUD may result from deficiency of any of the subunits. In PH s, the disease in caused by premature termination of translation, of the E1alpha subunit, that is induced by a cytidine to thymidine transition exon 2 (248C-->T), that converts the glutamine codon -6 to a stop codon (Q-6ST; Zhang et al 1990). We have shown that MSUD -affected PSxPH calves are heterozygous at the PH locus, illustrating molecular heterogeneity exists for bovine MSUD (Healy and Dennis 1994a). The fact that these crossbred calves are affected, indicates the PS, like the PH mutation, resides in the E1alpha subunit. Copyright 1999 Harcourt Publishers Ltd.

Codon usage bias and phylogenetic analysis of mitochondrial ND1 gene in pisces, aves, and mammals.

PubMed

Uddin, Arif; Choudhury, Monisha Nath; Chakraborty, Supriyo

2018-01-01

The mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1) gene is a subunit of the respiratory chain complex I and involved in the first step of the electron transport chain of oxidative phosphorylation (OXPHOS). To understand the pattern of compositional properties, codon usage and expression level of mitochondrial ND1 genes in pisces, aves, and mammals, we used bioinformatic approaches as no work was reported earlier. In this study, a perl script was used for calculating nucleotide contents and different codon usage bias parameters. The codon usage bias of MT-ND1 was low but the expression level was high as revealed from high ENC and CAI value. Correspondence analysis (COA) suggests that the pattern of codon usage for MT-ND1 gene is not same across species and that compositional constraint played an important role in codon usage pattern of this gene among pisces, aves, and mammals. From the regression equation of GC12 on GC3, it can be inferred that the natural selection might have played a dominant role while mutation pressure played a minor role in influencing the codon usage patterns. Further, ND1 gene has a discrepancy with cytochrome B (CYB) gene in preference of codons as evident from COA. The codon usage bias was low. It is influenced by nucleotide composition, natural selection, mutation pressure, length (number) of amino acids, and relative dinucleotide composition. This study helps in understanding the molecular biology, genetics, evolution of MT-ND1 gene, and also for designing a synthetic gene.

Anthranilate synthase subunit organization in Chromobacterium violaceum.

PubMed

Carminatti, C A; Oliveira, I L; Recouvreux, D O S; Antônio, R V; Porto, L M

2008-09-16

Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite of pharmacological interest. The genome analysis of this bacterium revealed that the genes trpA-F and pabA-B encode the enzymes of the tryptophan pathway in which the first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzyme complex. In the present study, the organization and structure of AS protein subunits from C. violaceum were analyzed using bioinformatics tools available on the Web. We showed by calculating molecular masses that AS in C. violaceum is composed of alpha (TrpE) and beta (PabA) subunits. This is in agreement with values determined experimentally. Catalytic and regulatory sites of the AS subunits were identified. The TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit is involved in the allosteric site. Protein models for the TrpE and PabA subunits were built by restraint-based homology modeling using AS enzyme, chains A and B, from Salmonella typhimurium (PDB ID 1I1Q).

Protein Flexibility Facilitates Quaternary Structure Assembly and Evolution

PubMed Central

Marsh, Joseph A.; Teichmann, Sarah A.

2014-01-01

The intrinsic flexibility of proteins allows them to undergo large conformational fluctuations in solution or upon interaction with other molecules. Proteins also commonly assemble into complexes with diverse quaternary structure arrangements. Here we investigate how the flexibility of individual protein chains influences the assembly and evolution of protein complexes. We find that flexibility appears to be particularly conducive to the formation of heterologous (i.e., asymmetric) intersubunit interfaces. This leads to a strong association between subunit flexibility and homomeric complexes with cyclic and asymmetric quaternary structure topologies. Similarly, we also observe that the more nonhomologous subunits that assemble together within a complex, the more flexible those subunits tend to be. Importantly, these findings suggest that subunit flexibility should be closely related to the evolutionary history of a complex. We confirm this by showing that evolutionarily more recent subunits are generally more flexible than evolutionarily older subunits. Finally, we investigate the very different explorations of quaternary structure space that have occurred in different evolutionary lineages. In particular, the increased flexibility of eukaryotic proteins appears to enable the assembly of heteromeric complexes with more unique components. PMID:24866000

Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matzuk, M.M.; Krieger, M.; Corless, C.L.

1987-09-01

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common ..cap alpha.. subunit but differ in their hormone-specific ..beta..-subunits. The ..beta.. subunit of hCG (hCG..beta..) is unique among the ..beta.. subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either hCG..beta.. gene alone or together with the hCG..cap alpha.. gene were transfected into a mutant Chinese hamster ovary cell line, 1d1D, which exhibits a reversible defect in O-glycosylation. The results revealmore » that hCG..beta.. can be secreted normally in the absence of its O-linked oligosaccharides. hCG..beta.. devoid of O-linked carbohydrate can also combine efficiently with hCG..cap alpha.. and be secreted as an intact dimer. The authors conclude that in Chinese hamster ovary cells, the hCG..beta.. O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG.« less

Respiratory chain complex II as general sensor for apoptosis.

PubMed

Grimm, Stefan

2013-05-01

I review here the evidence that complex II of the respiratory chain (RC) constitutes a general sensor for apoptosis induction. This concept emerged from work on neurodegenerative diseases and from recent data on metabolic alterations in cancer cells affecting the RC and in particular on mutations of complex II subunits. It is also supported by experiments with many anticancer compounds that compared the apoptosis sensitivities of complex II-deficient versus WT cells. These results are explained by the mechanistic understanding of how complex II mediates the diverse range of apoptosis signals. This protein aggregate is specifically activated for apoptosis by pH change as a common and early feature of dying cells. This leads to the dissociation of its SDHA and SDHB subunits from the remaining membrane-anchored subunits and the consequent block of it enzymatic SQR activity, while its SDH activity, which is contained in the SDHA/SDHB subcomplex, remains intact. The uncontrolled SDH activity then generates excessive amounts of reactive oxygen species for the demise of the cell. Future studies on these mitochondrial processes will help refine this model, unravel the contribution of mutations in complex II subunits as the cause of degenerative neurological diseases and tumorigenesis, and aid in discovering novel interference options. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease. Copyright © 2012 Elsevier B.V. All rights reserved.

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.

PubMed

Xie, Youming; Varshavsky, Alexander

2002-12-01

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.

The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas

PubMed Central

Reck, Jaimee; Schauer, Alexandria M.; VanderWaal Mills, Kristyn; Bower, Raqual; Tritschler, Douglas; Perrone, Catherine A.; Porter, Mary E.

2016-01-01

The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms. PMID:27251063

Straight-chain alcohols exhibit a cutoff in potency for the inhibition of recombinant glutamate receptor subunits

PubMed Central

Akinshola, B Emmanuel

2001-01-01

The effects of n-alcohols (methanol to 1-decanol) on kainate-activated AMPA receptor subunit GluR1 and GluR3 ion currents were studied in Xenopus oocytes using the two-electrode voltage-clamp recording technique. For short-chain alcohols from methanol to 1-hexanol, potency for inhibition of GluR1 and GluR3 receptor-mediated current increased in proportion to the chain length or hydrophobicity of the alcohol. The IC50 values of these alcohols for GluR1 were: methanol, 702 mM; ethanol, 170 mM; 1-propanol, 69 mM; 1-butanol, 20 mM; 1-pentanol, 17 mM; and 1-hexanol, 10 mM. For GluR3, IC50 values were: methanol, 712 mM; ethanol, 238 mM; 1-propanol, 50 mM; 1-butanol, 32 mM; 1-pentanol, 13 mM; and 1-hexanol, 7 mM. For long-chain alcohols, 1-heptanol was less potent than 1-hexanol (estimated IC50: 19 mM for GluR1 and 18 mM for GluR3), 1-octanol had little effect only on GluR3, and 1-nonanol and 1-decanol did not significantly inhibit both GluR1 and GluR3 responses. The observations indicate that straight-chain n-alcohols exhibit a cutoff in their potency for inhibition of the function of non-NMDA glutamate receptor subunits, GluR1 and GluR3. The cutoff in potency of n-alcohols for inhibition of non-NMDA glutamate receptor function is consistent with the interpretation that alcohols affect the function of these receptor-channels by interacting with an alcohol binding site of specific dimensions on the receptor protein. PMID:11429388

The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc(1) Complex of Yeast Mitochondria.

PubMed

Conte, Laura; Zara, Vincenzo

2011-01-01

The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc(1) complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc(1) complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc(1) complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain.

The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc 1 Complex of Yeast Mitochondria

PubMed Central

Conte, Laura; Zara, Vincenzo

2011-01-01

The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc 1 complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc 1 complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc 1 complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain. PMID:21716720

Human ferritin for tumor detection and therapy.

PubMed

Fan, Kelong; Gao, Lizeng; Yan, Xiyun

2013-01-01

Ferritin, a major iron storage protein found in most living organisms, is composed of a 24-subunit protein cage with a hollow interior cavity. Serum ferritin serves as a critical marker to detect total body iron status. However, recent research reveals a number of novel functions of ferritin besides iron storage; for example, a ferritin receptor, transferrin receptor 1 (TfR1), has been identified and serum ferritin levels are found to be elevated in tumors. A particular new finding is that magnetoferritin nanoparticles, biomimetically synthesized using H-chain ferritin to form a 24-subunit cage with an iron oxide core, possess intrinsic dual functionality, the protein shell specifically targeting tumors and the iron oxide core catalyzing peroxidase substrates to produce a color reaction allowing visualization of tumor tissues. Here we attempt to summarize current research on ferritin, particularly newly identified functions related to tumors, in order to address current challenges and highlight future directions. Copyright © 2013 Wiley Periodicals, Inc.

Growth differentiation factor 9 reverses activin A suppression of steroidogenic acute regulatory protein expression and progesterone production in human granulosa-lutein cells.

PubMed

Shi, Feng-Tao; Cheung, Anthony P; Klausen, Christian; Huang, He-Feng; Leung, Peter C K

2010-10-01

We have reported that growth differentiation factor 9 (GDF9) can enhance activin A (β(A)β(A))-induced inhibin B (αβ(B)) secretion in human granulosa-lutein (hGL) cells, but its effects on steroidogenic acute regulatory protein (StAR), ovarian steroidogenic enzymes, and progesterone production are unknown. We undertook this study to further evaluate GDF9 in this regard. hGL cells from women undergoing in vitro fertilization treatment were cultured with and without small interfering RNA (siRNA) transfection targeted at inhibin α-subunit or GDF9 before treatment with GDF9, activin A, FSH, or combinations. We compared StAR, P450 side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase expression in hGL cells and progesterone levels in culture media after these treatments. mRNA, protein, and hormone levels were assessed with real-time RT-PCR, immunoblotting, and ELISA, respectively. Data were analyzed by ANOVA followed by Tukey's test. Activin A alone reduced basal and FSH-induced progesterone production by decreasing the expression of StAR protein, which regulates the rate-limiting step in steroidogenesis but not P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase. GDF9 attenuated these activin A effects on StAR and progesterone. After transfection of α-subunit siRNA, activin A level increased (P < 0.001), whereas basal and activin A-induced inhibin B levels (with and without GDF9) decreased. Furthermore, the effects of GDF9 in reversing activin A suppression of progesterone production were attenuated (P < 0.001). Transfection of GDF9 siRNA decreased GDF9 as expected and led to lower StAR expression and progesterone secretion than those observed with activin A treatment alone. GDF9 attenuates the suppressive effects of activin A on StAR expression and progesterone production by increasing the expression of inhibin B, which acts as an activin A competitor.

The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.

PubMed Central

Myster, S H; Knott, J A; O'Toole, E; Porter, M E

1997-01-01

Multiple members of the dynein heavy chain (Dhc) gene family have been recovered in several organisms, but the relationships between these sequences and the Dhc isoforms that they encode are largely unknown. To identify Dhc loci and determine the specific functions of the individual Dhc isoforms, we have screened a collection of motility mutants generated by insertional mutagenesis in Chlamydomonas. In this report, we characterize one strain, pf9-3, in which the insertion event was accompanied by a deletion of approximately 13 kb of genomic DNA within the transcription unit of the Dhc1 gene. Northern blot analysis confirms that pf9-3 is a null mutation. Biochemical and structural studies of isolated axonemes demonstrate that the pf9-3 mutant fails to assemble the I1 inner arm complex, a two-headed dynein isoform composed of two Dhcs (1 alpha and 1 beta) and three intermediate chains. To determine if the Dhc1 gene product corresponds to one of the Dhcs of the I1 complex, antibodies were generated against a Dhc1-specific peptide sequence. Immunoblot analysis reveals that the Dhc1 gene encodes the 1 alpha Dhc subunit. These studies thus, identify the first inner arm Dhc locus to be described in any organism and further demonstrate that the 1 alpha Dhc subunit plays an essential role in the assembly of the I1 inner arm complex. Images PMID:9247642

Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain.

PubMed

Zhang, Yuxun; Bharathi, Sivakama S; Rardin, Matthew J; Lu, Jie; Maringer, Katherine V; Sims-Lucas, Sunder; Prochownik, Edward V; Gibson, Bradford W; Goetzman, Eric S

2017-06-16

SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro , succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5 -/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5 -/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5 -/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Biotechnology and Genetic Engineering Reviews. Volume 10

DTIC Science & Technology

1992-12-01

Johnson of Rice University for supplying the graphic of the ce-subunit haem pocket. and Yvonne Le Tellier and Conrad Wheeler of the Blood Research...human hemoglobin cross-linked between the c, chains. J. Bol. Chem. 266- 2697-2700. VANDEGRIFF, K.D., LE TELLIER , Y.C., WINSIoW, R.M., Roili.t:s, R.J...cross-linked between the ax subunits at lysine 99. J. Biol. Chenm. 266, 17 049-17 059. VANDEGRIFF, K.D.. LE TELLIER , Y.C., hESS. J.R. ANt) StIRAGER, R.I

Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1.

PubMed

Kida, Hiroshi; Sugano, Yuri; Iizuka, Ryo; Fujihashi, Masahiro; Yohda, Masafumi; Miki, Kunio

2008-11-14

Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of alpha and beta subunits and forms a "jellyfish-like" structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two alpha and two beta subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of alpha (alpha1 and alpha2) and beta subunits (beta1 and beta2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD beta1 subunit at 1.9 A resolution and its functional analysis. TsPFD beta1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The beta hairpin linkers of beta1 subunits assemble to form a beta barrel "body" around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the beta1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric beta1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD beta1 subunits act as molecular chaperones in living cells of some archaea.

Identical linkage and cooperativity of oxygen and carbon monoxide binding to Octopus dofleini hemocyanin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Connelly, P.R.; Gill, S.J.; Miller, K.I.

1989-02-21

Employment of high-precision thin-layer methods has enabled detailed functional characterization of oxygen and carbon monoxide binding for (1) the fully assembled form with 70 binding sites and (2) the isolated chains with 7 binding sites of octopus dofleini hemocyanin. The striking difference in the cooperativities of the two ligands for the assembled decamer is revealed through an examination of the binding capacities and the partition coefficient, determined as functions of the activities of both ligands. A global analysis of the data sets supported by a two-state allosteric model assuming an allosteric unit of 7. Higher level allosteric interactions were notmore » indicated. This contrasts to results obtained for arthropod hemocyanins. Oxygen and carbon monoxide experiments performed on the isolated subunit chain confirmed the presence of functional heterogeneity reported previously. The analysis shows two types of binding sites in the ratio of 4:3.« less

Oligonucleotide primers, probes and molecular methods for the environmental monitoring of methanogenic archaea

PubMed Central

Narihiro, Takashi; Sekiguchi, Yuji

2011-01-01

Summary For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α‐subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers. PMID:21375721

Human Immune Disorder Arising from Mutation of the α Chain of the Interleukin-2 Receptor

NASA Astrophysics Data System (ADS)

Sharfe, Nigel; Dadi, Harjit K.; Shahar, Michal; Roifman, Chaim M.

1997-04-01

Profound cellular immunodeficiency occurs as the result of mutations in proteins involved in both the differentiation and function of mature lymphoid cells. We describe here a novel human immune aberration arising from a truncation mutation of the interleukin-2 receptor α chain (CD25), a subunit of the tripartite high-affinity receptor for interleukin 2. This immunodeficiency is characterized by decreased numbers of peripheral T cells displaying abnormal proliferation but normal B cell development. Extensive lymphocytic infiltration of tissues, including lung, liver, gut, and bone, is observed, accompanied by tissue atrophy and inflammation. Although mature T cells are present, the absence of CD25 does affect the differentiation of thymocytes. While displaying normal development of CD2, CD3, CD4, and CD8 expression, CD25-deficient cortical thymocytes do not express CD1, and furthermore they fail to normally down-regulate levels of the anti-apoptotic protein bcl-2.

Collision-Induced Dissociation of Electrosprayed Protein Complexes: An All-Atom Molecular Dynamics Model with Mobile Protons.

PubMed

Popa, Vlad; Trecroce, Danielle A; McAllister, Robert G; Konermann, Lars

2016-06-16

Electrospray ionization mass spectrometry (ESI-MS) has become an indispensable technique for examining noncovalent protein complexes. Collision-induced dissociation (CID) of these multiply protonated gaseous ions usually culminates in ejection of a single subunit with a disproportionately large amount of charge. Experiments suggest that this process involves subunit unfolding prior to separation from the residual complex, as well as H(+) migration onto the unravelling chain. Molecular dynamics (MD) simulations are a promising avenue for gaining detailed insights into these CID events. Unfortunately, typical MD algorithms do not allow for mobile protons. Here we address this limitation by implementing a strategy that combines atomistic force fields (such as OPLS/AA and CHARMM36) with a proton hopping algorithm, focusing on the tetrameric complexes transthyretin and streptavidin. Protons are redistributed over all acidic and basic sites in 20 ps intervals, subject to an energy function that reflects electrostatic interactions and proton affinities. Our simulations predict that nativelike conformers at the onset of collisional heating contain multiple salt bridges. Collisional heating initially causes subtle structural changes that lead to a gradual decline of these zwitterionic patterns. Many of the MD runs show gradual unfolding of a single subunit in conjunction with H(+) migration, culminating in subunit separation from the complex. However, there are also instances where two or more chains start to unfold simultaneously, giving rise to charge competition. The scission point where the "winning" subunit separates from the complex can be attained for different degrees of unfolding, giving rise to product ions in various charge states. The simulated product ion distributions are in close agreement with experimental CID data. Proton enrichment in the departing subunit is driven by charge-charge repulsion, but the combination of salt bridge depletion, charge migration, and proton affinity causes surprising compensation effects among the various energy terms. It appears that this work provides the most detailed account to date of the mechanism whereby noncovalent protein complexes disassemble during CID.

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit.

PubMed

Chojnacki, Michal; Mansour, Wissam; Hameed, Dharjath S; Singh, Rajesh K; El Oualid, Farid; Rosenzweig, Rina; Nakasone, Mark A; Yu, Zanlin; Glaser, Fabian; Kay, Lewis E; Fushman, David; Ovaa, Huib; Glickman, Michael H

2017-04-20

Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (Ub PT ), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of Ub PT , and we expect that its future uses will help define and investigate the ubiquitin interactome. Copyright © 2017 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jiang; Malmirchegini, G. Reza; Clubb, Robert T.

Native mass spectrometry (MS) has become an invaluable tool for the characterization of proteins and non-covalent protein complexes under near physiological solution conditions. Here we report the structural characterization of human hemoglobin (Hb), a 64 kDa oxygen-transporting protein complex, by high resolution native top-down mass spectrometry using electrospray ionization (ESI) and a 15-Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Native MS preserves the non-covalent interactions between the globin subunits, and electron capture dissociation (ECD) produces fragments directly from the intact Hb complex without dissociating the subunits. Using activated ion ECD, we observe the gradual unfolding process of themore » Hb complex in the gas phase. Without protein ion activation, the native Hb shows very limited ECD fragmentation from the N-termini, suggesting a tightly packed structure of the native complex and therefore low fragmentation efficiency. Precursor ion activation allows steady increase of N-terminal fragment ions, while the C-terminal fragments remain limited (38 c ions and 4 z ions on the α chain; 36 c ions and 2 z ions on the β chain). This ECD fragmentation pattern suggests that upon activation, the Hb complex starts to unfold from the N-termini of both subunits, whereas the C-terminal regions and therefore the potential regions involved in the subunit binding interactions remain intact. ECD-MS of the Hb dimer show similar fragmentation patterns as the Hb tetramer, providing further evidence for the hypothesized unfolding process of the Hb complex in the gas phase. Native top-down ECD-MS allows efficient probing of the Hb complex structure and the subunit binding interactions in the gas phase. Finally, it may provide a fast and effective means to probe the structure of novel protein complexes that are intractable to traditional structural characterization tools.« less

Solar powered biohydrogen production requires specific localization of the hydrogenase

DOE PAGES

Burroughs, Nigel J.; Boehm, Marko; Eckert, Carrie; ...

2014-09-04

Cyanobacteria contain a bidirectional [NiFe] hydrogenase which transiently produces hydrogen upon exposure of anoxic cells to light, potentially acting as a “valve” releasing excess electrons from the electron transport chain. However, its interaction with the photosynthetic electron transport chain remains unclear. By GFP-tagging the HoxF diaphorase subunit we show that the hydrogenase is thylakoid associated, comprising a population dispersed uniformly through the thylakoids and a subpopulation localized to discrete puncta in the distal thylakoid. Thylakoid localisation of both the HoxH and HoxY hydrogenase subunits is confirmed by immunogold electron microscopy. The diaphorase HoxE subunit is essential for recruitment to themore » dispersed thylakoid population, potentially anchoring the hydrogenase to the membrane, but aggregation to puncta occurs through a distinct HoxE-independent mechanism. Membrane association does not require NDH-1. Localization is dynamic on a scale of minutes, with anoxia and high light inducing a significant redistribution between these populations in favour of puncta. Lastly, since HoxE is essential for access to its electron donor, electron supply to the hydrogenase depends on a physiologically controlled localization, potentially offering a new avenue to enhance photosynthetic hydrogen production by exploiting localization/aggregation signals.« less

Proximal tubule H-ferritin mediates iron trafficking in acute kidney injury

PubMed Central

Zarjou, Abolfazl; Bolisetty, Subhashini; Joseph, Reny; Traylor, Amie; Apostolov, Eugene O.; Arosio, Paolo; Balla, Jozsef; Verlander, Jill; Darshan, Deepak; Kuhn, Lukas C.; Agarwal, Anupam

2013-01-01

Ferritin plays a central role in iron metabolism and is made of 24 subunits of 2 types: heavy chain and light chain. The ferritin heavy chain (FtH) has ferroxidase activity that is required for iron incorporation and limiting toxicity. The purpose of this study was to investigate the role of FtH in acute kidney injury (AKI) and renal iron handling by using proximal tubule–specific FtH-knockout mice (FtHPT–/– mice). FtHPT–/– mice had significant mortality, worse structural and functional renal injury, and increased levels of apoptosis in rhabdomyolysis and cisplatin-induced AKI, despite significantly higher expression of heme oxygenase-1, an antioxidant and cytoprotective enzyme. While expression of divalent metal transporter-1 was unaffected, expression of ferroportin (FPN) was significantly lower under both basal and rhabdomyolysis-induced AKI in FtHPT–/– mice. Apical localization of FPN was disrupted after AKI to a diffuse cytosolic and basolateral pattern. FtH, regardless of iron content and ferroxidase activity, induced FPN. Interestingly, urinary levels of the iron acceptor proteins neutrophil gelatinase–associated lipocalin, hemopexin, and transferrin were increased in FtHPT–/– mice after AKI. These results underscore the protective role of FtH and reveal the critical role of proximal tubule FtH in iron trafficking in AKI. PMID:24018561

Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

PubMed Central

Ferrero, Rut; Torres, Magdalena

2002-01-01

Background Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability. PMID:12350235

Deletion of the low-molecular-weight glutenin subunit allele Glu-A3a of wheat (Triticum aestivum L.) significantly reduces dough strength and breadmaking quality.

PubMed

Zhen, Shoumin; Han, Caixia; Ma, Chaoying; Gu, Aiqin; Zhang, Ming; Shen, Xixi; Li, Xiaohui; Yan, Yueming

2014-12-19

Low-molecular-weight glutenin subunits (LMW-GS), encoded by Glu-3 complex loci in hexaploid wheat, play important roles in the processing quality of wheat flour. To date, the molecular characteristics and effects on dough quality of individual Glu-3 alleles and their encoding proteins have been poorly studied. We used a Glu-A3 deletion line of the Chinese Spring (CS-n) wheat variety to conduct the first comprehensive study on the molecular characteristics and functional properties of the LMW-GS allele Glu-A3a. The Glu-A3a allele at the Glu-A3 locus in CS and its deletion in CS-n were identified and characterized by proteome and molecular marker methods. The deletion of Glu-A3a had no significant influence on plant morphological and yield traits, but significantly reduced the dough strength and breadmaking quality compared to CS. The complete sequence of the Glu-A3a allele was cloned and characterized, which was found to encode a B-subunit with longer repetitive domains and an increased number of α-helices. The Glu-A3a-encoded B-subunit showed a higher expression level and accumulation rate during grain development. These characteristics of the Glu-A3a allele could contribute to achieving superior gluten quality and demonstrate its potential application to wheat quality improvement. Furthermore, an allele-specific polymerase chain reaction (AS-PCR) marker for the Glu-A3a allele was developed and validated using different bread wheat cultivars, including near-isogenic lines (NILs) and recombinant inbred lines (RILs), which could be used as an effective molecular marker for gluten quality improvement through marker-assisted selection. This work demonstrated that the LMW-GS allele Glu-A3a encodes a specific LMW-i type B-subunit that significantly affects wheat dough strength and breadmaking quality. The Glu-A3a-encoded B-subunit has a long repetitive domain and more α-helix structures as well as a higher expression level and accumulation rate during grain development, which could facilitate the formation of wheat with a stronger dough structure and superior breadmaking quality.

A case-control study on association of proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1) polymorphisms and their transcript levels in vitiligo from Gujarat.

PubMed

Jadeja, Shahnawaz D; Mansuri, Mohmmad Shoab; Singh, Mala; Dwivedi, Mitesh; Laddha, Naresh C; Begum, Rasheedunnisa

2017-01-01

Autoimmunity has been implicated in the destruction of melanocytes from vitiligo skin. Major histocompatibility complex (MHC) class-II linked genes proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1), involved in antigen processing and presentation have been reported to be associated with several autoimmune diseases including vitiligo. To explore PSMB8 rs2071464 and TAP1 rs1135216 single nucleotide polymorphisms and to estimate the expression of PSMB8 and TAP1 in patients with vitiligo and unaffected controls from Gujarat. PSMB8 rs2071464 polymorphism was genotyped using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and TAP1 rs1135216 polymorphism was genotyped by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in 378 patients with vitiligo and 509 controls. Transcript levels of PSMB8 and TAP1 were measured in the PBMCs of 91 patients and 96 controls by using qPCR. Protein levels of PSMB8 were also determined by Western blot analysis. The frequency of 'TT' genotype of PSMB8 polymorphism was significantly lowered in patients with generalized and active vitiligo (p = 0.019 and p = 0.005) as compared to controls suggesting its association with the activity of the disease. However, TAP1 polymorphism was not associated with vitiligo susceptibility. A significant decrease in expression of PSMB8 at both transcript level (p = 0.002) as well as protein level (p = 0.0460) was observed in vitiligo patients as compared to controls. No significant difference was observed between patients and controls for TAP1 transcripts (p = 0.553). Interestingly, individuals with the susceptible CC genotype of PSMB8 polymorphism showed significantly reduced PSMB8 transcript level as compared to that of CT and TT genotypes (p = 0.009 and p = 0.003 respectively). PSMB8 rs2071464 was associated with generalized and active vitiligo from Gujarat whereas TAP1 rs1135216 showed no association. The down-regulation of PSMB8 in patients with risk genotype 'CC' advocates the vital role of PSMB8 in the autoimmune basis of vitiligo.

Subunits of the Drosophila Actin-Capping Protein Heterodimer Regulate Each Other at Multiple Levels

PubMed Central

Amândio, Ana Rita; Gaspar, Pedro; Whited, Jessica L.; Janody, Florence

2014-01-01

The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth. PMID:24788460

Crystal structure of heterodimeric hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 reveals that the small subunit is directly involved in the product chain length regulation.

PubMed

Sasaki, Daisuke; Fujihashi, Masahiro; Okuyama, Naomi; Kobayashi, Yukiko; Noike, Motoyoshi; Koyama, Tanetoshi; Miki, Kunio

2011-02-04

Hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 (Ml-HexPPs) is a heterooligomeric type trans-prenyltransferase catalyzing consecutive head-to-tail condensations of three molecules of isopentenyl diphosphates (C(5)) on a farnesyl diphosphate (FPP; C(15)) to form an (all-E) hexaprenyl diphosphate (HexPP; C(30)). Ml-HexPPs is known to function as a heterodimer of two different subunits, small and large subunits called HexA and HexB, respectively. Compared with homooligomeric trans-prenyltransferases, the molecular mechanism of heterooligomeric trans-prenyltransferases is not yet clearly understood, particularly with respect to the role of the small subunits lacking the catalytic motifs conserved in most known trans-prenyltransferases. We have determined the crystal structure of Ml-HexPPs both in the substrate-free form and in complex with 7,11-dimethyl-2,6,10-dodecatrien-1-yl diphosphate ammonium salt (3-DesMe-FPP), an analog of FPP. The structure of HexB is composed of mostly antiparallel α-helices joined by connecting loops. Two aspartate-rich motifs (designated the first and second aspartate-rich motifs) and the other characteristic motifs in HexB are located around the diphosphate part of 3-DesMe-FPP. Despite the very low amino acid sequence identity and the distinct polypeptide chain lengths between HexA and HexB, the structure of HexA is quite similar to that of HexB. The aliphatic tail of 3-DesMe-FPP is accommodated in a large hydrophobic cleft starting from HexB and penetrating to the inside of HexA. These structural features suggest that HexB catalyzes the condensation reactions and that HexA is directly involved in the product chain length control in cooperation with HexB.

Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition.

PubMed

Doherty, Geoff P; Fogg, Mark J; Wilkinson, Anthony J; Lewis, Peter J

2010-12-01

Bacterial RNA polymerases (RNAPs) contain several small auxiliary subunits known to co-purify with the core α, β and β' subunits. The ω subunit is conserved between Gram-positive and Gram-negative bacteria, while the δ subunit is conserved within, but restricted to, Gram-positive bacteria. Although various functions have been assigned to these subunits via in vitro assays, very little is known about their in vivo roles. In this work we constructed a pair of vectors to investigate the subcellular localization of the δ and ω subunits in Bacillus subtilis with respect to the core RNAP. We found these subunits to be closely associated with RNAP involved in transcribing both mRNA and rRNA operons. Quantification of these subunits revealed δ to be present at equimolar levels with RNAP and ω to be present at around half the level of core RNAP. For comparison, the localization and quantification of RNAP β' and ω subunits in Escherichia coli was also investigated. Similar to B. subtilis, β' and ω closely associated with the nucleoid and formed subnucleoid regions of high green fluorescent protein intensity, but, unlike ω in B. subtilis, ω levels in E. coli were close to parity with those of β'. These results indicate that δ is likely to be an integral RNAP subunit in Gram-positives, whereas ω levels differ substantially between Gram-positives and -negatives. The ω subunit may be required for RNAP assembly and subsequently be turned over at different rates or it may play roles in Gram-negative bacteria that are performed by other factors in Gram-positives.

Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes.

PubMed

Duperray, A; Berthier, R; Chagnon, E; Ryckewaert, J J; Ginsberg, M; Plow, E; Marguerie, G

1987-06-01

Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected.

Purification of Ovine Respiratory Complex I Results in a Highly Active and Stable Preparation*

PubMed Central

Letts, James A.; Degliesposti, Gianluca; Fiedorczuk, Karol; Skehel, Mark; Sazanov, Leonid A.

2016-01-01

NADH-ubiquinone oxidoreductase (complex I) is the largest (∼1 MDa) and the least characterized complex of the mitochondrial electron transport chain. Because of the ease of sample availability, previous work has focused almost exclusively on bovine complex I. However, only medium resolution structural analyses of this complex have been reported. Working with other mammalian complex I homologues is a potential approach for overcoming these limitations. Due to the inherent difficulty of expressing large membrane protein complexes, screening of complex I homologues is limited to large mammals reared for human consumption. The high sequence identity among these available sources may preclude the benefits of screening. Here, we report the characterization of complex I purified from Ovis aries (ovine) heart mitochondria. All 44 unique subunits of the intact complex were identified by mass spectrometry. We identified differences in the subunit composition of subcomplexes of ovine complex I as compared with bovine, suggesting differential stability of inter-subunit interactions within the complex. Furthermore, the 42-kDa subunit, which is easily lost from the bovine enzyme, remains tightly bound to ovine complex I. Additionally, we developed a novel purification protocol for highly active and stable mitochondrial complex I using the branched-chain detergent lauryl maltose neopentyl glycol. Our data demonstrate that, although closely related, significant differences exist between the biochemical properties of complex I prepared from ovine and bovine mitochondria and that ovine complex I represents a suitable alternative target for further structural studies. PMID:27672209

Composition of the mitochondrial electron transport chain in acanthamoeba castellanii: structural and evolutionary insights.

PubMed

Gawryluk, Ryan M R; Chisholm, Kenneth A; Pinto, Devanand M; Gray, Michael W

2012-11-01

The mitochondrion, derived in evolution from an α-proteobacterial progenitor, plays a key metabolic role in eukaryotes. Mitochondria house the electron transport chain (ETC) that couples oxidation of organic substrates and electron transfer to proton pumping and synthesis of ATP. The ETC comprises several multiprotein enzyme complexes, all of which have counterparts in bacteria. However, mitochondrial ETC assemblies from animals, plants and fungi are generally more complex than their bacterial counterparts, with a number of 'supernumerary' subunits appearing early in eukaryotic evolution. Little is known, however, about the ETC of unicellular eukaryotes (protists), which are key to understanding the evolution of mitochondria and the ETC. We present an analysis of the ETC proteome from Acanthamoeba castellanii, an ecologically, medically and evolutionarily important member of Amoebozoa (sister to Opisthokonta). Data obtained from tandem mass spectrometric (MS/MS) analyses of purified mitochondria as well as ETC complexes isolated via blue native polyacrylamide gel electrophoresis are combined with the results of bioinformatic queries of sequence databases. Our bioinformatic analyses have identified most of the ETC subunits found in other eukaryotes, confirming and extending previous observations. The assignment of proteins as ETC subunits by MS/MS provides important insights into the primary structures of ETC proteins and makes possible, through the use of sensitive profile-based similarity searches, the identification of novel constituents of the ETC along with the annotation of highly divergent but phylogenetically conserved ETC subunits. © 2012 Elsevier B.V. All rights reserved.

Significance of two distinct types of tryptophan synthase beta chain in Bacteria, Archaea and higher plants.

PubMed

Xie, Gary; Forst, Christian; Bonner, Carol; Jensen, Roy A

2002-01-01

Tryptophan synthase consists of two subunits, alpha and beta. Two distinct subgroups of beta chain exist. The major group (TrpEb_1) includes the well-studied beta chain of Salmonella typhimurium. The minor group of beta chain (TrpEb_2) is most frequently found in the Archaea. Most of the amino-acid residues important for catalysis are highly conserved between both TrpE subfamilies. Conserved amino-acid residues of TrpEb_1 that make allosteric contact with the TrpEa subunit (the alpha chain) are absent in TrpEb_2. Representatives of Archaea, Bacteria and higher plants all exist that possess both TrpEb_1 and TrpEb_2. In those prokaryotes where two trpEb genes coexist, one is usually trpEb_1 and is adjacent to trpEa, whereas the second is trpEb_2 and is usually unlinked with other tryptophan-pathway genes. TrpEb_1 is nearly always partnered with TrpEa in the tryptophan synthase reaction. However, by default at least six lineages of the Archaea are likely to use TrpEb_2 as the functional beta chain, as TrpEb_1 is absent. The six lineages show a distinctive divergence within the overall TrpEa phylogenetic tree, consistent with the lack of selection for amino-acid residues in TrpEa that are otherwise conserved for interfacing with TrpEb_1. We suggest that the standalone function of TrpEb_2 might be to catalyze the serine deaminase reaction, an established catalytic capability of tryptophan synthase beta chains. A coincident finding of interest is that the Archaea seem to use the citramalate pathway, rather than threonine deaminase (IlvA), to initiate the pathway of isoleucine biosynthesis.

Chronic deficit in the expression of voltage-gated potassium channel Kv3.4 subunit in the hippocampus of pilocarpine-treated epileptic rats

PubMed Central

Pacheco Otalora, Luis F.; Skinner, Frank; Oliveira, Mauro S.; Dotson, Bianca Farrel; Arshadmansab, Massoud F.; Pandari, Tarun; Garcia, Ileana; Robles, Leslie; Rosas, Gerardo; Mello, Carlos F.; Ermolinsky, Boris S.; Garrido-Sanabria, Emilio R.

2010-01-01

Voltage gated K+ channels (Kv) are a highly diverse group of channels critical in determining neuronal excitability. Deficits of Kv channel subunit expression and function have been implicated in the pathogenesis of epilepsy. In this study, we investigate whether the expression of the specific subunit Kv3.4 is affected during epileptogenesis following pilocarpine-induced status epilepticus. For this purpose, we used immunohistochemistry, Western blotting assays and comparative analysis of gene expression using TaqMan-based probes and delta-delta cycle threshold (Δ ΔCT) method of quantitative real-time polymerase chain reaction (qPCR) technique in samples obtained from age-matched control and epileptic rats. A marked down-regulation of Kv3.4 immunoreactivity was detected in the stratum lucidum and hilus of dentate gyrus in areas corresponding to the mossy fiber system of chronically epileptic rats. Correspondingly, a 20% reduction of Kv3.4 protein levels was detected in the hippocampus of chronic epileptic rats. Real-time quantitative PCR analysis of gene expression revealed that a significant 33% reduction of transcripts for Kv3.4 (gene Kcnc4) occurred after 1 month of pilocarpine-induced status epilepticus and persisted during the chronic phase of the model. These data indicate a reduced expression of Kv3.4 channels at protein and transcript levels in the epileptic hippocampus. Down-regulation of Kv3.4 in mossy fibers may contribute to enhanced presynaptic excitability leading to recurrent seizures in the pilocarpine model of temporal lobe epilepsy. PMID:20971086

Altered expression of genes involved in GABAergic transmission and neuromodulation of granule cell activity in the cerebellum of schizophrenia patients.

PubMed

Bullock, W Michael; Cardon, Karen; Bustillo, Juan; Roberts, Rosalinda C; Perrone-Bizzozero, Nora I

2008-12-01

Deficits in gamma-aminobutyric acid (GABA) signaling have been described in the prefrontal cortex, limbic system, and cerebellum in individuals with schizophrenia. The purpose of the present study was to further investigate cerebellar gene expression alterations as they relate to decreases in GABAergic transmission by examining the expression of GABAergic markers, N-methyl-d-aspartic-acid (NMDA) receptor subunits, and cerebellum neuromodulators in individuals with schizophrenia. Subjects were postmortem men with a diagnosis of schizophrenia (N=13) and a postmortem interval-matched non-psychiatric male comparison group (N=13). The authors utilized real-time-quantitative polymerase chain reaction (PCR) to measure mRNA levels of the following GABAergic markers: glutamic acid decarboxylase (GAD) 65 and 67; GABA plasma membrane transporter-1 (GAT-1); GABA type A (GABA(A)) receptor subunits alpha(6), beta(3), and delta; and parvalbumin. In addition, real-time-quantitative PCR was utilized to assess mRNA levels of the NMDA receptor (NR) subunits NR1, NR2-A, NR2-B, NR2-C, and NR2-D as well as the cerebellar neuromodulators glutamate receptor (GluR)-6, kainate-preferring glutamate receptor subunit-2 (KA2), metabotropic glutamate receptor (mGluR)-2 and mGluR3, and neuronal nitric oxide synthase. Measurements for mRNA levels were determined using lateral cerebellar hemisphere tissue from both schizophrenia and comparison subjects. Schizophrenia subjects showed significant decreases in mRNA levels of GAD(67), GAD(65), GAT-1, mGluR2, and neuronal nitric oxide synthase. Increases in GABA(A)-alpha(6 )and GABA(A)-delta as well as GluR6 and KA2 were also observed. Medication effects on the expression of the same genes were examined in rats treated with either haloperidol (Sprague-Dawley rats [N=16]) or clozapine (Long-Evans rats [N=20]). Both haloperidol and clozapine increased the levels of GAD(67) in the cerebellum and altered the expression of other cerebellar mRNAs. These findings suggest that GABA transmission is decreased in the cerebellar cortices in individuals with schizophrenia and additional gene expression changes may reflect an attempt to increase GABA neurotransmission at the cerebellar glomerulus.

Antibiotics induce mitonuclear protein imbalance but fail to inhibit respiration and nutrient activation in pancreatic β-cells.

PubMed

Santo-Domingo, Jaime; Chareyron, Isabelle; Broenimann, Charlotte; Lassueur, Steve; Wiederkehr, Andreas

2017-08-15

Chloramphenicol and several other antibiotics targeting bacterial ribosomes inhibit mitochondrial protein translation. Inhibition of mitochondrial protein synthesis leads to mitonuclear protein imbalance and reduced respiratory rates as confirmed here in HeLa and PC12 cells. Unexpectedly, respiration in INS-1E insulinoma cells and primary human islets was unaltered in the presence of chloramphenicol. Resting respiratory rates and glucose stimulated acceleration of respiration were also not lowered when a range of antibiotics including, thiamphenicol, streptomycin, gentamycin and doxycycline known to interfere with bacterial protein synthesis were tested. However, chloramphenicol efficiently reduced mitochondrial protein synthesis in INS-1E cells, lowering expression of the mtDNA encoded COX1 subunit of the respiratory chain but not the nuclear encoded ATP-synthase subunit ATP5A. Despite a marked reduction of the essential respiratory chain subunit COX1, normal respiratory rates were maintained in INS-1E cells. ATP-synthase dependent respiration was even elevated in chloramphenicol treated INS-1E cells. Consistent with these findings, glucose-dependent calcium signaling reflecting metabolism-secretion coupling in beta-cells, was augmented. We conclude that antibiotics targeting mitochondria are able to cause mitonuclear protein imbalance in insulin secreting cells. We hypothesize that in contrast to other cell types, compensatory mechanisms are sufficiently strong to maintain normal respiratory rates and surprisingly even result in augmented ATP-synthase dependent respiration and calcium signaling following glucose stimulation. The result suggests that in insulin secreting cells only lowering COX1 below a threshold level may result in a measurable impairment of respiration. When focusing on mitochondrial function, care should be taken when including antibiotics targeting translation for long-term cell culture as depending on the sensitivity of the cell type analyzed, respiration, mitonuclear protein imbalance or down-stream signaling may be altered. Copyright © 2017 Elsevier Inc. All rights reserved.

Chlamydomonas Outer Arm Dynein Alters Conformation in Response to Ca2+

PubMed Central

Sakato, Miho; Sakakibara, Hitoshi

2007-01-01

We have previously shown that Ca2+ directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the β and γ heavy chains (HCs). The γ HC–associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca2+ with KCa = 3 × 10−5 M in vitro, suggesting it may act as a Ca2+ sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and γ HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the γ heavy chain. In vitro experiments indicate that LC4 undergoes a Ca2+-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca2+. The sedimentation profile of the γ HC subunit changed subtly upon Ca2+ addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated γ subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca2+ addition. We propose that Ca2+-dependent conformational change of LC4 has a direct effect on the stem domain of the γ HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm. PMID:17634291

GABRA2 Alcohol Dependence Risk Allele is Associated with Reduced Expression of Chromosome 4p12 GABAA Subunit Genes in Human Neural Cultures.

PubMed

Lieberman, Richard; Kranzler, Henry R; Joshi, Pujan; Shin, Dong-Guk; Covault, Jonathan

2015-09-01

Genetic variation in a region of chromosome 4p12 that includes the GABAA subunit gene GABRA2 has been reproducibly associated with alcohol dependence (AD). However, the molecular mechanisms underlying the association are unknown. This study examined correlates of in vitro gene expression of the AD-associated GABRA2 rs279858*C-allele in human neural cells using an induced pluripotent stem cell (iPSC) model system. We examined mRNA expression of chromosome 4p12 GABAA subunit genes (GABRG1, GABRA2, GABRA4, and GABRB1) in 36 human neural cell lines differentiated from iPSCs using quantitative polymerase chain reaction and next-generation RNA sequencing. mRNA expression in adult human brain was examined using the BrainCloud and BRAINEAC data sets. We found significantly lower levels of GABRA2 mRNA in neural cell cultures derived from rs279858*C-allele carriers. Levels of GABRA2 RNA were correlated with those of the other 3 chromosome 4p12 GABAA genes, but not other neural genes. Cluster analysis based on the relative RNA levels of the 4 chromosome 4p12 GABAA genes identified 2 distinct clusters of cell lines, a low-expression cluster associated with rs279858*C-allele carriers and a high-expression cluster enriched for the rs279858*T/T genotype. In contrast, there was no association of genotype with chromosome 4p12 GABAA gene expression in postmortem adult cortex in either the BrainCloud or BRAINEAC data sets. AD-associated variation in GABRA2 is associated with differential expression of the entire cluster of GABAA subunit genes on chromosome 4p12 in human iPSC-derived neural cell cultures. The absence of a parallel effect in postmortem human adult brain samples suggests that AD-associated genotype effects on GABAA expression, although not present in mature cortex, could have effects on regulation of the chromosome 4p12 GABAA cluster during neural development. Copyright © 2015 by the Research Society on Alcoholism.

Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli.

PubMed

Xie, Xi; Meesapyodsuk, Dauenpen; Qiu, Xiao

2017-05-01

Thraustochytrium sp. strain ATCC 26185 accumulates a high level of docosahexaenoic acid (DHA), a nutritionally important ω-3 very-long-chain polyunsaturated fatty acid (VLCPUFA) synthesized primarily by polyunsaturated fatty acid (PUFA) synthase, a type I polyketide synthase-like megaenzyme. The PUFA synthase in this species comprises three large subunits, each with multiple catalytic domains. It was hypothesized that among these domains, ketoacylsynthase (KS) domains might be critical for catalyzing the condensation of specific unsaturated acyl-acyl carrier proteins (ACPs) with malonyl-ACP, thereby retaining double bonds in an extended acyl chain. To investigate the functions of these putative KS domains, two segment sequences from subunit A (KS-A) and subunit B (KS-B) of the PUFA synthase were dissected and then expressed as stand-alone enzymes in Escherichia coli The results showed that both KS-A and KS-B domains could complement the defective phenotypes of both E. coli fabB and fabF mutants. Overexpression of these domains in wild-type E. coli led to increases in total fatty acid production. KS-B produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while KS-A could improve the overall production of fatty acids more effectively, particularly for the production of SFAs, implying that KS-A is more comparable to FabF, while KS-B is more similar to FabB in catalytic functions. Successful complementation and functional expression of the embedded KS domains in E. coli are the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs in Thraustochytrium IMPORTANCE Very-long-chain polyunsaturated fatty acids (VLCPUFAs) are important for human health. They can be biosynthesized in either an aerobic pathway or an anaerobic pathway in nature. However, abundant VLCPUFAs in marine microorganisms are primarily synthesized by polyunsaturated fatty acid (PUFA) synthase, a megaenzyme with multiple subunits, each with multiple catalytic domains. Furthermore, the fundamental mechanism for this enzyme to synthesize these fatty acids still remains unknown. This report started with dissecting the embedded KS domains of the PUFA synthase from marine protist Thraustochytrium sp. strain ATCC 26185 and then expressing them in wild-type E. coli and mutants defective in condensation of acyl-ACP with malonyl-ACP. Successful complementation of the mutants and improved fatty acid production in the overexpression experiments indicate that these KS domains can effectively function as stand-alone enzymes in E. coli This result has paved the way for further studying of molecular mechanisms of the PUFA synthase for the biosynthesis of VLCPUFAs. Copyright © 2017 American Society for Microbiology.

Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli

PubMed Central

Xie, Xi; Meesapyodsuk, Dauenpen

2017-01-01

ABSTRACT Thraustochytrium sp. strain ATCC 26185 accumulates a high level of docosahexaenoic acid (DHA), a nutritionally important ω-3 very-long-chain polyunsaturated fatty acid (VLCPUFA) synthesized primarily by polyunsaturated fatty acid (PUFA) synthase, a type I polyketide synthase-like megaenzyme. The PUFA synthase in this species comprises three large subunits, each with multiple catalytic domains. It was hypothesized that among these domains, ketoacylsynthase (KS) domains might be critical for catalyzing the condensation of specific unsaturated acyl-acyl carrier proteins (ACPs) with malonyl-ACP, thereby retaining double bonds in an extended acyl chain. To investigate the functions of these putative KS domains, two segment sequences from subunit A (KS-A) and subunit B (KS-B) of the PUFA synthase were dissected and then expressed as stand-alone enzymes in Escherichia coli. The results showed that both KS-A and KS-B domains could complement the defective phenotypes of both E. coli fabB and fabF mutants. Overexpression of these domains in wild-type E. coli led to increases in total fatty acid production. KS-B produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while KS-A could improve the overall production of fatty acids more effectively, particularly for the production of SFAs, implying that KS-A is more comparable to FabF, while KS-B is more similar to FabB in catalytic functions. Successful complementation and functional expression of the embedded KS domains in E. coli are the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs in Thraustochytrium. IMPORTANCE Very-long-chain polyunsaturated fatty acids (VLCPUFAs) are important for human health. They can be biosynthesized in either an aerobic pathway or an anaerobic pathway in nature. However, abundant VLCPUFAs in marine microorganisms are primarily synthesized by polyunsaturated fatty acid (PUFA) synthase, a megaenzyme with multiple subunits, each with multiple catalytic domains. Furthermore, the fundamental mechanism for this enzyme to synthesize these fatty acids still remains unknown. This report started with dissecting the embedded KS domains of the PUFA synthase from marine protist Thraustochytrium sp. strain ATCC 26185 and then expressing them in wild-type E. coli and mutants defective in condensation of acyl-ACP with malonyl-ACP. Successful complementation of the mutants and improved fatty acid production in the overexpression experiments indicate that these KS domains can effectively function as stand-alone enzymes in E. coli. This result has paved the way for further studying of molecular mechanisms of the PUFA synthase for the biosynthesis of VLCPUFAs. PMID:28213537

Effects of troxerutin on cognitive deficits and glutamate cysteine ligase subunits in the hippocampus of streptozotocin-induced type 1 diabetes mellitus rats.

PubMed

Zhang, Songyun; Li, Hongyan; Zhang, Lihui; Li, Jie; Wang, Ruiying; Wang, Mian

2017-02-15

Increasing evidence demonstrates an association between diabetes and hippocampal neuron damage. This study aimed to determine the effects of troxerutin on cognitive deficits and glutamate cysteine ligase subunits (GCLM and GCLC) in the hippocampus of streptozotocin-induced type 1 diabetes mellitus (T1DM) rats. At 12weeks after streptozotocin injection, T1DM rats were randomly divided into 4 groups (n=15 each group) to receive no treatment (T1DM), saline (T1DM+saline), alpha-lipoic acid (T1DM+alpha-lipoic acid), and troxerutin (T1DM+troxerutin), respectively, for 6weeks. Meanwhile, 10 control animals (NC group) were assessed in parallel. Learning performance was evaluated by the Morris water maze. After treatment, hippocampi were collected for pathological examination by hematoxylin and eosin (H&E) staining. Next, hippocampal superoxide dismutase (SOD) activity, and malondialdehyde (MDA) and glutathione (GSH) levels were assessed. Finally, glutamate cysteine ligase catalytic (GCLC) and glutamate cysteine ligase modifier (GCLM) subunit mRNA and protein levels were quantified by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. Compared with T1DM and T1DM+saline groups, escape latency was overtly reduced in T1DM+alpha-lipoic acid and T1DM+troxerutin groups. Significantly increased GCLM and GCLC mRNA levels, GCLC protein amounts, SOD activity, and GSH levels, and reduced MDA amounts were observed in T1DM+alpha-lipoic acid and T1DM+troxerutin groups. In T1DM and T1DM+saline groups, H&E staining showed less pyramidal cells in the hippocampus, with disorganized layers, karyopyknosis, decreased endochylema, and cavitation, effects relieved in T1DM+alpha-lipoic acid and T1DM+troxerutin groups. Troxerutin alleviates oxidative stress and promotes learning in streptozotocin-induced T1DM rats, a process involving GCLC expression. Copyright © 2016 Elsevier B.V. All rights reserved.

An Electrostatic Charge Partitioning Model for the Dissociation of Protein Complexes in the Gas Phase

NASA Astrophysics Data System (ADS)

Sciuto, Stephen V.; Liu, Jiangjiang; Konermann, Lars

2011-10-01

Electrosprayed multi-protein complexes can be dissociated by collisional activation in the gas phase. Typically, these processes follow a mechanism whereby a single subunit gets ejected with a disproportionately high amount of charge relative to its mass. This asymmetric behavior suggests that the departing subunit undergoes some degree of unfolding prior to being separated from the residual complex. These structural changes occur concomitantly with charge (proton) transfer towards the subunit that is being unraveled. Charge accumulation takes place up to the point where the subunit loses physical contact with the residual complex. This work develops a simple electrostatic model for studying the relationship between conformational changes and charge enrichment during collisional activation. Folded subunits are described as spheres that carry continuum surface charge. The unfolded chain is envisioned as random coil bead string. Simulations are guided by the principle that the system will adopt the charge configuration with the lowest potential energy for any backbone conformation. A finite-difference gradient algorithm is used to determine the charge on each subunit throughout the dissociation process. Both dimeric and tetrameric protein complexes are investigated. The model reproduces the occurrence of asymmetric charge partitioning for dissociation events that are preceded by subunit unfolding. Quantitative comparisons of experimental MS/MS data with model predictions yield estimates of the structural changes that occur during collisional activation. Our findings suggest that subunit separation can occur over a wide range of scission point structures that correspond to different degrees of unfolding.

Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery

PubMed Central

Lanfranco, Maria Fe; Gárate, Fernanda; Engdahl, Ashton J.; Maillard, Rodrigo A.

2017-01-01

Many allosteric proteins form homo-oligomeric complexes to regulate a biological function. In homo-oligomers, subunits establish communication pathways that are modulated by external stimuli like ligand binding. A challenge for dissecting the communication mechanisms in homo-oligomers is identifying intermediate liganded states, which are typically transiently populated. However, their identities provide the most mechanistic information on how ligand-induced signals propagate from bound to empty subunits. Here, we dissected the directionality and magnitude of subunit communication in a reengineered single-chain version of the homodimeric transcription factor cAMP receptor protein. By combining wild-type and mutant subunits in various asymmetric configurations, we revealed a linear relationship between the magnitude of cooperative effects and the number of mutant subunits. We found that a single mutation is sufficient to change the global allosteric behavior of the dimer even when one subunit was wild type. Dimers harboring two mutations with opposite cooperative effects had different allosteric properties depending on the arrangement of the mutations. When the two mutations were placed in the same subunit, the resulting cooperativity was neutral. In contrast, when placed in different subunits, the observed cooperativity was dominated by the mutation with strongest effects over cAMP affinity relative to wild type. These results highlight the distinct roles of intrasubunit interactions and intersubunit communication in allostery. Finally, dimers bound to either one or two cAMP molecules had similar DNA affinities, indicating that both asymmetric and symmetric liganded states activate DNA interactions. These studies have revealed the multiple communication pathways that homo-oligomers employ to transduce signals. PMID:28188293

Mechanism of polyphosphate kinase from Propionibacterium shermanii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robinson, N.A.

1986-01-01

Polyphosphate kinase, which catalyzes the reaction shown below, is one of two enzymes which have been reported to catalyze the synthesis of polyphosphate. Purification performed by ammonium sulfate precipitation (0-40% fraction) was followed by chromatography. The enzyme represents 70% of the protein in the hydroxylapatite pool and is stable at this level of purity. The subunit molecular weight was determined by SDS polyacrylamide gel analysis, (83,000 +/- 3000), nondenaturing polyacrylamide gel electrophoresis, (80,000 and 86,000 daltons), gel filtration (Biogel A 0.5m column was 85,000 +/- 4000.) Polyphosphate kinase appears to be a monomeric enzyme of approx.83,000 daltons. Four assays weremore » developed for polyphosphate kinase. Basic proteins such as polylysine stimulate the synthesis of polyphosphate, these proteins cause precipitation of polyphosphate kinase from relatively impure enzyme extracts: Synthesized polyphosphate interacts noncovalently with the basic protein-enzyme precipitate. Efficient synthesis of polyphosphate requires the addition of either phosphate or short chain polyphosphate. Synthesis did occur at 1/10 the rate when neither of these two compounds were included. Initiation, elongation, and termination events of polyphosphate synthesis were examined. Short chain polyphosphate acts as a primer, with (/sup 32/P) short-chain polyphosphate incorporation into long chain polyphosphate by the kinase.« less

Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

PubMed Central

Neely, Alan; Hidalgo, Patricia

2014-01-01

Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

PDB-NMA of a protein homodimer reproduces distinct experimental motility asymmetry.

PubMed

Tirion, Monique M; Ben-Avraham, Daniel

2018-01-16

We have extended our analytically derived PDB-NMA formulation, Atomic Torsional Modal Analysis or ATMAN (Tirion and ben-Avraham 2015 Phys. Rev. E 91 032712), to include protein dimers using mixed internal and Cartesian coordinates. A test case on a 1.3 [Formula: see text] resolution model of a small homodimer, ActVA-ORF6, consisting of two 112-residue subunits identically folded in a compact 50 [Formula: see text] sphere, reproduces the distinct experimental Debye-Waller motility asymmetry for the two chains, demonstrating that structure sensitively selects vibrational signatures. The vibrational analysis of this PDB entry, together with biochemical and crystallographic data, demonstrates the cooperative nature of the dimeric interaction of the two subunits and suggests a mechanical model for subunit interconversion during the catalytic cycle.

PDB-NMA of a protein homodimer reproduces distinct experimental motility asymmetry

NASA Astrophysics Data System (ADS)

Tirion, Monique M.; ben-Avraham, Daniel

2018-03-01

We have extended our analytically derived PDB-NMA formulation, Atomic Torsional Modal Analysis or ATMAN (Tirion and ben-Avraham 2015 Phys. Rev. E 91 032712), to include protein dimers using mixed internal and Cartesian coordinates. A test case on a 1.3 {\\mathringA} resolution model of a small homodimer, ActVA-ORF6, consisting of two 112-residue subunits identically folded in a compact 50 {\\mathringA} sphere, reproduces the distinct experimental Debye-Waller motility asymmetry for the two chains, demonstrating that structure sensitively selects vibrational signatures. The vibrational analysis of this PDB entry, together with biochemical and crystallographic data, demonstrates the cooperative nature of the dimeric interaction of the two subunits and suggests a mechanical model for subunit interconversion during the catalytic cycle.

In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction

PubMed Central

Chen, Cai-Ping; Chen, Xin; Qiao, Yan-Ning; Wang, Pei; He, Wei-Qi; Zhang, Cheng-Hai; Zhao, Wei; Gao, Yun-Qian; Chen, Chen; Tao, Tao; Sun, Jie; Wang, Ye; Gao, Ning; Kamm, Kristine E; Stull, James T; Zhu, Min-Sheng

2015-01-01

Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle. Key points Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. PMID:25433069

In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction.

PubMed

Chen, Cai-Ping; Chen, Xin; Qiao, Yan-Ning; Wang, Pei; He, Wei-Qi; Zhang, Cheng-Hai; Zhao, Wei; Gao, Yun-Qian; Chen, Chen; Tao, Tao; Sun, Jie; Wang, Ye; Gao, Ning; Kamm, Kristine E; Stull, James T; Zhu, Min-Sheng

2015-02-01

Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

1-3-A Resolution Structure of Human Glutathione S-Transferase With S-Hexyl Glutathione Bound Reveals Possible Extended Ligandin Binding Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trong, I.Le; Stenkamp, R.E.; Ibarra, C.

2005-08-22

Cytosolic glutathione S-transferases (GSTs) play a critical role in xenobiotic binding and metabolism, as well as in modulation of oxidative stress. Here, the high-resolution X-ray crystal structures of homodimeric human GSTA1-1 in the apo form and in complex with S-hexyl glutathione (two data sets) are reported at 1.8, 1.5, and 1.3A respectively. At this level of resolution, distinct conformations of the alkyl chain of S-hexyl glutathione are observed, reflecting the nonspecific nature of the hydrophobic substrate binding site (H-site). Also, an extensive network of ordered water, including 75 discrete solvent molecules, traverses the open subunit-subunit interface and connects the glutathionemore » binding sites in each subunit. In the highest-resolution structure, three glycerol moieties lie within this network and directly connect the amino termini of the glutathione molecules. A search for ligand binding sites with the docking program Molecular Operating Environment identified the ordered water network binding site, lined mainly with hydrophobic residues, suggesting an extended ligand binding surface for nonsubstrate ligands, the so-called ligandin site. Finally, detailed comparison of the structures reported here with previously published X-ray structures reveal a possible reaction coordinate for ligand-dependent conformational changes in the active site and the C-terminus.« less

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

PubMed

Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

1995-06-01

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves.

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

PubMed Central

Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

1995-01-01

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves. PMID:7610168

Acute high-altitude hypoxic brain injury: Identification of ten differential proteins

PubMed Central

Li, Jianyu; Qi, Yuting; Liu, Hui; Cui, Ying; Zhang, Li; Gong, Haiying; Li, Yaxiao; Li, Lingzhi; Zhang, Yongliang

2013-01-01

Hypobaric hypoxia can cause severe brain damage and mitochondrial dysfunction, and is involved in hypoxic brain injury. However, little is currently known about the mechanisms responsible for mitochondrial dysfunction in hypobaric hypoxic brain damage. In this study, a rat model of hypobaric hypoxic brain injury was established to investigate the molecular mechanisms associated with mitochondrial dysfunction. As revealed by two-dimensional electrophoresis analysis, 16, 21, and 36 differential protein spots in cerebral mitochondria were observed at 6, 12, and 24 hours post-hypobaric hypoxia, respectively. Furthermore, ten protein spots selected from each hypobaric hypoxia subgroup were similarly regulated and were identified by mass spectrometry. These detected proteins included dihydropyrimidinase-related protein 2, creatine kinase B-type, isovaleryl-CoA dehydrogenase, elongation factor Ts, ATP synthase beta-subunit, 3-mercaptopyruvate sulfurtransferase, electron transfer flavoprotein alpha-subunit, Chain A of 2-enoyl-CoA hydratase, NADH dehydrogenase iron-sulfur protein 8 and tropomyosin beta chain. These ten proteins are all involved in the electron transport chain and the function of ATP synthase. Our findings indicate that hypobaric hypoxia can induce the differential expression of several cerebral mitochondrial proteins, which are involved in the regulation of mitochondrial energy production. PMID:25206614

Purification of Ovine Respiratory Complex I Results in a Highly Active and Stable Preparation.

PubMed

Letts, James A; Degliesposti, Gianluca; Fiedorczuk, Karol; Skehel, Mark; Sazanov, Leonid A

2016-11-18

NADH-ubiquinone oxidoreductase (complex I) is the largest (∼1 MDa) and the least characterized complex of the mitochondrial electron transport chain. Because of the ease of sample availability, previous work has focused almost exclusively on bovine complex I. However, only medium resolution structural analyses of this complex have been reported. Working with other mammalian complex I homologues is a potential approach for overcoming these limitations. Due to the inherent difficulty of expressing large membrane protein complexes, screening of complex I homologues is limited to large mammals reared for human consumption. The high sequence identity among these available sources may preclude the benefits of screening. Here, we report the characterization of complex I purified from Ovis aries (ovine) heart mitochondria. All 44 unique subunits of the intact complex were identified by mass spectrometry. We identified differences in the subunit composition of subcomplexes of ovine complex I as compared with bovine, suggesting differential stability of inter-subunit interactions within the complex. Furthermore, the 42-kDa subunit, which is easily lost from the bovine enzyme, remains tightly bound to ovine complex I. Additionally, we developed a novel purification protocol for highly active and stable mitochondrial complex I using the branched-chain detergent lauryl maltose neopentyl glycol. Our data demonstrate that, although closely related, significant differences exist between the biochemical properties of complex I prepared from ovine and bovine mitochondria and that ovine complex I represents a suitable alternative target for further structural studies. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Steady-state levels of G-protein beta-subunit expression are regulated by treatment of cells with bacterial toxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watkins, D.C.; Northup, J.K.; Malbon, C.C.

1987-05-01

Cultures of 3T3-L1 cells were incubated with either 10 ng/ml cholera toxin or 10 ng/ml pertussis toxin from 4 days prior to the initiation of differentiation and throughout the subsequent incubation. Toxin concentrations were sufficient to completely prevent the labelling of alpha-subunits with (/sup 32/P)NAD/sup +/ and pertussis toxin and to prevent by more than 90% the labelling with (/sup 32/P)NAD/sup +/ and cholera toxin in membranes prepared from these cells. Neither toxin prevented the differentiation to the adipocyte phenotype. Neither toxin prevented the increases in the relative amounts of G-proteins which occur upon differentiation. Both toxins dramatically decreased themore » amount of beta-subunits. As measured by quantitative immunoblotting with antisera specific for both the 35 kDa and 36 kDa beta-subunits, levels of beta-subunit were decreased by more than 50% of steady-state level of control cells. Thus, bacterial toxins which modifies G-protein alpha-subunits are capable of modulating the levels of beta-subunits in vivo. The basis for the regulation of G-protein subunit expression by bacterial toxins is under study.« less

MspA Nanopores from Subunit Dimers

PubMed Central

Pavlenok, Mikhail; Derrington, Ian M.; Gundlach, Jens H.; Niederweis, Michael

2012-01-01

Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA. PMID:22719928

Meta-Analyses of Dehalococcoides mccartyi Strain 195 Transcriptomic Profiles Identify a Respiration Rate-Related Gene Expression Transition Point and Interoperon Recruitment of a Key Oxidoreductase Subunit

PubMed Central

Mansfeldt, Cresten B.; Rowe, Annette R.; Heavner, Gretchen L. W.; Zinder, Stephen H.

2014-01-01

A cDNA-microarray was designed and used to monitor the transcriptomic profile of Dehalococcoides mccartyi strain 195 (in a mixed community) respiring various chlorinated organics, including chloroethenes and 2,3-dichlorophenol. The cultures were continuously fed in order to establish steady-state respiration rates and substrate levels. The organization of array data into a clustered heat map revealed two major experimental partitions. This partitioning in the data set was further explored through principal component analysis. The first two principal components separated the experiments into those with slow (1.6 ± 0.6 μM Cl−/h)- and fast (22.9 ± 9.6 μM Cl−/h)-respiring cultures. Additionally, the transcripts with the highest loadings in these principal components were identified, suggesting that those transcripts were responsible for the partitioning of the experiments. By analyzing the transcriptomes (n = 53) across experiments, relationships among transcripts were identified, and hypotheses about the relationships between electron transport chain members were proposed. One hypothesis, that the hydrogenases Hup and Hym and the formate dehydrogenase-like oxidoreductase (DET0186-DET0187) form a complex (as displayed by their tight clustering in the heat map analysis), was explored using a nondenaturing protein separation technique combined with proteomic sequencing. Although these proteins did not migrate as a single complex, DET0112 (an FdhB-like protein encoded in the Hup operon) was found to comigrate with DET0187 rather than with the catalytic Hup subunit DET0110. On closer inspection of the genome annotations of all Dehalococcoides strains, the DET0185-to-DET0187 operon was found to lack a key subunit, an FdhB-like protein. Therefore, on the basis of the transcriptomic, genomic, and proteomic evidence, the place of the missing subunit in the DET0185-to-DET0187 operon is likely filled by recruiting a subunit expressed from the Hup operon (DET0112). PMID:25063656

Regulation of protein phosphatase 2A during embryonic diapause process in the silkworm, Bombyx mori.

PubMed

Gu, Shi-Hong; Hsieh, Hsiao-Yen; Lin, Pei-Ling

2017-11-01

Regulation of protein phosphorylation requires coordinated interactions between protein kinases and protein phosphatases. In the present study, we investigated regulation of protein phosphatase 2A (PP2A) during the embryonic diapause process of B. mori. An immunoblotting analysis showed that Bombyx eggs contained a catalytic C subunit, a major regulatory B subunit (B55/PR55 subunit), and a structural A subunit, with the A and B subunits undergoing differential changes between diapause and non-diapause eggs during embryonic process. In non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5°C for 70days and then were transferred to 25°C, protein levels of the A and B subunits of PP2A gradually increased toward embryonic development. However, protein levels of the A and B subunits in diapause eggs remained at low levels during the first 8days after oviposition. The direct determination of PP2A enzymatic activity showed that the activity remained at low levels in diapause eggs during the first 8days after oviposition. However, in non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling, PP2A enzymatic activity sharply increased during the first several days, reached a peak during the middle embryonic development, and then greatly decreased 3 or 4days before hatching. Examination of temporal changes in mRNA expression levels of the catalytic β subunit and regulatory subunit of PP2A showed high levels in eggs whose diapause initiation was prevented by HCl compared to those in diapause eggs. These results demonstrate that the higher PP2A gene expression and PP2A A and B subunit protein levels and increased enzymatic activity are related to embryonic development of B. mori. Copyright © 2017 Elsevier Ltd. All rights reserved.

Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor

PubMed Central

Assadi-Porter, Fariba M.; Maillet, Emeline L.; Radek, James T.; Quijada, Jeniffer; Markley, John L.; Max, Marianna

2010-01-01

The sweet protein brazzein activates the human sweet receptor, a heterodimeric G-protein coupled receptor (GPCR) composed of subunits T1R2 and T1R3. In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by the in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: Site 1 (Loop43), Site 2 (N- and C-terminus and adjacent Glu36, Loop33), and Site 3 (Loop9–19). Basic residues in Site 1 and acidic residues in Site 2 were essential for positive responses from each assay. Mutation of Y39A (Site 1) greatly reduced positive responses. A bulky side chain at position 54 (Site 2), rather than a side chain with hydrogen bonding potential, was required for positive responses as was the presence of the native disulfide bond in Loop 9–19 (Site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus fly trap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in the brazzein response. The exception, hT1R2:R217A-hT1R3, which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site in involved in subunit-subunit interaction rather than direct brazzein binding. Results from this study support a multipoint interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models. PMID:20302879

Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the alpha6-integrin subunit.

PubMed

Kashimata, M; Gresik, E W

1997-02-01

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) regulate branching morphogenesis of fetal mouse submandibular gland (SMG) rudiments in vitro. The EGF system (EGF, TGF-alpha, and their shared receptor, EGFR) also regulates expression of integrins and their ligands in the extracellular matrix. We show here that inhibition of EGFR tyrosine-kinase activity by a tyrphostin retards in vitro development of SMGs. Using total RNA isolated from pooled SMGs taken from intact mouse fetuses, mRNA transcripts for EGF, TGF-alpha, and EGFR were detected by reverse transcription-polymerase chain reaction (RT-PCR), and age-dependent variations in the levels of these mRNA were quantitatively determined by nuclease protection assays. These findings suggest that the EGF system is operative in the in vivo development of this gland. alpha6-Integrin subunit was localized by immunofluorescence at the basal surface of epithelial cells. Branching morphogenesis of cultured SMG rudiments was inhibited by anti-alpha6 antibodies. Synthesis of alpha6-subunit in cultured SMGs, detected by metabolic labeling and immunoprecipitation, was increased by EGF and drastically reduced by tyrphostin. RT-PCR revealed that mRNAs for alpha6- and beta1- and beta4-integrin subunits are expressed at all ages between embryonic day 13 and postnatal day 7. These findings suggest that 1) the EGF system is a physiologic regulator of development of fetal mouse SMG, and 2) one mechanism by which it acts may be by regulating expression of integrins, which in turn control interaction of epithelial cells with the extracellular matrix.

Increased Expression of Laminin Subunit Alpha 1 Chain by dCas9-VP160.

PubMed

Perrin, Arnaud; Rousseau, Joël; Tremblay, Jacques P

2017-03-17

Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adult human muscle, but laminin subunit alpha 1 (LAMA1) gene is expressed only during embryogenesis. We thus developed an alternative method to laminin-111 protein repeated administration by inducing expression of the endogenous mouse Lama1 gene. This was done with the CRSPR/Cas9 system, i.e., by targeting the Lama1 promoter with one or several gRNAs and a dCas9 coupled with the VP160 transcription activation domain. Lama1 mRNA (qRT-PCR) and proteins (immunohistochemistry and western blot) were not detected in the control C2C12 myoblasts and in control muscles. However, significant expression was observed in cells transfected and in mouse muscles electroporated with plasmids coding for dCas9-VP160 and a gRNA. Larger synergic increases were observed by using two or three gRNAs. The increased Lama1 expression did not modify the expression of the α7 and β1 integrins. Increased expression of Lama1 by the CRISPR/Cas9 system will have to be further investigated by systemic delivery of the CRISPR/Cas9 components to verify whether this could be a treatment for several myopathies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Native top-down mass spectrometry for the structural characterization of human hemoglobin

DOE PAGES

Zhang, Jiang; Malmirchegini, G. Reza; Clubb, Robert T.; ...

2015-06-09

Native mass spectrometry (MS) has become an invaluable tool for the characterization of proteins and non-covalent protein complexes under near physiological solution conditions. Here we report the structural characterization of human hemoglobin (Hb), a 64 kDa oxygen-transporting protein complex, by high resolution native top-down mass spectrometry using electrospray ionization (ESI) and a 15-Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Native MS preserves the non-covalent interactions between the globin subunits, and electron capture dissociation (ECD) produces fragments directly from the intact Hb complex without dissociating the subunits. Using activated ion ECD, we observe the gradual unfolding process of themore » Hb complex in the gas phase. Without protein ion activation, the native Hb shows very limited ECD fragmentation from the N-termini, suggesting a tightly packed structure of the native complex and therefore low fragmentation efficiency. Precursor ion activation allows steady increase of N-terminal fragment ions, while the C-terminal fragments remain limited (38 c ions and 4 z ions on the α chain; 36 c ions and 2 z ions on the β chain). This ECD fragmentation pattern suggests that upon activation, the Hb complex starts to unfold from the N-termini of both subunits, whereas the C-terminal regions and therefore the potential regions involved in the subunit binding interactions remain intact. ECD-MS of the Hb dimer show similar fragmentation patterns as the Hb tetramer, providing further evidence for the hypothesized unfolding process of the Hb complex in the gas phase. Native top-down ECD-MS allows efficient probing of the Hb complex structure and the subunit binding interactions in the gas phase. Finally, it may provide a fast and effective means to probe the structure of novel protein complexes that are intractable to traditional structural characterization tools.« less

Molecular basis for potentiation of Cx36 gap junction channel conductance by n-alcohols and general anesthetics.

PubMed

Raškevičius, Vytautas; Jotautis, Vaidas; Rimkutė, Lina; Marandykina, Alina; Kazokaitė, Mintautė; Kairys, Visvaldas; Skeberdis, Vytenis Arvydas

2018-02-28

In our recent study, we have demonstrated that short carbon chain n -alcohols (up to octanol) stimulated while long carbon chain n -alcohols inhibited the conductance of connexin (Cx) 36 (Cx36) gap junction (GJ) channels. In contrast, GJ channels composed of other types of Cxs all were inhibited by n -alcohols independent of their carbon chain length. To identify the putative structural domains of Cx36, responsible for the dual effect of n -alcohols, we performed structural modeling of Cx36 protein docking with hexanol and isoflurane that stimulated as well as nonanol and carbenoxolone that inhibited the conductance of Cx36 GJs and revealed their multiple common docking sites and a single pocket accessible only to hexanol and isoflurane. The pocket is located in the vicinity of three unique cysteine residues, namely C264 in the fourth, and C92 and C87 in the second transmembrane domain of the neighboring Cx36 subunits. To examine the hypothesis that disulphide bonding might be involved in the stimulatory effect of hexanol and isoflurane, we generated cysteine substitutions in Cx36 and demonstrated by a dual whole-cell patch-clamp technique that in HeLa (human cervix carcinoma cell line) and N2A (mouse neuroblastoma cell line) cells these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical of other Cxs that lack respective cysteines and a specific docking pocket for these compounds. Our findings suggest that the stimulatory effect of hexanol and isoflurane on Cx36 GJ conductance could be achieved by re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87. © 2018 The Author(s).

Increased IL-27/IL-27R expression in association with the immunopathology of murine ocular toxoplasmosis.

PubMed

Tong, Xinxin; Chen, Shengjie; Zheng, Huanqin; Huang, Shiguang; Lu, Fangli

2018-05-19

Interleukin 27 (IL-27) is a member of the IL-6/IL-12 family, and IL-27 receptor (IL-27R) consists of WSX-1 (the IL-27Rα subunit) and the signal-transducing subunit gp130. Human and mouse mast cells (MCs) express the IL-27R. To explore the expressions of IL-27/IL-27R subunits (WSX-1 and gp130) during acute ocular toxoplasmosis (OT), we established mouse model by intraocular injection of 500 Toxoplasma gondii RH strain tachyzoites. Histopathological changes were analyzed, MCs were counted by toluidine blue staining, and tryptase + /IL-27 + MCs were examined by immunofluorescence double-staining in the eyes and cervical lymph nodes (CLNs) of T. gondii-infected mice. The mRNA expressions of IL-27p28, WSX-1, gp130, and tachyzoite specific surface antigen 1 (SAG1) in the eyes and CLNs of T. gondii-infected mice, and the expressions of WSX-1 and gp130 in the murine mastocytoma cell line P815 infected with T. gondii tachyzoites in vitro were examined by using quantitative real-time reverse transcription-polymerase chain reaction. Our results showed that, after T. gondii infection, severe histopathological changes, increased numbers of total MCs and degranulated MCs, elevated expressions of IL-27p28, WSX-1, and gp130 were found in the eyes and CLNs, and significant correlations between the levels of IL-27 and SAG1 existed in the eyes and CLNs of T. gondii-infected mice. In addition, increased levels of WSX-1 and gp130 were examined in T. gondii-infected P815 cells. Our data suggested that IL-27/IL-27R expression induced by T. gondii infection may regulate MC-mediated immune response during acute OT in mouse model.

Molecular Modeling and Imaging of Initial Stages of Cellulose Fibril Assembly: Evidence for a Disordered Intermediate Stage

PubMed Central

Haigler, Candace H.; Grimson, Mark J.; Gervais, Julien; Le Moigne, Nicolas; Höfte, Herman; Monasse, Bernard; Navard, Patrick

2014-01-01

The remarkable mechanical strength of cellulose reflects the arrangement of multiple β-1,4-linked glucan chains in a para-crystalline fibril. During plant cellulose biosynthesis, a multimeric cellulose synthesis complex (CSC) moves within the plane of the plasma membrane as many glucan chains are synthesized from the same end and in close proximity. Many questions remain about the mechanism of cellulose fibril assembly, for example must multiple catalytic subunits within one CSC polymerize cellulose at the same rate? How does the cellulose fibril bend to align horizontally with the cell wall? Here we used mathematical modeling to investigate the interactions between glucan chains immediately after extrusion on the plasma membrane surface. Molecular dynamics simulations on groups of six glucans, each originating from a position approximating its extrusion site, revealed initial formation of an uncrystallized aggregate of chains from which a protofibril arose spontaneously through a ratchet mechanism involving hydrogen bonds and van der Waals interactions between glucose monomers. Consistent with the predictions from the model, freeze-fracture transmission electron microscopy using improved methods revealed a hemispherical accumulation of material at points of origination of apparent cellulose fibrils on the external surface of the plasma membrane where rosette-type CSCs were also observed. Together the data support the possibility that a zone of uncrystallized chains on the plasma membrane surface buffers the predicted variable rates of cellulose polymerization from multiple catalytic subunits within the CSC and acts as a flexible hinge allowing the horizontal alignment of the crystalline cellulose fibrils relative to the cell wall. PMID:24722535

Apical Plasma Membrane Mispolarization of NaK-ATPase in Polycystic Kidney Disease Epithelia Is Associated with Aberrant Expression of the β2 Isoform

PubMed Central

Wilson, Patricia D.; Devuyst, Olivier; Li, Xiaohong; Gatti, Laura; Falkenstein, Doris; Robinson, Shawn; Fambrough, Douglas; Burrow, Christopher R.

2000-01-01

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease of the kidney, characterized by cystic enlargement of renal tubules, aberrant epithelial proliferation, and ion and fluid secretion into the lumen. Previous studies have shown abnormalities in polarization of membrane proteins, including mislocalization of the NaK-ATPase to the apical plasma membranes of cystic epithelia. Apically located NaK-ATPase has previously been shown to be fully functional in vivo and in membrane-grown ADPKD epithelial cells in vitro, where basal-to-apical 22Na transport was inhibited by application of ouabain to the apical membrane compartment. Studies were conducted with polymerase chain reaction-generated specific riboprobes and polyclonal peptide antibodies against human sequences of α1, α3, β1, and β2 subunits of NaK-ATPase. High levels of expression of α1 and β1 messenger RNA were detected in ADPKD and age-matched normal adult kidneys in vivo, whereas β2 messenger RNA was detected only in ADPKD kidneys. Western blot analysis and immunocytochemical studies showed that, in normal adult kidneys, peptide subunit-specific antibodies against α1 and β1 localized to the basolateral membranes of normal renal tubules, predominantly thick ascending limbs of Henle’s loop. In ADPKD kidneys, α1 and β2 subunits were localized to the apical epithelial cell membranes, whereas β1 was distributed throughout the cytoplasm and predominantly in the endoplasmic reticulum, but was not seen associated with cystic epithelial cell membranes or in cell membrane fractions. Polarizing, renal-derived epithelial Madin Darby canine kidney cells, stably expressing normal or N-terminally truncated chicken β1 subunits, showed selective accumulation in the basolateral Madin Darby canine kidney cell surface, whereas c-myc epitope-tagged chicken β2 or human β2 subunits accumulated selectively in the apical cell surface. Similarly, human ADPKD epithelial cell lines, which endogenously expressed α1 and β2 NaK-ATPase subunits, showed colocalization at the apical cell surface and coassociation by immunoprecipitation analysis. These results are consistent with a model in which the additional transcription and translation of the β2 subunit of NaK-ATPase may result in the apical mislocalization of NaK-ATPase in ADPKD cystic epithelia. PMID:10623674

Magnolol Inhibits Human Glioblastoma Cell Migration by Regulating N-Cadherin.

PubMed

Cheng, Yu-Chen; Tsao, Min-Jen; Chiu, Chen-Yang; Kan, Po-Chieh; Chen, Ying

2018-06-01

Glioblastoma is a primary malignant brain tumor with a poor prognosis. An effective treatment for glioblastoma is needed. Magnolol is a natural compound from Magnolia officinalis suggested to have antiproliferative activity. The aim of this research was to investigate the anticancer effects of magnolol in glioma, with an emphasis on migration and the underlying mechanism. Magnolol decreased the expression of focal adhesion-related proteins and inhibited LN229 and U87MG glioma cell migration. The levels of phosphorylated myosin light chain (p-MLC), phosphorylated myosin light chain kinase and myosin phosphatase target subunit 1 were reduced in response to magnolol treatment. In addition, immunostaining and membrane fractionation showed that the distribution of N-cadherin at the glioma cell membrane was decreased by magnolol. In an orthotropic xenograft animal model, magnolol treatment not only inhibited tumor progression but also reduced p-MLC and N-cadherin protein expression. In conclusion, magnolol reduces cell migration, potentially through regulating focal adhesions and N-cadherin in glioma cells. Magnolol is a potential candidate for glioma treatment.

Potential involvement of F0F1-ATP(synth)ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines : a mechanism for induction of apoptosis via the 18 kDa mitochondrial translocator protein.

PubMed

Veenman, Leo; Alten, Julia; Linnemannstöns, Karen; Shandalov, Yulia; Zeno, Sivan; Lakomek, Max; Gavish, Moshe; Kugler, Wilfried

2010-07-01

Erucylphosphohomocholine (ErPC3, Erufosine) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial F(O)F(1)-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the F(O) and F(1) subunits of the mitochondrial F(O)F(1)-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Deltapsi(m)). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Deltapsi(m), normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the F(O) subunit of the mitochondrial F(O)F(1)-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Deltapsi(m) as well as ROS generation by ErPC3 and TSPO.

The functional interplay between the HIF pathway and the ubiquitin system - more than a one-way road.

PubMed

Günter, Julia; Ruiz-Serrano, Amalia; Pickel, Christina; Wenger, Roland H; Scholz, Carsten C

2017-07-15

The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue functions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIFα subunits leads to the recruitment of an E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIFα subunits, demonstrating the regulation of the HIF pathway by the ubiquitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the best-studied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway. Understanding these mechanisms and their relevance for the activity of each other is of major importance for the comprehensive elucidation of the oxygen-dependent regulation of cellular processes. This review describes the current knowledge of the functional bidirectional interplay between the HIF pathway and the ubiquitin system on the protein level. Copyright © 2017 Elsevier Inc. All rights reserved.

Essential arginine in subunit a and aspartate in subunit c of FoF1 ATP synthase: effect of repositioning within helix 4 of subunit a and helix 2 of subunit c.

PubMed

Langemeyer, Lars; Engelbrecht, Siegfried

2007-07-01

FoF1 ATP synthase couples proton flow through the integral membrane portion Fo (ab2c10) to ATP-synthesis in the extrinsic F1-part ((alphabeta)3gammadeltaepsilon) (Escherichia coli nomenclature and stoichiometry). Coupling occurs by mechanical rotation of subunits c10gammaepsilon relative to (alphabeta)3deltaab2. Two residues were found to be essential for proton flow through ab2c10, namely Arg210 in subunit a (aR210) and Asp61 in subunits c (cD61). Their deletion abolishes proton flow, but "horizontal" repositioning, by anchoring them in adjacent transmembrane helices, restores function. Here, we investigated the effects of "vertical" repositioning aR210, cD61, or both by one helical turn towards the N- or C-termini of their original helices. Other than in the horizontal the vertical displacement changes the positions of the side chains within the depth of the membrane. Mutant aR210A/aN214R appeared to be short-circuited in that it supported proton conduction only through EF1-depleted EFo, but not in EFoEF1, nor ATP-driven proton pumping. Mutant cD61N/cM65D grew on succinate, retained the ability to synthesize ATP and supported passive proton conduction but apparently not ATP hydrolysis-driven proton pumping.

SLP-2 interacts with prohibitins in the mitochondrial inner membrane and contributes to their stability.

PubMed

Da Cruz, Sandrine; Parone, Philippe A; Gonzalo, Philippe; Bienvenut, Willy V; Tondera, Daniel; Jourdain, Alexis; Quadroni, Manfredo; Martinou, Jean-Claude

2008-05-01

Stomatin is a member of a large family of proteins including prohibitins, HflK/C, flotillins, mechanoreceptors and plant defense proteins, that are thought to play a role in protein turnover. Using different proteomic approaches, we and others have identified SLP-2, a member of the stomatin gene family, as a component of the mitochondria. In this study, we show that SLP-2 is strongly associated with the mitochondrial inner membrane and that it interacts with prohibitins. Depleting HeLa cells of SLP-2 lead to increased proteolysis of prohibitins and of subunits of the respiratory chain complexes I and IV. Further supporting the role of SLP-2 in regulating the stability of specific mitochondrial proteins, we found that SLP-2 is up-regulated under conditions of mitochondrial stress leading to increased protein turnover. These data indicate that SLP-2 plays a role in regulating the stability of mitochondrial proteins including prohibitins and subunits of respiratory chain complexes.

Bridging cobalt-calixarene subunits into a Co8 entity or a chain with 4,4‧-bipyridyl

NASA Astrophysics Data System (ADS)

Liu, Wei; Liu, Mei; Du, Shangchao; Li, Yafeng; Liao, Wuping

2014-02-01

Two novel calixarene-based compounds, {[Co4Cl(TC4A)(HCOO)3]2(4,4‧-bpy)2} (CIAC-206) and {[Co3(H2O)(SC4A-SO2)(HCOO)2]2(4,4‧-bpy)}n (CIAC-207) (H4TC4A = p-tert-butylthiacalix[4]arene, SC4A-SO2 = p-tert-butylsulfonylcalix[4]arene, 4,4‧-bpy = 4,4‧-bipyridyl) were synthesized under solvothermal conditions, and characterized by single crystal X-ray diffraction analysis, TG-DSC analysis, elemental analysis and IR spectroscopy. These two structures are featured with isolated Z-shaped Co8 entities containing two Co4-TC4A subunits bridged by two parallel 4,4‧-bpy (CIAC-206) and some zigzag chains with [Co3-SC4A-SO2]2 dimers bridged by single 4,4‧-bpy (CIAC-207), respectively. In order to evaluate their properties, the N2 sorption behavior and magnetic property were examined.

Crystal Structure of Alcohol Oxidase from Pichia pastoris

PubMed Central

Valerius, Oliver; Feussner, Ivo; Ficner, Ralf

2016-01-01

FAD-dependent alcohol oxidases (AOX) are key enzymes of methylotrophic organisms that can utilize lower primary alcohols as sole source of carbon and energy. Here we report the crystal structure analysis of the methanol oxidase AOX1 from Pichia pastoris. The crystallographic phase problem was solved by means of Molecular Replacement in combination with initial structure rebuilding using Rosetta model completion and relaxation against an averaged electron density map. The subunit arrangement of the homo-octameric AOX1 differs from that of octameric vanillyl alcohol oxidase and other dimeric or tetrameric alcohol oxidases, due to the insertion of two large protruding loop regions and an additional C-terminal extension in AOX1. In comparison to other alcohol oxidases, the active site cavity of AOX1 is significantly reduced in size, which could explain the observed preference for methanol as substrate. All AOX1 subunits of the structure reported here harbor a modified flavin adenine dinucleotide, which contains an arabityl chain instead of a ribityl chain attached to the isoalloxazine ring. PMID:26905908

Structural and Mechanistic Insight into the Listeria monocytogenes Two-enzyme Lipoteichoic Acid Synthesis System*

PubMed Central

Campeotto, Ivan; Percy, Matthew G.; MacDonald, James T.; Förster, Andreas; Freemont, Paul S.; Gründling, Angelika

2014-01-01

Lipoteichoic acid (LTA) is an important cell wall component required for proper cell growth in many Gram-positive bacteria. In Listeria monocytogenes, two enzymes are required for the synthesis of this polyglycerolphosphate polymer. The LTA primase LtaPLm initiates LTA synthesis by transferring the first glycerolphosphate (GroP) subunit onto the glycolipid anchor and the LTA synthase LtaSLm extends the polymer by the repeated addition of GroP subunits to the tip of the growing chain. Here, we present the crystal structures of the enzymatic domains of LtaPLm and LtaSLm. Although the enzymes share the same fold, substantial differences in the cavity of the catalytic site and surface charge distribution contribute to enzyme specialization. The eLtaSLm structure was also determined in complex with GroP revealing a second GroP binding site. Mutational analysis confirmed an essential function for this binding site and allowed us to propose a model for the binding of the growing chain. PMID:25128528

A Bond-Fluctuation Model of Translational Dynamics of Chain-like Particles through Mucosal Scaffolds.

PubMed

Bajd, Franci; Serša, Igor

2018-06-05

Mucus scaffolds represent one of the most common barriers in targeted drug delivery and can remarkably reduce the outcome of pharmacological therapies. An efficient transport of drug particles through a mucus barrier is a precondition for an efficient drug delivery. Understanding the transport mechanism is particularly important for treatment of disorders such as cystic fibrosis. These are characterized by an onset of high-density mucus scaffolds imposing an increased steric filtering. In this study, we employed the bond-fluctuation model to analyze the effect of steric interactions on slowing the translational dynamics of compound chain-like particles traversing through scaffolds of different configurations (regular isotropic and anisotropic versus irregular random). The model, which accounts for both the geometry-imposed steric interaction as well as the intrachain steric interaction between the chain subunits, yields a transient subdiffusive motional pattern persists between the short-time and long-time Gaussian diffusion limits. The motion is analyzed in terms of a mean-squared displacement, diffusion coefficient, and radius of gyration. With higher levels of restriction or larger particles, the subdiffusive motional regime persists longer. The study also demonstrates that an important feature of the motion is also geometry-induced chain accommodation. The presented model is generic and could also be applied to studying the translational dynamics of other particles with more complex architecture such as dendrites or chain-decorated nanoparticles. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A case-control study on association of proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1) polymorphisms and their transcript levels in vitiligo from Gujarat

PubMed Central

Jadeja, Shahnawaz D.; Mansuri, Mohmmad Shoab; Singh, Mala; Dwivedi, Mitesh; Laddha, Naresh C.

2017-01-01

Background Autoimmunity has been implicated in the destruction of melanocytes from vitiligo skin. Major histocompatibility complex (MHC) class-II linked genes proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1), involved in antigen processing and presentation have been reported to be associated with several autoimmune diseases including vitiligo. Objectives To explore PSMB8 rs2071464 and TAP1 rs1135216 single nucleotide polymorphisms and to estimate the expression of PSMB8 and TAP1 in patients with vitiligo and unaffected controls from Gujarat. Methods PSMB8 rs2071464 polymorphism was genotyped using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and TAP1 rs1135216 polymorphism was genotyped by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in 378 patients with vitiligo and 509 controls. Transcript levels of PSMB8 and TAP1 were measured in the PBMCs of 91 patients and 96 controls by using qPCR. Protein levels of PSMB8 were also determined by Western blot analysis. Results The frequency of ‘TT’ genotype of PSMB8 polymorphism was significantly lowered in patients with generalized and active vitiligo (p = 0.019 and p = 0.005) as compared to controls suggesting its association with the activity of the disease. However, TAP1 polymorphism was not associated with vitiligo susceptibility. A significant decrease in expression of PSMB8 at both transcript level (p = 0.002) as well as protein level (p = 0.0460) was observed in vitiligo patients as compared to controls. No significant difference was observed between patients and controls for TAP1 transcripts (p = 0.553). Interestingly, individuals with the susceptible CC genotype of PSMB8 polymorphism showed significantly reduced PSMB8 transcript level as compared to that of CT and TT genotypes (p = 0.009 and p = 0.003 respectively). Conclusions PSMB8 rs2071464 was associated with generalized and active vitiligo from Gujarat whereas TAP1 rs1135216 showed no association. The down-regulation of PSMB8 in patients with risk genotype ‘CC’ advocates the vital role of PSMB8 in the autoimmune basis of vitiligo. PMID:28700671

Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity.

PubMed

Matsunaga, Toshiyuki; Endo, Satoshi; Maeda, Satoshi; Ishikura, Shuhei; Tajima, Kazuo; Tanaka, Nobutada; Nakamura, Kazuo T; Imamura, Yorishige; Hara, Akira

2008-09-15

Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and alpha-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C(19)/C(21)-steroids into 3beta-hydroxysteroids. The stereospecific conversion to 3beta-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor alpha ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3beta-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.

n-Alcohols Inhibit Voltage-Gated Na+ Channels Expressed in Xenopus Oocytes

PubMed Central

Horishita, Takafumi; Harris, R. Adron

2008-01-01

Voltage-gated sodium channels are essential for the initiation and propagation of action potentials in excitable cells and are known as a target of local anesthetics. In addition, inhibition of sodium channels by volatile anesthetics has been proposed as a mechanism of general anesthesia. The n-alcohols produce anesthesia, and their potency increases with carbon number until a “cut-off” is reached. In this study, we examined effects of a range of n-alcohols on Nav1.2 subunits to determine the alcohol cut-off for this channel. We also studied the effect of a short-chain alcohol (ethanol) and a long-chain alcohol (octanol) on Nav1.2, Nav1.4, Nav1.6, and Nav1.8 subunits, and we investigated the effects of alcohol on channel kinetics. Ethanol and octanol inhibited sodium currents of all subunits, and the inhibition of the Nav1.2 channel by n-alcohols indicated a cut-off at nonanol. Ethanol and octanol produced open-channel block, which was more pronounced for Nav1.8 than for the other sodium channels. Inhibition of Nav1.2 was due to decreased activation and increased inactivation. These results suggest that sodium channels may have a hydrophobic binding site for n-alcohols and demonstrate the differences in the kinetic mechanisms of inhibition for n-alcohols and inhaled anesthetics. PMID:18434586

The Essential tacF Gene Is Responsible for the Choline-Dependent Growth Phenotype of Streptococcus pneumoniae▿

PubMed Central

Damjanovic, Marlen; Kharat, Arun S.; Eberhardt, Alice; Tomasz, Alexander; Vollmer, Waldemar

2007-01-01

Streptococcus pneumoniae has an absolute nutritional requirement for choline, and the choline molecules are known to incorporate exclusively into the cell wall and membrane teichoic acids of the bacterium. We describe here the isolation of a mutant of strain R6 in which a single G→T point mutation in the gene tacF (formerly designated spr1150) is responsible for generating a choline-independent phenotype. The choline-independent phenotype could be transferred to the laboratory strain R6 and to the encapsulated strain D39 by genetic transformation with a PCR product or with a plasmid carrying the mutated tacF gene. The tacF gene product belongs to the protein family of polysaccharide transmembrane transporters (flippases). A model is presented in which TacF is required for the transport of the teichoic acid subunits across the cytoplasmic membrane. According to this model, wild-type TacF has a strict specificity for choline-containing subunits, whereas the TacF present in the choline-independent mutant strain is able to transport both choline-containing and choline-free teichoic acid chains. The proposed transport specificity of parental-type TacF for choline-containing subunits would ensure the loading of the cell wall with teichoic acid chains decorated with choline residues, which appear to be essential for the virulence of this pathogen. PMID:17660291

Caffeic acid attenuates rat liver reperfusion injury through sirtuin 3-dependent regulation of mitochondrial respiratory chain.

PubMed

Mu, Hong-Na; Li, Quan; Pan, Chun-Shui; Liu, Yu-Ying; Yan, Li; Hu, Bai-He; Sun, Kai; Chang, Xin; Zhao, Xin-Rong; Fan, Jing-Yu; Han, Jing-Yan

2015-08-01

Sirtuin 3 (Sirt3) plays critical roles in regulating mitochondrial oxidative metabolism. However, whether Sirt3 is involved in liver ischemia and reperfusion (I/R) injury remains elusive. Caffeic acid (CA) is a natural antioxidant derived from Salvia miltiorrhiza. Whether CA protects against liver I/R injury through regulating Sirt3 and the mitochondrial respiratory chain (MRC) is unclear. This study investigated the effect of CA on liver I/R injury, microcirculatory disturbance, and potential mechanisms, particularly focusing on Sirt3-dependent MRC. Liver I/R of male Sprague-Dawley rats was established by occlusion of portal area vessels for 30 min followed by 120 min of reperfusion. CA (15 mg/kg/h) was continuously infused via the femoral vein starting 30 min before ischemia. After I/R, Sirt3 expression, and MRC activity decreased, acetylation of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 and succinate dehydrogenase complex, subunit A, flavoprotein variant provoked, and the liver microcirculatory disturbance and injury were observed. Treatment with CA attenuated liver injury, inhibited Sirt3 down-expression, and up-regulated MRC activity. CA attenuated rat liver microcirculatory disturbance and oxidative injury through regulation of Sirt3 and the mitochondrial respiratory chain. Copyright © 2015 Elsevier Inc. All rights reserved.

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

PubMed Central

Sauvé, K; Nachman, M; Spence, C; Bailon, P; Campbell, E; Tsien, W H; Kondas, J A; Hakimi, J; Ju, G

1991-01-01

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction. PMID:2052547

Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.

PubMed

Elson, G C; Graber, P; Losberger, C; Herren, S; Gretener, D; Menoud, L N; Wells, T N; Kosco-Vilbois, M H; Gauchat, J F

1998-08-01

In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R alpha-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-alpha, IL-6, and IFN-gamma. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.

Primary structure of the hemoglobin alpha-chain of rose-ringed parakeet (Psittacula krameri).

PubMed

Islam, A; Beg, O U; Persson, B; Zaidi, Z H; Jörnvall, H

1988-10-01

The structure of the hemoglobin alpha-chain of Rose-ringed Parakeet was determined by sequence degradations of the intact subunit, the CNBr fragments, and peptides obtained by digestion with staphylococcal Glu-specific protease and trypsin. Using this analysis, the complete alpha-chain structure of 21 avian species is known, permitting comparisons of the protein structure and of avian relationships. The structure exhibits differences from previously established avian alpha-chains at a total of 61 positions, five of which have residues unique to those of the parakeet (Ser-12, Gly-65, Ser-67, Ala-121, and Leu-134). The analysis defines hemoglobin variation within an additional avian order (Psittaciformes), demonstrates distant patterns for evaluation of relationships within other avian orders, and lends support to taxonomic conclusions from molecular data.

Sodium valproate induces mitochondrial respiration dysfunction in HepG2 in vitro cell model.

PubMed

Komulainen, Tuomas; Lodge, Tiffany; Hinttala, Reetta; Bolszak, Maija; Pietilä, Mika; Koivunen, Peppi; Hakkola, Jukka; Poulton, Joanna; Morten, Karl J; Uusimaa, Johanna

2015-05-04

Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system

PubMed Central

Huang, Zhong; Phoolcharoen, Waranyoo; Lai, Huafang; Piensook, Khanrat; Cardineau, Guy; Zeitlin, Larry; Whaley, Kevin J.; Arntzen, Charles J.

2010-01-01

Plant viral vectors have great potential in rapid production of important pharmaceutical proteins. However, high-yield production of heterooligomeric proteins that require the expression and assembly of two or more protein subunits often suffers problems due to the “competing” nature of viral vectors derived from the same virus. Previously we reported that a bean yellow dwarf virus (BeYDV)-derived, three-component DNA replicon system allows rapid production of single recombinant proteins in plants (Huang et al. 2009). In this article, we report further development of this expression system for its application in high-yield production of oligomeric protein complexes including monoclonal antibodies (mAbs) in plants. We showed that the BeYDV replicon system permits simultaneous efficient replication of two DNA replicons and thus, high-level accumulation of two recombinant proteins in the same plant cell. We also demonstrated that a single vector that contains multiple replicon cassettes was as efficient as the three-component system in driving the expression of two distinct proteins. Using either the non-competing, three-vector system or the multi-replicon single vector, we produced both the heavy and light chain subunits of a protective IgG mAb 6D8 against Ebola virus GP1 (Wilson et al. 2000) at 0.5 mg of mAb per gram leaf fresh weight within 4 days post infiltration of Nicotiana benthamiana leaves. We further demonstrated that full-size tetrameric IgG complex containing two heavy and two light chains was efficiently assembled and readily purified, and retained its functionality in specific binding to inactivated Ebola virus. Thus, our single-vector replicon system provides high-yield production capacity for heterooligomeric proteins, yet eliminates the difficult task of identifying non-competing virus and the need for co-infection of multiple expression modules. The multi-replicon vector represents a significant advance in transient expression technology for antibody production in plants. PMID:20047189

Identification of a small molecule that inhibits herpes simplex virus DNA Polymerase subunit interactions and viral replication.

PubMed

Pilger, Beatrice D; Cui, Can; Coen, Donald M

2004-05-01

The interaction between the catalytic subunit Pol and the processivity subunit UL42 of herpes simplex virus DNA polymerase has been characterized structurally and mutationally and is a potential target for novel antiviral drugs. We developed and validated an assay for small molecules that could disrupt the interaction of UL42 and a Pol-derived peptide and used it to screen approximately 16,000 compounds. Of 37 "hits" identified, four inhibited UL42-stimulated long-chain DNA synthesis by Pol in vitro, of which two exhibited little inhibition of polymerase activity by Pol alone. One of these specifically inhibited the physical interaction of Pol and UL42 and also inhibited viral replication at concentrations below those that caused cytotoxic effects. Thus, a small molecule can inhibit this protein-protein interaction, which provides a starting point for the discovery of new antiviral drugs.

Functional role of the MrpA- and MrpD-homologous protein subunits in enzyme complexes evolutionary related to respiratory chain complex I.

PubMed

Moparthi, Vamsi K; Kumar, Brijesh; Al-Eryani, Yusra; Sperling, Eva; Górecki, Kamil; Drakenberg, Torbjörn; Hägerhäll, Cecilia

2014-01-01

NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na(+)/H(+) antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity. © 2013. Published by Elsevier B.V. All rights reserved.

Arginine 26 and aspartic acid 69 of the regulatory subunit are key residues of subunits interaction of acetohydroxyacid synthase isozyme III from E. coli.

PubMed

Zhao, Yuefang; Wen, Xin; Niu, Congwei; Xi, Zhen

2012-11-05

Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched-chain amino acids, is composed of catalytic and regulatory subunits. The enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). However, the crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and RSU is also unknown. Herein, we introduced a global-surface, site-directed labeling scanning method to determine the potential interaction region of the RSU. This approach relies on the insertion of a bulky fluorescent probe at the designated site on the surface of the RSU to cause a dramatic change in holoenzyme activity by perturbing subunit interaction. Then, the key amino acid residues in the potential interaction regions were identified by site-directed mutagenesis. Compared to the wild-type, the single-point mutants R26A and D69A showed 54 and 64 % activity, respectively, whereas the double mutant (R26A+D69A) gave 14 %, thus suggesting that residues Arg26 and Asp69 are the key residues of subunit interaction with cooperative action. Additionally, the results of GST pull-down assays and pH-dependence experiments suggested that polar interaction is the main force for subunits interaction. A plausible protein-protein interaction model of the holoenzyme of Escherichia coli AHAS III is proposed, based on the mutagenesis and protein docking studies. The protocol established here should be useful for the identification of the molecular interactions between proteins. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanism of the modulation of BK potassium channel complexes with different auxiliary subunit compositions by the omega-3 fatty acid DHA.

PubMed

Hoshi, Toshinori; Tian, Yutao; Xu, Rong; Heinemann, Stefan H; Hou, Shangwei

2013-03-19

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are well known for their functional versatility, which is bestowed in part by their rich modulatory repertoire. We recently showed that long-chain omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) found in oily fish lower blood pressure by activating vascular BK channels made of Slo1+β1 subunits. Here we examined the action of DHA on BK channels with different auxiliary subunit compositions. Neuronal Slo1+β4 channels were just as well activated by DHA as vascular Slo1+β1 channels. In contrast, the stimulatory effect of DHA was much smaller in Slo1+β2, Slo1+LRRC26 (γ1), and Slo1 channels without auxiliary subunits. Mutagenesis of β1, β2, and β4 showed that the large effect of DHA in Slo1+β1 and Slo1+β4 is conferred by the presence of two residues, one in the N terminus and the other in the first transmembrane segment of the β1 and β4 subunits. Transfer of this amino acid pair from β1 or β4 to β2 introduces a large response to DHA in Slo1+β2. The presence of a pair of oppositely charged residues at the aforementioned positions in β subunits is associated with a large response to DHA. The Slo1 auxiliary subunits are expressed in a highly tissue-dependent fashion. Thus, the subunit composition-dependent stimulation by DHA demonstrates that BK channels are effectors of omega-3 fatty acids with marked tissue specificity.

Disordered organic electronic materials based on non-benzenoid 1,6-methano[10]annulene rings

DOEpatents

Tovar, John D; Streifel, Benjamin C; Peart, Patricia A

2014-10-07

Conjugated polymers and small molecules including the nonplanar aromatic 1,6-methano[10]annulene ring structure along with aromatic subunits, such as diketopyrrolopyrrole, and 2,1,3-benzothiadiazole, substituted with alkyl chains in a "Tail In," "Tail Out," or "No Tail" regiochemistry are disclosed.

[Effect of electroacupuncture on expression of ionotropic glutamate receptor subunits and their genes in lumbar segments of spinal cord in rats with neuropathic pain].

PubMed

Ma, Cheng; Yu, Li; Yan, Li-ping

2010-12-01

To observe the effect of electroacupuncture (EA) on the expression of ionotropic glutamate receptor (iGluR) subunits and their mRNAs in the lumbar segments of spinal cord in rats with neuropathic pain, so as to explore its underlying mechanism in relieving spinal hyperalgesia. Thirty SD rats were randomly divided into control, model, and EA groups, with 10 rats in each. The spared nerve injury (SNI) model was established by ligature of the sural nerve after cutting off the common peroneal nerve and anterior tibial nerve. EA (2 Hz, 1 mA) was applied to "Huantiao" (GB 30) and "Weizhong" (BL 40) for 30 min, once daily for 7 days. Mechanical pain threshold was detected before and after modeling and before and after EA treatment. The expression levels of N-methyl-d-aspartic acid (NMDA) receptor subunits NR1 and NR 2 B,and AMPA receptor subunit GluR 1 of iGluR and their genes were assayed by Western blot and reverse transcription polymerase chain reaction (RT-PCR) separately. In comparison with control group, the mechanical pain thresholds were decreased significantly on day 2, 7 and day 14 following modeling in the model group (P < 0.05, P < 0.01). While compared with the model group, the pain threshold was increased considerably on day 14 in the EA group (P < 0.01). Compared with the control group, the expression levels of lumbar spinal cord NR 2 B and NR 2 B mRNA in the model group were increased significantly (P < 0.05), and those of lumbar spinal cord NR 1 and NR 1 mRNA, GluR 1 and GluR 1 mRNA in the model group increased slightly (P > 0.05). In comparison with the model group, the expression levels of lumbar spinal cord NR 2 B and NR 2 B mRNA in the EA group were downregulated remarkably (P < 0.05), and those of lumbar spinal cord NR 1 and NR 1 mRNA, GluR 1 and GluR 1 mRNA in the EA group down-regulated slightly (P > 0.05). EA can significantly suppress pain reaction in rats with neuropathic pain probably through down-regulating the expression of lumbar spinal cord NR 2 B protein and NR 2 B mRNA.

Inhalation of Roman chamomile essential oil attenuates depressive-like behaviors in Wistar Kyoto rats.

PubMed

Kong, Yingying; Wang, Ting; Wang, Rong; Ma, Yichuan; Song, Shanshan; Liu, Juan; Hu, Weiwei; Li, Shengtian

2017-06-01

The idea of aromatherapy, using essential oils, has been considered as an alternative antidepressant treatment. In the present study, we investigated the effect of Roman chamomile essential oil inhalation for two weeks on depressive-like behaviors in Wistar-Kyoto (WKY) rats. We found that inhalation of either Roman chamomile or one of its main components α-pinene, attenuated depressive-like behavior in WKY rats in the forced swim test. Using isobaric tags for relative and absolute quantitation analysis (iTRAQ), we found that inhalation of α-pinene increased expression of proteins that are involved in oxidative phosphorylation, such as cytochrome c oxidase subunit 6C-2, cytochrome c oxidase subunit 7A2, ATPase inhibitor in the hippocampus, and cytochrome c oxidase subunit 6C-2, ATP synthase subunit e, Acyl carrier protein, and Cytochrome b-c1 complex subunit 6 in the PFC (prefrontal cortex). In addition, using the quantitative real-time polymerase chain reaction technique, we confirmed an increase of parvalbumin mRNA expression in the hippocampus, which was shown to be upregulated by 2.8-fold in iTRAQ analysis, in α-pinene treated WKY rats. These findings collectively suggest the involvement of mitochondrial functions and parvalbumin-related signaling in the antidepressant effect of α-pinene inhalation.

Purification and Functional Reconstitution of a Seven-Subunit Mrp-Type Na+/H+ Antiporter

PubMed Central

Morino, Masato; Suzuki, Toshiharu; Ito, Masahiro

2014-01-01

Mrp antiporters and their homologues in the cation/proton antiporter 3 family of the Membrane Transporter Database are widely distributed in bacteria. They have major roles in supporting cation and cytoplasmic pH homeostasis in many environmental, extremophilic, and pathogenic bacteria. These antiporters require six or seven hydrophobic proteins that form hetero-oligomeric complexes, while most other cation/proton antiporters require only one membrane protein for their activity. The resemblance of three Mrp subunits to membrane-embedded subunits of the NADH:quinone oxidoreductase of respiratory chains and to subunits of several hydrogenases has raised interest in the evolutionary path and commonalities of their proton-translocating domains. In order to move toward a greater mechanistic understanding of these unusual antiporters and to rigorously demonstrate that they function as secondary antiporters, powered by an imposed proton motive force, we established a method for purification and functional reconstitution of the seven-subunit Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4. Na+/H+ antiporter activity was demonstrated by a fluorescence-based assay with proteoliposomes in which the Mrp complex was coreconstituted with a bacterial FoF1-ATPase. Proton pumping by the ATPase upon addition of ATP generated a proton motive force across the membranes that powered antiporter activity upon subsequent addition of Na+. PMID:24142251

Purification and functional reconstitution of a seven-subunit mrp-type na+/h+ antiporter.

PubMed

Morino, Masato; Suzuki, Toshiharu; Ito, Masahiro; Krulwich, Terry Ann

2014-01-01

Mrp antiporters and their homologues in the cation/proton antiporter 3 family of the Membrane Transporter Database are widely distributed in bacteria. They have major roles in supporting cation and cytoplasmic pH homeostasis in many environmental, extremophilic, and pathogenic bacteria. These antiporters require six or seven hydrophobic proteins that form hetero-oligomeric complexes, while most other cation/proton antiporters require only one membrane protein for their activity. The resemblance of three Mrp subunits to membrane-embedded subunits of the NADH:quinone oxidoreductase of respiratory chains and to subunits of several hydrogenases has raised interest in the evolutionary path and commonalities of their proton-translocating domains. In order to move toward a greater mechanistic understanding of these unusual antiporters and to rigorously demonstrate that they function as secondary antiporters, powered by an imposed proton motive force, we established a method for purification and functional reconstitution of the seven-subunit Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4. Na(+)/H(+) antiporter activity was demonstrated by a fluorescence-based assay with proteoliposomes in which the Mrp complex was coreconstituted with a bacterial FoF1-ATPase. Proton pumping by the ATPase upon addition of ATP generated a proton motive force across the membranes that powered antiporter activity upon subsequent addition of Na(+).

Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions.

PubMed

Römling, Ute; Galperin, Michael Y

2015-09-01

Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host. Copyright © 2015 Elsevier Ltd. All rights reserved.

6-Shogaol induces caspase-independent paraptosis in cancer cells via proteasomal inhibition.

PubMed

Nedungadi, Divya; Binoy, Anupama; Pandurangan, Nanjan; Pal, Sanjay; Nair, Bipin G; Mishra, Nandita

2018-03-15

An α, β-unsaturated carbonyl compound of ginger, 6-Shogaol (6S), induced extensive cytoplasmic vacuolation and cell death in breast cancer cell (MDA-MB-231) and non-small lung cancer (A549) cells. In the presence of autophagic inhibitors the cells continued to exhibit cytoplasmic vacuolation and cell death clearly distinguishing it from the classic autophagic process. 6S induced death did not exhibit the characteristic apoptotic features like caspase cleavage, phosphatidyl serine exposure and DNA fragmentation. The immunofluorescence with the Endoplasmic Reticulum (ER) resident protein, calreticulin indicated that the vacuoles were of ER origin, typical of paraptosis. This was supported by the increase in level of microtubule associated protein light chain 3B (LC3 I and LC3 II) and polyubiquitin binding protein, p62. The level of ER stress markers like polyubiquitinated proteins, Bip and CHOP also consistently increased. We have found that 6S inhibits the 26S proteasome. The proteasomal inhibitory activity was elucidated by a) molecular docking of 6S onto the active site of β5 subunit and b) reduced fluorescence by the fluorogenic substrate of the chymotrypsin-like subunit. In conclusion these studies demonstrate for the first time that proteasomal inhibition by 6S induces cell death via paraptosis. So 6-shogaol may act as a template for anti-cancer lead discovery against the apoptosis resistant cancer cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Molecular Cloning of Pituitary Glycoprotein α-Subunit and Follicle Stimulating Hormone and Chorionic Gonadotropin β-Subunits from New World Squirrel Monkey and Owl Monkey

PubMed Central

Scammell, Jonathan G.; Funkhouser, Jane D.; Moyer, Felricia S.; Gibson, Susan V.; Willis, Donna L.

2008-01-01

The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone α-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature β subunits of follicle stimulating hormone (FSHβ) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHβ. New World primate glycoprotein hormone α-polypeptides and FSHβ subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the β-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the β-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGβ are 143 and 144 amino acids in length and 77% homologous with human CGβ. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGβ, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGβs. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG. PMID:17897645

Molecular cloning of pituitary glycoprotein alpha-subunit and follicle stimulating hormone and chorionic gonadotropin beta-subunits from New World squirrel monkey and owl monkey.

PubMed

Scammell, Jonathan G; Funkhouser, Jane D; Moyer, Felricia S; Gibson, Susan V; Willis, Donna L

2008-02-01

The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone alpha-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature beta subunits of follicle stimulating hormone (FSHbeta) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHbeta. New World primate glycoprotein hormone alpha-polypeptides and FSHbeta subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the beta-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the beta-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGbeta are 143 and 144 amino acids in length and 77% homologous with human CGbeta. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGbeta, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGbetas. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG.

Selection of Single-Chain Antibodies against the VP8* Subunit of Rotavirus VP4 Outer Capsid Protein and Their Expression in Lactobacillus casei

PubMed Central

Monedero, Vicente; Rodríguez-Díaz, Jesús; Viana, Rosa; Buesa, Javier; Pérez-Martínez, Gaspar

2004-01-01

Single-chain antibodies (scFv) recognizing the VP8* fraction of rotavirus outer capsid and blocking rotavirus infection in vitro were isolated by phage display. Vectors for the extracellular expression in Lactobacillus casei of one of the scFv were constructed. L. casei was able to secrete active scFv to the growth medium, showing the potential of probiotic bacteria to be engineered to express molecules suitable for in vivo antirotavirus therapies. PMID:15528568

Radiation inactivation of ricin occurs with transfer of destructive energy across a disulfide bridge

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haigler, H.T.; Woodbury, D.J.; Kempner, E.S.

1985-08-01

The ionizing radiation sensitivity of ricin, a disulfide-linked heterodimeric protein, was studied as a model to determine the ability of disulfide bonds to transmit destructive energy. The radiation-dependent loss of A chain enzymatic activity after irradiation of either intact ricin or ricin in which the interchain disulfide bond was disrupted gave target sizes corresponding to the molecular size of dimeric ricin or monomeric A chain, respectively. These results clearly show that a disulfide bond can transmit destructive energy between protein subunits.

Structural basis for the mechanism and substrate specificity of glycocyamine kinase, a phosphagen kinase family member

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Kap; Pullalarevu, Sadhana; Surabian, Karen Talin

2010-03-12

Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg{sup 2+}-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phosphoglycocyamine to ADP to yield glycocyamine and ATP. This reaction helps to maintain the energy homeostasis of the cell in some multicelullar organisms that encounter high and variable energy turnover. GK from the marine worm Namalycastis sp. is heterodimeric, with two homologous polypeptide chains, {alpha} and {beta}, derived from a common pre-mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GK{beta} encodes a peptide that is different in sequence and is 16 amino acids longermore » than that encoded by the N-terminal exon of GK{alpha}. The crystal structures of recombinant GK{alpha}{beta} and GK{beta}{beta} from Namalycastis sp. were determined at 2.6 and 2.4 {angstrom} resolution, respectively. In addition, the structure of the GK{beta}{beta} was determined at 2.3 {angstrom} resolution in complex with a transition state analogue, Mg{sup 2+}-ADP-NO{sub 3}{sup -}-glycocyamine. Consistent with the sequence homology, the GK subunits adopt the same overall fold as that of other phosphagen kinases of known structure (the homodimeric creatine kinase (CK) and the monomeric arginine kinase (AK)). As with CK, the GK N-termini mediate the dimer interface. In both heterodimeric and homodimeric GK forms, the conformations of the two N-termini are asymmetric, and the asymmetry is different than that reported previously for the homodimeric CKs from several organisms. The entire polypeptide chains of GK{alpha}{beta} are structurally defined, and the longer N-terminus of the {beta} subunit is anchored at the dimer interface. In GK{beta}{beta} the 24 N-terminal residues of one subunit and 11 N-terminal residues of the second subunit are disordered. This observation is consistent with a proposal that the GK{alpha}{beta} amino acids involved in the interface formation were optimized once a heterodimer emerged as the physiological form of the enzyme. As a consequence, the homodimer interface (either solely {alpha} or solely {beta} chains) has been corrupted. In the unbound state, GK exhibits an open conformation analogous to that observed with ligand-free CK or AK. Upon binding the transition state analogue, both subunits of GK undergo the same closure motion that clasps the transition state analogue, in contrast to the transition state analogue complexes of CK, where the corresponding transition state analogue occupies only one subunit, which undergoes domain closure. The active site environments of the GK, CK, and AK at the bound states reveal the structural determinants of substrate specificity. Despite the equivalent binding in both active sites of the GK dimer, the conformational asymmetry of the N-termini is retained. Thus, the coupling between the structural asymmetry and negative cooperativity previously proposed for CK is not supported in the case of GK.« less

The electrophoretically 'slow' and 'fast' forms of the alpha 2-macroglobulin molecule.

PubMed Central

Barrett, A J; Brown, M A; Sayers, C A

1979-01-01

alpha 2-Macroglobulin (alpha 2M) was isolated from human plasma by a four-step procedure: poly(ethylene glyco) fractionation, gel chromatography, euglobulin precipitation and immunoadsorption. No contaminants were detected in the final preparations by electrophoresis or immunoprecipitation. The protein ran as a single slow band in gel electrophoresis, and was designated 'S-alpha 2M'. S-alpha 2M bound about 2 mol of trypsin/mol. Treatment of S-alpha 2M with a proteinase or ammonium salts produced a form of the molecule more mobile in electrophoresis, and lacking proteinase-binding activity (F-alpha 2M). The electrophoretic mobility of the F-alpha 2M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. We attribute the change in electrophoretic mobility of the alpha 2M to a conformation change, but there was no evidence of a change in pI or Strokes radius. Electrophoresis of S-alpha 2M in the presence of sodium dodecylsulphate gave results consistent with the view that the alpha 2M molecule is a tetramer of identical subunits, assembled as a non-covalent pair of disulphide-linked dimers. Some of the subunits seemed to be 'nicked' into two-thires-length and one-third-length chains, however. This was not apparent with F-alpha 2M produced by ammonium salts. F-alpha 2M produced by trypsin showed two new bands attributable to cleavage of the subunit polypeptide chain near the middle. Immunoassays of F-alpha 2M gave 'rockets' 12-29% lower than those with S-alpha 2M. The nature of the interactions between subunits in S-alpha 2M and F-alpha 2M was investigated by treating each form with glutaraldehyde before electrophoresis in the presence of sodium dodecyl sulphate. A much greater degree of cross-linking was observed with the F-alpha 2M, indicating that the subunits interact most closely in this form of the molecule. Exposure of S-alpha 2M to 3 M-urea or pH3 resulted in dissociation to the disulphide-bonded half-molecules; these did not show the proteinase-binding activity characteristic of the intact alpha 2M. F-alpha 2M was less easily dissociated than was S-alpha 2M. S-alpha 2M was readily dissociated to the quarter-subunits by mild reduction, with the formation of 3-4 new thiol groups per subunit. Inact reactive alpha 2M could then be regenerated in high yield by reoxidation of the subunits. F-alpha 2M formed by reaction with a proteinase or ammonium salts was not dissociated under the same conditions, although the interchain disulphide bonds were reduced. If the thiol groups of the quarter-subunits of S-alpha 2M were blocked by carboxymethylation, oxidative reassociation did not occur. Nevertheless treatment of these subunits with methylammonium salts or a proteinase caused the reassembly of half-molecules and intact (F-) tetramers. It is emphasized that F-alpha 2M does not have the properties of a denatured form of the protein... Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:91367

Morphological and molecular identification of nasopharyngeal bot fly larvae infesting red deer (Cervus elaphus) in Austria.

PubMed

Leitner, Natascha; Schwarzmann, Laurin; Zittra, Carina; Palmieri, Nicola; Eigner, Barbara; Otranto, Domenico; Glawischnig, Walter; Fuehrer, Hans-Peter

2016-11-01

Nasopharyngeal myiases are caused by larvae of bot flies (Diptera: Oestridae), which have evolved a high specificity for their hosts. Bot flies (n = 916) were collected from 137 (57.6 %) out of 238 red deer (Cervus elaphus) hunted in Vorarlberg and Tyrol (Western Austria). After being stored in 75 % ethanol, larvae were identified to species level and developmental stage using morphological and morphometric keys. Larvae were also molecularly characterized by polymerase chain reaction (PCR) amplification and partial sequencing of the mitochondrial cytochrome oxidase subunit I gene. Morphological and molecular analysis allowed identification of larvae as Cephenemyia auribarbis and Pharyngomyia picta. Genetic variations were also examined within the specimens collected in both geographical locations.

The A-chain of insulin contacts the insert domain of the insulin receptor. Photo-cross-linking and mutagenesis of a diabetes-related crevice.

PubMed

Huang, Kun; Chan, Shu Jin; Hua, Qing-xin; Chu, Ying-Chi; Wang, Run-ying; Klaproth, Birgit; Jia, Wenhua; Whittaker, Jonathan; De Meyts, Pierre; Nakagawa, Satoe H; Steiner, Donald F; Katsoyannis, Panayotis G; Weiss, Michael A

2007-11-30

The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of Val(A3), which projects within a crevice between the A- and B-chains. Engineered receptor alpha-subunits containing specific protease sites ("midi-receptors") are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a Pap(B25)-insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and alpha-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (Gly(A3) and Ser(A3)) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of Aba(A3) and allo-Thr(A3) analogs are reduced at least 10-fold; the activity of Thr(A3)-DKP-insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (Aba(A3)) or by altered polarity (allo-Thr(A3) and Thr(A3)). Our results provide evidence that Val(A3), a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the alpha-subunit in a hormone-receptor complex.

Glycosylation and processing of high-mannose oligosaccharides of thyroid-stimulating hormone subunits: comparison to nonsecretory cell glycoproteins.

PubMed

Ronin, C; Stannard, B S; Rosenbloom, I L; Magner, J A; Weintraub, B D

1984-09-25

Thyroid-stimulating hormone (TSH) subunit glycosylation was compared to that of total cell glycoproteins in mouse thyrotropic tumors. Lipid-linked oligosaccharides, total cell glycoproteins, and TSH subunits were labeled with either [3H]mannose, [3H]galactose, or [3H]glucose in pulse and pulse-chase experiments. The various oligosaccharides were isolated respectively by lipid extraction and mild acid hydrolysis, by selective immunoprecipitation, or by acid precipitation followed by trypsin and endoglycosidase H treatment. The nature of the oligosaccharides was assessed by their migration in paper chromatography, their relative incorporation of different precursors, and also their resistance to alpha-mannosidase. At 60 min, lipid-linked oligosaccharides were found to be composed of Glc3-2Man9GlcNAc2, Man9-8GlcNAc2, and Man5GlcNAc2. At 10 or 60 min of labeling, total cell proteins contained Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, Man9GlcNAc2, Glc1Man8GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2. The largest oligosaccharide, Glc3Man9GlcNAc2, had an unusually long half-life of about 2 h. In contrast, no Glc3Man9GlcNAc2 was found either on TSH + alpha subunits or on free beta subunits isolated either by immunoprecipitation or by sodium dodecyl sulfate gel electrophoresis. Instead, primarily Man9GlcNAc2 was found after a 10-min pulse both on TSH + alpha subunits and on beta subunits. When the pulse was followed by a chase up to 2 h, there was a progressive increase in Man8GlcNAc2 in higher amounts on TSH + alpha-subunit carbohydrate chains than on beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Na+/K+-ATPase α-subunit in swimming crab Portunus trituberculatus: molecular cloning, characterization, and expression under low salinity stress

NASA Astrophysics Data System (ADS)

Han, Xiaolin; Liu, Ping; Gao, Baoquan; Wang, Haofeng; Duan, Yafei; Xu, Wenfei; Chen, Ping

2015-07-01

Na+/K+-ATPases are membrane-associated enzymes responsible for the active transport of Na+ and K+ ions across cell membranes, generating chemical and electrical gradients. These enzymes' α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na+/K+-ATPase α-subunit cDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end methods. Analysis of the nucleotide sequence revealed that the cDNA had a full-length of 3 833 base pairs (bp), with an open reading frame of 3 120 bp, 5' untranslated region (UTR) of 317 bp, and 3' UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na+/K+-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of amino acid sequences showed that the P. trituberculatus α-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit's transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab's Na+/K+-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.

Phylogeographic patterns of Armillaria ostoyae in the western United States

Treesearch

J. W. Hanna; N. B. Klopfenstein; M. -S. Kim; G. I. McDonald; J. A. Moore

2007-01-01

Nuclear ribosomal DNA regions (i.e. large subunit, internal transcribed spacer, 5.8S and intergenic spacer) were sequenced using a direct-polymerase chain reaction method from Armillaria ostoyae genets collected from the western USA. Many of the A. ostoyae genets contained heterogeneity among rDNA repeats, indicating intragenomic variation and likely intraspecific...

Improving flavour and quality of tomatoes by expression of synthetic gene encoding sweet protein monellin.

PubMed

Reddy, Chinreddy Subramanyam; Vijayalakshmi, Muvva; Kaul, Tanushri; Islam, Tahmina; Reddy, Malireddy K

2015-05-01

Monellin a sweet-tasting protein exists naturally as a heterodimer of two non-covalently linked subunits chain A and B, which loses its sweetness on denaturation. In this study, we validated the expression of a synthetic monellin gene encoding a single polypeptide chain covalently linking the two subunits under T7 and fruit-ripening-specific promoters in Escherichia coli and tomato fruits, respectively. Purified recombinant monellin protein retained its sweet flavour at 70 °C and pH 2. We developed 15 transgenic T0 tomato plants overexpressing monellin, which were devoid of any growth penalty or phenotypic abnormalities during greenhouse conditions. T-DNA integration and fruit-specific heterologous expression of monellin had occurred in these transgenic tomato lines. ELISA revealed that expression of monellin was 4.5% of the total soluble fruit protein. Functional analyses of transgenic tomatoes of T2-5 and T2-14 lines revealed distinctly strong sweetness compared with wild type. Monellin a potential non-carbohydrate sweetener, if expressed in high amounts in fruits and vegetables, would enhance their flavour and quality.

Mitochondrial genes are altered in blood early in Alzheimer's disease.

PubMed

Lunnon, Katie; Keohane, Aoife; Pidsley, Ruth; Newhouse, Stephen; Riddoch-Contreras, Joanna; Thubron, Elisabeth B; Devall, Matthew; Soininen, Hikka; Kłoszewska, Iwona; Mecocci, Patrizia; Tsolaki, Magda; Vellas, Bruno; Schalkwyk, Leonard; Dobson, Richard; Malik, Afshan N; Powell, John; Lovestone, Simon; Hodges, Angela

2017-05-01

Although mitochondrial dysfunction is a consistent feature of Alzheimer's disease in the brain and blood, the molecular mechanisms behind these phenomena are unknown. Here we have replicated our previous findings demonstrating reduced expression of nuclear-encoded oxidative phosphorylation (OXPHOS) subunits and subunits required for the translation of mitochondrial-encoded OXPHOS genes in blood from people with Alzheimer's disease and mild cognitive impairment. Interestingly this was accompanied by increased expression of some mitochondrial-encoded OXPHOS genes, namely those residing closest to the transcription start site of the polycistronic heavy chain mitochondrial transcript (MT-ND1, MT-ND2, MT-ATP6, MT-CO1, MT-CO2, MT-C03) and MT-ND6 transcribed from the light chain. Further we show that mitochondrial DNA copy number was unchanged suggesting no change in steady-state numbers of mitochondria. We suggest that an imbalance in nuclear and mitochondrial genome-encoded OXPHOS transcripts may drive a negative feedback loop reducing mitochondrial translation and compromising OXPHOS efficiency, which is likely to generate damaging reactive oxygen species. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

11S Storage globulin from pumpkin seeds: regularities of proteolysis by papain.

PubMed

Rudakova, A S; Rudakov, S V; Kakhovskaya, I A; Shutov, A D

2014-08-01

Limited proteolysis of the α- and β-chains and deep cleavage of the αβ-subunits by the cooperative (one-by-one) mechanism was observed in the course of papain hydrolysis of cucurbitin, an 11S storage globulin from seeds of the pumpkin Cucurbita maxima. An independent analysis of the kinetics of the limited and cooperative proteolyses revealed that the reaction occurs in two successive steps. In the first step, limited proteolysis consisting of detachments of short terminal peptides from the α- and β-chains was observed. The cooperative proteolysis, which occurs as a pseudo-first order reaction, started at the second step. Therefore, the limited proteolysis at the first step plays a regulatory role, impacting the rate of deep degradation of cucurbitin molecules by the cooperative mechanism. Structural alterations of cucurbitin induced by limited proteolysis are suggested to generate its susceptibility to cooperative proteolysis. These alterations are tentatively discussed on the basis of the tertiary structure of the cucurbitin subunit pdb|2EVX in comparison with previously obtained data on features of degradation of soybean 11S globulin hydrolyzed by papain.

cis-Prenyltransferase: New Insights into Protein Glycosylation, Rubber Synthesis, and Human Diseases.

PubMed

Grabińska, Kariona A; Park, Eon Joo; Sessa, William C

2016-08-26

cis-Prenyltransferases (cis-PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. cis-PTs catalyze consecutive condensation reactions of allylic diphosphate acceptor with isopentenyl diphosphate (IPP) in the cis (Z) configuration to generate linear polyprenyl diphosphate. The chain lengths of isoprenoid carbon skeletons vary widely from neryl pyrophosphate (C10) to natural rubber (C>10,000). The homo-dimeric bacterial enzyme, undecaprenyl diphosphate synthase (UPPS), has been structurally and mechanistically characterized in great detail and serves as a model for understanding the mode of action of eukaryotic cis-PTs. However, recent experiments have revealed that mammals, fungal, and long-chain plant cis-PTs are heteromeric enzymes composed of two distantly related subunits. In this review, the classification, function, and evolution of cis-PTs will be discussed with a special emphasis on the role of the newly described NgBR/Nus1 subunit and its plants' orthologs as essential, structural components of the cis-PTs activity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

PubMed

Chin, Randall M; Fu, Xudong; Pai, Melody Y; Vergnes, Laurent; Hwang, Heejun; Deng, Gang; Diep, Simon; Lomenick, Brett; Meli, Vijaykumar S; Monsalve, Gabriela C; Hu, Eileen; Whelan, Stephen A; Wang, Jennifer X; Jung, Gwanghyun; Solis, Gregory M; Fazlollahi, Farbod; Kaweeteerawat, Chitrada; Quach, Austin; Nili, Mahta; Krall, Abby S; Godwin, Hilary A; Chang, Helena R; Faull, Kym F; Guo, Feng; Jiang, Meisheng; Trauger, Sunia A; Saghatelian, Alan; Braas, Daniel; Christofk, Heather R; Clarke, Catherine F; Teitell, Michael A; Petrascheck, Michael; Reue, Karen; Jung, Michael E; Frand, Alison R; Huang, Jing

2014-06-19

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

Biochemical Characterization of Mutants in Chaperonin Proteins CCT4 and CCT5 Associated with Hereditary Sensory Neuropathy*

PubMed Central

Sergeeva, Oksana A.; Tran, Meme T.; Haase-Pettingell, Cameron; King, Jonathan A.

2014-01-01

Hereditary sensory neuropathies are a class of disorders marked by degeneration of the nerve fibers in the sensory periphery neurons. Recently, two mutations were identified in the subunits of the eukaryotic cytosolic chaperonin TRiC, a protein machine responsible for folding actin and tubulin in the cell. C450Y CCT4 was identified in a stock of Sprague-Dawley rats, whereas H147R CCT5 was found in a human Moroccan family. As with many genetically identified mutations associated with neuropathies, the underlying molecular basis of the mutants was not defined. We investigated the biochemical properties of these mutants using an expression system in Escherichia coli that produces homo-oligomeric rings of CCT4 and CCT5. Full-length versions of both mutant protein chains were expressed in E. coli at levels approaching that of the WT chains. Sucrose gradient centrifugation revealed chaperonin-sized complexes of both WT and mutant chaperonins, but with reduced recovery of C450Y CCT4 soluble subunits. Electron microscopy of negatively stained samples of C450Y CCT4 revealed few ring-shaped species, whereas WT CCT4, H147R CCT5, and WT CCT5 revealed similar ring structures. CCT5 complexes were assayed for their ability to suppress aggregation of and refold the model substrate γd-crystallin, suppress aggregation of mutant huntingtin, and refold the physiological substrate β-actin in vitro. H147R CCT5 was not as efficient in chaperoning these substrates as WT CCT5. The subtle effects of these mutations are consistent with the homozygous disease phenotype, in which most functions are carried out during development and adulthood, but some selective function is lost or reduced. PMID:25124038

Hypoxic augmentation of Ca2+ channel currents requires a functional electron transport chain.

PubMed

Brown, Stephen T; Scragg, Jason L; Boyle, John P; Hudasek, Kristin; Peers, Chris; Fearon, Ian M

2005-06-10

The incidence of Alzheimer disease is increased following ischemic episodes, and we previously demonstrated that following chronic hypoxia (CH), amyloid beta (Abeta) peptide-mediated increases in voltage-gated L-type Ca(2+) channel activity contribute to the Ca(2+) dyshomeostasis seen in Alzheimer disease. Because in certain cell types mitochondria are responsible for detecting altered O(2) levels we examined the role of mitochondrial oxidant production in the regulation of recombinant Ca(2+) channel alpha(1C) subunits during CH and exposure to Abeta-(1-40). In wild-type (rho(+)) HEK 293 cells expressing recombinant L-type alpha(1C) subunits, Ca(2+) currents were enhanced by prolonged (24 h) exposure to either CH (6% O(2)) or Abeta-(1-40) (50 nm). By contrast the response to CH was absent in rho(0) cells in which the mitochondrial electron transport chain (ETC) was depleted following long term treatment with ethidium bromide or in rho(+) cells cultured in the presence of 1 microm rotenone. CH was mimicked in rho(0) cells by the exogenous production of O2(-.). by xanthine/xanthine oxidase. Furthermore Abeta-(1-40) enhanced currents in rho(0) cells to a degree similar to that seen in cells with an intact ETC. The antioxidants ascorbate (200 microm) and Trolox (500 microm) ablated the effect of CH in rho(+) cells but were without effect on Abeta-(1-40)-mediated augmentation of Ca(2+) current in rho(0) cells. Thus oxidant production in the mitochondrial ETC is a critical factor, acting upstream of amyloid beta peptide production in the up-regulation of Ca(2+) channels in response to CH.

Development of a Freeze-Dried, Heat-Stable Influenza Subunit Vaccine Formulation

PubMed Central

Flood, Alexander; Chen, Dexiang

2016-01-01

An influenza pandemic remains a major public health concern. A key strategy to prevent a pandemic is to stockpile and pre-position stable influenza vaccine to allow rapid deployment in response to an outbreak. However, most influenza vaccines today are formulated as liquids that are stable only within a temperature range of 2°C to 8°C and require use of a cold chain, making vaccine transportation, distribution, and storage complicated and expensive, particularly for developing countries. To support the National Strategy for Pandemic Influenza preparedness in the United States and internationally, we developed two lead dry formulations of stable H1N1 influenza subunit vaccines using freeze-drying technology. The stable formulations contain an excipient combination of a disaccharide, such as sucrose or trehalose, and glycine, in addition to a surfactant and phosphate buffer. The freeze-dried vaccines were shown to be safe and remained immunogenic in an in vivo study in mice. Moreover, the lead formulations demonstrated no significant loss of activity after 40 months at storage temperatures of 25°C and 37°C. This stability can be particularly attractive as it could eliminate the need to use a cold chain for vaccine deployment and facilitate integration of vaccine distribution with general drug distribution where appropriate. These freeze-dried thermostable influenza subunit vaccines could also reduce the frequency of vaccine stockpile turnover, offering a cost-effective option for pandemic preparedness. PMID:27851765

Chromosome localization of human genes for clathrin adaptor polypeptides AP2{beta} and AP50 and the clathrin-binding protein, VCP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Druck, T.; Gu, Y.; Prabhala, G.

1995-11-01

Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed {open_quotes}assembly proteins,{close_quotes} or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2{alpha} and AP2{beta}, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2{beta} (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with A1`s. Chromosomal in situ hybridization of a human genomic clonemore » demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2{beta} fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34. 13 refs., 2 figs.« less

Development of a Freeze-Dried, Heat-Stable Influenza Subunit Vaccine Formulation.

PubMed

Flood, Alexander; Estrada, Marcus; McAdams, David; Ji, Yuhua; Chen, Dexiang

2016-01-01

An influenza pandemic remains a major public health concern. A key strategy to prevent a pandemic is to stockpile and pre-position stable influenza vaccine to allow rapid deployment in response to an outbreak. However, most influenza vaccines today are formulated as liquids that are stable only within a temperature range of 2°C to 8°C and require use of a cold chain, making vaccine transportation, distribution, and storage complicated and expensive, particularly for developing countries. To support the National Strategy for Pandemic Influenza preparedness in the United States and internationally, we developed two lead dry formulations of stable H1N1 influenza subunit vaccines using freeze-drying technology. The stable formulations contain an excipient combination of a disaccharide, such as sucrose or trehalose, and glycine, in addition to a surfactant and phosphate buffer. The freeze-dried vaccines were shown to be safe and remained immunogenic in an in vivo study in mice. Moreover, the lead formulations demonstrated no significant loss of activity after 40 months at storage temperatures of 25°C and 37°C. This stability can be particularly attractive as it could eliminate the need to use a cold chain for vaccine deployment and facilitate integration of vaccine distribution with general drug distribution where appropriate. These freeze-dried thermostable influenza subunit vaccines could also reduce the frequency of vaccine stockpile turnover, offering a cost-effective option for pandemic preparedness.

Reduced Chrna7 expression in C3H mice is associated with increases in hippocampal parvalbumin and glutamate decarboxylase-67 (GAD67) as well as altered levels of GABAA receptor subunits

PubMed Central

Bates, Ryan C.; Stith, Bradley J.; Stevens, Karen E.; Adams, Catherine E.

2014-01-01

Decreased expression of CHRNA7, the gene encoding the α7* subtype of nicotinic receptor, may contribute to the cognitive dysfunction observed in schizophrenia by disrupting the inhibitory/excitatory balance in the hippocampus. C3H mice with reduced Chrna7 expression have significant reductions in hippocampal α7* receptor density, deficits in hippocampal auditory gating, increased hippocampal activity as well as significant decreases in hippocampal glutamate decarboxylase-65 (GAD65) and γ-aminobutyric acid-A (GABAA) receptor levels. The current study investigated whether altered Chrna7 expression is associated with changes in the levels of parvalbumin, GAD67 and/or GABAA receptor subunits in hippocampus from male and female C3H Chrna7 wildtype, C3H Chrna7 heterozygous and C3H Chrna7 knockout mice using quantitative western immunoblotting. Reduced Chrna7 expression was associated with significant increases in hippocampal parvalbumin and GAD67 and with complex alterations in GABAA receptor subunits. A decrease in α3 subunit protein was seen in both female C3H Chrna7 Het and KO mice while a decrease in α4 subunit protein was also detected in C3H Chrna7 KO mice with no sex difference. In contrast, an increase in δ subunit protein was observed in C3H Chrna7 Het mice while a decrease in this subunit was observed in C3H Chrna7 KO mice, with δ subunit protein levels being greater in males than in females. Finally, an increase in γ2 subunit protein was found in C3H Chrna7 KO mice with the levels of this subunit again being greater in males than in females. The increases in hippocampal parvalbumin and GAD67 observed in C3H Chrna7 mice are contrary to reports of reductions in these proteins in postmortem hippocampus from schizophrenic individuals. We hypothesize that the disparate results may occur because of the influence of factors other than CHRNA7 that have been found to be abnormal in schizophrenia. PMID:24836856

Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control.

PubMed

Yau, Richard G; Doerner, Kerstin; Castellanos, Erick R; Haakonsen, Diane L; Werner, Achim; Wang, Nan; Yang, X William; Martinez-Martin, Nadia; Matsumoto, Marissa L; Dixit, Vishva M; Rape, Michael

2017-11-02

Posttranslational modification with ubiquitin chains controls cell fate in all eukaryotes. Depending on the connectivity between subunits, different ubiquitin chain types trigger distinct outputs, as seen with K48- and K63-linked conjugates that drive protein degradation or complex assembly, respectively. Recent biochemical analyses also suggested roles for mixed or branched ubiquitin chains, yet without a method to monitor endogenous conjugates, the physiological significance of heterotypic polymers remained poorly understood. Here, we engineered a bispecific antibody to detect K11/K48-linked chains and identified mitotic regulators, misfolded nascent polypeptides, and pathological Huntingtin variants as their endogenous substrates. We show that K11/K48-linked chains are synthesized and processed by essential ubiquitin ligases and effectors that are mutated across neurodegenerative diseases; accordingly, these conjugates promote rapid proteasomal clearance of aggregation-prone proteins. By revealing key roles of K11/K48-linked chains in cell-cycle and quality control, we establish heterotypic ubiquitin conjugates as important carriers of biological information. Copyright © 2017 Elsevier Inc. All rights reserved.

Semisynthetic protein nanoreactor for single-molecule chemistry

PubMed Central

Lee, Joongoo; Bayley, Hagan

2015-01-01

The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane β-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach. PMID:26504203

Proteome Imbalance of Mitochondrial Electron Transport Chain in Brown Adipocytes Leads to Metabolic Benefits.

PubMed

Masand, Ruchi; Paulo, Esther; Wu, Dongmei; Wang, Yangmeng; Swaney, Danielle L; Jimenez-Morales, David; Krogan, Nevan J; Wang, Biao

2018-03-06

Brown adipose tissue (BAT) thermogenesis is critical for thermoregulation and contributes to total energy expenditure. However, whether BAT has non-thermogenic functions is largely unknown. Here, we describe that BAT-specific liver kinase b1 knockout (Lkb1 BKO ) mice exhibited impaired BAT mitochondrial respiration and thermogenesis but reduced adiposity and liver triglyceride accumulation under high-fat-diet feeding at room temperature. Importantly, these metabolic benefits were also present in Lkb1 BKO mice at thermoneutrality, where BAT thermogenesis was not required. Mechanistically, decreased mRNA levels of mtDNA-encoded electron transport chain (ETC) subunits and ETC proteome imbalance led to defective BAT mitochondrial respiration in Lkb1 BKO mice. Furthermore, reducing mtDNA gene expression directly in BAT by removing mitochondrial transcription factor A (Tfam) in BAT also showed ETC proteome imbalance and the trade-off between BAT thermogenesis and systemic metabolism at room temperature and thermoneutrality. Collectively, our data demonstrate that ETC proteome imbalance in BAT regulates systemic metabolism independently of thermogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

Symmetrical refolding of protein domains and subunits: example of the dimeric two-domain 3-isopropylmalate dehydrogenases.

PubMed

Gráczer, Eva; Varga, Andrea; Melnik, Bogdan; Semisotnov, Gennady; Závodszky, Péter; Vas, Mária

2009-02-10

The refolding mechanism of the homodimeric two-domain 3-isopropylmalate dehydrogenase (IPMDH) from the organisms adapted to different temperatures, Thermus thermophilus (Tt), Escherichia coli (Ec), and Vibrio sp. I5 (Vib), is described. In all three cases, instead of a self-template mechanism, the high extent of symmetry and cooperativity in folding of subunits and domains have been concluded from the following experimental findings: The complex time course of refolding, monitored by Trp fluorescence, consists of a fast (the rate constant varies as 16.5, 25.0, and 11.7 min-1 in the order of Tt, Ec, and Vib IPMDHs) and a slow (the rate constants are 0.11, 0.80, and 0.23 min-1 for the three different species) first-order process. However, a burst increase of Trp fluorescence anisotropy to the value of the native states indicates that in all three cases the association of the two polypeptide chains occurs at the beginning of refolding. This dimeric species binds the substrate IPM, but the native-like interactions of the tertiary and quaternary structures are only formed during the slow phase of refolding, accompanied by further increase of protein fluorescence and appearance of FRET between Trp side chain(s) and the bound NADH. Joining the contacting arms of each subunit also takes place exclusively during this slow phase. To monitor refolding of each domain within the intact molecule of T. thermophilus IPMDH, Trp's (located in separate domains) were systematically replaced with Phe's. The refolding processes of the mutants were followed by measuring changes in Trp fluorescence and in FRET between the particular Trp and NADH. The high similarity of time courses (both in biphasicity and in their rates) strongly suggests cooperative folding of the domains during formation of the native three-dimensional structure of IPMDH.

[A new mechanism of ubiquitin-dependent proteolytic pathway--polyubiquitin chain recognition and proteasomal targeting].

PubMed

Kawahara, Hiroyuki; Yokosawa, Hideyoshi

2008-01-01

The RPN10 subunit of 26S proteasome and several UBA domain proteins can bind to the polyubiquitin chain and play a role as ubiquitin receptors of the 26S proteasome. Although it was thought that substrate recognition is an essential step in the proteasome-mediated protein degradation, deletion of rpn10 genes in yeast does not influence the viability of cells but instead causes only a mild phenotype, suggesting that the above ubiquitin receptors are redundantly involved in substrate delivery to the proteasome. However, their functional difference is still enigmatic. In this review, we summarize recent advances in polyubiquitin chain recognition/delivery system and provide potential applications to modulate this system as a probable target for drug development.

NADH:ubiquinone oxidoreductase from bovine heart mitochondria. cDNA sequences of the import precursors of the nuclear-encoded 39 kDa and 42 kDa subunits.

PubMed Central

Fearnley, I M; Finel, M; Skehel, J M; Walker, J E

1991-01-01

The 39 kDa and 42 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria are nuclear-coded components of the hydrophobic protein fraction of the enzyme. Their amino acid sequences have been deduced from the sequences of overlapping cDNA clones. These clones were amplified from total bovine heart cDNA by means of the polymerase chain reaction, with the use of complex mixtures of oligonucleotide primers based upon fragments of protein sequence determined at the N-terminals of the proteins and at internal sites. The protein sequences of the 39 kDa and 42 kDa subunits are 345 and 320 amino acid residues long respectively, and their calculated molecular masses are 39,115 Da and 36,693 Da. Both proteins are predominantly hydrophilic, but each contains one or two hydrophobic segments that could possibly be folded into transmembrane alpha-helices. The bovine 39 kDa protein sequence is related to that of a 40 kDa subunit from complex I from Neurospora crassa mitochondria; otherwise, it is not related significantly to any known sequence, including redox proteins and two polypeptides involved in import of proteins into mitochondria, known as the mitochondrial processing peptidase and the processing-enhancing protein. Therefore the functions of the 39 kDa and 42 kDa subunits of complex I are unknown. The mitochondrial gene product, ND4, a hydrophobic component of complex I with an apparent molecular mass of about 39 kDa, has been identified in preparations of the enzyme. This subunit stains faintly with Coomassie Blue dye, and in many gel systems it is not resolved from the nuclearcoded 36 kDa subunit. Images Fig. 1. PMID:1832859

C-Type Lectin-Like Receptor 2 Suppresses AKT Signaling and Invasive Activities of Gastric Cancer Cells by Blocking Expression of Phosphoinositide 3-Kinase Subunits.

PubMed

Wang, Lan; Yin, Jie; Wang, Xuefei; Shao, Miaomiao; Duan, Fangfang; Wu, Weicheng; Peng, Peike; Jin, Jing; Tang, Yue; Ruan, Yuanyuan; Sun, Yihong; Gu, Jianxin

2016-05-01

C-type lectin-like receptor 2 (CLEC2) is a transmembrane receptor expressed on platelets and several hematopoietic cells. CLEC2 regulates platelet aggregation and the immune response. We investigated its expression and function in normal and transformed gastric epithelial cells from human tissues. We performed tissue microarray analyses of gastric carcinoma samples collected from 96 patients who underwent surgery at Zhongshan Hospital of Fudan University in Shanghai, China and performed real-time polymerase chain reaction assays from an independent group of 60 patients; matched nontumor gastric mucosa tissues were used as the control. Full-length and mutant forms of CLEC2 were expressed in gastric cancer cell line (MGC80-3), or CLEC2 protein was knocked down using small-hairpin RNAs in gastric cancer cell lines (NCI-N87 and AGS). CLEC2 signaling was stimulated by incubation of cells with recombinant human podoplanin or an antibody agonist of CLEC2; cell migration and invasion were assessed by transwell and wound-healing assays. Immunoblot, immunofluorescence microscopy, and real-time polymerase chain reaction assays were used to measure expression of markers of the epithelial to mesenchymal transition and activation of signaling pathways. Immunoprecipitation experiments were performed with an antibody against spleen tyrosine kinase (SYK). Cells were injected into lateral tail vein of BALB/C nude mice; some mice were also given injections of the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Lung and liver tissues were collected and analyzed for metastases. Levels of CLEC2 were higher in nontumor gastric mucosa (control) than in gastric tumor samples. Levels of CLEC2 protein in gastric tumor tissues correlated with depth of tumor invasion, metastasis to lymph node, tumor TNM stage, and 5-year survival of patients. Activation of CLEC2 in gastric cancer cells reduced their invasive activities in vitro and expression of epithelial to mesenchymal transition markers; these tumor-suppressive effects of CLEC2 required SYK. CLEC2 and SYK interacted physically, and SYK maintained the stability of CLEC2 in cells. AGS cells with CLEC2 knockdown had increased levels of phosphorylated AKT and glycogen synthase kinase-3 beta, increased expression of Snail, reduced levels of E-cadherin, and formed more metastases in mice than AGS cells that expressed CLEC2; these knockdown changes were prevented by the PI3K inhibitor LY294002. Activation of CLEC2 in AGS cells reduced protein and messenger RNA levels of PI3K subunits p85 and p110; this effect was blocked by SYK inhibitor R406. Levels of CLEC2 and SYK proteins and messenger RNAs correlated in gastric tumor samples. CLEC2 suppresses metastasis of gastric cancer cells injected into mice, and prevents activation of AKT and glycogen synthase kinase-3 beta signaling, as well as invasiveness and expression of epithelial to mesenchymal transition markers in gastric cancer cell lines. CLEC2 prevents expression of PI3K subunits, in a SYK-dependent manner. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

Crystal structure of a two-subunit TrkA octameric gating ring assembly

DOE PAGES

Deller, Marc C.; Johnson, Hope A.; Miller, Mitchell D.; ...

2015-03-31

The TM1088 locus of T. maritima codes for two proteins designated TM1088A and TM1088B, which combine to form the cytosolic portion of a putative Trk K⁺ transporter. We report the crystal structure of this assembly to a resolution of 3.45 Å. The high resolution crystal structures of the components of the assembly, TM1088A and TM1088B, were also determined independently to 1.50 Å and 1.55 Å, respectively. The TM1088 proteins are structurally homologous to each other and to other K⁺ transporter proteins, such as TrkA. These proteins form a cytosolic gating ring assembly that controls the flow of K⁺ ions acrossmore » the membrane. TM1088 represents the first structure of a two-subunit Trk assembly. Despite the atypical genetics and chain organization of the TM1088 assembly, it shares significant structural homology and an overall quaternary organization with other single-subunit K⁺ gating ring assemblies. This structure provides the first structural insights into what may be an evolutionary ancestor of more modern single-subunit K⁺ gating ring assemblies.« less

Point Mutations in the Stem Region and the Fourth AAA Domain of Cytoplasmic Dynein Heavy Chain Partially Suppress the Phenotype of NUDF/LIS1 Loss in Aspergillus nidulans

PubMed Central

Zhuang, Lei; Zhang, Jun; Xiang, Xin

2007-01-01

Cytoplasmic dynein performs multiple cellular tasks but its regulation remains unclear. The dynein heavy chain has a N-terminal stem that binds to other subunits and a C-terminal motor unit that contains six AAA (ATPase associated with cellular activities) domains and a microtubule-binding site located between AAA4 and AAA5. In Aspergillus nidulans, NUDF (a LIS1 homolog) functions in the dynein pathway, and two nudF6 partial suppressors were mapped to the nudA dynein heavy chain locus. Here we identified these two mutations. The nudAL1098F mutation resides in the stem region, and nudAR3086C is in the end of AAA4. These mutations partially suppress the phenotype of nudF deletion but do not suppress the phenotype exhibited by mutants of dynein intermediate chain and Arp1. Surprisingly, the stronger ΔnudF suppressor, nudAR3086C, causes an obvious decrease in the basal level of dynein's ATPase activity and an increase in dynein's distribution along microtubules. Thus, suppression of the ΔnudF phenotype may result from mechanisms other than simply the enhancement of dynein's ATPase activity. The fact that a mutation in the end of AAA4 negatively regulates dynein's ATPase activity but partially compensates for NUDF loss indicates the importance of the AAA4 domain in dynein regulation in vivo. PMID:17237507

Curcumin suppresses the production of interleukin-6 in Prevotella intermedia lipopolysaccharide-activated RAW 264.7 cells

PubMed Central

2011-01-01

Purpose Curcumin is known to exert numerous biological effects including anti-inflammatory activity. In this study, we investigated the effects of curcumin on the production of interleukin-6 (IL-6) by murine macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a major cause of inflammatory periodontal disease, and sought to determine the underlying mechanisms of action. Methods LPS was prepared from lyophilized P. intermedia ATCC 25611 cells by the standard hot phenol-water method. Culture supernatants were collected and assayed for IL-6. We used real-time polymerase chain reaction to detect IL-6 mRNA expression. IκB-α degradation, nuclear translocation of NF-κB subunits, and STAT1 phosphorylation were characterized via immunoblotting. DNA-binding of NF-κB was also analyzed. Results Curcumin strongly suppressed the production of IL-6 at both gene transcription and translation levels in P. intermedia LPS-activated RAW 264.7 cells. Curcumin did not inhibit the degradation of IκB-α induced by P. intermedia LPS. Curcumin blocked NF-κB signaling through the inhibition of nuclear translocation of NF-κB p50 subunit. Curcumin also attenuated DNA binding activity of p50 and p65 subunits and suppressed STAT1 phosphorylation. Conclusions Although further study is required to explore the detailed mechanism of action, curcumin may contribute to blockade of the host-destructive processes mediated by IL-6 and appears to have potential therapeutic values in the treatment of inflammatory periodontal disease. PMID:21811692

Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

PubMed Central

Römling, Ute; Galperin, Michael Y.

2015-01-01

Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

Decrease in neuronal nicotinic acetylcholine receptor subunit and PSD-93 transcript levels in the male mouse MPG after cavernous nerve injury or explant culture.

PubMed

Girard, Beatrice M; Merriam, Laura A; Tompkins, John D; Vizzard, Margaret A; Parsons, Rodney L

2013-11-15

Quantitative real-time PCR was used to test whether cavernous nerve injury leads to a decrease in major pelvic ganglia (MPG) neuronal nicotinic ACh receptor (nAChR) subunit and postsynaptic density (PSD)-93 transcript levels. Subunits α3, β4, and α7, commonly expressed in the MPG, were selected for analysis. After 72 h in explant culture, MPG transcript levels for α3, β4, α7, and PSD-93 were significantly depressed. Three days after cavernous nerve axotomy or crush in vivo, transcript levels for α3, β4, and PSD-93, but not for α7, were significantly depressed. Three days after dissection of the cavernous nerve free of underlying tissue and application of a 5-mm lateral stretch (manipulation), transcript levels for α3 and PSD-93 were also significantly decreased. Seven days after all three surgical procedures, α3 transcript levels remained depressed, but PSD-93 transcript levels were still decreased only after axotomy or nerve crush. At 30 days postsurgery, transcript levels for the nAChR subunits and PSD-93 had recovered. ACh-induced currents were significantly smaller in MPG neurons dissociated from 3-day explant cultured ganglia than from those recorded in neurons dissociated from acutely isolated ganglia; this observation provides direct evidence showing that a decrease in nAChR function was coincident with a decrease in nAChR subunit transcript levels. We conclude that a downregulation of nAChR subunit and PSD-93 expression after cavernous nerve injury, or even manipulation, could interrupt synaptic transmission within the MPG and thus contribute to the loss of neural control of urogenital organs after pelvic surgeries.

Crystallization of the photosystem II core complex and its chlorophyll binding subunit CP43 from transplastomic plants of Nicotiana tabacum.

PubMed

Piano, Dario; El Alaoui, Sabah; Korza, Henryk J; Filipek, Renata; Sabala, Izabela; Haniewicz, Patrycja; Buechel, Claudia; De Sanctis, Daniele; Bochtler, Matthias

2010-12-01

Photosystem II from transplastomic plants of Nicotiana tabacum with a hexahistidine tag at the N-terminal end of the PsbE subunit (α-chain of the cytochrome b(559)) was purified according to the protocol of Fey et al. (BBA 12:1501-1509, 2008). The protein sample was then subjected to two additional gel filtration runs in order to increase its homogeneity and to standardize the amount of detergent. Large three dimensional crystals of the core complex were obtained. Crystals of one of its chlorophyll binding subunits (CP43) in isolation grew in very similar conditions that differed only in the concentration of the detergent. Diffraction of Photosystem II and CP43 crystals at various synchrotron beamlines was limited to a resolution of 7 and 14 Å, respectively. In both cases the diffraction quality was insufficient for an unambiguous assignment of the crystallographic lattice or space group.

The life of plant mitochondrial complex I.

PubMed

Braun, Hans-Peter; Binder, Stefan; Brennicke, Axel; Eubel, Holger; Fernie, Alisdair R; Finkemeier, Iris; Klodmann, Jennifer; König, Ann-Christine; Kühn, Kristina; Meyer, Etienne; Obata, Toshihiro; Schwarzländer, Markus; Takenaka, Mizuki; Zehrmann, Anja

2014-11-01

The mitochondrial NADH dehydrogenase complex (complex I) of the respiratory chain has several remarkable features in plants: (i) particularly many of its subunits are encoded by the mitochondrial genome, (ii) its mitochondrial transcripts undergo extensive maturation processes (e.g. RNA editing, trans-splicing), (iii) its assembly follows unique routes, (iv) it includes an additional functional domain which contains carbonic anhydrases and (v) it is, indirectly, involved in photosynthesis. Comprising about 50 distinct protein subunits, complex I of plants is very large. However, an even larger number of proteins are required to synthesize these subunits and assemble the enzyme complex. This review aims to follow the complete "life cycle" of plant complex I from various molecular perspectives. We provide arguments that complex I represents an ideal model system for studying the interplay of respiration and photosynthesis, the cooperation of mitochondria and the nucleus during organelle biogenesis and the evolution of the mitochondrial oxidative phosphorylation system. Copyright © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

The insecticide target in the PSST subunit of complex I.

PubMed

Schuler, F; Casida, J E

2001-10-01

Current insecticides have been selected by sifting and winnowing hundreds of thousands of synthetic chemicals and natural products to obtain commercial preparations of optimal effectiveness and safety. This process has often ended up with compounds of high potency as inhibitors of the electron transport chain and more specifically of complex I (NADH:ubiquinone oxidoreductase). Many classes of chemicals are involved and the enzyme is one of the most complicated known, with 43 subunits catalyzing electron transfer from NADH to ubiquinone through flavin mononucleotide and up to eight iron-sulfur clusters. We used a potent photoaffinity ligand, (trifluoromethyl)diazirinyl[3H]pyridaben, to localize the insecticide target to a single high-affinity site in the PSST subunit that couples electron transfer from iron-sulfur cluster N2 to ubiquinone. Most importantly, all of the potent complex I-inhibiting pesticides, despite their great structural diversity, compete for this same specific binding domain in PSST. Finding their common mode of action and target provides insight into shared toxicological features and potential selection for resistant pests.

Immunochemical Proof that a Novel Rearranging Gene Encodes the T Cell Receptor δ Subunit

NASA Astrophysics Data System (ADS)

Band, Hamid; Hochstenbach, Frans; McLean, Joanne; Hata, Shingo; Krangel, Michael S.; Brenner, Michael B.

1987-10-01

The T cell receptor (TCR) δ protein is expressed as part of a heterodimer with TCR γ , in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR δ was produced that binds specifically to the surface of several TCR γ δ cell lines and immunoprecipitates the TCR γ δ as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR δ subunit alone after chain separation. A candidate human TCR δ complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR γ δ cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR δ . This complementary DNA clone thus corresponds to the gene that encodes the TCR δ subunit.

Quercetin protects against aluminium induced oxidative stress and promotes mitochondrial biogenesis via activation of the PGC-1α signaling pathway.

PubMed

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Verma, Deepika; Priyanka, Kumari; Bal, Amanjit; Gill, Kiran Dip

2015-12-01

The present investigation was carried out to elucidate a possible molecular mechanism related to the protective effect of quercetin administration against aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of PGC-1α and its downstream targets, i.e. NRF-1, NRF-2 and Tfam in mitochondrial biogenesis. Aluminium lactate (10mg/kg b.wt./day) was administered intragastrically to rats, which were pre-treated with quercetin 6h before aluminium (10mg/kg b.wt./day, intragastrically) for 12 weeks. We found a decrease in ROS levels, mitochondrial DNA oxidation and citrate synthase activity in the hippocampus (HC) and corpus striatum (CS) regions of rat brain treated with quercetin. Besides this an increase in the mRNA levels of the mitochondrial encoded subunits - ND1, ND2, ND3, Cyt b, COX1, COX3 and ATPase6 along with increased expression of nuclear encoded subunits COX4, COX5A and COX5B of electron transport chain (ETC). In quercetin treated group an increase in the mitochondrial DNA copy number and mitochondrial content in both the regions of rat brain was observed. The PGC-1α was up regulated in quercetin treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α. Electron microscopy results revealed a significant decrease in the mitochondrial cross-section area, mitochondrial perimeter length and increase in mitochondrial number in case of quercetin treated rats as compared to aluminium treated ones. Therefore it seems quercetin increases mitochondrial biogenesis and makes it an almost ideal flavanoid to control or limit the damage that has been associated with the defective mitochondrial function seen in many neurodegenerative diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of p67phox on the mitochondrial oxidative state in the kidney of Dahl salt-sensitive rats: optical fluorescence 3-D cryoimaging

PubMed Central

Salehpour, F.; Ghanian, Z.; Yang, C.; Zheleznova, N. N.; Kurth, T.; Dash, R. K.; Cowley, A. W.

2015-01-01

The goal of the present study was to quantify and correlate the contribution of the cytosolic p67phox subunit of NADPH oxidase 2 to mitochondrial oxidative stress in the kidneys of the Dahl salt-sensitive (SS) hypertensive rat. Whole kidney redox states were uniquely assessed using a custom-designed optical fluorescence three-dimensional cryoimager to acquire multichannel signals of the intrinsic fluorophores NADH and FAD. SS rats were compared with SS rats in which the cytosolic subunit p67phox was rendered functionally inactive by zinc finger nuclease mutation of the gene (SSp67phox-null rats). Kidneys of SS rats fed a 0.4% NaCl diet exhibited significantly (P = 0.023) lower tissue redox ratio (NADH/FAD; 1.42 ± 0.06, n = 5) than SSp67phox-null rats (1.64 ± 0.07, n = 5), indicating reduced levels of mitochondrial electron transport chain metabolic activity and enhanced oxidative stress in SS rats. When fed a 4.0% salt diet for 21 days, both strains exhibited significantly lower tissue redox ratios (P < 0.001; SS rats: 1.03 ± 0.05, n = 9, vs. SSp67phox-null rats: 1.46 ± 0.04, n = 7) than when fed a 0.4% salt, but the ratio was still significantly higher in SSp67phox rats at the same salt level as SS rats. These results are consistent with results from previous studies that found elevated medullary interstitial fluid concentrations of superoxide and H2O2 in the medulla of SS rats. We conclude that the p67phox subunit of NADPH oxidase 2 plays an important role in the excess production of ROS from mitochondria in the renal medulla of the SS rat. PMID:26062875

Effects of p67phox on the mitochondrial oxidative state in the kidney of Dahl salt-sensitive rats: optical fluorescence 3-D cryoimaging.

PubMed

Salehpour, F; Ghanian, Z; Yang, C; Zheleznova, N N; Kurth, T; Dash, R K; Cowley, A W; Ranji, M

2015-08-15

The goal of the present study was to quantify and correlate the contribution of the cytosolic p67(phox) subunit of NADPH oxidase 2 to mitochondrial oxidative stress in the kidneys of the Dahl salt-sensitive (SS) hypertensive rat. Whole kidney redox states were uniquely assessed using a custom-designed optical fluorescence three-dimensional cryoimager to acquire multichannel signals of the intrinsic fluorophores NADH and FAD. SS rats were compared with SS rats in which the cytosolic subunit p67(phox) was rendered functionally inactive by zinc finger nuclease mutation of the gene (SS(p67phox)-null rats). Kidneys of SS rats fed a 0.4% NaCl diet exhibited significantly (P = 0.023) lower tissue redox ratio (NADH/FAD; 1.42 ± 0.06, n = 5) than SS(p67phox)-null rats (1.64 ± 0.07, n = 5), indicating reduced levels of mitochondrial electron transport chain metabolic activity and enhanced oxidative stress in SS rats. When fed a 4.0% salt diet for 21 days, both strains exhibited significantly lower tissue redox ratios (P < 0.001; SS rats: 1.03 ± 0.05, n = 9, vs. SS(p67phox)-null rats: 1.46 ± 0.04, n = 7) than when fed a 0.4% salt, but the ratio was still significantly higher in SS(p67phox) rats at the same salt level as SS rats. These results are consistent with results from previous studies that found elevated medullary interstitial fluid concentrations of superoxide and H2O2 in the medulla of SS rats. We conclude that the p67(phox) subunit of NADPH oxidase 2 plays an important role in the excess production of ROS from mitochondria in the renal medulla of the SS rat. Copyright © 2015 the American Physiological Society.

Dysfunctional C8 beta chain in patients with C8 deficiency.

PubMed

Tschopp, J; Penea, F; Schifferli, J; Späth, P

1986-12-01

Two sera from unrelated individuals, each lacking C8 activity, were examined by Western blot analysis. Using antisera raised against whole C8, the two sera are shown to lack the C8 beta chain, indicating a C8 beta deficiency, which is frequently observed in cases of dysfunctional C8. In contrast, by means of a specific anti-C8-beta antiserum, a C8 beta-like polypeptide chain of apparently identical molecular weight compared to normal C8 beta was detected. Digestion of normal and dysfunctional C8 beta with Staphylococcus aureus V8 protease revealed distinct differences in the enzymatic digestion pattern. We conclude that the dysfunction in the C8 protein in these two patients resides in the dysfunctional C8 beta chain, and that this form of C8 deficiency is distinct from C8 deficiencies previously reported, in which one or both C8 subunits are lacking.

The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2

PubMed Central

Wickliffe, Katherine E.; Lorenz, Sonja; Wemmer, David E.; Kuriyan, John; Rape, Michael

2011-01-01

Ubiquitin chains of different topologies trigger distinct functional consequences, including protein degradation and reorganization of complexes. The assembly of most ubiquitin chains is promoted by E2s, yet how these enzymes achieve linkage specificity is poorly understood. We have discovered that the K11-specific Ube2S orients the donor ubiquitin through an essential non-covalent interaction that occurs in addition to the thioester bond at the E2 active site. The E2-donor ubiquitin complex transiently recognizes the acceptor ubiquitin, primarily through electrostatic interactions. The recognition of the acceptor ubiquitin surface around Lys11, but not around other lysines, generates a catalytically competent active site, which is composed of residues of both Ube2S and ubiquitin. Our studies suggest that monomeric E2s promote linkage-specific ubiquitin chain formation through substrate-assisted catalysis. PMID:21376237

Quantitative RT-PCR for inhibin/activin subunits: measurements of rat hypothalamic and ovarian inhibin/activin subunit mRNAs during the estrous cycle.

PubMed

Murata, T; Takizawa, T; Funaba, M; Fujimura, H; Murata, E; Takahashi, M; Torii, K

1997-02-01

Inhibins (alpha-beta(A) and alpha-beta(B)) and activins (beta(A)-beta(A), beta(A)-beta(B) and beta(B)-beta(B)) were originally isolated from ovarian follicular fluids as FSH secretion modifiers. Inhibin/activin subunits, alpha, beta(A) and beta(B), are widely distributed in several tissues, including gonads and brain, and inhibins and activins have been reported to be involved in ovarian or hypothalamic functions. In this study, we established and employed a competitive RT-PCR assay system for rat inhibin/activin subunits by capillary electrophoresis to determine rat hypothalamic and ovarian inhibin/activin subunit mRNA levels during the estrous cycle. Linearity of standards for alpha, beta(A), and beta(B) subunit assays were between 0.01-0.3 amol, 0.003-0.09 amol and 0.002-0.02 amol of each fragment DNA as a standard, respectively. Hypothalamic beta(A) subunit mRNA during the estrous morning (1000 h) tended to be increased compared with that of the proestrous evening (1700 h), although they were not significantly different. Ovarian alpha subunit mRNA levels tended to be increased during the proestrous morning (1000 h) and were significantly increased in the proestrous evening (1700 h), compared with diestrus and estrus (P < 0.05). Ovarian beta(A) subunit mRNA was also significantly higher in the proestrous evening, compared with diestrus and estrus (P < 0.05), but in the case of beta(B) subunit mRNA there was no difference among diestrus, proestrus and estrus. We thus established a sensitive competitive RT-PCR system for the measurement of inhibin/activin alpha, beta(A) and beta(B) subunits, and this assay system would be helpful for the study of inhibin/activin action in brain and other tissues where these factors are expressed at low levels.

Evolution of the eukaryotic dynactin complex, the activator of cytoplasmic dynein

PubMed Central

2012-01-01

Background Dynactin is a large multisubunit protein complex that enhances the processivity of cytoplasmic dynein and acts as an adapter between dynein and the cargo. It is composed of eleven different polypeptides of which eight are unique to this complex, namely dynactin1 (p150Glued), dynactin2 (p50 or dynamitin), dynactin3 (p24), dynactin4 (p62), dynactin5 (p25), dynactin6 (p27), and the actin-related proteins Arp1 and Arp10 (Arp11). Results To reveal the evolution of dynactin across the eukaryotic tree the presence or absence of all dynactin subunits was determined in most of the available eukaryotic genome assemblies. Altogether, 3061 dynactin sequences from 478 organisms have been annotated. Phylogenetic trees of the various subunit sequences were used to reveal sub-family relationships and to reconstruct gene duplication events. Especially in the metazoan lineage, several of the dynactin subunits were duplicated independently in different branches. The largest subunit repertoire is found in vertebrates. Dynactin diversity in vertebrates is further increased by alternative splicing of several subunits. The most prominent example is the dynactin1 gene, which may code for up to 36 different isoforms due to three different transcription start sites and four exons that are spliced as differentially included exons. Conclusions The dynactin complex is a very ancient complex that most likely included all subunits in the last common ancestor of extant eukaryotes. The absence of dynactin in certain species coincides with that of the cytoplasmic dynein heavy chain: Organisms that do not encode cytoplasmic dynein like plants and diplomonads also do not encode the unique dynactin subunits. The conserved core of dynactin consists of dynactin1, dynactin2, dynactin4, dynactin5, Arp1, and the heterodimeric actin capping protein. The evolution of the remaining subunits dynactin3, dynactin6, and Arp10 is characterized by many branch- and species-specific gene loss events. PMID:22726940

Modulation of smooth muscle tonus in the lower urinary tract: interplay of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP).

PubMed

Lin, Guiting; Fandel, Thomas M; Shindel, Alan W; Wang, Guifang; Banie, Lia; Ning, Hongxiu; Lue, Tom F; Lin, Ching-Shwun

2011-07-01

To assess and compare the expression and activity of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP) in rat bladder and urethra. Bladder and urethral smooth muscles were obtained from 2-month-old female Sprague-Dawley rats. They were analysed by real-time polymerase chain reaction for the mRNA expression of MLCK and myosin phosphatase-targeting subunit of protein phosphatase type 1 (MYPT1, a subunit of MLCP). Levels of MLCK and MYPT1 mRNA expression were determined as a ratio to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The tissues were also analysed by Western blotting for MLCK and MYPT1 protein expression as a ratio to the expression of β-actin. A two-step enzymatic activity assay using phosphorylated and dephosphorylated smooth muscle myosin was used to assess MLCK and MLCP activity. MLCK mRNA expression was higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 0.26 (0.17) vs 0.14 (0.12); P = 0.09]. MYPT1 mRNA expression was significantly higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 2.31 (1.04) vs 0.56 (0.36); P = 0.001]. Expression of both MLCK and MYPT1 protein was significantly higher in the bladder compared with the urethra [mean (sd) ratio to β-actin: 1.63 (0.25) vs 0.91 (0.29) and 0.97 (0.10) vs 0.37 (0.29), respectively; both P < 0.001]. Enzymatic assay identified significantly greater MLCK activity in the bladder than in the urethra. While, MLCP activity was lower in the bladder than in the urethra. In healthy young female rats, MLCK activity is higher and MLCP activity is lower in the bladder relative to the urethra. These differences probably play a role in modulating the functional differences between bladder and urethral smooth muscle tone. © 2010 THE AUTHORS. BJU INTERNATIONAL © 2010 BJU INTERNATIONAL.

The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit

PubMed Central

Jalah, Rashmi; Rosati, Margherita; Ganneru, Brunda; Pilkington, Guy R.; Valentin, Antonio; Kulkarni, Viraj; Bergamaschi, Cristina; Chowdhury, Bhabadeb; Zhang, Gen-Mu; Beach, Rachel Kelly; Alicea, Candido; Broderick, Kate E.; Sardesai, Niranjan Y.; Pavlakis, George N.; Felber, Barbara K.

2013-01-01

IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ∼1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy. PMID:23297419

Study on relationship between expression level and molecular conformations of gene drugs targeting to hepatoma cells in vitro

PubMed Central

Yang, Dong-Ye; Lu, Fang-Gen; Tang, Xi-Xiang; Zhao, Shui-Ping; Ouyang, Chun-Hui; Wu, Xiao-Ping; Liu, Xiao-Wei; Wu, Xiao-Ying

2003-01-01

AIM: To increase exogenous gene expression level by modulating molecular conformations of targeting gene drugs. METHODS: The full length cDNAs of both P40 and P35 subunits of human interleukin 12 were amplified through polymerase chain reaction (PCR) and cloned into eukaryotic expressing vectors pcDNA3.1 (±) to construct plasmids of P (+)/IL-12, P (+)/P40 and P (-)/P35. These plasmids were combined with ASOR-PLL to form two targeting gene drugs [ASOR-PLL-P (+)/IL-12 and ASOR-PLL-P (+)/P40 + ASOR-PLL-P (-)/P35] in optimal ratios. The conformations of these two drugs at various concentrations adjuvant were examined under electron microscope (EM) and the drugs were transfected into HepG2 (ASGr+) cells. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed with total RNA extracted from the transfected cells to determine the hIL12 mRNA transcript level. The hIL12 protein in the cultured supernatant was measured with enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection. RESULTS: Targeting gene drugs, whose structures were granular and circle-like and diameters ranged from 25 nm to 150 nm, had the highest hIL-12 expression level. The hIL-12 expression level in the group co-transfected with ASOR-PLL-P (+)/P40 and ASOR-PLL-P (-)/P35 was higher than that of ASOR-PLL-P (+)/IL-12 transfected group. CONCLUSION: The molecular conformations of targeting gene drugs play an important role in exogenous gene expression level, the best structures are granular and circle-like and their diameters range from 25 nm to 150 nm. The sizes and linking styles of exogenous genes also have some effects on their expression level. PMID:12970883

GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

NASA Technical Reports Server (NTRS)

Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

1995-01-01

The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of schizophrenics, the previously reported upregulation of muscimol binding sites and downregulation of benzodiazepine binding sites in the prefrontal and adjacent cingulate cortex of schizophrenics are possibly due to posttranscriptional modifications of mRNAs and their translated polypeptides.

The importance of immunohistochemical analyses in evaluating the phenotype of Kv channel knockout mice.

PubMed

Menegola, Milena; Clark, Eliana; Trimmer, James S

2012-06-01

To gain insights into the phenotype of voltage-gated potassium (Kv)1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze the expression of component principal or α subunits and auxiliary subunits of neuronal Kv channels in knockout mouse brains. Genetic ablation of the Kv1.1 α subunit did not result in compensatory changes in the expression levels or subcellular distribution of related ion channel subunits in hippocampal medial perforant path and mossy fiber nerve terminals, where high levels of Kv1.1 are normally expressed. Genetic ablation of the Kv4.2 α subunit did not result in altered neuronal cytoarchitecture of the hippocampus. Although Kv4.2 knockout mice did not exhibit compensatory changes in the expression levels or subcellular distribution of the related Kv4.3 α subunit, we found dramatic decreases in the cellular and subcellular expression of specific Kv channel interacting proteins (KChIPs) that reflected their degree of association and colocalization with Kv4.2 in wild-type mouse and rat brains. These studies highlight the insights that can be gained by performing detailed immunohistochemical analyses of Kv channel knockout mouse brains. Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.

GammaM23K, gammaM232K, and gammaL77K single substitutions in the TF1-ATPase lower ATPase activity by disrupting a cluster of hydrophobic side chains.

PubMed

Bandyopadhyay, Sanjay; Allison, William S

2004-07-27

In crystal structures of the bovine F(1)-ATPase (MF(1)), the side chains of gammaMet(23), gammaMet(232), and gammaLeu(77) interact in a cluster. Substitution of the corresponding residues in the alpha(3)beta(3)gamma subcomplex of TF(1) with lysine lowers the ATPase activity to 2.3, 11, and 15%, respectively, of that displayed by wild-type. In contrast, TF(1) subcomplexes containing the gammaM(23)C, gammaM(232)C, and gammaL(77)C substitutions display 36, 36, and 130%, respectively, of the wild-type ATPase activity. The ATPase activity of the gammaM(23)C/gammaM(232)C double mutant subcomplex is 36% that of the wild-type subcomplex before and after cross-linking the introduced cysteines, whereas the ATPase activity of the gammaM(23)C/L(77)C double mutant increased from 50 to 85% that of wild-type after cross-linking the introduced cysteines. Only beta-beta cross-links formed when the alpha(3)(betaE(395)C)(3)gammaM(23)C double mutant was inactivated with CuCl(2). The overall results suggest that the attenuated ATPase of the mutant subcomplexes containing the gammaM(23)K, gammaL(77)K, and gammaM(232)K substitutions is caused by disruption of the cluster of hydrophobic amino acid side chains and that the midregion of the coiled-coil comprised of the amino- and carboxyl-terminal alpha helices of the gamma subunit does not undergo unwinding or major displacement from the side chain of gammaLeu(77) during ATP-driven rotation of the gamma subunit.

Ferulic Acid Attenuates the Injury-Induced Decrease of Protein Phosphatase 2A Subunit B in Ischemic Brain Injury

PubMed Central

Koh, Phil-Ok

2013-01-01

Background Ferulic acid provides a neuroprotective effect during cerebral ischemia through its anti-oxidant function. Protein phosphatase 2A (PP2A) is a serine and threonine phosphatase that contributes broadly to normal brain function. This study investigated whether ferulic acid regulates PP2A subunit B in a middle cerebral artery occlusion (MCAO) animal model and glutamate toxicity-induced neuronal cell death. Methodology/Principal Findings MCAO was surgically induced to yield permanent cerebral ischemic injury in rats. The rats were treated with either vehicle or ferulic acid (100 mg/kg, i.v.) immediately after MCAO, and cerebral cortex tissues were collected 24 h after MCAO. A proteomics approach, RT-PCR, and Western blot analyses performed to identification of PP2A subunit B expression levels. Ferulic acid significantly reduced the MCAO-induced infarct volume of the cerebral cortex. A proteomics approach elucidated the reduction of PP2A subunit B in MCAO-induced animals, and ferulic acid treatment prevented the injury-induced reduction in PP2A subunit B levels. RT-PCR and Western blot analyses also showed that ferulic acid treatment attenuates the injury-induced decrease in PP2A subunit B levels. Moreover, the number of PP2A subunit B-positive cells was reduced in MCAO-induced animals, and ferulic acid prevented these decreases. In cultured neuronal cells, ferulic acid treatment protected cells against glutamate toxicity and prevented the glutamate-induced decrease in PP2A subunit B. Conclusions/Significance These results suggest that the maintenance of PP2A subunit B by ferulic acid in ischemic brain injury plays an important role for the neuroprotective function of ferulic acid. PMID:23349830

Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana

PubMed Central

Magnotta, Scot M; Gogarten, Johann Peter

2002-01-01

Background Vacuolar type H+-ATPases play a critical role in the maintenance of vacuolar homeostasis in plant cells. V-ATPases are also involved in plants' defense against environmental stress. This research examined the expression and regulation of the catalytic subunit of the vacuolar type H+-ATPase in Arabidopsis thaliana and the effect of environmental stress on multiple transcripts generated by this gene. Results Evidence suggests that subunit A of the vacuolar type H+-ATPase is encoded by a single gene in Arabidopsis thaliana. Genome blot analysis showed no indication of a second subunit A gene being present. The single gene identified was shown by whole RNA blot analysis to be transcribed in all organs of the plant. Subunit A was shown by sequencing the 3' end of multiple cDNA clones to exhibit multi site polyadenylation. Four different poly (A) tail attachment sites were revealed. Experiments were performed to determine the response of transcript levels for subunit A to environmental stress. A PCR based strategy was devised to amplify the four different transcripts from the subunit A gene. Conclusions Amplification of cDNA generated from seedlings exposed to cold, salt stress, and etiolation showed that transcript levels for subunit A of the vacuolar type H+-ATPase in Arabidopsis were responsive to stress conditions. Cold and salt stress resulted in a 2–4 fold increase in all four subunit A transcripts evaluated. Etiolation resulted in a slight increase in transcript levels. All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation. PMID:11985780

Transfer in SDS of biotinylated proteins from acrylamide gels to an avidin-coated membrane filter.

PubMed

Karlin, Arthur; Wang, Chaojian; Li, Jing; Xu, Qiang

2004-06-01

Avidin was covalently linked to aldehyde-derivatized polyethersulfone membrane filters. These filters were used in Western blot analysis of proteins reacted with biotinylation reagents and electrophoresed in sodium dodecyl sulfate (SDS) on polyacrylamide gels. Electrophoretic transfer from the gels to these filters was in 0.1% SDS, in which the covalently bound avidin retained its biotin-binding capacity. We compared Western blots on avidin-coated membrane filters of biotinylated and nonbiotinylated forms of mouse immunoglobulin G (IgG), mouse IgG heavy chain, muscle-type acetylcholine receptor alpha subunit, and fused alpha and beta subunits of receptor. Biotinylated proteins were captured with high specificity compared to their nonbiotinylated counterparts and sensitively detected on the avidin-coated membranes.

Metabolic functions of AMPK: aspects of structure and of natural mutations in the regulatory gamma subunits.

PubMed

Moffat, Cynthia; Harper, Mary Ellen

2010-10-01

AMP-activated protein kinase, AMPK, is widely accepted as the master regulator of energy levels within the cell. Responding quickly to changing energy demands, AMPK works to restore levels of ATP during times of cellular stress by promoting ATP producing catabolic pathways and inhibiting ATP consuming anabolic ones. As a heterotrimeric protein complex, AMPK's subunits each act in unique and crucial ways to control AMPK function and its localization within the cell. Research in the last decade has identified and begun to characterize the impact of naturally occurring mutations in the gamma regulatory subunits. Mutations in the γ2 subunit have implications for cardiac function and disease, while the R225W mutation in the γ3 subunit have implications for skeletal muscle fuel metabolism and resistance to fatigue. Research focused on structure-function aspects of AMPK regulatory subunits will lead to a better understanding of the roles of AMPK in health and disease.

Extricating Manual and Non-Manual Features for Subunit Level Medical Sign Modelling in Automatic Sign Language Classification and Recognition.

PubMed

R, Elakkiya; K, Selvamani

2017-09-22

Subunit segmenting and modelling in medical sign language is one of the important studies in linguistic-oriented and vision-based Sign Language Recognition (SLR). Many efforts were made in the precedent to focus the functional subunits from the view of linguistic syllables but the problem is implementing such subunit extraction using syllables is not feasible in real-world computer vision techniques. And also, the present recognition systems are designed in such a way that it can detect the signer dependent actions under restricted and laboratory conditions. This research paper aims at solving these two important issues (1) Subunit extraction and (2) Signer independent action on visual sign language recognition. Subunit extraction involved in the sequential and parallel breakdown of sign gestures without any prior knowledge on syllables and number of subunits. A novel Bayesian Parallel Hidden Markov Model (BPaHMM) is introduced for subunit extraction to combine the features of manual and non-manual parameters to yield better results in classification and recognition of signs. Signer independent action aims in using a single web camera for different signer behaviour patterns and for cross-signer validation. Experimental results have proved that the proposed signer independent subunit level modelling for sign language classification and recognition has shown improvement and variations when compared with other existing works.

Regulation of PSMB5 Protein and β Subunits of Mammalian Proteasome by Constitutively Activated Signal Transducer and Activator of Transcription 3 (STAT3)

PubMed Central

Vangala, Janakiram Reddy; Dudem, Srikanth; Jain, Nishant; Kalivendi, Shasi V.

2014-01-01

The ubiquitin-proteasome system facilitates the degradation of ubiquitin-tagged proteins and performs a regulatory role in cells. Elevated proteasome activity and subunit expression are found in several cancers. However, the inherent molecular mechanisms responsible for increased proteasome function in cancers remain unclear despite the well investigated and defined role of the mammalian proteasome. This study was initiated to elucidate the mechanisms involved in the regulation of β subunits of the mammalian proteasome. Suppression of STAT3 tyrosine phosphorylation coordinately decreased the mRNA and protein levels of the β subunits of the 20 S core complex in DU145 cells. Notably, PSMB5, a molecular target of bortezomib, was shown to be a target of STAT3. Knockdown of STAT3 decreased PSMB5 protein. Inhibition of phospho-STAT3 substantially reduced PSMB5 protein levels in cells expressing constitutively active-STAT3. Accumulation of activated STAT3 resulted in the induction of PSMB5 promoter and protein levels. In addition, a direct correlation was observed between the endogenous levels of PSMB5 and constitutively active STAT3. PSMB5 and STAT3 protein levels remained unaltered following the inhibition of proteasome activity. The EGF-induced concerted increase of β subunits was blocked by inhibition of the EGF receptor or STAT3 but not by the PI3K/AKT or MEK/ERK pathways. Decreased proteasome activities were due to reduced protein levels of catalytic subunits of the proteasome in STAT3-inhibited cells. Combined treatments with bortezomib and inhibitor of STAT3 abrogated proteasome activity and enhanced cellular apoptosis. Overall, we demonstrate that aberrant activation of STAT3 regulates the expression of β subunits, in particular PSMB5, and the catalytic activity of the proteasome. PMID:24627483

Expression of laminin-5 and integrins in actinic cheilitis and superficially invasive squamous cell carcinomas of the lip.

PubMed

Peixoto da-Silva, Janaína; Lourenço, Silvia; Nico, Marcello; Silva, Filomena H; Martins, Marília Trierveiler; Costa-Neves, Adriana

2012-10-15

The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5γ2 chain as well as α3, β1 subunits of α3β1 heterodimer and β4 subunit of integrin α6β4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of β1, β4 and α3 integrins, and SISCCs lacked β1, β4 and α3 integrins in the invasive front. AC cases were negative for the Ln-5γ2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5γ2 chain expression in the invasive front of the tumor. Integrin β1, β4 and α3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5γ2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype. Copyright © 2012 Elsevier GmbH. All rights reserved.

Mutations in the Gene Encoding IFT Dynein Complex Component WDR34 Cause Jeune Asphyxiating Thoracic Dystrophy

PubMed Central

Schmidts, Miriam; Vodopiutz, Julia; Christou-Savina, Sonia; Cortés, Claudio R.; McInerney-Leo, Aideen M.; Emes, Richard D.; Arts, Heleen H.; Tüysüz, Beyhan; D’Silva, Jason; Leo, Paul J.; Giles, Tom C.; Oud, Machteld M.; Harris, Jessica A.; Koopmans, Marije; Marshall, Mhairi; Elçioglu, Nursel; Kuechler, Alma; Bockenhauer, Detlef; Moore, Anthony T.; Wilson, Louise C.; Janecke, Andreas R.; Hurles, Matthew E.; Emmet, Warren; Gardiner, Brooke; Streubel, Berthold; Dopita, Belinda; Zankl, Andreas; Kayserili, Hülya; Scambler, Peter J.; Brown, Matthew A.; Beales, Philip L.; Wicking, Carol; Duncan, Emma L.; Mitchison, Hannah M.

2013-01-01

Bidirectional (anterograde and retrograde) motor-based intraflagellar transport (IFT) governs cargo transport and delivery processes that are essential for primary cilia growth and maintenance and for hedgehog signaling functions. The IFT dynein-2 motor complex that regulates ciliary retrograde protein transport contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other less well functionally defined subunits. Deficiency of IFT proteins, including DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome sequencing and a targeted next-generation sequencing panel, we identified a total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40 repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein intermediate chain associated with the retrograde intraflagellar transport motor. Three-dimensional protein modeling suggests that the identified mutations all affect residues critical for WDR34 protein-protein interactions. We find that WDR34 concentrates around the centrioles and basal bodies in mammalian cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the dynein-1 light chain DYNLL1 in vitro, and mining of proteomics data suggests that WDR34 could represent a previously unrecognized link between the cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that WDR34 is critical for ciliary functions essential to normal development and survival, most probably as a previously unrecognized component of the mammalian dynein-IFT machinery. PMID:24183451

Development and characterization of six monoclonal antibodies to hemagglutinin-70 (HA70) of Clostridium botulinum and their application in a sandwich ELISA

USDA-ARS?s Scientific Manuscript database

Botulinum neurotoxins (BoNT),produced by Clostridium botulinum, cause severe neuroparalytic disease that if not treated quickly is often fatal. The toxin is synthesized as a 150 kDa precursor protein (holotoxin), which is then enzymatically cleaved to form two subunit chains linked by a single disul...

Respiromics - An integrative analysis linking mitochondrial bioenergetics to molecular signatures.

PubMed

Walheim, Ellen; Wiśniewski, Jacek R; Jastroch, Martin

2018-03-01

Energy metabolism is challenged upon nutrient stress, eventually leading to a variety of metabolic diseases that represent a major global health burden. Here, we combine quantitative mitochondrial respirometry (Seahorse technology) and proteomics (LC-MS/MS-based total protein approach) to understand how molecular changes translate to changes in mitochondrial energy transduction during diet-induced obesity (DIO) in the liver. The integrative analysis reveals that significantly increased palmitoyl-carnitine respiration is supported by an array of proteins enriching lipid metabolism pathways. Upstream of the respiratory chain, the increased capacity for ATP synthesis during DIO associates strongest to mitochondrial uptake of pyruvate, which is routed towards carboxylation. At the respiratory chain, robust increases of complex I are uncovered by cumulative analysis of single subunit concentrations. Specifically, nuclear-encoded accessory subunits, but not mitochondrial-encoded or core units, appear to be permissive for enhanced lipid oxidation. Our integrative analysis, that we dubbed "respiromics", represents an effective tool to link molecular changes to functional mechanisms in liver energy metabolism, and, more generally, can be applied for mitochondrial analysis in a variety of metabolic and mitochondrial disease models. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

A simplified method for identification of human cardiac myosin heavy-chain isoforms.

PubMed

Piao, Shengfu; Yu, Fushun; Mihm, Michael J; Reiser, Peter J; McCarthy, Patrick M; Van Wagoner, David R; Bauer, John Anthony

2003-02-01

Cardiac myosin is a central participant in the cross-bridge cycling that mediates myocyte contraction and consists of multiple subunits that mediate both hydrolysis of ATP and mechanical production of contractile force Two isoforms of myosin heavy chain (MHC- alpha and MHC- beta ) are known to exist in mammalian cardiac tissue, and it is within this myosin subunit that ATPase activity resides. These isoforms differ by less than 0.2% in total molecular mass and amino acid sequence, but, strikingly, influence the rate and efficiency of energy utilization for generation of contractile force. Changes in the MHC- alpha /MHC- beta ratio has been classically viewed as an adaptation of a failing myocyte in both animal models and humans; however, their measurement has traditionally required specialized preparations and materials for sufficient resolution. Here we describe a greatly simplified method for routine assessments of myosin isoform composition in human cardiac tissues. The primary advantages of our approach include higher throughput and reduced supply costs with no apparent loss of statistical power, reproducibility or achieved results. Use of this more convenient method may provide enhanced access to an otherwise specialized technique and could provide additional opportunity for investigation of cardiac myocyte adaptive changes.

Expression of Inwardly Rectifying Potassium Channel Subunits in Native Human Retinal Pigment Epithelium

PubMed Central

Yang, Dongli; Zhang, Xiaoming; Hughes, Bret A.

2008-01-01

Previously, we demonstrated that the inwardly rectifying K+ (Kir) channel subunit Kir7.1 is highly expressed in bovine and human retinal pigment epithelium (RPE). The purpose of this study was to determine whether any of the 14 other members of the Kir gene family are expressed in native human RPE. Conventional reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that in addition to Kir7.1, 7 other Kir channel subunits (Kir1.1, Kir2.1, Kir2.2, Kir3.1, Kir3.4, Kir4.2 and Kir6.1) are expressed in the RPE, whereas in neural retina, all 14 of the Kir channel subunits examined are expressed. The identities of RT-PCR products in the RPE were confirmed by DNA sequencing. Real-time RT-PCR analysis showed, however, that transcripts of these channels are significantly less abundant than Kir7.1 in the RPE. Western blot analysis of the Kir channel subunits detected in the RPE by RT-PCR revealed the expression of Kir2.1, Kir3.1, Kir3.4, Kir4.2, Kir6.1, and possibly Kir2.2, but not Kir1.1, in both human RPE and neural retina. Our results indicate that human RPE expresses at least 5 other Kir channel subtypes in addition to Kir7.1, suggesting that multiple members of the Kir channel family may function in this epithelium. PMID:18653180

Impaired Muscle Mitochondrial Biogenesis and Myogenesis in Spinal Muscular Atrophy

PubMed Central

Ripolone, Michela; Ronchi, Dario; Violano, Raffaella; Vallejo, Dionis; Fagiolari, Gigliola; Barca, Emanuele; Lucchini, Valeria; Colombo, Irene; Villa, Luisa; Berardinelli, Angela; Balottin, Umberto; Morandi, Lucia; Mora, Marina; Bordoni, Andreina; Fortunato, Francesco; Corti, Stefania; Parisi, Daniela; Toscano, Antonio; Sciacco, Monica; DiMauro, Salvatore; Comi, Giacomo P.; Moggio, Maurizio

2016-01-01

IMPORTANCE The important depletion of mitochondrial DNA (mtDNA) and the general depression of mitochondrial respiratory chain complex levels (including complex II) have been confirmed, implying an increasing paucity of mitochondria in the muscle from patients with types I, II, and III spinal muscular atrophy (SMA-I, -II, and -III, respectively). OBJECTIVE To investigate mitochondrial dysfunction in a large series of muscle biopsy samples from patients with SMA. DESIGN, SETTING, AND PARTICIPANTS We studied quadriceps muscle samples from 24 patients with genetically documented SMA and paraspinal muscle samples from 3 patients with SMA-II undergoing surgery for scoliosis correction. Postmortem muscle samples were obtained from 1 additional patient. Age-matched controls consisted of muscle biopsy specimens from healthy children aged 1 to 3 years who had undergone analysis for suspected myopathy. Analyses were performed at the Neuromuscular Unit, Istituto di Ricovero e Cura a Carattere Scientifico Foundation Ca’ Granda Ospedale Maggiore Policlinico-Milano, from April 2011 through January 2015. EXPOSURES We used histochemical, biochemical, and molecular techniques to examine the muscle samples. MAIN OUTCOMES AND MEASURES Respiratory chain activity and mitochondrial content. RESULTS Results of histochemical analysis revealed that cytochrome-c oxidase (COX) deficiency was more evident in muscle samples from patients with SMA-I and SMA-II. Residual activities for complexes I, II, and IV in muscles from patients with SMA-I were 41%, 27%, and 30%, respectively, compared with control samples (P < .005). Muscle mtDNA content and cytrate synthase activity were also reduced in all 3 SMA types (P < .05). We linked these alterations to downregulation of peroxisome proliferator–activated receptor coactivator 1α, the transcriptional activators nuclear respiratory factor 1 and nuclear respiratory factor 2, mitochondrial transcription factor A, and their downstream targets, implying depression of the entire mitochondrial biogenesis. Results of Western blot analysis confirmed the reduced levels of the respiratory chain subunits that included mitochondrially encoded COX1 (47.5%; P = .004), COX2 (32.4%; P < .001), COX4 (26.6%; P < .001), and succinate dehydrogenase complex subunit A (65.8%; P = .03) as well as the structural outer membrane mitochondrial porin (33.1%; P < .001). Conversely, the levels of expression of 3 myogenic regulatory factors—muscle-specificmyogenic factor 5, myoblast determination 1, and myogenin—were higher in muscles from patients with SMA compared with muscles from age-matched controls (P < .05). CONCLUSIONS AND RELEVANCE Our results strongly support the conclusion that an altered regulation of myogenesis and a downregulated mitochondrial biogenesis contribute to pathologic change in the muscle of patients with SMA. Therapeutic strategies should aim at counteracting these changes. PMID:25844556

Structure of the membrane domain of respiratory complex I.

PubMed

Efremov, Rouslan G; Sazanov, Leonid A

2011-08-07

Complex I is the first and largest enzyme of the respiratory chain, coupling electron transfer between NADH and ubiquinone to the translocation of four protons across the membrane. It has a central role in cellular energy production and has been implicated in many human neurodegenerative diseases. The L-shaped enzyme consists of hydrophilic and membrane domains. Previously, we determined the structure of the hydrophilic domain. Here we report the crystal structure of the Esherichia coli complex I membrane domain at 3.0 Å resolution. It includes six subunits, NuoL, NuoM, NuoN, NuoA, NuoJ and NuoK, with 55 transmembrane helices. The fold of the homologous antiporter-like subunits L, M and N is novel, with two inverted structural repeats of five transmembrane helices arranged, unusually, face-to-back. Each repeat includes a discontinuous transmembrane helix and forms half of a channel across the membrane. A network of conserved polar residues connects the two half-channels, completing the proton translocation pathway. Unexpectedly, lysines rather than carboxylate residues act as the main elements of the proton pump in these subunits. The fourth probable proton-translocation channel is at the interface of subunits N, K, J and A. The structure indicates that proton translocation in complex I, uniquely, involves coordinated conformational changes in six symmetrical structural elements.

Marker-assisted selection for recognizing wheat mutant genotypes carrying HMW glutenin alleles related to baking quality.

PubMed

Zamani, Mohammad Javad; Bihamta, Mohammad Reza; Naserian Khiabani, Behnam; Tahernezhad, Zahra; Hallajian, Mohammad Taher; Shamsi, Marzieh Varasteh

2014-01-01

Allelic diversity of HMW glutenin loci in several studies revealed that allelic combinations affect dough quality. Dx5 + Dy10 subunits are related to good baking quality and Dx2 + Dy12 are related to undesirable baking quality. One of the most regular methods to evaluate the baking quality is SDS-PAGE which is used to improve baking quality labs. Marker-assisted selection is the method which can recognize the alleles related to baking quality and this method is based on polymerase chain reaction. 10 pairs of specific primers related to Dx2, Dx2.1, Dx5, Dy10, and Dy12 subunits were used for recognizing baking quality of some wheat varieties and some mutant genotypes. Only 5 pairs of them could show the specific bands. All subunits were recognized by the primers except Dx2.1. Some of the primers were extracted from previous studies and the others were designed based on D genome subunits of wheat. SDS-PAGE method accomplished having confidence in these marker's results. To realize the effect of mutation, seed storage proteins were measured. It showed that mutation had effect on the amount of seed storage protein on the mutant seeds (which showed polymorphism).

PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

PubMed

Villand, P; Aalen, R; Olsen, O A; Lüthi, E; Lönneborg, A; Kleczkowski, L A

1992-06-01

Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

PubMed

Wieczorek, Anna; McHenry, Charles S

2006-05-05

The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

Subunit III-depleted cytochrome c oxidase provides insight into the process of proton uptake by proteins

PubMed Central

Varanasi, Lakshman; Hosler, Jonathan P.

2011-01-01

We review studies of subunit III-depleted cytochrome c oxidase (CcO III (−)) that elucidate the structural basis of steady-state proton uptake from solvent into an internal proton transfer pathway. The removal of subunit III from R. sphaeroides CcO makes proton uptake into the D pathway a rate-determining step, such that measurements of the pH dependence of steady-state O2 consumption can be used to compare the rate and functional pKa of proton uptake by D pathways containing different initial proton acceptors. The removal of subunit III also promotes spontaneous suicide inactivation by CcO, greatly shortening its catalytic lifespan. Because the probability of suicide inactivation is controlled by the rate at which the D pathway delivers protons to the active site, measurements of catalytic lifespan provide a second method to compare the relative efficacy of proton uptake by engineered CcO III (−) forms. These simple experimental systems have been used to explore general questions of proton uptake by proteins, such as the functional value of an initial proton acceptor, whether an initial acceptor must be surface-exposed, which side chains will function as initial proton acceptors and whether multiple acceptors can speed proton uptake. PMID:22023935

Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development.

PubMed

Dezfulian, Mohammad H; Foreman, Curtis; Jalili, Espanta; Pal, Mrinal; Dhaliwal, Rajdeep K; Roberto, Don Karl A; Imre, Kathleen M; Kohalmi, Susanne E; Crosby, William L

2017-04-07

Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. Acetolactate synthase (ALS) is the first enzyme in the BCAA synthesis pathway. Although the functional contribution of ALS to BCAA biosynthesis has been extensively characterized, a comprehensive understanding of the regulation of this pathway at the molecular level is still lacking. To characterize the regulatory processes governing ALS activity we utilized several complementary approaches. Using the ALS catalytic protein subunit as bait we performed a yeast two-hybrid (Y2H) screen which resulted in the identification of a set of interacting proteins, two of which (denoted as ALS-INTERACTING PROTEIN1 and 3 [AIP1 and AIP3, respectively]) were found to be evolutionarily conserved orthologues of bacterial feedback-regulatory proteins and therefore implicated in the regulation of ALS activity. To investigate the molecular role AIPs might play in BCAA synthesis in Arabidopsis thaliana, we examined the functional contribution of aip1 and aip3 knockout alleles to plant patterning and development and BCAA synthesis under various growth conditions. Loss-of-function genetic backgrounds involving these two genes exhibited differential aberrant growth responses in valine-, isoleucine-, and sodium chloride-supplemented media. While BCAA synthesis is believed to be localized to the chloroplast, both AIP1 and AIP3 were found to localize to the peroxisome in addition to the chloroplast. Analysis of free amino acid pools in the mutant backgrounds revealed that they differ in the absolute amount of individual BCAAs accumulated and exhibit elevated levels of BCAAs in leaf tissues. Despite the phenotypic differences observed in aip1 and aip3 backgrounds, functional redundancy between these loci was suggested by the finding that aip1/aip3 double knockout mutants are severely developmentally compromised. Taken together the data suggests that the two regulatory proteins, in conjunction with ALS, have overlapping but distinct functions in BCAA synthesis, and also play a role in pathways unrelated to BCAA synthesis such as sodium-ion homeostasis, extending to broader aspects of patterning and development.

Purification and characterization of an anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol.

PubMed

Meng, Fantao; Xu, Yan

2010-04-01

An anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol was purified by 26-fold to homogeneity. The enzyme had a homodimeric structure consisting of 49 kDa subunits, required NADPH, but not NADH, as a cofactor and was a Zn-independent short-chain dehydrogenase. Aliphatic methyl ketones (chain length > or =6 carbon atoms) and aromatic methyl ketones were the preferred substrates for the enzyme, the best being 2-octanone. Maximum enzyme activity with 2-octanone was at 45 degrees C and at pH 8.0.

Quantifying protein interface footprinting by hydroxyl radical oxidation and molecular dynamics simulation: application to galectin-1.

PubMed

Charvátová, Olga; Foley, B Lachele; Bern, Marshall W; Sharp, Joshua S; Orlando, Ron; Woods, Robert J

2008-11-01

Biomolecular surface mapping methods offer an important alternative method for characterizing protein-protein and protein-ligand interactions in cases in which it is not possible to determine high-resolution three-dimensional (3D) structures of complexes. Hydroxyl radical footprinting offers a significant advance in footprint resolution compared with traditional chemical derivatization. Here we present results of footprinting performed with hydroxyl radicals generated on the nanosecond time scale by laser-induced photodissociation of hydrogen peroxide. We applied this emerging method to a carbohydrate-binding protein, galectin-1. Since galectin-1 occurs as a homodimer, footprinting was employed to characterize the interface of the monomeric subunits. Efficient analysis of the mass spectrometry data for the oxidized protein was achieved with the recently developed ByOnic (Palo Alto, CA) software that was altered to handle the large number of modifications arising from side-chain oxidation. Quantification of the level of oxidation has been achieved by employing spectral intensities for all of the observed oxidation states on a per-residue basis. The level of accuracy achievable from spectral intensities was determined by examination of mixtures of synthetic peptides related to those present after oxidation and tryptic digestion of galectin-1. A direct relationship between side-chain solvent accessibility and level of oxidation emerged, which enabled the prediction of the level of oxidation given the 3D structure of the protein. The precision of this relationship was enhanced through the use of average solvent accessibilities computed from 10 ns molecular dynamics simulations of the protein.

Isolation and characterization of the stage-specific cytochrome b small subunit (CybS) of Ascaris suum complex II from the aerobic respiratory chain of larval mitochondria.

PubMed

Amino, Hisako; Osanai, Arihiro; Miyadera, Hiroko; Shinjyo, Noriko; Tomitsuka, Eriko; Taka, Hikari; Mineki, Reiko; Murayama, Kimie; Takamiya, Shinzaburo; Aoki, Takashi; Miyoshi, Hideto; Sakamoto, Kimitoshi; Kojima, Somei; Kita, Kiyoshi

2003-05-01

We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron-sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS(L)) is distinct from the adult CybS (CybS(A)). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS(L) and CybS(A) in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K(m) values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host.

Molecular cloning and nucleotide sequence of the alpha and beta subunits of allophycocyanin from the cyanelle genome of Cyanophora paradoxa.

PubMed Central

Bryant, D A; de Lorimier, R; Lambert, D H; Dubbs, J M; Stirewalt, V L; Stevens, S E; Porter, R D; Tam, J; Jay, E

1985-01-01

The genes for the alpha- and beta-subunit apoproteins of allophycocyanin (AP) were isolated from the cyanelle genome of Cyanophora paradoxa and subjected to nucleotide sequence analysis. The AP beta-subunit apoprotein gene was localized to a 7.8-kilobase-pair Pst I restriction fragment from cyanelle DNA by hybridization with a tetradecameric oligonucleotide probe. Sequence analysis using that oligonucleotide and its complement as primers for the dideoxy chain-termination sequencing method confirmed the presence of both AP alpha- and beta-subunit genes on this restriction fragment. Additional oligonucleotide primers were synthesized as sequencing progressed and were used to determine rapidly the nucleotide sequence of a 1336-base-pair region of this cloned fragment. This strategy allowed the sequencing to be completed without a detailed restriction map and without extensive and time-consuming subcloning. The sequenced region contains two open reading frames whose deduced amino acid sequences are 81-85% homologous to cyanobacterial and red algal AP subunits whose amino acid sequences have been determined. The two open reading frames are in the same orientation and are separated by 39 base pairs. AP alpha is 5' to AP beta and both coding sequences are preceded by a polypurine, Shine-Dalgarno-type sequence. Sequences upstream from AP alpha closely resemble the Escherichia coli consensus promoter sequences and also show considerable homology to promoter sequences for several chloroplast-encoded psbA genes. A 56-base-pair palindromic sequence downstream from the AP beta gene could play a role in the termination of transcription or translation. The allophycocyanin apoprotein subunit genes are located on the large single-copy region of the cyanelle genome. PMID:2987916

Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer, Chilo suppressalis (Lepidoptera: Crambidae).

PubMed

Xu, Gang; Wu, Shun-Fan; Teng, Zi-Wen; Yao, Hong-Wei; Fang, Qi; Huang, Jia; Ye, Gong-Yin

2017-06-01

Nicotinic acetylcholine receptors (nAChRs) are members of the cys-loop ligand-gated ion channel (cysLGIC) superfamily, mediating fast synaptic cholinergic transmission in the central nervous system in insects. Insect nAChRs are the molecular targets of economically important insecticides, such as neonicotinoids and spinosad. Identification and characterization of the nAChR gene family in the rice striped stem borer, Chilo suppressalis, could provide beneficial information about this important receptor gene family and contribute to the investigation of the molecular modes of insecticide action and resistance for current and future chemical control strategies. We searched our C. suppressalis transcriptome database using Bombyx mori nAChR sequences in local BLAST searches and obtained the putative nAChR subunit complementary DNAs (cDNAs) via reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. Similar to B. mori, C. suppressalis possesses 12 nAChR subunits, including nine α-type and three β-type subunits. Quantitative RT-PCR analysis revealed the expression profiles of the nAChR subunits in various tissues, including the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules. Developmental expression analyses showed clear differential expression of nAChR subunits throughout the C. suppressalis life cycle. The identification of nAChR subunits in this study will provide a foundation for investigating the diverse roles played by nAChRs in C. suppressalis and for exploring specific target sites for chemicals that control agricultural pests while sparing beneficial species. ©2016 The Authors Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.

Preservation of skeletal muscle mitochondrial content in older adults: relationship between mitochondria, fibre type and high-intensity exercise training.

PubMed

Wyckelsma, Victoria L; Levinger, Itamar; McKenna, Michael J; Formosa, Luke E; Ryan, Michael T; Petersen, Aaron C; Anderson, Mitchell J; Murphy, Robyn M

2017-06-01

Ageing is associated with an upregulation of mitochondrial dynamics proteins mitofusin 2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) in human skeletal muscle with the increased abundance of Mfn2 being exclusive to type II muscle fibres. These changes occur despite a similar content of mitochondria, as measured by COXIV, NDUFA9 and complexes in their native states (Blue Native PAGE). Following 12 weeks of high-intensity training (HIT), older adults exhibit a robust increase in mitochondria content, while there is a decline in Mfn2 in type II fibres. We propose that the upregulation of Mfn2 and MiD49 with age may be a protective mechanism to protect against mitochondrial dysfunction, in particularly in type II skeletal muscle fibres, and that exercise may have a unique protective effect negating the need for an increased turnover of mitochondria. Mitochondrial dynamics proteins are critical for mitochondrial turnover and maintenance of mitochondrial health. High-intensity interval training (HIT) is a potent training modality shown to upregulate mitochondrial content in young adults but little is known about the effects of HIT on mitochondrial dynamics proteins in older adults. This study investigated the abundance of protein markers for mitochondrial dynamics and mitochondrial content in older adults compared to young adults. It also investigated the adaptability of mitochondria to 12 weeks of HIT in older adults. Both older and younger adults showed a higher abundance of mitochondrial respiratory chain subunits COXIV and NDUFA9 in type I compared with type II fibres, with no difference between the older adults and young groups. In whole muscle homogenates, older adults had higher mitofusin-2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) contents compared to the young group. Also, older adults had higher levels of Mfn2 in type II fibres compared with young adults. Following HIT in older adults, MiD49 and Mfn2 levels were not different in whole muscle and Mfn2 content decreased in type II fibres. Increases in citrate synthase activity (55%) and mitochondrial respiratory chain subunits COXIV (37%) and NDUFA9 (48%) and mitochondrial respiratory chain complexes (∼70-100%) were observed in homogenates and/or single fibres. These findings reveal (i) a similar amount of mitochondria in muscle from young and healthy older adults and (ii) a robust increase of mitochondrial content following 12 weeks of HIT exercise in older adults. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Transcriptional regulators of Na,K-ATPase subunits

PubMed Central

Li, Zhiqin; Langhans, Sigrid A.

2015-01-01

The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic α-subunit, the β-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids, and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits has been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease. PMID:26579519

Cloning, promoter analysis and expression in response to bacterial exposure of sea bass (Dicentrarchus labrax L.) interleukin-12 p40 and p35 subunits.

PubMed

Nascimento, Diana S; do Vale, Ana; Tomás, Ana M; Zou, Jun; Secombes, Christopher J; dos Santos, Nuno M S

2007-03-01

Interleukin-12 (IL-12) is a heterodimeric cytokine pivotal in resistance to microbial and viral infections. In the search for immunoregulatory genes in sea bass the genes for the two IL-12 subunits p40 and p35 were cloned and sequenced. Molecular characterization of these two genes was performed at both the cDNA and genomic levels. Sea bass IL-12 p40 and p35 conserve most cysteines involved in the intra-chain disulfide bonds of human IL-12 subunits as well as the important structural residues for human IL-12 heterodimerization. The gene organization of sea bass IL-12 p40 is similar to the human orthologue, whilst the sea bass IL-12 p35 gene structure, as reported for pufferfish, differs from the human one in containing an additional exon and lacking a second copy of a duplicated exon present in the mammalian genes. The promoter analysis of both sea bass and pufferfish IL-12 genes showed the presence of the main cis-acting elements involved in the transcriptional regulation of human and mouse orthologues. The involvement of IL-12 in sea bass anti-bacterial immune responses was demonstrated by investigating the expression profiles of IL-1beta, IL-12 p40 and p35 in the head-kidney and spleen following intraperitoneal injection of UV-killed and live Photobacterium damselae ssp. piscicida (Phdp). Finally, the importance of nuclear factor (NF)-kappaB on UV-killed Phdp-induced IL-12 p40 and p35 gene transcription was shown by the use of pyrrolidine dithiocarbamate (PDTC).

Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene.

PubMed

Bai, Y; Hájek, P; Chomyn, A; Chan, E; Seo, B B; Matsuno-Yagi, A; Yagi, T; Attardi, G

2001-10-19

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

Lipid functions in cytochrome bc complexes: an odd evolutionary transition in a membrane protein structure

PubMed Central

Hasan, S. Saif; Cramer, William A.

2012-01-01

Lipid-binding sites and properties were compared in the hetero-oligomeric cytochrome (cyt) b6f and the yeast bc1 complexes that function, respectively, in photosynthetic and respiratory electron transport. Seven lipid-binding sites in the monomeric unit of the dimeric cyanobacterial b6f complex overlap four sites in the Chlamydomonas reinhardtii algal b6f complex and four in the yeast bc1 complex. The proposed lipid functions include: (i) interfacial–interhelix mediation between (a) the two 8-subunit monomers of the dimeric complex, (b) between the core domain (cyt b, subunit IV) and the six trans membrane helices of the peripheral domain (cyt f, iron–sulphur protein (ISP), and four small subunits in the boundary ‘picket fence’); (ii) stabilization of the ISP domain-swapped trans-membrane helix; (iii) neutralization of basic residues in the single helix of cyt f and of the ISP; (iv) a ‘latch’ to photosystem I provided by the β-carotene chain protruding through the ‘picket fence’; (v) presence of a lipid and chlorophyll a chlorin ring in b6f in place of the eighth helix in the bc1 cyt b polypeptide. The question is posed of the function of the lipid substitution in relation to the evolutionary change between the eight and seven helix structures of the cyt b polypeptide. On the basis of the known n-side activation of light harvesting complex II (LHCII) kinase by the p-side level of plastoquinol, one possibility is that the change was directed by the selective advantage of p- to n-side trans membrane signalling functions in b6f, with the lipid either mediating this function or substituting for the trans membrane helix of a signalling protein lost in crystallization. PMID:23148267

Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein.

PubMed

Son, Marjatta; Leary, Scot C; Romain, Nadine; Pierrel, Fabien; Winge, Dennis R; Haller, Ronald G; Elliott, Jeffrey L

2008-05-02

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

Bicuculline reverts the neuroprotective effects of meloxicam in an oxygen and glucose deprivation (OGD) model of organotypic hippocampal slice cultures.

PubMed

Landucci, Elisa; Llorente, Irene L; Anuncibay-Soto, Berta; Pellegrini-Giampietro, Domenico E; Fernández-López, Arsenio

2018-06-24

We previously demonstrated that the non-steroidal anti-inflammatory agent meloxicam has neuroprotective effects in an oxygen and glucose deprivation model (OGD) of rat organotypic hippocampal slice cultures. We wondered if GABAergic transmission changed the neuroprotective effects of meloxicam and if meloxicam was able to modulate endoplasmic reticulum stress (ER stress) in this model. Mortality was measured using propidium iodide. Western blot assays were performed to measure levels of cleaved and non-cleaved caspase-3 to quantify apoptosis, while levels of GRP78, GRP94 and phosphorylated eIF2α were used to detect unfolded protein response (UPR). Transcript levels of GRP78, GRP94 and GABAergic receptor α, β, and γ subunits were measured by real-time quantitative polymerase chain reaction (qPCR). In the present study, we show that the presence of meloxicam in a 30 min OGD assay, followed by 24 h of normoxic conditions, presented an antiapoptotic effect. The simultaneous presence of the GABA A receptor antagonist, bicuculline, in combination with meloxicam blocked the neuroprotective effect provided by the latter. However, in light of its effects on caspase 3 and PARP, bicuculline did not seem to promote the apoptotic pathway. Our results also showed that meloxicam modified the unfolded protein response (UPR), as well as the transcriptional response of different genes, including the GABA A receptor, alpha1, beta3 and gamma2 subunits. We concluded that meloxicam has a neuroprotective anti-apoptotic action, is able to enhance the UPR independently of the systemic anti-inflammatory response and its neuroprotective effect can be inhibited by blocking GABA A receptors. Copyright © 2018. Published by Elsevier Ltd.

Localization of type IV collagen a 1 to a 6 chains in basement membrane during mouse molar germ development.

PubMed

Nagai, N; Nakano, K; Sado, Y; Naito, I; Gunduz, M; Tsujigiwa, H; Nagatsuka, H; Ninomiya, Y; Siar, C H

2001-10-01

The dental basement membrane (BM) putatively mediates epithelial-mesenchymal interactions during tooth morphogenesis and cytodifferentiation. Type IV collagen alpha chains, a major network-forming protein of the dental BM, was studied and results disclosed distinct expression patterns at different stages of mouse molar germ development. At the dental placode and bud stage, the BM of the oral epithelium expressed alpha 1, alpha 2, alpha 5 and alpha 6 chains while the gubernaculum dentis, in addition to the above four chains, also expressed a 4 chain. An asymmetrical expression for alpha 4, alpha 5 and alpha 6 chains was observed at the bud stage. At the early bell stage, the BM associated with the inner enamel epithelium (IEE) of molar germ expressed alpha 1, alpha 2 and alpha 4 chains while the BM of the outer enamel epithelium (OEE) expressed only alpha 1 and a 2 chains. With the onset of dentinogenesis, the collagen a chain profile of the IEE BM gradually disappeared. Howeverfrom the early to late bell stage, the gubernaculum dentis consistently expressed alpha 1, alpha 2, alpha 5 and a 6 chains resembling fetal oral mucosa. These findings suggest that stage- and position-specific distribution of type IV collagen alpha subunits occur during molar germ development and that these changes are essential for molar morphogenesis and cytodifferentiation.

5-Fluoroindole Resistance Identifies Tryptophan Synthase Beta Subunit Mutants in Arabidopsis Thaliana

PubMed Central

Barczak, A. J.; Zhao, J.; Pruitt, K. D.; Last, R. L.

1995-01-01

A study of the biochemical genetics of the Arabidopsis thaliana tryptophan synthase beta subunit was initiated by characterization of mutants resistant to the inhibitor 5-fluoroindole. Thirteen recessive mutations were recovered that are allelic to trp2-1, a mutation in the more highly expressed of duplicate tryptophan synthase beta subunit genes (TSB1). Ten of these mutations (trp2-2 through trp2-11) cause a tryptophan requirement (auxotrophs), whereas three (trp2-100 through trp2-102) remain tryptophan prototrophs. The mutations cause a variety of changes in tryptophan synthase beta expression. For example, two mutations (trp2-5 and trp2-8) cause dramatically reduced accumulation of TSB mRNA and immunologically detectable protein, whereas trp2-10 is associated with increased mRNA and protein. A correlation exists between the quantity of mutant beta and wild-type alpha subunit levels in the trp2 mutant plants, suggesting that the synthesis of these proteins is coordinated or that the quantity or structure of the beta subunit influences the stability of the alpha protein. The level of immunologically detectable anthranilate synthase alpha subunit protein is increased in the trp2 mutants, suggesting the possibility of regulation of anthranilate synthase levels in response to tryptophan limitation. PMID:7635295

Propofol effectively inhibits lithium-pilocarpine- induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

PubMed Central

Wang, Henglin; Wang, Zhuoqiang; Mi, Weidong; Zhao, Cong; Liu, Yanqin; Wang, Yongan; Sun, Haipeng

2012-01-01

Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine. The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior, electroencephalography and 24-hour survival rate. Propofol (12.5–100 mg/kg) improved status epilepticus in a dose-dependent manner, and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection. Western blot results showed that, 24 hours after induction of status epilepticus, the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus. Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels, but not the increase in N-methyl-D-aspartate receptor 2A subunit levels. The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine. This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures. PMID:25737709

Heterotrimeric G Stimulatory Protein α Subunit Is Required for Intestinal Smooth Muscle Contraction in Mice.

PubMed

Qin, Xiaoteng; Liu, Shangming; Lu, Qiulun; Zhang, Meng; Jiang, Xiuxin; Hu, Sanyuan; Li, Jingxin; Zhang, Cheng; Gao, Jiangang; Zhu, Min-Sheng; Feil, Robert; Li, Huashun; Chen, Min; Weinstein, Lee S; Zhang, Yun; Zhang, Wencheng

2017-04-01

The α subunit of the heterotrimeric G stimulatory protein (Gsa), encoded by the guanine nucleotide binding protein, α-stimulating gene (Gnas, in mice), is expressed ubiquitously and mediates receptor-stimulated production of cyclic adenosine monophosphate and activation of the protein kinase A signaling pathway. We investigated the roles of Gsa in vivo in smooth muscle cells of mice. We performed studies of mice with Cre recombinase-mediated disruption of Gnas in smooth muscle cells (Gsa SMKO and SM22-CreER T2 , induced in adult mice by tamoxifen). Intestinal tissues were collected for histologic, biochemical, molecular, cell biology, and physiology analyses. Intestinal function was assessed in mice using the whole-gut transit time test. We compared gene expression patterns of intestinal smooth muscle from mice with vs without disruption of Gnas. Biopsy specimens from ileum of patients with chronic intestinal pseudo-obstruction and age-matched control biopsies were analyzed by immunohistochemistry. Disruption of Gnas in smooth muscle of mice reduced intestinal motility and led to death within 4 weeks. Tamoxifen-induced disruption of Gnas in adult mice impaired contraction of intestinal smooth muscle and peristalsis. More than 80% of these died within 3 months of tamoxifen exposure, with features of intestinal pseudo-obstruction characterized by chronic intestinal dilation and dysmotility. Gsa deficiency reduced intestinal levels of cyclic adenosine monophosphate and transcriptional activity of the cyclic adenosine monophosphate response element binding protein 1 (CREB1); this resulted in decreased expression of the forkhead box F1 gene (Foxf1) and protein, and contractile proteins, such as myosin heavy chain 11; actin, α2, smooth muscle, aorta; calponin 1; and myosin light chain kinase. We found decreased levels of Gsa, FOXF1, CREB1, and phosphorylated CREB1 proteins in intestinal muscle layers of patients with chronic intestinal pseudo-obstruction, compared with tissues from controls. Gsa is required for intestinal smooth muscle contraction in mice, and its levels are reduced in ileum biopsies of patients with chronic intestinal pseudo-obstruction. Mice with disruption of Gnas might be used to study human chronic intestinal pseudo-obstruction. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Linear ubiquitin chains: enzymes, mechanisms and biology

PubMed Central

2017-01-01

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases. PMID:28446710

Linear ubiquitin chains: enzymes, mechanisms and biology.

PubMed

Rittinger, Katrin; Ikeda, Fumiyo

2017-04-01

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases. © 2017 The Authors.

Expression of NMDA receptor subunits in human blood lymphocytes: A peripheral biomarker in online computer game addiction.

PubMed

Sadat-Shirazi, Mitra-Sadat; Vousooghi, Nasim; Alizadeh, Bentolhoda; Makki, Seyed Mohammad; Zarei, Seyed Zeinolabedin; Nazari, Shahrzad; Zarrindast, Mohammad Reza

2018-05-23

Background and aims Repeated performance of some behaviors such as playing computer games could result in addiction. The NMDA receptor is critically involved in the development of behavioral and drug addictions. It has been claimed that the expression level of neurotransmitter receptors in the brain may be reflected in peripheral blood lymphocytes (PBLs). Methods Here, using a real-time PCR method, we have investigated the mRNA expression of GluN2A, GluN2D, GluN3A, and GluN3B subunits of the NMDA receptor in PBLs of male online computer game addicts (n = 25) in comparison with normal subjects (n = 26). Results Expression levels of GluN2A, GluN2D, and GluN3B subunits were not statistically different between game addicts and the control group. However, the mRNA expression of the GluN3A subunit was downregulated in PBLs of game addicts. Discussion and conclusions Transcriptional levels of GluN2A and GluN2D subunits in online computer game addicts are similar to our previously reported data of opioid addiction and are not different from the control group. However, unlike our earlier finding of drug addiction, the mRNA expression levels of GluN3A and GluN3B subunits in PBLs of game addicts are reduced and unchanged, respectively, compared with control subjects. It seems that the downregulated state of the GluN3A subunit of NMDA receptor in online computer game addicts is a finding that deserves more studies in the future to see whether it can serve as a peripheral biomarker in addiction studies, where the researcher wants to rule out the confusing effects of abused drugs.

Decreased GRK3 but not GRK2 expression in frontal cortex from bipolar disorder patients

PubMed Central

Rao, Jagadeesh S; Rapoport, Stanley I; Kim, Hyung-Wook

2009-01-01

Overactivation of G-protein mediated functions and altered G-protein regulation have been reported in bipolar disorder (BD) brain. Further, drugs effective in treating BD are reported to upregulate expression of G-protein receptor kinase (GRK) 3 in rat frontal cortex. We therefore hypothesized that some G-protein subunits and GRK levels would be reduced in the brains of BD patients. We determined protein and mRNA levels of G-protein β and γ subunits, GRK2, and GRK3 in postmortem frontal cortex from 10 BD patients and 10 age-matched controls by using immunoblots and real-time RT-PCR. There were the statistically significant decreases in protein and mRNA levels of G-protein subunits β and γ and of GRK3 in the BD brains but not a significant difference in the GRK2 level. Decreased expression of G-protein subunits and of GRK3 may alter neurotransmission, leading to disturbed cognition and behavior in BD. PMID:19400979

Change of subunit composition of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in Ascaris suum during the migration in the experimental host.

PubMed

Iwata, Fumiko; Shinjyo, Noriko; Amino, Hisako; Sakamoto, Kimitoshi; Islam, M Khyrul; Tsuji, Naotoshi; Kita, Kiyoshi

2008-03-01

The mitochondrial metabolic pathway of the parasitic nematode Ascaris suum changes dramatically during its life cycle, to adapt to changes in the environmental oxygen concentration. We previously showed that A. suum mitochondria express stage-specific isoforms of complex II (succinate-ubiquinone reductase: SQR/quinol-fumarate reductase: QFR). The flavoprotein (Fp) and small subunit of cytochrome b (CybS) in adult complex II differ from those of infective third stage larval (L3) complex II. However, there is no difference in the iron-sulfur cluster (Ip) or the large subunit of cytochrome b (CybL) between adult and L3 isoforms of complex II. In the present study, to clarify the changes that occur in the respiratory chain of A. suum larvae during their migration in the host, we examined enzymatic activity, quinone content and complex II subunit composition in mitochondria of lung stage L3 (LL3) A. suum larvae. LL3 mitochondria showed higher QFR activity ( approximately 160 nmol/min/mg) than mitochondria of A. suum at other stages (L3: approximately 80 nmol/min/mg; adult: approximately 70 nmol/min/mg). Ubiquinone content in LL3 mitochondria was more abundant than rhodoquinone ( approximately 1.8 nmol/mg versus approximately 0.9 nmol/mg). Interestingly, the results of two-dimensional bule-native/sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses showed that LL3 mitochondria contained larval Fp (Fp(L)) and adult Fp (Fp(A)) at a ratio of 1:0.56, and that most LL3 CybS subunits were of the adult form (CybS(A)). This clearly indicates that the rearrangement of complex II begins with a change in the isoform of the anchor CybS subunit, followed by a similar change in the Fp subunit.

Electronic cigarette aerosols and copper nanoparticles induce mitochondrial stress and promote DNA fragmentation in lung fibroblasts.

PubMed

Lerner, Chad A; Rutagarama, Pierrot; Ahmad, Tanveer; Sundar, Isaac K; Elder, Alison; Rahman, Irfan

2016-09-02

Oxidants or nanoparticles have recently been identified as constituents of aerosols released from various styles of electronic cigarettes (E-cigs). Cells in the lung may be directly exposed to these constituents and harbor reactive properties capable of incurring acute cell injury. Our results show mitochondria are sensitive to both E-cig aerosols and aerosol containing copper nanoparticles when exposed to human lung fibroblasts (HFL-1) using an Air-Liquid Interface culture system, evident by elevated levels of mitochondrial ROS (mtROS). Increased mtROS after aerosol exposure is associated with reduced stability of OxPhos electron transport chain (ETC) complex IV subunit and nuclear DNA fragmentation. Increased levels of IL-8 and IL-6 in HFL-1 conditioned media were also observed. These findings reveal both mitochondrial, genotoxic, and inflammatory stresses are features of direct cell exposure to E-cig aerosols which are ensued by inflammatory duress, raising a concern on deleterious effect of vaping. Copyright © 2016 Elsevier Inc. All rights reserved.

The sodium pump α1 sub-unit: a disease progression–related target for metastatic melanoma treatment

PubMed Central

Mathieu, Véronique; Pirker, Christine; Martin de Lassalle, Elisabeth; Vernier, Mathieu; Mijatovic, Tatjana; DeNeve, Nancy; Gaussin, Jean-François; Dehoux, Mischael; Lefranc, Florence; Berger, Walter; Kiss, Robert

2009-01-01

Melanomas remain associated with dismal prognosis because they are naturally resistant to apoptosis and they markedly metastasize. Up-regulated expression of sodium pump α sub-units has previously been demonstrated when comparing metastatic to non-metastatic melanomas. Our previous data revealed that impairing sodium pump α1 activity by means of selective ligands, that are cardiotonic steroids, markedly impairs cell migration and kills apoptosis-resistant cancer cells. The objective of this study was to determine the expression levels of sodium pump α sub-units in melanoma clinical samples and cell lines and also to characterize the role of α1 sub-units in melanoma cell biology. Quantitative RT-PCR, Western blotting and immunohistochemistry were used to determine the expression levels of sodium pump α sub-units. In vitro cytotoxicity of various cardenolides and of an anti-α1 siRNA was evaluated by means of MTT assay, quantitative videomicroscopy and through apoptosis assays. The in vivo activity of a novel cardenolide UNBS1450 was evaluated in a melanoma brain metastasis model. Our data show that all investigated human melanoma cell lines expressed high levels of the α1 sub-unit, and 33% of human melanomas displayed significant α1 sub-unit expression in correlation with the Breslow index. Furthermore, cardenolides (notably UNBS1450; currently in Phase I clinical trials) displayed marked anti-tumour effects against melanomas in vitro. This activity was closely paralleled by decreases in cMyc expression and by increases in apoptotic features. UNBS1450 also displayed marked anti-tumour activity in the aggressive human metastatic brain melanoma model in vivo. The α1 sodium pump sub-unit could represent a potential novel target for combating melanoma. PMID:19243476

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2.

PubMed

Neviani, E; Boquien, C Y; Monnet, V; Thanh, L P; Gripon, J C

1989-09-01

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.

7 CFR 275.7 - Selection of sub-units for review.

Code of Federal Regulations, 2014 CFR

2014-01-01

... situations, the sub-unit would be classified based upon its primary function exclusively. However, when any... classification and if selected for review, all functions performed shall be examined. For example, if a sub-unit... functions related to more than one of the areas. For example, coupon issuers must maintain a level of coupon...

7 CFR 275.7 - Selection of sub-units for review.

Code of Federal Regulations, 2011 CFR

2011-01-01

... situations, the sub-unit would be classified based upon its primary function exclusively. However, when any... classification and if selected for review, all functions performed shall be examined. For example, if a sub-unit... functions related to more than one of the areas. For example, coupon issuers must maintain a level of coupon...

7 CFR 275.7 - Selection of sub-units for review.

Code of Federal Regulations, 2012 CFR

2012-01-01

... situations, the sub-unit would be classified based upon its primary function exclusively. However, when any... classification and if selected for review, all functions performed shall be examined. For example, if a sub-unit... functions related to more than one of the areas. For example, coupon issuers must maintain a level of coupon...

7 CFR 275.7 - Selection of sub-units for review.

Code of Federal Regulations, 2013 CFR

2013-01-01

... situations, the sub-unit would be classified based upon its primary function exclusively. However, when any... classification and if selected for review, all functions performed shall be examined. For example, if a sub-unit... functions related to more than one of the areas. For example, coupon issuers must maintain a level of coupon...

A traffic signal for heterodimeric amino acid transporters to transfer from the ER to the Golgi.

PubMed

Ganapathy, Vadivel

2009-01-15

Heterodimeric amino acid transporters represent a unique class of transport systems that consist of a light chain that serves as the 'transporter proper' and a heavy chain that is necessary for targeting the complex to the plasma membrane. The currently prevailing paradigm assigns no role for the light chains in the cellular processing of these transporters. In this issue of the Biochemical Journal, Sakamoto et al. provide evidence contrary to this paradigm. Their studies with the rBAT -b(0,+)AT (related to b(0,+) amino acid transporter-b(0,+)-type amino acid transporter) heterodimeric amino acid transporter show that the C-terminus of the light chain b(0,+)AT contains a sequence motif that serves as the traffic signal for the transfer of the heterodimeric complex from the endoplasmic reticulum to the Golgi. This is a novel function for the light chain in addition to its already established role as the subunit responsible for the transport activity. These new findings also seem to be applicable to other heterodimeric amino acid transporters as well.

Congenitally learned helpless rats show abnormalities in intracellular signaling.

PubMed

Kohen, Ruth; Neumaier, John F; Hamblin, Mark W; Edwards, Emmeline

2003-03-15

Affective disorders and the drugs used to treat them lead to changes in intracellular signaling. We used a genetic animal model to investigate to what extent changes in intracellular signal transduction confer a vulnerability to mood or anxiety disorders. Levels of gene expression in a selectively bred strain of rats with a high vulnerability to develop congenitally learned helplessness (cLH), a strain highly resistant to the same behavior (cNLH) and outbred Sprague-Dawley (SD) control animals were compared using quantitative reverse transcription polymerase chain reaction. Congenitally learned helpless animals had a 24%-30% reduced expression of the cyclic adenosine monophosphate response element binding protein messenger ribonucleic acid (mRNA) in the hippocampus and a 40%-41% increased level of the antiapoptotic protein bcl-2 mRNA in the prefrontal cortex compared to cNLH and SD rats. Other significant changes included changes in the expression levels of the alpha catalytic subunit of protein kinase A, glycogen synthase kinase 3beta, and protein kinase C epsilon. Congenitally learned helpless animals show evidence of altered signal transduction and regulation of apoptosis compared to cNLH and SD control animals.

The devil is in the details: comparison between COP9 signalosome (CSN) and the LID of the 26S proteasome.

PubMed

Meister, Cindy; Gulko, Miriam Kolog; Köhler, Anna M; Braus, Gerhard H

2016-02-01

The COP9 signalosome (CSN) and the proteasomal LID are conserved macromolecular complexes composed of at least eight subunits with molecular weights of approximately 350 kDa. CSN and LID are part of the ubiquitin–proteasome pathway and cleave isopeptide linkages of lysine side chains on target proteins. CSN cleaves the isopeptide bond of ubiquitin-like protein Nedd8 from cullins, whereas the LID cleaves ubiquitin from target proteins sentenced for degradation. CSN and LID are structurally and functionally similar but the order of the assembly pathway seems to be different. The assembly differs in at least the last subunit joining the pre-assembled subcomplex. This review addresses the similarities and differences in structure, function and assembly of CSN and LID.

QIL1 mutation causes MICOS disassembly and early onset fatal mitochondrial encephalopathy with liver disease

PubMed Central

Guarani, Virginia; Jardel, Claude; Chrétien, Dominique; Lombès, Anne; Bénit, Paule; Labasse, Clémence; Lacène, Emmanuelle; Bourillon, Agnès; Imbard, Apolline; Benoist, Jean-François; Dorboz, Imen; Gilleron, Mylène; Goetzman, Eric S; Gaignard, Pauline; Slama, Abdelhamid; Elmaleh-Bergès, Monique; Romero, Norma B; Rustin, Pierre; Ogier de Baulny, Hélène; Paulo, Joao A; Harper, J Wade; Schiff, Manuel

2016-01-01

Previously, we identified QIL1 as a subunit of mitochondrial contact site (MICOS) complex and demonstrated a role for QIL1 in MICOS assembly, mitochondrial respiration, and cristae formation critical for mitochondrial architecture (Guarani et al., 2015). Here, we identify QIL1 null alleles in two siblings displaying multiple clinical symptoms of early-onset fatal mitochondrial encephalopathy with liver disease, including defects in respiratory chain function in patient muscle. QIL1 absence in patients’ fibroblasts was associated with MICOS disassembly, abnormal cristae, mild cytochrome c oxidase defect, and sensitivity to glucose withdrawal. QIL1 expression rescued cristae defects, and promoted re-accumulation of MICOS subunits to facilitate MICOS assembly. MICOS assembly and cristae morphology were not efficiently rescued by over-expression of other MICOS subunits in patient fibroblasts. Taken together, these data provide the first evidence of altered MICOS assembly linked with a human mitochondrial disease and confirm a central role for QIL1 in stable MICOS complex formation. DOI: http://dx.doi.org/10.7554/eLife.17163.001 PMID:27623147

Mutant NDUFS3 subunit of mitochondrial complex I causes Leigh syndrome.

PubMed

Bénit, P; Slama, A; Cartault, F; Giurgea, I; Chretien, D; Lebon, S; Marsac, C; Munnich, A; Rötig, A; Rustin, P

2004-01-01

Respiratory chain complex I deficiency represents a genetically heterogeneous group of diseases resulting from mutations in mitochondrial or nuclear genes. Mutations have been reported in 13 of the 14 subunits encoding the core of complex I (seven mitochondrial and six nuclear genes) and these result in Leigh or Leigh-like syndromes or cardiomyopathy. In this study, a combination of denaturing high performance liquid chromatography and sequence analysis was used to study the NDUFS3 gene in a series of complex I deficient patients. Mutations found in this gene (NADH dehydrogenase iron-sulphur protein 3), coding for the seventh and last subunit of complex I core, were shown to cause late onset Leigh syndrome, optic atrophy, and complex I deficiency. A biochemical diagnosis of complex I deficiency on cultured amniocytes from a later pregnancy was confirmed through the identification of disease causing NDUFS3 mutations in these cells. While mutations in the NDUFS3 gene thus result in Leigh syndrome, a dissimilar clinical phenotype is observed in mutations in the NDUFV2 and NDUFS2 genes, resulting in encephalomyopathy and cardiomyopathy. The reasons for these differences are uncertain.

A novel function of twins, B subunit of protein phosphatase 2A, in regulating actin polymerization.

PubMed

Yeh, Po-An; Chang, Ching-Jin

2017-01-01

Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation.

A novel function of twins, B subunit of protein phosphatase 2A, in regulating actin polymerization

PubMed Central

Chang, Ching-Jin

2017-01-01

Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation. PMID:28977036

Crystal structure of bovine Cu,Zn superoxide dismutase at 3 A resolution: chain tracing and metal ligands.

PubMed Central

Richardson, J; Thomas, K A; Rubin, B H; Richardson, D C

1975-01-01

An electron density map at 3 angstrom resolution has been calculated for Cu2+, Zn2+ superoxide dismutase from bovine erythrocytes, and the course of the main chain has been traced. The dominant structural feature is an 8-stranded barrel of antiparallel beta-pleated sheet. There is one very short helical section and two long loops of non-repetitive structure. The Cu and Zn are bound between the loops and one side of the beta barrel and are about 6 Angstrom apart, with a common histidine ligand. The Cu has four histidine ligands in a somewhat distorted square plane, and the Zn has three histidines and an aspartate in approximately tetrahedral arrangement. The two coppers of a dimer are about 34 Angstrom apart. The two subunits have essentially the same conformation and have an extensive contact area that mainly involves hydrophobic side chain interactions. The overall folding pattern of the polypeptide chain is very similar to that of an immunoglobulin domain. Images PMID:1055410

The nicotinic acetylcholine receptor gene family of the silkworm, Bombyx mori

PubMed Central

Shao, Ya-Ming; Dong, Ke; Zhang, Chuan-Xi

2007-01-01

Background Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic cholinergic transmission in the insect central nervous system. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Like mammalian nAChRs, insect nAChRs are considered to be made up of five subunits, coded by homologous genes belonging to the same family. The nAChR subunit genes of Drosophila melanogaster, Apis mellifera and Anopheles gambiae have been cloned previously based on their genome sequences. The silkworm Bombyx mori is a model insect of Lepidoptera, among which are many agricultural pests. Identification and characterization of B. mori nAChR genes could provide valuable basic information for this important family of receptor genes and for the study of the molecular mechanisms of neonicotinoid action and resistance. Results We searched the genome sequence database of B. mori with the fruit fly and honeybee nAChRs by tBlastn and cloned all putative silkworm nAChR cDNAs by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. B. mori appears to have the largest known insect nAChR gene family to date, including nine α-type subunits and three β-type subunits. The silkworm possesses three genes having low identity with others, including one α and two β subunits, α9, β2 and β3. Like the fruit fly and honeybee counterparts, silkworm nAChR gene α6 has RNA-editing sites, and α4, α6 and α8 undergo alternative splicing. In particular, alternative exon 7 of Bmα8 may have arisen from a recent duplication event. Truncated transcripts were found for Bmα4 and Bmα5. Conclusion B. mori possesses a largest known insect nAChR gene family characterized to date, including nine α-type subunits and three β-type subunits. RNA-editing, alternative splicing and truncated transcripts were found in several subunit genes, which might enhance the diversity of the gene family. PMID:17868469

Structure of the aspartic protease from Rous sarcoma retrovirus refined at 2-A resolution.

PubMed

Jaskólski, M; Miller, M; Rao, J K; Leis, J; Wlodawer, A

1990-06-26

The structure of Rous sarcoma virus protease has been solved by multiple isomorphous replacement in the crystal form belonging to space group P3(1)21, with unit-cell parameters a = 88.95 A and c = 78.90 A. The enzyme belongs to the family of aspartic proteases with two identical subunits composing the active homodimer. The noncrystallographic dyad relating these two subunits was identified after preliminary tracing in the MIR map and was used for phase improvement by electron-density averaging. Structure refinement resulted in a model that included 1772 protein atoms and 252 water molecules, with an R factor of 0.144 for data extending to 2 A. The secondary structure of a retroviral protease molecule closely resembles that of a single domain in pepsin-like aspartic proteases and consists of several beta-strands and of one well-defined and one distorted alpha-helix. The dimer interface is composed of the N- and C-terminal chains from both subunits which are intertwined to form a well-ordered four-stranded antiparallel beta-sheet. In each monomer, the catalytic triad (Asp-Ser-Gly) is located in a loop that forms a part of the psi-structure characteristic to all aspartic proteases. The position of a water molecule between the active-site aspartate residues and the general scheme of H bonding within the active site bear close resemblance to those in pepsin-like aspartic proteases and therefore suggest a similar enzymatic mechanism. The binding cleft over the active site is covered by two flap arms, one from each monomer, which are partially disordered. The retroviral protease dimer has been compared with several enzymes of cellular origin, with chains aligning to an rms deviation of 1.90 A or better.

NADPH oxidase and redox status in amygdala, hippocampus and cortex of male Wistar rats in an animal model of post-traumatic stress disorder.

PubMed

Petrovic, Romana; Puskas, Laslo; Jevtic Dozudic, Gordana; Stojkovic, Tihomir; Velimirovic, Milica; Nikolic, Tatjana; Zivkovic, Milica; Djorovic, Djordje J; Nenadovic, Milutin; Petronijevic, Natasa

2018-05-26

Post-traumatic stress disorder (PTSD) is a highly prevalent and impairing disorder. Oxidative stress is implicated in its pathogenesis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important source of free radicals. The aim of the study was to assess oxidative stress parameters, activities of respiratory chain enzymes, and the expression of NADPH oxidase subunits (gp91phox, p22phox, and p67phox) in the single prolonged stress (SPS) animal model of PTSD. Twenty-four (12 controls; 12 subjected to SPS), 9-week-old, male Wistar rats were used. SPS included physical restraint, forced swimming, and ether exposure. The rats were euthanized seven days later. Cortex, hippocampus, amygdala, and thalamus were dissected. Malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), Complex I, and cytochrome C oxidase were measured using spectrophotometric methods, while the expression of NADPH oxidase subunits was determined by Western blot. Increased MDA and decreased GSH concentrations were found in the amygdala and hippocampus of the SPS rats. SOD activity was decreased in amygdala and GPx was decreased in hippocampus. Increased expression of the NADPH oxidase subunits was seen in amygdala, while mitochondrial respiratory chain enzyme expression was unchanged both in amygdala and hippocampus. In the cortex concentrations of MDA and GSH were unchanged despite increased Complex I and decreased GPx, while in the thalamus no change of any parameter was noticed. We conclude that oxidative stress is present in hippocampus and amygdala seven days after the SPS procedure. NADPH oxidase seems to be a main source of free radicals in the amygdala.

The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction.

PubMed

Wang, L M; Michieli, P; Lie, W R; Liu, F; Lee, C C; Minty, A; Sun, X J; Levine, A; White, M F; Pierce, J H

1995-12-01

Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3-dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin-like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL-13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.

Structure of the alternative complex III in a supercomplex with cytochrome oxidase.

PubMed

Sun, Chang; Benlekbir, Samir; Venkatakrishnan, Padmaja; Wang, Yuhang; Hong, Sangjin; Hosler, Jonathan; Tajkhorshid, Emad; Rubinstein, John L; Gennis, Robert B

2018-05-01

Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria 1-3 . Like complex III (also known as the bc 1 complex), ACIII catalyses the oxidation of membrane-bound quinol and the reduction of cytochrome c or an equivalent electron carrier. However, the two complexes have no structural similarity 4-7 . Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron-sulfur molybdoenzyme (CISM) superfamily 8 , including the proton pump polysulfide reductase 9,10 . We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer 11-14 , both as an independent enzyme and as a functional 1:1 supercomplex with an aa 3 -type cytochrome c oxidase (cyt aa 3 ). We determined the structure of ACIII to 3.4 Å resolution by cryo-electron microscopy and constructed an atomic model for its six subunits. The structure, which contains a [3Fe-4S] cluster, a [4Fe-4S] cluster and six haem c units, shows that ACIII uses known elements from other electron transport complexes arranged in a previously unknown manner. Modelling of the cyt aa 3 component of the supercomplex revealed that it is structurally modified to facilitate association with ACIII, illustrating the importance of the supercomplex in this electron transport chain. The structure also resolves two of the subunits of ACIII that are anchored to the lipid bilayer with N-terminal triacylated cysteine residues, an important post-translational modification found in numerous prokaryotic membrane proteins that has not previously been observed structurally in a lipid bilayer.

Huntingtin-interacting protein 1 (Hip1) and Hip1-related protein (Hip1R) bind the conserved sequence of clathrin light chains and thereby influence clathrin assembly in vitro and actin distribution in vivo.

PubMed

Chen, Chih-Ying; Brodsky, Frances M

2005-02-18

Clathrin heavy and light chains form triskelia, which assemble into polyhedral coats of membrane vesicles that mediate transport for endocytosis and organelle biogenesis. Light chain subunits regulate clathrin assembly in vitro by suppressing spontaneous self-assembly of the heavy chains. The residues that play this regulatory role are at the N terminus of a conserved 22-amino acid sequence that is shared by all vertebrate light chains. Here we show that these regulatory residues and others in the conserved sequence mediate light chain interaction with Hip1 and Hip1R. These related proteins were previously found to be enriched in clathrin-coated vesicles and to promote clathrin assembly in vitro. We demonstrate Hip1R binding preference for light chains associated with clathrin heavy chain and show that Hip1R stimulation of clathrin assembly in vitro is blocked by mutations in the conserved sequence of light chains that abolish interaction with Hip1 and Hip1R. In vivo overexpression of a fragment of clathrin light chain comprising the Hip1R-binding region affected cellular actin distribution. Together these results suggest that the roles of Hip1 and Hip1R in affecting clathrin assembly and actin distribution are mediated by their interaction with the conserved sequence of clathrin light chains.

Permethrin Induces Overexpression of Cytochrome c Oxidase Subunit 3 in Aedes aegypti

USDA-ARS?s Scientific Manuscript database

Using quantitative PCR (QPCR), the relative transcriptional levels of cytochrome c oxidase subunit 3 (CO3) were studied in Aedes aegypti (L.) in response to treatments with acetone, permethrin, or fipronil. The transcriptional levels of CO3 were significantly (p <0.05) higher in acetone-treated Ae. ...

A novel deficiency of mitochondrial ATPase of nuclear origin.

PubMed

Houstek, J; Klement, P; Floryk, D; Antonická, H; Hermanská, J; Kalous, M; Hansíková, H; Hout'ková, H; Chowdhury, S K; Rosipal, T; Kmoch, S; Stratilová, L; Zeman, J

1999-10-01

We report a new type of fatal mitochondrial disorder caused by selective deficiency of mitochondrial ATP synthase (ATPase). A hypotrophic newborn from a consanguineous marriage presented severe lactic acidosis, cardiomegaly and hepatomegaly and died from heart failure after 2 days. The activity of oligomycin-sensitive ATPase was only 31-34% of the control, both in muscle and heart, but the activities of cytochrome c oxidase, citrate synthase and pyruvate dehydrogenase were normal. Electrophoretic and western blot analysis revealed selective reduction of ATPase complex but normal levels of the respiratory chain complexes I, III and IV. The same selective deficiency of ATPase was found in cultured skin fibroblasts which showed similar decreases in ATPase content, ATPase hydrolytic activity and level of substrate-dependent ATP synthesis (20-25, 18 and 29-33% of the control, respectively). Pulse-chase labelling of patient fibroblasts revealed low incorporation of [(35)S]methionine into assembled ATPase complexes, but increased incorporation into immunoprecipitated ATPase subunit beta, which had a very short half-life. In contrast, no difference was found in the size and subunit composition of the assembled and newly produced ATPase complex. Transmitochondrial cybrids prepared from enucleated fibroblasts of the patient and rho degrees cells derived from 143B. TK(-)human osteosarcoma cells fully restored the ATPase activity, ATP synthesis and ATPase content, when compared with control cybrids. Likewise, the pattern of [(35)S]methionine labelling of ATPase was found to be normal in patient cybrids. We conclude that the generalized deficiency of mitochondrial ATPase described is of nuclear origin and is caused by altered biosynthesis of the enzyme.

Differential Expression of Glutamate Receptors in Avian Neural Pathways for Learned Vocalization

PubMed Central

WADA, KAZUHIRO; SAKAGUCHI, HIRONOBU; JARVIS, ERICH D.; HAGIWARA, MASATOSHI

2008-01-01

Learned vocalization, the substrate for human language, is a rare trait. It is found in three distantly related groups of birds—parrots, hummingbirds, and songbirds. These three groups contain cerebral vocal nuclei for learned vocalization not found in their more closely related vocal nonlearning relatives. Here, we cloned 21 receptor subunits/subtypes of all four glutamate receptor families (AMPA, kainate, NMDA, and metabotropic) and examined their expression in vocal nuclei of songbirds. We also examined expression of a subset of these receptors in vocal nuclei of hummingbirds and parrots, as well as in the brains of dove species as examples of close vocal nonlearning relatives. Among the 21 subunits/subtypes, 19 showed higher and/or lower prominent differential expression in songbird vocal nuclei relative to the surrounding brain subdivisions in which the vocal nuclei are located. This included relatively lower levels of all four AMPA subunits in lMAN, strikingly higher levels of the kainite subunit GluR5 in the robust nucleus of the arcopallium (RA), higher and lower levels respectively of the NMDA subunits NR2A and NR2B in most vocal nuclei and lower levels of the metabotropic group I subtypes (mGluR1 and -5) in most vocal nuclei and the group II subtype (mGluR2), showing a unique expression pattern of very low levels in RA and very high levels in HVC. The splice variants of AMPA subunits showed further differential expression in vocal nuclei. Some of the receptor subunits/subtypes also showed differential expression in hummingbird and parrot vocal nuclei. The magnitude of differential expression in vocal nuclei of all three vocal learners was unique compared with the smaller magnitude of differences found for nonvocal areas of vocal learners and vocal nonlearners. Our results suggest that evolution of vocal learning was accompanied by differential expression of a conserved gene family for synaptic transmission and plasticity in vocal nuclei. They also suggest that neural activity and signal transduction in vocal nuclei of vocal learners will be different relative to the surrounding brain areas. PMID:15236466

The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension

PubMed Central

Smit, Judith J; Monteferrario, Davide; Noordermeer, Sylvie M; van Dijk, Willem J; van der Reijden, Bert A; Sixma, Titia K

2012-01-01

Activation of the NF-κB pathway requires the formation of Met1-linked ‘linear' ubiquitin chains on NEMO, which is catalysed by the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of HOIP, HOIL-1L and Sharpin. Here, we show that both LUBAC catalytic activity and LUBAC specificity for linear ubiquitin chain formation are embedded within the RING-IBR-RING (RBR) ubiquitin ligase subunit HOIP. Linear ubiquitin chain formation by HOIP proceeds via a two-step mechanism involving both RING and HECT E3-type activities. RING1-IBR catalyses the transfer of ubiquitin from the E2 onto RING2, to transiently form a HECT-like covalent thioester intermediate. Next, the ubiquitin is transferred from HOIP onto the N-terminus of a target ubiquitin. This transfer is facilitated by a unique region in the C-terminus of HOIP that we termed ‘Linear ubiquitin chain Determining Domain' (LDD), which may coordinate the acceptor ubiquitin. Consistent with this mechanism, the RING2-LDD region was found to be important for NF-κB activation in cellular assays. These data show how HOIP combines a general RBR ubiquitin ligase mechanism with unique, LDD-dependent specificity for producing linear ubiquitin chains. PMID:22863777

Actin binding by Hip1 (huntingtin-interacting protein 1) and Hip1R (Hip1-related protein) is regulated by clathrin light chain.

PubMed

Wilbur, Jeremy D; Chen, Chih-Ying; Manalo, Venus; Hwang, Peter K; Fletterick, Robert J; Brodsky, Frances M

2008-11-21

The huntingtin-interacting protein family members (Hip1 and Hip1R in mammals and Sla2p in yeast) link clathrin-mediated membrane traffic to actin cytoskeleton dynamics. Genetic data in yeast have implicated the light chain subunit of clathrin in regulating this link. To test this hypothesis, the biophysical properties of mammalian Hip1 and Hip1R and their interaction with clathrin light chain and actin were analyzed. The coiled-coil domains (clathrin light chain-binding) of Hip1 and Hip1R were found to be stable homodimers with no propensity to heterodimerize in vitro. Homodimers were also predominant in vivo, accounting for cellular segregation of Hip1 and Hip1R functions. Coiled-coil domains of Hip1 and Hip1R differed in their stability and flexibility, correlating with slightly different affinities for clathrin light chain and more markedly with effects of clathrin light chain binding on Hip protein-actin interactions. Clathrin light chain binding induced a compact conformation of both Hip1 and Hip1R and significantly reduced actin binding by their THATCH domains. Thus, clathrin is a negative regulator of Hip-actin interactions. These observations necessarily change models proposed for Hip protein function.

Actin Binding by Hip1 (Huntingtin-interacting Protein 1) and Hip1R (Hip1-related Protein) Is Regulated by Clathrin Light Chain*S⃞

PubMed Central

Wilbur, Jeremy D.; Chen, Chih-Ying; Manalo, Venus; Hwang, Peter K.; Fletterick, Robert J.; Brodsky, Frances M.

2008-01-01

The huntingtin-interacting protein family members (Hip1 and Hip1R in mammals and Sla2p in yeast) link clathrin-mediated membrane traffic to actin cytoskeleton dynamics. Genetic data in yeast have implicated the light chain subunit of clathrin in regulating this link. To test this hypothesis, the biophysical properties of mammalian Hip1 and Hip1R and their interaction with clathrin light chain and actin were analyzed. The coiled-coil domains (clathrin light chain-binding) of Hip1 and Hip1R were found to be stable homodimers with no propensity to heterodimerize in vitro. Homodimers were also predominant in vivo, accounting for cellular segregation of Hip1 and Hip1R functions. Coiled-coil domains of Hip1 and Hip1R differed in their stability and flexibility, correlating with slightly different affinities for clathrin light chain and more markedly with effects of clathrin light chain binding on Hip protein-actin interactions. Clathrin light chain binding induced a compact conformation of both Hip1 and Hip1R and significantly reduced actin binding by their THATCH domains. Thus, clathrin is a negative regulator of Hip-actin interactions. These observations necessarily change models proposed for Hip protein function. PMID:18790740

ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype.

PubMed Central

Muñoz, R; De La Campa, A G

1996-01-01

The genes encoding the ParC and ParE subunits of topoisomerase IV of Streptococcus pneumoniae, together with the region encoding amino acids 46 to 172 (residue numbers are as in Escherichia coli) of the pneumococcal GyrA subunit, were partially characterized. The gyrA gene maps to a physical location distant from the gyrB and parC loci on the chromosome, whereas parC is closely linked to parE. Ciprofloxacin-resistant (Cpr) clinical isolates of S. pneumoniae had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level Cpr) or in both quinolone resistance-determining regions of ParC and GyrA (high-level Cpr). Mutations were found in residue positions equivalent to the serine at position 83 and the aspartic acid at position 87 of the E. coli GyrA subunit. Transformation experiments suggest that ParC is the primary target of ciprofloxacin. Mutation in parC appears to be a prerequisite before mutations in gyrA can influence resistance levels. PMID:8891124

Differences in α and β polypeptide chains of tubulin resolved by electron microscopy with image reconstruction

PubMed Central

Crepeau, Richard H.; McEwen, Bruce; Edelstein, Stuart J.

1978-01-01

Electron microscopic techniques have been used to reveal two classes of subunits of tubulin in ordered arrays. Presumably the two classes correspond to the α and β polypeptide chains of tubulin that have been distinguished by chemical criteria. The two types of subunits alternate along individual protofilaments in microtubules, microtubule-precursor sheets, and extended zinc-tubulin sheets. The resolution of the two types of polypeptide chains is achieved by improved negative staining methods which produce micrographs with layer lines at 28 Å-1 and 84 Å-1 in optical or computed transforms, in addition to the layer lines at 21 Å-1 and 42 Å-1 described previously [Crepeau, R. H., McEwen, B., Dykes, G. & Edelstein, S. J. (1977) J Mol. Biol. 116, 301-315]. In microtubules or microtubule-precursor sheets, adjacent protofilaments are staggered by about 10 Å, but parallel, in the sense that the α-β vector points in the same direction for all of the protofilaments of the microtubule. However, for the sheets assembled in the presence of zinc, adjacent protofilaments are staggered by about 21 Å and oriented in an antiparallel arrangement with alternate protofilaments related by a 2-fold screw axis. The antiparallel alignment of the protofilaments in the zinc-tubulin sheets accounts for their planarity (no tubular structures are found in the presence of moderate concentrations of zinc), since the intrinsic curvature found with parallel alignment of protofilaments in the absence of zinc would be cancelled by the antiparallel arrangement. Images PMID:283410

Cardiolipin deficiency causes a dissociation of the b 6 c:caa 3 megacomplex in B. subtilis membranes.

PubMed

García Montes de Oca, Led Yered Jafet; Cabellos Avelar, Tecilli; Picón Garrido, Gerardo Ignacio; Chagoya-López, Alicia; González de la Vara, Luis; Delgado Buenrostro, Norma Laura; Chirino-López, Yolanda Irasema; Gómez-Lojero, Carlos; Gutiérrez-Cirlos, Emma Berta

2016-08-01

The associations among respiratory complexes in energy-transducing membranes have been established. In fact, it is known that the Gram-negative bacteria Paracoccus denitrificans and Escherichia coli have respiratory supercomplexes in their membranes. These supercomplexes are important for channeling substrates between enzymes in a metabolic pathway, and the assembly of these supercomplexes depends on the protein subunits and membrane lipids, mainly cardiolipin, which is present in both the mitochondrial inner membrane and bacterial membranes. The Gram-positive bacterium Bacillus subtilis has a branched respiratory chain, in which some complexes generate proton motive force whereas others constitute an escape valve of excess reducing power. Some peculiarities of this respiratory chain are the following: a type II NADH dehydrogenase, a unique b 6 c complex that has a b 6 type cytochrome with a covalently bound heme, and a c-type heme attached to the third subunit, which is similar to subunit IV of the photosynthetic b 6 f complex. Cytochrome c oxygen reductase (caa 3 ) contains a c-type cytochrome on subunit I. We previously showed that the b 6 c and the caa 3 complexes form a supercomplex. Both the b 6 c and the caa 3 together with the quinol oxygen reductase aa 3 generate the proton motive force in B. subtilis. In order to seek proof that this supercomplex is important for bacterial growth in aerobic conditions we compared the b 6 c: caa 3 supercomplex from wild type membranes with membranes from two mutants lacking cardiolipin. Both mutant complexes were found to have similar activity and heme content as the wild type. Clear native electrophoresis showed that mutants lacking cardiolipin had b 6 c:caa 3 supercomplexes of lower mass or even individual complexes after membrane solubilization with digitonin. The use of dodecyl maltoside revealed a more evident difference between wild-type and mutant supercomplexes. Here we provide evidence showing that cardiolipin plays a role in the stability of the b 6 c:caa 3 supercomplex in B. subtilis.

Identification and characterisation of the IL-27 p28 subunits in fish: Cloning and comparative expression analysis of two p28 paralogues in Atlantic salmon Salmo salar.

PubMed

Husain, Mansourah; Martin, Samuel A M; Wang, Tiehui

2014-11-01

Interleukin (IL)-27 is an IL-6/IL-12 family member with pro-inflammatory and anti-inflammatory properties. It is a heterodimeric cytokine composed of an α-chain p28 and a β-chain Ebi3 (Epstein-Barr virus induce gene 3). The p28 subunit can also be secreted as a monomer and function as IL-30 that acts as an inhibitor of IL-27 signalling. At present, the p28 gene has only been described in mammals. Thus, for the first time outwith mammals, we have identified seven p28 molecules in six divergent teleost fish species, Atlantic salmon, two cichlids, two cyprinids and a yellowtail. The fish p28 molecules have higher similarities to mammalian p28 than other IL-6/12 family members. Critical residues involved in the interaction with Ebi3 and the receptor gp130 are highly conserved. The prediction that these are true orthologues is supported by phylogenetic and synteny analysis. Two p28 paralogues (p28a and p28b) sharing 72% aa identity have been identified and characterised in Atlantic salmon. There are multiple upstream ATGs in the 5'-UTR and ATTTA motifs in the 3'-UTR of both cDNA sequences, suggesting regulation at the post-transcriptional and translational level. Both salmon p28 genes are highly expressed in immune relevant tissues, such as thymus, gills, spleen and head kidney. Conversely salmon Ebi3 is highly expressed in other organs, such as liver and caudal kidney. The expression of p28 but not Ebi3 was induced by PAMPs and recombinant cytokines in head kidney cells, and in spleen by Poly I:C challenge in vivo. The dissociation of the expression and modulation of p28 and Ebi3 suggest that both p28 and Ebi3 may be secreted alone or with other partners. Copyright © 2014 Elsevier Ltd. All rights reserved.

NMDA receptor subunits change in the prefrontal cortex of pure-opioid and multi-drug abusers: a post-mortem study.

PubMed

Daneshparvar, Hamidreza; Sadat-Shirazi, Mitra-Sadat; Fekri, Monir; Khalifeh, Solmaz; Ziaie, Ali; Esfahanizadeh, Nasrin; Vousooghi, Nasim; Zarrindast, Mohammad-Reza

2018-05-16

Addiction is a chronic relapsing disorder and is one of the most important issues in the world. Changing the level of neurotransmitters and the activities of their receptors, play a major role in the pathophysiology of substance abuse disorders. It is well-established that N-methyl-D-aspartate receptors (NMDARs) play a significant role in the molecular basis of addiction. NMDAR has two obligatory GluN1 and two regionally localized GluN2 subunits. This study investigated changes in the protein level of GluN1, GluN2A, and GluN2B in the prefrontal cortex of drug abusers. The medial prefrontal cortex (mPFC), lateral prefrontal cortex (lPFC), and orbitofrontal cortex (OFC) were dissected from the brain of 101 drug addicts brains and were compared with the brains of non-addicts (N = 13). Western blotting technique was used to show the alteration in NMDAR subunits level. Data obtained using Western blotting technique showed a significant increase in the level of GluN1 and GluN2B, but not in GluN2A subunits in all the three regions (mPFC, lPFC, and OFC) of men whom suffered from addiction as compared to the appropriate controls. These findings showed a novel role for GluN1, GluN2B subunits, rather than the GluN2A subunit of NMDARs, in the pathophysiology of addiction and suggested their role in the drug-induced plasticity of NMDARs.

AMPA, NMDA and kainate glutamate receptor subunits are expressed in human peripheral blood mononuclear cells (PBMCs) where the expression of GluK4 is altered by pregnancy and GluN2D by depression in pregnant women.

PubMed

Bhandage, Amol K; Jin, Zhe; Hellgren, Charlotte; Korol, Sergiy V; Nowak, Krzysztof; Williamsson, Louise; Sundström-Poromaa, Inger; Birnir, Bryndis

2017-04-15

The amino acid glutamate opens cation permeable ion channels, the iGlu receptors. These ion channels are abundantly expressed in the mammalian brain where glutamate is the main excitatory neurotransmitter. The neurotransmitters and their receptors are being increasingly detected in the cells of immune system. Here we examined the expression of the 18 known subunits of the iGlu receptors families; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, N-methyl-d-aspartate (NMDA) and delta in human peripheral blood mononuclear cells (PBMCs). We compared the expression of the subunits between four groups: men, non-pregnant women, healthy pregnant women and depressed pregnant women. Out of 18 subunits of the iGlu receptors, mRNAs for 11 subunits were detected in PBMCs from men and non-pregnant women; AMPA: GluA3, GluA4, kainate: GluK2, GluK4, GluK5, NMDA: GluN1, GluN2C, GluN2D, GluN3A, GluN3B, and delta: GluD1. In the healthy and the depressed pregnant women, in addition, the delta GluD2 subunit was identified. The mRNAs for GluK4, GluK5, GluN2C and GluN2D were expressed at a higher level than other subunits. Gender, pregnancy or depression during pregnancy altered the expression of GluA3, GluK4, GluN2D, GluN3B and GluD1 iGlu subunit mRNAs. The greatest changes recorded were the lower GluA3 and GluK4 mRNA levels in pregnant women and the higher GluN2D mRNA level in healthy but not in depressed pregnant women as compared to non-pregnant individuals. Using subunit specific antibodies, the GluK4, GluK5, GluN1, GluN2C and GluN2D subunit proteins were identified in the PBMCs. The results show expression of specific iGlu receptor subunit in the PBMCs and support the idea of physiology-driven changes of iGlu receptors subtypes in the immune cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Meisheng; Tran, V.T.; Fong, H.K.W.

1991-05-01

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha}more » protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.« less

Changes in Whole-Body Oxygen Consumption and Skeletal Muscle Mitochondria During Linezolid-Induced Lactic Acidosis.

PubMed

Protti, Alessandro; Ronchi, Dario; Bassi, Gabriele; Fortunato, Francesco; Bordoni, Andreina; Rizzuti, Tommaso; Fumagalli, Roberto

2016-07-01

To better clarify the pathogenesis of linezolid-induced lactic acidosis. Case report. ICU. A 64-year-old man who died with linezolid-induced lactic acidosis. Skeletal muscle was sampled at autopsy to study mitochondrial function. Lactic acidosis developed during continuous infusion of linezolid while oxygen consumption and oxygen extraction were diminishing from 172 to 52 mL/min/m and from 0.27 to 0.10, respectively. Activities of skeletal muscle respiratory chain complexes I, III, and IV, encoded by nuclear and mitochondrial DNA, were abnormally low, whereas activity of complex II, entirely encoded by nuclear DNA, was not. Protein studies confirmed stoichiometric imbalance between mitochondrial (cytochrome c oxidase subunits 1 and 2) and nuclear (succinate dehydrogenase A) DNA-encoded respiratory chain subunits. These findings were not explained by defects in mitochondrial DNA or transcription. There were no compensatory mitochondrial biogenesis (no induction of nuclear respiratory factor 1 and mitochondrial transcript factor A) or adaptive unfolded protein response (reduced concentration of heat shock proteins 60 and 70). Linezolid-induced lactic acidosis is associated with diminished global oxygen consumption and extraction. These changes reflect selective inhibition of mitochondrial protein synthesis (probably translation) with secondary mitonuclear imbalance. One novel aspect of linezolid toxicity that needs to be confirmed is blunting of reactive mitochondrial biogenesis and unfolded protein response.

Triclosan-Evoked Neurotoxicity Involves NMDAR Subunits with the Specific Role of GluN2A in Caspase-3-Dependent Apoptosis.

PubMed

Szychowski, Konrad A; Wnuk, Agnieszka; Rzemieniec, Joanna; Kajta, Małgorzata; Leszczyńska, Teresa; Wójtowicz, Anna K

2018-04-19

Triclosan (TCS) is an antimicrobial agent that is used extensively in personal care and in sanitising products. A number of studies have shown the presence of TCS in different human tissues such as blood, adipose tissue, the liver, brain as well as in breast milk and urine. N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that are widely expressed in the central nervous system and which play key roles in excitatory synaptic transmission. There is, however, no data on the involvement of NMDAR subunits in the apoptotic and neurotoxic effects of TCS. Our experiments are the first to show that TCS used at environmentally relevant concentrations evoked NMDA-dependent effects in neocortical neurons in primary cultures, as MK-801, an uncompetitive NMDA receptor antagonist, reduced the levels of TCS-induced ROS production as well as caspase-3 activity and LDH release. TCS caused a decrease in protein expression of all the studied NMDA receptor subunits (GluN1, GluN2A, GluN2B) that were measured at 3, 6 and 24 h post-treatment. However, at 48 h of the experiment, the level of the GluN1 subunit returned to the control level, and the levels of the other subunits showed a tendency to increase. In TCS-treated neocortical cells, protein profiles of NMDAR subunits measured up to 24 h were similar to mRNA expression of GluN1 and GluN2A, but not to GluN2B mRNA. In this study, cells transiently transfected with GluN1, GluN2A or GluN2B siRNA exhibited reduced levels of LDH release, which suggests the involvement of all of the studied NMDAR subunits in the neurotoxic action of TCS. According to our data, GluN1 and GluN2A were mainly responsible for neuronal cell death as evidenced by neutral red uptake, whereas GluN2A was involved in TCS-induced caspase-3-dependent apoptosis. We suggest that TCS-evoked apoptosis and neurotoxicity could be related to transient degradation of NMDAR subunits in mouse neurons. Furthermore, recycling of NMDAR subunits in response to TCS is possible. Because transfections with specific siRNA did not completely abolish the effects of TCS as compared to cells transfected with negative siRNA in this study, other NMDAR-independent mechanisms of TCS action are also possible.

High dosage of cannabidiol (CBD) alleviates pentylenetetrazole-induced epilepsy in rats by exerting an anticonvulsive effect

PubMed Central

Mao, Ke; You, Chao; Lei, Ding; Zhang, Heng

2015-01-01

The study was designed to investigate the effect of various concentrations of cannabidiol (CBD) in rats with chronic epilepsy. The chronic epilepsy rat model was prepared by intraperitoneally injecting pentylenetetrazole to the rats pre-treated with CBD (10, 20 and 50 mg/kg) for 28 consecutive days. Behavioral measurements of convulsion following pentylenetetrazole treatment and morphological changes of the hippocampal neurons with hematoxylin and eosin staining were used to observe the epileptic behaviour. Immunohistochemistry was used to detect the expression levels of glial fibrillary acidic protein and inducible nitric oxide synthase (iNOS) in the hippocampus. The mRNA expression of N-methyl-D-aspartic acid (NMDA) receptor subunits (NR1 and NR2B) was detected by reverse transcription polymerase chain reaction. The results revealed a significant decrease in the daily average grade of epileptic seizures on treatment with CBD (50 mg/kg). The neuronal loss and astrocyte hyperplasia in the hippocampal area were also decreased. CBD treatment did not affect the expression of iNOS in the hippocampus; however, the expression of NR1 was decreased significantly. Thus, CBD administration inhibited the effect of pentylenetetrazole in rats, decreased the astrocytic hyperplasia, decreased neuronal damage in the hippocampus caused by seizures and selectively reduced the expression of the NR1 subunit of NMDA. Therefore, CBD exhibits an anticonvulsive effect in the rats with chronic epilepsy. PMID:26309534

Redox and Reactive Oxygen Species Regulation of Mitochondrial Cytochrome c Oxidase Biogenesis

PubMed Central

Bourens, Myriam; Fontanesi, Flavia; Soto, Iliana C.; Liu, Jingjing

2013-01-01

Abstract Significance: Cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is the major oxygen consumer enzyme in the cell. COX biogenesis involves several redox-regulated steps. The process is highly regulated to prevent the formation of pro-oxidant intermediates. Recent Advances: Regulation of COX assembly involves several reactive oxygen species and redox-regulated steps. These include: (i) Intricate redox-controlled machineries coordinate the expression of COX isoenzymes depending on the environmental oxygen concentration. (ii) COX is a heme A-copper metalloenzyme. COX copper metallation involves the copper chaperone Cox17 and several other recently described cysteine-rich proteins, which are oxidatively folded in the mitochondrial intermembrane space. Copper transfer to COX subunits 1 and 2 requires concomitant transfer of redox power. (iii) To avoid the accumulation of reactive assembly intermediates, COX is regulated at the translational level to minimize synthesis of the heme A-containing Cox1 subunit when assembly is impaired. Critical Issues: An increasing number of regulatory pathways converge to facilitate efficient COX assembly, thus preventing oxidative stress. Future Directions: Here we will review on the redox-regulated COX biogenesis steps and will discuss their physiological relevance. Forthcoming insights into the precise regulation of mitochondrial COX biogenesis in normal and stress conditions will likely open future perspectives for understanding mitochondrial redox regulation and prevention of oxidative stress. Antioxid. Redox Signal. 19, 1940–1952. PMID:22937827

High dosage of cannabidiol (CBD) alleviates pentylenetetrazole-induced epilepsy in rats by exerting an anticonvulsive effect.

PubMed

Mao, Ke; You, Chao; Lei, Ding; Zhang, Heng

2015-01-01

The study was designed to investigate the effect of various concentrations of cannabidiol (CBD) in rats with chronic epilepsy. The chronic epilepsy rat model was prepared by intraperitoneally injecting pentylenetetrazole to the rats pre-treated with CBD (10, 20 and 50 mg/kg) for 28 consecutive days. Behavioral measurements of convulsion following pentylenetetrazole treatment and morphological changes of the hippocampal neurons with hematoxylin and eosin staining were used to observe the epileptic behaviour. Immunohistochemistry was used to detect the expression levels of glial fibrillary acidic protein and inducible nitric oxide synthase (iNOS) in the hippocampus. The mRNA expression of N-methyl-D-aspartic acid (NMDA) receptor subunits (NR1 and NR2B) was detected by reverse transcription polymerase chain reaction. The results revealed a significant decrease in the daily average grade of epileptic seizures on treatment with CBD (50 mg/kg). The neuronal loss and astrocyte hyperplasia in the hippocampal area were also decreased. CBD treatment did not affect the expression of iNOS in the hippocampus; however, the expression of NR1 was decreased significantly. Thus, CBD administration inhibited the effect of pentylenetetrazole in rats, decreased the astrocytic hyperplasia, decreased neuronal damage in the hippocampus caused by seizures and selectively reduced the expression of the NR1 subunit of NMDA. Therefore, CBD exhibits an anticonvulsive effect in the rats with chronic epilepsy.

Phenyl-alpha-tert-butyl nitrone reverses mitochondrial decay in acute Chagas' disease.

PubMed

Wen, Jian-Jun; Bhatia, Vandanajay; Popov, Vsevolod L; Garg, Nisha Jain

2006-12-01

In this study, we investigated the mechanism(s) of mitochondrial functional decline in acute Chagas' disease. Our data show a substantial decline in respiratory complex activities (39 to 58%) and ATP (38%) content in Trypanosoma cruzi-infected murine hearts compared with normal controls. These metabolic alterations were associated with an approximately fivefold increase in mitochondrial reactive oxygen species production rate, substantial oxidative insult of mitochondrial membranes and respiratory complex subunits, and >60% inhibition of mtDNA-encoded transcripts for respiratory complex subunits in infected myocardium. The antioxidant phenyl-alpha-tert-butyl nitrone (PBN) arrested the oxidative damage-mediated loss in mitochondrial membrane integrity, preserved redox potential-coupled mitochondrial gene expression, and improved respiratory complex activities (47 to 95% increase) and cardiac ATP level (>or=40% increase) in infected myocardium. Importantly, PBN resulted twofold decline in mitochondrial reactive oxygen species production rate in infected myocardium. Taken together, our data demonstrate the pathological significance of oxidative stress in metabolic decay and energy homeostasis in acute chagasic myocarditis and further suggest that oxidative injuries affecting mitochondrial integrity-dependent expression and activity of the respiratory complexes initiate a feedback cycle of electron transport chain inefficiency, increased reactive oxygen species production, and energy homeostasis in acute chagasic hearts. PBN and other mitochondria-targeted antioxidants may be useful in altering mitochondrial decay and oxidative pathology in Chagas' disease.

ATP Synthase Repression in Tobacco Restricts Photosynthetic Electron Transport, CO2 Assimilation, and Plant Growth by Overacidification of the Thylakoid Lumen[OA

PubMed Central

Rott, Markus; Martins, Nádia F.; Thiele, Wolfram; Lein, Wolfgang; Bock, Ralph; Kramer, David M.; Schöttler, Mark A.

2011-01-01

Tobacco (Nicotiana tabacum) plants strictly adjust the contents of both ATP synthase and cytochrome b6f complex to the metabolic demand for ATP and NADPH. While the cytochrome b6f complex catalyzes the rate-limiting step of photosynthetic electron flux and thereby controls assimilation, the functional significance of the ATP synthase adjustment is unknown. Here, we reduced ATP synthase accumulation by an antisense approach directed against the essential nuclear-encoded γ-subunit (AtpC) and by the introduction of point mutations into the translation initiation codon of the plastid-encoded atpB gene (encoding the essential β-subunit) via chloroplast transformation. Both strategies yielded transformants with ATP synthase contents ranging from 100 to <10% of wild-type levels. While the accumulation of the components of the linear electron transport chain was largely unaltered, linear electron flux was strongly inhibited due to decreased rates of plastoquinol reoxidation at the cytochrome b6f complex (photosynthetic control). Also, nonphotochemical quenching was triggered at very low light intensities, strongly reducing the quantum efficiency of CO2 fixation. We show evidence that this is due to an increased steady state proton motive force, resulting in strong lumen overacidification, which in turn represses photosynthesis due to photosynthetic control and dissipation of excitation energy in the antenna bed. PMID:21278125

The over-expression of the β2 catalytic subunit of the proteasome decreases homologous recombination and impairs DNA double-strand break repair in human cells.

PubMed

Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

2011-01-01

By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

PubMed Central

Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

2011-01-01

By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p. PMID:21660142

Functional metabolomics as a tool to analyze Mediator function and structure in plants.

PubMed

Davoine, Celine; Abreu, Ilka N; Khajeh, Khalil; Blomberg, Jeanette; Kidd, Brendan N; Kazan, Kemal; Schenk, Peer M; Gerber, Lorenz; Nilsson, Ove; Moritz, Thomas; Björklund, Stefan

2017-01-01

Mediator is a multiprotein transcriptional co-regulator complex composed of four modules; Head, Middle, Tail, and Kinase. It conveys signals from promoter-bound transcriptional regulators to RNA polymerase II and thus plays an essential role in eukaryotic gene regulation. We describe subunit localization and activities of Mediator in Arabidopsis through metabolome and transcriptome analyses from a set of Mediator mutants. Functional metabolomic analysis based on the metabolite profiles of Mediator mutants using multivariate statistical analysis and heat-map visualization shows that different subunit mutants display distinct metabolite profiles, which cluster according to the reported localization of the corresponding subunits in yeast. Based on these results, we suggest localization of previously unassigned plant Mediator subunits to specific modules. We also describe novel roles for individual subunits in development, and demonstrate changes in gene expression patterns and specific metabolite levels in med18 and med25, which can explain their phenotypes. We find that med18 displays levels of phytoalexins normally found in wild type plants only after exposure to pathogens. Our results indicate that different Mediator subunits are involved in specific signaling pathways that control developmental processes and tolerance to pathogen infections.

Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.

PubMed

Agardh, Elisabet; Gustavsson, Carin; Hagert, Per; Nilsson, Marie; Agardh, Carl-David

2006-02-01

The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

Chronic intermittent ethanol exposure selectively alters the expression of Gα subunit isoforms and RGS subtypes in rat prefrontal cortex.

PubMed

Luessen, D J; Sun, H; McGinnis, M M; McCool, B A; Chen, R

2017-10-01

Chronic alcohol exposure induces pronounced changes in GPCR-mediated G-protein signaling. Recent microarray and RNA-seq analyses suggest associations between alcohol abuse and the expression of genes involved in G-protein signaling. The activity of G-proteins (e.g. Gαi/o and Gαq) is negatively modulated by regulator of G-protein signaling (RGS) proteins which are implicated in drugs of abuse including alcohol. The present study used 7days of chronic intermittent ethanol exposure followed by 24h withdrawal (CIE) to investigate changes in mRNA and protein levels of G-protein subunit isoforms and RGS protein subtypes in rat prefrontal cortex, a region associated with cognitive deficit attributed to excessive alcohol drinking. We found that this ethanol paradigm induced differential expression of Gα subunits and RGS subtypes. For example, there were increased mRNA and protein levels of Gαi1/3 subunits and no changes in the expression of Gαs and Gαq subunits in ethanol-treated animals. Moreover, CIE increased the mRNA but not the protein levels of Gαo. Additionally, a modest increase in Gαi2 mRNA level by CIE was accompanied by a pronounced increase in its protein level. Interestingly, we found that CIE increased mRNA and protein levels of RGS2, RGS4, RGS7 and RGS19 but had no effect on the expression of RGS5, RGS6, RGS8, RGS12 or RGS17. Changes in the expression of Gα subunits and RGS subtypes could contribute to the functional alterations of certain GPCRs following chronic ethanol exposure. The present study suggests that RGS proteins may be potential new targets for intervention of alcohol abuse via modification of Gα-mediated GPCR function. Copyright © 2017 Elsevier B.V. All rights reserved.

Ultraviolet B Radiation Stimulates the Interaction between Nuclear Factor of Activated T Cells 5 (NFAT5) and Nuclear Factor-Kappa B (NF-κB) in Human Lens Epithelial Cells.

PubMed

Chung, Inyoung; Hah, Young-Sool; Ju, SunMi; Kim, Ji-Hye; Yoo, Woong-Sun; Cho, Hee-Young; Yoo, Ji-Myong; Seo, Seong-Wook; Choi, Wan-Sung; Kim, Seong-Jae

2017-07-01

Nuclear factor-kappa B (NF-κB) has been proposed as a therapeutic target for the treatment of cataracts. The authors investigated the relationship between nuclear factor of activated T cells 5 (NFAT5) and NF-κB in ultraviolet B (UVB)-irradiated human lens epithelial (HLE) cells. Human lens epithelial B-3 (HLE-B3) cells were exposed to UVB light at a dose of 10 mJ/cm 2 and then incubated for 24 h. Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) assay. Gene expression level of NFAT5 was determined using real-time quantitative polymerase chain reaction (qPCR). Protein expression levels of NFAT5, NF-κB p65, and α-smooth muscle actin (α-SMA) and the association of NFAT5 with the NF-κB p65 subunit were measured by Western blot analysis and a co-immunoprecipitation assay, respectively. The cellular distribution of NFAT5 and NF-κB p65 was examined by triple immunofluorescence staining. At 24 h after UVB exposure, cell viability significantly decreased in a dose-dependent manner, and UVB light (15 and 20 mJ/cm 2 ) significantly increased the ROS generation. UVB irradiation increased NFAT5 mRNA and protein levels and increased phosphorylation of NF-κB in HLE-B3 cells. α-SMA protein levels were increased in the irradiated cells. In addition, NFAT5 and NF-κB translocated from the cytoplasm to the nucleus, and binding between the p65 subunit and NFAT5 was increased. Exposure to UVB radiation induces nuclear translocation and stimulates binding between NFAT5 and NF-κB proteins in HLE-B3 cells. These interactions may form part of the biochemical mechanism of cataractogenesis in UVB-irradiated HLECs.

Phylogenetic position of parabasalid symbionts from the termite Calotermes flavicollis based on small subunit rRNA sequences.

PubMed

Gerbod, D; Edgcomb, V P; Noël, C; Delgado-Viscogliosi, P; Viscogliosi, E

2000-09-01

Small subunit rDNA genes were amplified by polymerase chain reaction using specific primers from mixed-population DNA obtained from the whole hindgut of the termite Calotermes flavicollis. Comparative sequence analysis of the clones revealed two kinds of sequences that were both from parabasalid symbionts. In a molecular tree inferred by distance, parsimony and likelihood methods, and including 27 parabasalid sequences retrieved from the data bases, the sequences of the group II (clones Cf5 and Cf6) were closely related to the Devescovinidae/Calonymphidae species and thus were assigned to the Devescovinidae Foaina. The sequence of the group I (clone Cf1) emerged within the Trichomonadinae and strongly clustered with Tetratrichomonas gallinarum. On the basis of morphological data, the Monocercomonadidae Hexamastix termitis might be the most likely origin of this sequence.

SH3-like motif-containing C-terminal domain of staphylococcal teichoic acid transporter suggests possible function.

PubMed

Ko, Tzu-Ping; Tseng, Shih-Ting; Lai, Shu-Jung; Chen, Sheng-Chia; Guan, Hong-Hsiang; Shin Yang, Chia; Jung Chen, Chun; Chen, Yeh

2016-09-01

The negatively charged bacterial polysaccharides-wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising a transmembrane subunit TagG and an ATPase subunit TagH. We determined the crystal structure of the C-terminal domain of TagH (TagH-C) to investigate its function. The structure shows an N-terminal SH3-like subdomain wrapped by a C-terminal subdomain with an anti-parallel β-sheet and an outer shell of α-helices. A stretch of positively charged surface across the subdomain interface is flanked by two negatively charged regions, suggesting a potential binding site for negatively charged polymers, such as WTAs or acidic peptide chains. Proteins 2016; 84:1328-1332. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The Evolution of COP9 Signalosome in Unicellular and Multicellular Organisms.

PubMed

Barth, Emanuel; Hübler, Ron; Baniahmad, Aria; Marz, Manja

2016-05-02

The COP9 signalosome (CSN) is a highly conserved protein complex, recently being crystallized for human. In mammals and plants the COP9 complex consists of nine subunits, CSN 1-8 and CSNAP. The CSN regulates the activity of culling ring E3 ubiquitin and plays central roles in pleiotropy, cell cycle, and defense of pathogens. Despite the interesting and essential functions, a thorough analysis of the CSN subunits in evolutionary comparative perspective is missing. Here we compared 61 eukaryotic genomes including plants, animals, and yeasts genomes and show that the most conserved subunits of eukaryotes among the nine subunits are CSN2 and CSN5. This may indicate a strong evolutionary selection for these two subunits. Despite the strong conservation of the protein sequence, the genomic structures of the intron/exon boundaries indicate no conservation at genomic level. This suggests that the gene structure is exposed to a much less selection compared with the protein sequence. We also show the conservation of important active domains, such as PCI (proteasome lid-CSN-initiation factor) and MPN (MPR1/PAD1 amino-terminal). We identified novel exons and alternative splicing variants for all CSN subunits. This indicates another level of complexity of the CSN. Notably, most COP9-subunits were identified in all multicellular and unicellular eukaryotic organisms analyzed, but not in prokaryotes or archaeas. Thus, genes encoding CSN subunits present in all analyzed eukaryotes indicate the invention of the signalosome at the root of eukaryotes. The identification of alternative splice variants indicates possible "mini-complexes" or COP9 complexes with independent subunits containing potentially novel and not yet identified functions. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The Evolution of COP9 Signalosome in Unicellular and Multicellular Organisms

PubMed Central

Barth, Emanuel; Hübler, Ron; Baniahmad, Aria; Marz, Manja

2016-01-01

The COP9 signalosome (CSN) is a highly conserved protein complex, recently being crystallized for human. In mammals and plants the COP9 complex consists of nine subunits, CSN 1–8 and CSNAP. The CSN regulates the activity of culling ring E3 ubiquitin and plays central roles in pleiotropy, cell cycle, and defense of pathogens. Despite the interesting and essential functions, a thorough analysis of the CSN subunits in evolutionary comparative perspective is missing. Here we compared 61 eukaryotic genomes including plants, animals, and yeasts genomes and show that the most conserved subunits of eukaryotes among the nine subunits are CSN2 and CSN5. This may indicate a strong evolutionary selection for these two subunits. Despite the strong conservation of the protein sequence, the genomic structures of the intron/exon boundaries indicate no conservation at genomic level. This suggests that the gene structure is exposed to a much less selection compared with the protein sequence. We also show the conservation of important active domains, such as PCI (proteasome lid-CSN-initiation factor) and MPN (MPR1/PAD1 amino-terminal). We identified novel exons and alternative splicing variants for all CSN subunits. This indicates another level of complexity of the CSN. Notably, most COP9-subunits were identified in all multicellular and unicellular eukaryotic organisms analyzed, but not in prokaryotes or archaeas. Thus, genes encoding CSN subunits present in all analyzed eukaryotes indicate the invention of the signalosome at the root of eukaryotes. The identification of alternative splice variants indicates possible “mini-complexes” or COP9 complexes with independent subunits containing potentially novel and not yet identified functions. PMID:27044515

Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line.

PubMed

Kimmich, Tanja; Brüning, Ansgar; Käufl, Stephanie D; Makovitzky, Josef; Kuhn, Christina; Jeschke, Udo; Friese, Klaus; Mylonas, Ioannis

2010-08-01

Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC and betaE, have been identified and shown to be expressed in several human tissues. However, only limited data on the expression of these novel inhibin subunits in normal human endometrial tissue and endometrial adenocarcinoma cell lines exist. Samples of proliferative and secretory human endometrium were obtained from five premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Normal endometrial tissue and Ishikawa endometrial adenocarcinoma cell lines were analyzed by immunohistochemistry, immunofluorescence and RT-PCR. Expression of the inhibin betaC and betaE subunits could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by establishing a betaC- and betaE-specific RT-PCR analysis in normal human endometrial tissue and the parental Ishikawa cell line. Interestingly, in a highly de-differentiated subclone of the Ishikawa cell line lacking estrogen receptor expression, the expression of the inhibin-betaC subunit appeared strongly reduced. Here, we show for the first time that the novel inhibin/activin-betaC and -betaE subunits are expressed in normal human endometrium and the estrogen receptor positive human endometrial carcinoma cell line Ishikawa using RT-PCR and immunohistochemical detection methods. Interestingly, the Ishikawa minus cell line (lacking estrogen receptor expression) demonstrated no to minimal expression of the betaC subunit as observed with immunofluorescence and RT-PCR, suggesting a possible hormone- dependency of this subunit in human endometrial cancer cells. Moreover, because the Ishikawa cell line minus is thought to be a more malignant endometrial cell line than its estrogen receptor positive counterpart, inhibin-betaC subunit might be substantially involved in the pathogenesis and malignant transformation in human endometrium.

NMR and SAXS characterization of the denatured state of the chemotactic protein Che Y: Implications for protein folding initiation

PubMed Central

Garcia, Pascal; Serrano, Luis; Durand, Dominique; Rico, Manuel; Bruix, Marta

2001-01-01

The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X-ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies show that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexible links. A comparison of the contour length obtained for the chain at 5 M urea with the one expected for a fully expanded chain suggests that ∼25% of the residues are involved in residual structures. Conformational shifts of the α-protons, heteronuclear 15N-{1H} NOEs and 15N relaxation properties have been used to identify some regions in the protein that deviate from a random coil behavior. According to these NMR data, the protein can be divided into two subdomains, which largely coincide with the two folding subunits identified in a previous kinetic study of the folding of the protein. The first of these subdomains, spanning residues 1–70, is shown here to exhibit a restricted mobility as compared to the rest of the protein. Two regions, one in each subdomain, were identified as deviating from the random coil chemical shifts. Peptides corresponding to these sequences were characterized by NMR and their backbone 1H chemical shifts were compared to those in the intact protein under identical denaturing conditions. For the region located in the first subdomain, this comparison shows that the observed deviation from random coil parameters is caused by interactions with the rest of the molecule. The restricted flexibility of the first subdomain and the transient collapse detected in that subunit are consistent with the conclusions obtained by applying the protein engineering method to the characterization of the folding reaction transition state. PMID:11369848

Identification of an Inhibitory Alcohol Binding Site in GABAA ρ1 Receptors

PubMed Central

Borghese, Cecilia M.; Ruiz, Carlos I.; Lee, Ui S.; Cullins, Madeline A.; Bertaccini, Edward J.; Trudell, James R.; Harris, R. Adron

2016-01-01

Alcohols inhibit γ-aminobutyric acid type A ρ1 receptor function. After introducing mutations in several positions of the second transmembrane helix in ρ1, we studied the effects of ethanol and hexanol on GABA responses using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. The 6′ mutations produced the following effects on ethanol and hexanol responses: small increase or no change (T6′M), increased inhibition (T6′V) and small potentiation (T6′Y and T6′F). The 5′ mutations produced mainly increases in hexanol inhibition. Other mutations produced small (3′ and 9′) or no changes (2′ and L277 in the first transmembrane domain) in alcohol effects. These results suggest an inhibitory alcohol binding site near the 6′ position. Homology models of ρ1 receptors based on the X-ray structure of GluCl showed that the 2′, 5′, 6’ and 9′ residues were easily accessible from the ion pore, with 5′ and 6′ residues from neighboring subunits facing each other; L3′ and L277 also faced the neighboring subunit. We tested ethanol through octanol on single and double mutated ρ1 receptors [ρ1(I15′S), ρ1(T6′Y) and ρ1(T6′Y,I15′S)] to further characterize the inhibitory alcohol pocket in the wild-type ρ1 receptor. The pocket can only bind relatively short-chain alcohols and is eliminated by introducing Y in the 6’ position. Replacing the bulky 15′ residue with a smaller side chain introduced a potentiating binding site, more sensitive to long-chain than to short-chain alcohols. In conclusion, the net alcohol effect on the ρ1 receptor is determined by the sum of its actions on inhibitory and potentiating sites. PMID:26571107

Distribution of the branched-chain α-ketoacid dehydrogenase complex E1α subunit and glutamate dehydrogenase in the human brain and their role in neuro-metabolism.

PubMed

Hull, Jonathon; Usmari Moraes, Marcela; Brookes, Emma; Love, Seth; Conway, Myra E

2018-01-01

Glutamate is the major excitatory neurotransmitter of the central nervous system, with the branched-chain amino acids (BCAAs) acting as key nitrogen donors for de novo glutamate synthesis. Despite the importance of these major metabolites, their metabolic pathway in the human brain is still not well characterised. The metabolic pathways that influence the metabolism of BCAAs have been well characterised in rat models. However, the expression of key proteins such as the branched-chain α-ketoacid dehydrogenase (BCKD) complex and glutamate dehydrogenase isozymes (GDH) in the human brain is still not well characterised. We have used specific antibodies to these proteins to analyse their distribution within the human brain and report, for the first time, that the E1α subunit of the BCKD is located in both neurons and vascular endothelial cells. We also demonstrate that GDH is localised to astrocytes, although vascular immunolabelling does occur. The labelling of GDH was most intense in astrocytes adjacent to the hippocampus, in keeping with glutamatergic neurotransmission in this region. GDH was also present in astrocyte processes abutting vascular endothelial cells. Previously, we demonstrated that the branched-chain aminotransferase (hBCAT) proteins were most abundant in vascular cells (hBCATm) and neurons (hBCATc). Present findings are further evidence that BCAAs are metabolised within both the vasculature and neurons in the human brain. We suggest that GDH, hBCAT and the BCKD proteins operate in conjunction with astrocytic glutamate transporters and glutamine synthetase to regulate the availability of glutamate. This has important implications given that the dysregulation of glutamate metabolism, leading to glutamate excitotoxicity, is an important contributor to the pathogenesis of several neurodegenerative conditions such as Alzheimer's disease. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

A point mutation in the human Slo1 channel that impairs its sensitivity to omega-3 docosahexaenoic acid

PubMed Central

Xu, Rong; Hou, Shangwei; Heinemann, Stefan H.; Tian, Yutao

2013-01-01

Long-chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA) at nanomolar concentrations reversibly activate human large-conductance Ca2+- and voltage-gated K+ (Slo1 BK) channels containing auxiliary β1 or β4 subunits in cell-free patches. Here we examined the action of DHA on the Slo1 channel without any auxiliary subunit and sought to elucidate the biophysical mechanism and the molecular determinants of the DHA sensitivity. Measurements of ionic currents through human Slo1 (hSlo1) channels reveal that the stimulatory effect of DHA does not require activation of the voltage or Ca2+ sensors. Unlike gating of the hSlo1 channel, that of the Drosophila melanogaster Slo1 (dSlo1) channel is unaltered by DHA. Our mutagenesis study based on the differential responses of human and dSlo1 channels to DHA pinpoints that Y318 near the cytoplasmic end of S6 in the hSlo1 channel is a critical determinant of the stimulatory action of DHA. The mutation Y318S in hSlo1, which replaces Y with S as found in dSlo1, greatly diminishes the channel’s response to DHA with a 22-carbon chain whether β1 or β4 is absent or present. However, the responses to α-linolenic acid, an omegea-3 fatty acid with an 18-carbon chain, and to arachidonic acid, an omega-6 fatty acid with a 20-carbon chain, remain unaffected by the mutation. Y318 in the S6 segment of hSlo1 is thus an important determinant of the electrophysiological response of the channel to DHA. Furthermore, the mutation Y318S may prove to be useful in dissecting out the complex lipid-mediated modulation of Slo1 BK channels. PMID:24127525

Tyrosine Phosphorylation of GABAA Receptor γ2-Subunit Regulates Tonic and Phasic Inhibition in the Thalamus

PubMed Central

Nani, Francesca; Bright, Damian P.; Revilla-Sanchez, Raquel; Tretter, Verena; Moss, Stephen J.

2013-01-01

GABA-mediated tonic and phasic inhibition of thalamic relay neurons of the dorsal lateral geniculate nucleus (dLGN) was studied after ablating tyrosine (Y) phosphorylation of receptor γ2-subunits. As phosphorylation of γ2 Y365 and Y367 reduces receptor internalization, to understand their importance for inhibition we created a knock-in mouse in which these residues are replaced by phenylalanines. On comparing wild-type (WT) and γ2Y365/367F+/− (HT) animals (homozygotes are not viable in utero), the expression levels of GABAA receptor α4-subunits were increased in the thalamus of female, but not male mice. Raised δ-subunit expression levels were also observed in female γ2Y365/367F +/− thalamus. Electrophysiological analyses revealed no difference in the level of inhibition in male WT and HT dLGN, while both the spontaneous inhibitory postsynaptic activity and the tonic current were significantly augmented in female HT relay cells. The sensitivity of tonic currents to the δ-subunit superagonist THIP, and the blocker Zn2+, were higher in female HT relay cells. This is consistent with upregulation of extrasynaptic GABAA receptors containing α4- and δ-subunits to enhance tonic inhibition. In contrast, the sensitivity of GABAA receptors mediating inhibition in the female γ2Y356/367F +/− to neurosteroids was markedly reduced compared with WT. We conclude that disrupting tyrosine phosphorylation of the γ2-subunit activates a sex-specific increase in tonic inhibition, and this most likely reflects a genomic-based compensation mechanism for the reduced neurosteroid sensitivity of inhibition measured in female HT relay neurons. PMID:23904608

Determinants in the β and δ subunit cytoplasmic loop regulate Golgi trafficking and surface expression of the muscle acetylcholine receptor.

PubMed

Rudell, Jolene Chang; Borges, Lucia S; Rudell, John B; Beck, Kenneth A; Ferns, Michael J

2014-01-03

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-β and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-β and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25-28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, βK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined βK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the β and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.

Function of fusion regulatory proteins (FRPs) in immune cells and virus-infected cells.

PubMed

Tsurudome, M; Ito, Y

2000-01-01

Two molecules that regulate cell fusion have been identified and designated fusion regulatory protein-1 (FRP-1) and FRP-2. FRP-1 is a complex composed of a glycosylated heavy chain and a nonglycosylated light chain that are disulfide linked. FRP-1 heavy chain is identical to 4F2/CD98 heavy chain, whereas FRP-2 is identical to integrin alpha3 subunit. The FRP-1 heavy chain is a multifunctional molecule: that is, fusion regulator, amino acid transporter, integrin regulator, comitogenic factor, Na+-Ca2+ exchanger, oncogenic protein, and so on. Several aspects of the structure and function of the FRP-1 system are reviewed: fusion regulatory molecular mechanisms, cross-talk between the FRP-1 and integrin, the FRP-1 system as amino acid transporter, and FRP-1-mediated T-cell activation. The FRP-1 system is involved in virus-mediated cell fusion and multinucleated giant cell formation of blood monocytes. Monoclonal antibodies against human FRP-1 heavy chain induce polykaryocytes that have properties as osteoclasts. Multiple steps participate in molecular mechanisms regulating cell fusion. The FRP-1 heavy chain supports amino acid transport activity and the FRP-1 light chains have recently been cloned as amino acid transporters that require association with the heavy chain to exhibit their activity. Novel pathways for monocyte-dependent regulation of T-cell activation have recently been found that are mediated by the FRP-1 system. In conclusion, the FRP-1 molecules are essential factors for basic cellular functions.

Molecular tools for investigating microbial community structure and function in oxygen-deficient marine waters.

PubMed

Hawley, Alyse K; Kheirandish, Sam; Mueller, Andreas; Leung, Hilary T C; Norbeck, Angela D; Brewer, Heather M; Pasa-Tolic, Ljiljana; Hallam, Steven J

2013-01-01

Water column oxygen (O2)-deficiency shapes food-web structure by progressively directing nutrients and energy away from higher trophic levels into microbial community metabolism resulting in fixed nitrogen loss and greenhouse gas production. Although respiratory O2 consumption during organic matter degradation is a natural outcome of a productive surface ocean, global-warming-induced stratification intensifies this process leading to oxygen minimum zone (OMZ) expansion. Here, we describe useful tools for detection and quantification of potential key microbial players and processes in OMZ community metabolism including quantitative polymerase chain reaction primers targeting Marine Group I Thaumarchaeota, SUP05, Arctic96BD-19, and SAR324 small-subunit ribosomal RNA genes and protein extraction methods from OMZ waters compatible with high-resolution mass spectrometry for profiling microbial community structure and functional dynamics. © 2013 Elsevier Inc. All rights reserved.

2.2 A resolution structure analysis of two refined N-acetylneuraminyl-lactose--wheat germ agglutinin isolectin complexes.

PubMed

Wright, C S

1990-10-20

The crystal structures of complexes of isolectins 1 and 2 of wheat germ agglutinin (WGA1 and WGA2) with N-acetylneuraminyl-lactose (NeuNAc-alpha(2-3)-Gal-beta(1-4)-Glc) have been refined on the basis of data in the 8 to 2.2 A resolution range to final crystallographic R-factors of 17.2% and 15.3% (Fo greater than 1 sigma), respectively. Specific binding interactions and water association, as well as changes in conformation and mobility of the structure upon ligand binding, were compared in the two complexes. The temperature factors (B = 16.3 A2 and 18.4 A2) were found to be much lower compared with those of their respective native structures (19 to 22 A2). Residues involved in sugar binding, dimerization and in lattice contacts exhibit the largest decreases in B-value, suggesting that sugar binding reduces the overall mobility of the protein molecules in the crystal lattice. The binding mode of this sialyl-trisaccharide, an important cell receptor analogue, has been compared in the two isolectins. Only one of the two unique binding sites (4 per dimer), located in the subunit/subunit interface, is occupied in the crystals. This site, termed the "primary" binding site, contains one of the five amino acid substitutions that differentiate WGA1 and WGA2. Superposition of the refined models in each of the independent crystallographic environments indicates a close match only of the terminal non-reducing NeuNAc residue (root-mean-square delta r of 0.5 to 0.6 A). The Gal-Glc portion was found to superimpose poorly, lack electron density, and possess high atomic thermal factors. In both complexes NeuNAc is stabilized through contact with six amino acid side-chains (Ser114 and Glu115 of subunit 1 and Ser62, Tyr64, Tyr(His)66 and Tyr73 of subunit 2), involving all NeuNAc ring substituents. Refinement has allowed accurate assessment of the contact distances for four hydrogen bonds, a strong buried non-polar contact with the acetamido CH3 group and a large number of van der Waals' interactions with the three aromatic side-chains. The higher affinity of N-acetylneuraminyl-lactose observed by nuclear magnetic resonance studies for WGA1 can be explained by the more favorable binding interactions that occur when residue 66 is a Tyr. The tyrosyl side-chain provides a larger surface for van der Waals' stacking against the NeuNAc pyranose ring than His66 and a hydrogen bond contact with Gal (C2-OH), not possible in WGA2.(ABSTRACT TRUNCATED AT 400 WORDS)

The AMPK β2 subunit is required for energy homeostasis during metabolic stress.

PubMed

Dasgupta, Biplab; Ju, Jeong Sun; Sasaki, Yo; Liu, Xiaona; Jung, Su-Ryun; Higashida, Kazuhiko; Lindquist, Diana; Milbrandt, Jeffrey

2012-07-01

AMP activated protein kinase (AMPK) plays a key role in the regulatory network responsible for maintaining systemic energy homeostasis during exercise or nutrient deprivation. To understand the function of the regulatory β2 subunit of AMPK in systemic energy metabolism, we characterized β2 subunit-deficient mice. Using these mutant mice, we demonstrated that the β2 subunit plays an important role in regulating glucose, glycogen, and lipid metabolism during metabolic stress. The β2 mutant animals failed to maintain euglycemia and muscle ATP levels during fasting. In addition, β2-deficient animals showed classic symptoms of metabolic syndrome, including hyperglycemia, glucose intolerance, and insulin resistance when maintained on a high-fat diet (HFD), and were unable to maintain muscle ATP levels during exercise. Cell surface-associated glucose transporter levels were reduced in skeletal muscle from β2 mutant animals on an HFD. In addition, they displayed poor exercise performance and impaired muscle glycogen metabolism. These mutant mice had decreased activation of AMPK and deficits in PGC1α-mediated transcription in skeletal muscle. Our results highlight specific roles of AMPK complexes containing the β2 subunit and suggest the potential utility of AMPK isoform-specific pharmacological modulators for treatment of metabolic, cardiac, and neurological disorders.

Design of polymer conjugated 3-helix micelles as nanocarriers with tunable shapes.

PubMed

Ma, Dan; DeBenedictis, Elizabeth P; Lund, Reidar; Keten, Sinan

2016-11-24

Amphiphilic peptide-polymer conjugates have the ability to form stable nanoscale micelles, which show great promise for drug delivery and other applications. A recent design has utilized the end-conjugation of alkyl chains to 3-helix coiled coils to achieve amphiphilicity, combined with the side-chain conjugation of polyethylene glycol (PEG) to tune micelle size through entropic confinement forces. Here we investigate this phenomenon in depth, using coarse-grained dissipative particle dynamics (DPD) simulations in an explicit solvent and micelle theory. We analyze the conformations of PEG chains conjugated to three different positions on 3-helix bundle peptides to ascertain the degree of confinement upon assembly, as well as the ordering of the subunits making up the micelle. We discover that the micelle size and stability is dictated by a competition between the entropy of PEG chain conformations in the assembled state, as well as intermolecular cross-interactions among PEG chains that promote cohesion between neighboring conjugates. Our analyses build on the role of PEG molecular weight and conjugation site and lead to computational phase diagrams that can be used to design 3-helix micelles. This work opens pathways for the design of multifunctional micelles with tunable size, shape and stability.

Evolutionary comparison of prenylation pathway in kinetoplastid Leishmania and its sister Leptomonas.

PubMed

Chauhan, Indira Singh; Kaur, Jaspreet; Krishna, Shagun; Ghosh, Arpita; Singh, Prashant; Siddiqi, Mohammad Imran; Singh, Neeloo

2015-11-21

Leptomonas is monogenetic kinetoplastid parasite of insects and is primitive in comparison to Leishmania. Comparative studies of these two kinetoplastid may share light on the evolutionary transition to dixenous parasitism in Leishmania. In order to adapt and survive within two hosts, Leishmania species must have acquired virulence factors in addition to mechanisms that mediate susceptibility/resistance to infection in the pathology associated with disease. Rab proteins are key mediators of vesicle transport and contribute greatly to the evolution of complexity of membrane transport system. In this study we used our whole genome sequence data of these two divergent kinetoplastids to analyze the orthologues/paralogues of Rab proteins. During change of lifestyle from monogenetic (Leptomonas) to digenetic (Leishmania), we found that the prenyl machinery remained unchanged. Geranylgeranyl transferase-I (GGTase-I) was absent in both Leishmania and its sister Leptomonas. Farnesyltransferase (FTase) and geranylgeranyl transferase-II (GGTase-II) were identified for protein prenylation. We predict that activity of the missing alpha-subunit (α-subunit) of GGTase-II in Leptomonas was probably contributed by the α-subunit of FTase, while beta-subunit (β-subunit) of GGTase-II was conserved and indicated functional conservation in the evolution of these two kinetoplastids. Therefore the β-subunit emerges as an excellent target for compounds inhibiting parasite activity in clinical cases of co-infections. We also confirmed that during the evolution to digenetic life style in Leishmania, the parasite acquired capabilities to evade drug action and maintain parasite virulence in the host with the incorporation of short-chain dehydrogenase/reductase (SDR/MDR) superfamily in Rab genes. Our study based on whole genome sequences is the first to build comparative evolutionary analysis and identification of prenylation proteins in Leishmania and its sister Leptomonas. The information presented in our present work has importance for drug design targeted to kill L. donovani in humans but not affect the human form of the prenylation enzymes.

Mitochondrial respiratory chain complexes as sources and targets of thiol-based redox-regulation.

PubMed

Dröse, Stefan; Brandt, Ulrich; Wittig, Ilka

2014-08-01

The respiratory chain of the inner mitochondrial membrane is a unique assembly of protein complexes that transfers the electrons of reducing equivalents extracted from foodstuff to molecular oxygen to generate a proton-motive force as the primary energy source for cellular ATP-synthesis. Recent evidence indicates that redox reactions are also involved in regulating mitochondrial function via redox-modification of specific cysteine-thiol groups in subunits of respiratory chain complexes. Vice versa the generation of reactive oxygen species (ROS) by respiratory chain complexes may have an impact on the mitochondrial redox balance through reversible and irreversible thiol-modification of specific target proteins involved in redox signaling, but also pathophysiological processes. Recent evidence indicates that thiol-based redox regulation of the respiratory chain activity and especially S-nitrosylation of complex I could be a strategy to prevent elevated ROS production, oxidative damage and tissue necrosis during ischemia-reperfusion injury. This review focuses on the thiol-based redox processes involving the respiratory chain as a source as well as a target, including a general overview on mitochondria as highly compartmentalized redox organelles and on methods to investigate the redox state of mitochondrial proteins. This article is part of a Special Issue entitled: Thiol-Based Redox Processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.

PubMed

McLachlan, Sandra M; Aliesky, Holly A; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil

2012-01-01

Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the TSHR) displaying strong self-tolerance.

Breaking Tolerance in Transgenic Mice Expressing the Human TSH Receptor A-Subunit: Thyroiditis, Epitope Spreading and Adjuvant as a ‘Double Edged Sword’

PubMed Central

McLachlan, Sandra M.; Aliesky, Holly A.; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil

2012-01-01

Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a “double-edged sword”. On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the TSHR) displaying strong self-tolerance. PMID:22970131

Characterization of two forms of mouse salivary androgen-binding protein (ABP): implications for evolutionary relationships and ligand-binding function.

PubMed

Karn, Robert C; Laukaitis, Christina M

2003-06-17

Mouse salivary androgen-binding protein (ABP) is a member of the secretoglobin family produced in the submaxillary glands of house mice (Mus musculus). We report the cDNA sequences and amino acid sequences of the beta and gamma subunits of ABP from a mouse cDNA library, identifying the two subunits by their pIs and molecular weights. An anomalously high molecular weight of the alpha subunit is likely due to glycosylation at a single site. A phylogenetic comparison of the three subunits of ABP with the chains of other mammalian secretoglobins shows that ABP is most closely related to mouse lachrymal protein and to the major cat allergen Fel dI. An evaluation of the most conserved residues in ABP and the other secretoglobins, in light of structural data reported by others [Callebaut, I., Poupon, A., Bally, R., Demaret, J.-P., Housset, D., Delettre, J., Hossenlopp, P., and Mornon, J.-P. (2000) Ann. N.Y. Acad. Sci. 923, 90-112; Pattabiraman, N., Matthews, J., Ward, K., Mantile-Selvaggi, G., Miele, L., and Mukherjee, A. (2000) Ann. N.Y. Acad. Sci. 923, 113-127], allows us to draw conclusions about the critical residues important in ligand binding by the two different ABP dimers and to assess the importance of ligand binding in the function of the molecule. In addition to the cDNAs, which represent those of the musculus subspecies of Mus musculus, we also report the coding regions of the beta and gamma subunit cDNAs from two other mouse inbred strains which represent the other two subspecies: M. musculus domesticus and M. musculus castaneus. The high nonsynonymous/synonymous substitution rate ratios (K(a)/K(s)) for both the beta and gamma subunits suggest that these two proteins are evolving under strong directional selection, as has been reported for the alpha subunit [Hwang, J., Hofstetter, J., Bonhomme, F., and Karn, R. (1997) J. Hered. 88, 93-97; Karn, R., and Clements, M. (1999) Biochem. Genet. 37, 187-199].

Homologous kappa-neurotoxins exhibit residue-specific interactions with the alpha 3 subunit of the nicotinic acetylcholine receptor: a comparison of the structural requirements for kappa-bungarotoxin and kappa-flavitoxin binding.

PubMed

McLane, K E; Weaver, W R; Lei, S; Chiappinelli, V A; Conti-Tronconi, B M

1993-07-13

kappa-Flavotoxin (kappa-FTX), a snake neurotoxin that is a selective antagonist of certain neuronal nicotinic acetylcholine receptors (AChRs), has recently been isolated and characterized [Grant, G. A., Frazier, M. W., & Chiappinelli, V. A. (1988) Biochemistry 27, 1532-1537]. Like the related snake toxin kappa-bungarotoxin (kappa-BTX), kappa-FTX binds with high affinity to alpha 3 subtypes of neuronal AChRs, even though there are distinct sequence differences between the two toxins. To further characterize the sequence regions of the neuronal AChR alpha 3 subunit involved in formation of the binding site for this family of kappa-neurotoxins, we investigated kappa-FTX binding to overlapping synthetic peptides screening the alpha 3 subunit sequence. A sequence region forming a "prototope" for kappa-FTX was identified within residues alpha 3 (51-70), confirming the suggestions of previous studies on the binding of kappa-BTX to the alpha 3 subunit [McLane, K. E., Tang, F., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 1537-1544] and alpha-bungarotoxin to the Torpedo AChR alpha subunit [Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230] that this sequence region is involved in formation of a cholinergic site. Single residue substituted analogues, where each residue of the sequence alpha 3 (51-70) was sequentially replaced by a glycine, were used to identify the amino acid side chains involved in the interaction of this prototope with kappa-FTX.(ABSTRACT TRUNCATED AT 250 WORDS)

Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation.

PubMed

Zhu, Shaotong; Vik, Steven B

2015-08-21

Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441-445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu(2+) ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50-90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10-20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Dizocilpine (MK-801) induces distinct changes of N-methyl-D-aspartic acid receptor subunits in parvalbumin-containing interneurons in young adult rat prefrontal cortex.

PubMed

Xi, Dong; Zhang, Wentong; Wang, Huai-Xing; Stradtman, George G; Gao, Wen-Jun

2009-11-01

N-methyl-D-aspartic acid receptor (NMDAR) hypofunction has long been implicated in schizophrenia and NMDARs on gamma-aminobutyric acid (GABA)ergic interneurons are proposed to play an essential role in the pathogenesis. However, controversial results have been reported regarding the regulation of NMDAR expression, and direct evidence of how NMDAR antagonists act on specific subpopulations of prefrontal interneurons is missing. We investigated the effects of the NMDAR antagonist dizocilpine (MK-801) on the expression of NMDAR subtypes in the identified interneurons in young adult rat prefrontal cortex (PFC) by using laser microdissection and real-time polymerase chain reaction, combined with Western blotting and immunofluorescent staining. We found that MK-801 induced distinct changes of NMDAR subunits in the parvalbumin-immunoreactive (PV-ir) interneurons vs. pyramidal neurons in the PFC circuitry. The messenger RNA (mRNA) expression of all NMDAR subtypes, including NR1 and NR2A to 2D, exhibited inverted-U dose-dependent changes in response to MK-801 treatment in the PFC. In contrast, subunit mRNAs of NMDARs in PV-ir interneurons were significantly down-regulated at low doses, unaltered at medium doses, and significantly decreased again at high doses, suggesting a biphasic dose response to MK-801. The differential effects of MK-801 in mRNA expression of NMDAR subunits were consistent with the protein expression of NR2A and NR2B subunits revealed with Western blotting and double immunofluorescent staining. These results suggest that PV-containing interneurons in the PFC exhibit a distinct responsiveness to NMDAR antagonism and that NMDA antagonist can differentially and dose-dependently regulate the functions of pyramidal neurons and GABAergic interneurons in the prefrontal cortical circuitry.

Gene Duplication and Evolutionary Innovations in Hemoglobin-Oxygen Transport

PubMed Central

2016-01-01

During vertebrate evolution, duplicated hemoglobin (Hb) genes diverged with respect to functional properties as well as the developmental timing of expression. For example, the subfamilies of genes that encode the different subunit chains of Hb are ontogenetically regulated such that functionally distinct Hb isoforms are expressed during different developmental stages. In some vertebrate taxa, functional differentiation between co-expressed Hb isoforms may also contribute to physiologically important divisions of labor. PMID:27053736

Fasciola hepatica Infection in an Indigenous Community of the Peruvian Jungle

PubMed Central

Cabada, Miguel M.; Castellanos-Gonzalez, Alejandro; Lopez, Martha; Caravedo, María Alejandra; Arque, Eulogia; White, Arthur Clinton

2016-01-01

Fasciola hepatica is a zoonotic infection with a worldwide distribution. Autochthonous cases have not been reported in the Amazon region of Peru. Operculated eggs resembling F. hepatica were identified in the stools of five out of 215 subjects in a remote indigenous community of the Peruvian jungle. Polymerase chain reaction targeting Fasciola hepatica cytochrome oxidase subunit 1 (COI) gene and sequencing of the products confirmed Fasciola infection. PMID:26976892

A Transcript-Specific eIF3 Complex Mediates Global Translational Control of Energy Metabolism.

PubMed

Shah, Meera; Su, Dan; Scheliga, Judith S; Pluskal, Tomáš; Boronat, Susanna; Motamedchaboki, Khatereh; Campos, Alexandre Rosa; Qi, Feng; Hidalgo, Elena; Yanagida, Mitsuhiro; Wolf, Dieter A

2016-08-16

The multi-subunit eukaryotic translation initiation factor eIF3 is thought to assist in the recruitment of ribosomes to mRNA. The expression of eIF3 subunits is frequently disrupted in human cancers, but the specific roles of individual subunits in mRNA translation and cancer remain elusive. Using global transcriptomic, proteomic, and metabolomic profiling, we found a striking failure of Schizosaccharomyces pombe cells lacking eIF3e and eIF3d to synthesize components of the mitochondrial electron transport chain, leading to a defect in respiration, endogenous oxidative stress, and premature aging. Energy balance was maintained, however, by a switch to glycolysis with increased glucose uptake, upregulation of glycolytic enzymes, and strict dependence on a fermentable carbon source. This metabolic regulatory function appears to be conserved in human cells where eIF3e binds metabolic mRNAs and promotes their translation. Thus, via its eIF3d-eIF3e module, eIF3 orchestrates an mRNA-specific translational mechanism controlling energy metabolism that may be disrupted in cancer. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Ca2+ signalling, voltage-gated Ca2+ channels and praziquantel in flatworm neuromusculature.

PubMed

Greenberg, R M

2005-01-01

Transient changes in calcium (Ca2+) levels regulate a wide variety of cellular processes, and cells employ both intracellular and extracellular sources of Ca2+ for signalling. Praziquantel, the drug of choice against schistosomiasis, disrupts Ca2+ homeostasis in adult worms. This review will focus on voltage-gated Ca2+ channels, which regulate levels of intracellular Ca2+ by coupling membrane depolarization to entry of extracellular Ca2+. Ca2+ channels are members of the ion channel superfamily and represent essential components of neurons, muscles and other excitable cells. Ca2+ channels are membrane protein complexes in which the pore-forming alpha1 subunit is modulated by auxiliary subunits such as beta and alpha2delta. Schistosomes express two Ca2+ channel beta subunit subtypes: a conventional subtype similar to beta subunits found in other vertebrates and invertebrates and a novel variant subtype with unusual structural and functional properties. The variant schistosome beta subunit confers praziquantel sensitivity to an otherwise praziquantel-insensitive mammalian Ca2+ channel, implicating it as a mediator of praziquantel action.

Molecular characterization of maple syrup urine disease patients from Tunisia.

PubMed

Jaafar, N; Moleirinho, A; Kerkeni, E; Monastiri, K; Seboui, H; Amorim, A; Prata, M J; Quental, S

2013-03-15

Maple syrup urine disease (MSUD) is a rare disorder of branched-chain amino acids (BCAA) metabolism caused by the defective function of branched-chain α-ketoacid dehydrogenase complex (BCKD). The disease causal mutations can occur either in BCKDHA, BCKDHB or DBT genes encoding respectively the E1α, E1β and E2 subunits of the complex. In this study we report the molecular characterization of 3 Tunisian patients with the classic form of MSUD. Two novel putative mutations have been identified: the alteration c.716A>G (p.Glu239Gly) in BCKDHB and a small deletion (c.1333_1336delAATG; p.Asn445X) detected in DBT gene. Copyright © 2012 Elsevier B.V. All rights reserved.

Functional conservation of RNA polymerase II in fission and budding yeasts.

PubMed

Shpakovski, G V; Gadal, O; Labarre-Mariotte, S; Lebedenko, E N; Miklos, I; Sakurai, H; Proshkin, S A; Van Mullem, V; Ishihama, A; Thuriaux, P

2000-02-04

The complementary DNAs of the 12 subunits of fission yeast (Schizosaccharomyces pombe) RNA polymerase II were expressed from strong promoters in Saccharomyces cerevisiae and tested for heterospecific complementation by monitoring their ability to replace in vivo the null mutants of the corresponding host genes. Rpb1 and Rpb2, the two largest subunits and Rpb8, a small subunit shared by all three polymerases, failed to support growth in S. cerevisiae. The remaining nine subunits were all proficient for heterospecific complementation and led in most cases to a wild-type level of growth. The two alpha-like subunits (Rpb3 and Rpb11), however, did not support growth at high (37 degrees C) or low (25 degrees C) temperatures. In the case of Rpb3, growth was restored by increasing the gene dosage of the host Rpb11 or Rpb10 subunits, confirming previous evidence of a close genetic interaction between these three subunits. Copyright 2000 Academic Press.

Protein Kinase A Subunit Balance Regulates Lipid Metabolism in Caenorhabditis elegans and Mammalian Adipocytes*

PubMed Central

Lee, Jung Hyun; Han, Ji Seul; Kong, Jinuk; Ji, Yul; Lv, Xuchao; Lee, Junho; Li, Peng; Kim, Jae Bum

2016-01-01

Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. Here we demonstrated that the stoichiometric balance between catalytic and regulatory subunits is crucial for maintaining basal PKA activity and lipid homeostasis. To uncover the potential roles of each PKA subunit, Caenorhabditis elegans was used to investigate the effects of PKA subunit deficiency. In worms, suppression of PKA via RNAi resulted in severe phenotypes, including shortened life span, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits RIα and RIIβ via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti-lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism. PMID:27496951

Immunohistochemical analyses of alpha1 and alpha3 Na+/K+-ATPase subunit expression in medulloblastomas.

PubMed

Suñol, Mariona; Cusi, Victoria; Cruz, Ofelia; Kiss, Robert; Lefranc, Florence

2011-03-01

The levels of expression of the α1 and α3 subunits of the Na(+)/K(+)-ATPase (the NaK sodium pump) in medulloblastomas are unclear. This study investigated the expression of the NaK subunits using immunohistochemical methods in 29 medulloblastomas including 23 classic, three large-cell/anaplastic and three nodular/desmoplastic medulloblastomas, as well as in three atypical teratoid/rhabdoid tumors (AT/RTs). There was overexpression of the α1 or α3 NaK subunits in more than half of the medulloblastomas and atypical AT/RTs, with about one-third of these tumours displaying overexpression of both subunits. These preliminary data suggest that targeting these subunits in AT/RTs and medulloblastomas that overexpress these proteins may lead to therapeutic benefit. These findings warrant confirmation in larger numbers of patients than those used in this study. Moreover, it should be determined whether inhibition of the α1/α3 NaK subunits can be integrated into the risk stratification schemes already in use for medulloblastoma patients.

Basal Levels of AMPA Receptor GluA1 Subunit Phosphorylation at Threonine 840 and Serine 845 in Hippocampal Neurons

ERIC Educational Resources Information Center

Babiec, Walter E.; Guglietta, Ryan; O'Dell, Thomas J.

2016-01-01

Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are…

Alcoholytic Cleavage of Polyhydroxyalkanoate Chains by Class IV Synthases Induced by Endogenous and Exogenous Ethanol

PubMed Central

Hyakutake, Manami; Tomizawa, Satoshi; Mizuno, Kouhei; Abe, Hideki

2014-01-01

Polyhydroxyalkanoate (PHA)-producing Bacillus strains express class IV PHA synthase, which is composed of the subunits PhaR and PhaC. Recombinant Escherichia coli expressing PHA synthase from Bacillus cereus strain YB-4 (PhaRCYB-4) showed an unusual reduction of the molecular weight of PHA produced during the stationary phase of growth. Nuclear magnetic resonance analysis of the low-molecular-weight PHA revealed that its carboxy end structure was capped by ethanol, suggesting that the molecular weight reduction was the result of alcoholytic cleavage of PHA chains by PhaRCYB-4 induced by endogenous ethanol. This scission reaction was also induced by exogenous ethanol in both in vivo and in vitro assays. In addition, PhaRCYB-4 was observed to have alcoholysis activity for PHA chains synthesized by other synthases. The PHA synthase from Bacillus megaterium (PhaRCBm) from another subgroup of class IV synthases was also assayed and was shown to have weak alcoholysis activity for PHA chains. These results suggest that class IV synthases may commonly share alcoholysis activity as an inherent feature. PMID:24334666

Interstrain differences of ionotropic glutamate receptor subunits in the hippocampus and induction of hippocampal sclerosis with pilocarpine in mice.

PubMed

Dobó, Endre; Török, Ibolya; Mihály, András; Károly, Norbert; Krisztin-Péva, Beáta

2015-01-01

Rodent strains used in epilepsy research have various neurological characteristics. These differences were suggested to be attributed to the diverse densities of the ionotropic glutamate receptor (iGluR) subunits. However, previous studies failed to find interstrain differences in the hippocampal receptor levels. We supposed that a detailed layer-to-layer analysis of the iGluR subunits in the hippocampus might reveal strain-dependent differences in their base lines and reactions induced by pilocarpine (PILO) between two mouse strains without documented ancestors. Levels of iGluR subunits in Balb/c and NMRI mice were compared using semiquantitative immunohistochemistry. The alterations in the neuronal circuitry were validated by neuropeptide Y (NPY) and neuronal nuclear antigen (NeuN) immunostainings. Immunohistochemistry showed interstrain laminar differences in some subunits of both the control and PILO-treated animals. The seizure-induced irreversible neuronal changes were accompanied by reduced GluA1 and GluA2 levels. Their changes were inversely correlated in the individual NMRI mice by Pearson's method. Increase in NPY immunoreactivity showed positive correlation with GluA1, and negative correlation with GluA2. The NMRI strain was susceptible to PILO-induced hippocampal sclerosis, while the Balb/c animals showed resistance. Basal levels of iGluRs differ in mouse strains, which may account for the interstrain differences in their reactions to the convulsant. Copyright © 2015 Elsevier B.V. All rights reserved.

Alpha subunit of glycoprotein hormones in the sera of acromegalic patients and its mRNA in the tumors.

PubMed

Machiavelli, G A; Artese, R; Benencia, H; Bruno, O; Guerra, L; Basso, A; Burdman, J A

1999-04-01

Within a population of 16 pituitary adenomas we found high levels of glycoprotein alpha subunits in the sera of patients with somatotrophic tumors. This finding was correlated with the presence of mRNA alpha subunit in these tumors indicating the adenomas themselves as the origin of the circulating alpha-subunit. Synthesis of these two hormones, which are chemically very different, by the same tumor cells indicates a high degree of differentiation of these cells. We are unable at this time to conclusively correlate differentiation of these tumors aggressively.

A Cyclin Dependent Kinase Regulatory Subunit (CKS) Gene of Pigeonpea Imparts Abiotic Stress Tolerance and Regulates Plant Growth and Development in Arabidopsis

PubMed Central

Tamirisa, Srinath; Vudem, Dashavantha R.; Khareedu, Venkateswara R.

2017-01-01

Frequent climatic changes in conjunction with other extreme environmental factors are known to affect growth, development and productivity of diverse crop plants. Pigeonpea, a major grain legume of the semiarid tropics, endowed with an excellent deep-root system, is known as one of the important drought tolerant crop plants. Cyclin dependent kinases (CDKs) are core cell cycle regulators and play important role in different aspects of plant growth and development. The cyclin-dependent kinase regulatory subunit gene (CKS) was isolated from the cDNA library of pigeonpea plants subjected to drought stress. Pigeonpea CKS (CcCKS) gene expression was detected in both the root and leaf tissues of pigeonpea and was upregulated by polyethylene glycol (PEG), mannitol, NaCl and abscisic acid (ABA) treatments. The overexpression of CcCKS gene in Arabidopsis significantly enhanced tolerance of transgenics to drought and salt stresses as evidenced by different physiological parameters. Under stress conditions, transgenics showed higher biomass, decreased rate of water loss, decreased MDA levels, higher free proline contents, and glutathione levels. Moreover, under stress conditions transgenics exhibited lower stomatal conductance, lower transpiration, and higher photosynthetic rates. However, under normal conditions, CcCKS-transgenics displayed decreased plant growth rate, increased cell size and decreased stomatal number compared to those of wild-type plants. Real-time polymerase chain reaction revealed that CcCKS could regulate the expression of both ABA-dependent and ABA-independent genes associated with abiotic stress tolerance as well as plant growth and development. As such, the CcCKS seems promising and might serve as a potential candidate gene for enhancing the abiotic stress tolerance of crop plants. PMID:28239388

A Cyclin Dependent Kinase Regulatory Subunit (CKS) Gene of Pigeonpea Imparts Abiotic Stress Tolerance and Regulates Plant Growth and Development in Arabidopsis.

PubMed

Tamirisa, Srinath; Vudem, Dashavantha R; Khareedu, Venkateswara R

2017-01-01

Frequent climatic changes in conjunction with other extreme environmental factors are known to affect growth, development and productivity of diverse crop plants. Pigeonpea, a major grain legume of the semiarid tropics, endowed with an excellent deep-root system, is known as one of the important drought tolerant crop plants. Cyclin dependent kinases (CDKs) are core cell cycle regulators and play important role in different aspects of plant growth and development. The cyclin-dependent kinase regulatory subunit gene ( CKS ) was isolated from the cDNA library of pigeonpea plants subjected to drought stress. Pigeonpea CKS ( CcCKS ) gene expression was detected in both the root and leaf tissues of pigeonpea and was upregulated by polyethylene glycol (PEG), mannitol, NaCl and abscisic acid (ABA) treatments. The overexpression of CcCKS gene in Arabidopsis significantly enhanced tolerance of transgenics to drought and salt stresses as evidenced by different physiological parameters. Under stress conditions, transgenics showed higher biomass, decreased rate of water loss, decreased MDA levels, higher free proline contents, and glutathione levels. Moreover, under stress conditions transgenics exhibited lower stomatal conductance, lower transpiration, and higher photosynthetic rates. However, under normal conditions, CcCKS -transgenics displayed decreased plant growth rate, increased cell size and decreased stomatal number compared to those of wild-type plants. Real-time polymerase chain reaction revealed that Cc CKS could regulate the expression of both ABA-dependent and ABA-independent genes associated with abiotic stress tolerance as well as plant growth and development. As such, the CcCKS seems promising and might serve as a potential candidate gene for enhancing the abiotic stress tolerance of crop plants.

Rubisco small subunit, chlorophyll a/b-binding protein and sucrose:fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light.

PubMed

Lu, Chungui; Koroleva, Olga A; Farrar, John F; Gallagher, Joe; Pollock, Chris J; Tomos, A Deri

2002-11-01

We describe a highly efficient two-step single-cell reverse transcriptase-polymerase chain reaction technique for analyzing gene expression at the single-cell level. Good reproducibility and a linear dose response indicated that the technique has high specificity and sensitivity for detection and quantification of rare RNA. Actin could be used as an internal standard. The expression of message for Rubisco small subunit (RbcS), chlorophyll a/b-binding protein (Cab), sucrose (Suc):fructan-6-fructosyl transferase (6-SFT), and Actin were measured in individual photosynthetic cells of the barley (Hordeum vulgare) leaf. Only Actin was found in the non-photosynthetic epidermal cells. Cab, RbcS, and 6-SFT genes were expressed at a low level in mesophyll and parenchymatous bundle sheath (BS) cells when sampled from plants held in dark for 40 h. Expression increased considerably after illumination. The amount of 6-SFT, Cab, and RbcS transcript increased more in mesophyll cells than in the parenchymatous BS cells. The difference may be caused by different chloroplast structure and posttranscriptional control in mesophyll and BS cells. When similar single-cell samples were assayed for Suc, glucose, and fructan, there was high correlation between 6-SFT gene expression and Suc and glucose concentrations. This is consistent with Suc concentration being the trigger for transcription. Together with earlier demonstrations that the mesophyll cells have a higher sugar threshold for fructan polymerization, our data may indicate separate control of transcription and enzyme activity. Values for the sugar concentrations of the individual cell types are reported.

Branch pattern of starch internal structure influences the glucogenesis by mucosal Nt-maltase-glucoamylase.

PubMed

Lin, Amy Hui-Mei; Ao, Zihua; Quezada-Calvillo, Roberto; Nichols, Buford L; Lin, Chi-Tien; Hamaker, Bruce R

2014-10-13

To produce sufficient amounts of glucose from food starch, both α-amylase and mucosal α-glucosidases are required. We found previously that the digestion rate of starch is influenced by its susceptibility to mucosal α-glucosidases. In the present study, six starches and one glycogen were pre-hydrolyzed by α-amylase for various time periods, and then further hydrolyzed with the mucosal α-glucosidase, the N-terminal subunit of maltase-glucoamylase (Nt-MGAM), to generate free glucose. Results showed that α-amylase amplified the Nt-MGAM glucogenesis, and that the amplifications differed in various substrates. The amount of branches within α-amylase hydrolysate substrates was highly related to the rate of Nt-MGAM glucogenesis. After de-branching, the hydrolysates showed three fractions, Fraction 1, 2, and 3, in size exclusion chromatographs. We found that the α-amylase hydrolysates with higher quantity of the Fraction 3 (molecules with relatively short chain-length) and shorter average chain-length of this fraction had lower rates of Nt-MGAM glucogenesis. This study revealed that the branch pattern of α-amylase hydrolysates modulates glucose release by Nt-MGAM. It further supported the hypothesis that the internal structure of starch affects its digestibility at the mucosal α-glucosidase level. Published by Elsevier Ltd.

Estimating hydraulic parameters of a heterogeneous aquitard using long-term multi-extensometer and groundwater level data

NASA Astrophysics Data System (ADS)

Zhuang, Chao; Zhou, Zhifang; Illman, Walter A.; Guo, Qiaona; Wang, Jinguo

2017-09-01

The classical aquitard-drainage model COMPAC has been modified to simulate the compaction process of a heterogeneous aquitard consisting of multiple sub-units (Multi-COMPAC). By coupling Multi-COMPAC with the parameter estimation code PEST++, the vertical hydraulic conductivity ( K v) and elastic ( S ske) and inelastic ( S skp) skeletal specific-storage values of each sub-unit can be estimated using observed long-term multi-extensometer and groundwater level data. The approach was first tested through a synthetic case with known parameters. Results of the synthetic case revealed that it was possible to accurately estimate the three parameters for each sub-unit. Next, the methodology was applied to a field site located in Changzhou city, China. Based on the detailed stratigraphic information and extensometer data, the aquitard of interest was subdivided into three sub-units. Parameters K v, S ske and S skp of each sub-unit were estimated simultaneously and then were compared with laboratory results and with bulk values and geologic data from previous studies, demonstrating the reliability of parameter estimates. Estimated S skp values ranged within the magnitude of 10-4 m-1, while K v ranged over 10-10-10-8 m/s, suggesting moderately high heterogeneity of the aquitard. However, the elastic deformation of the third sub-unit, consisting of soft plastic silty clay, is masked by delayed drainage, and the inverse procedure leads to large uncertainty in the S ske estimate for this sub-unit.

Comparative studies of rat recombinant purple acid phosphatase and bone tartrate-resistant acid phosphatase.

PubMed Central

Ek-Rylander, B; Barkhem, T; Ljusberg, J; Ohman, L; Andersson, K K; Andersson, G

1997-01-01

The tartrate-resistant acid phosphatase (TRAP) of rat osteoclasts has been shown to exhibit high (85-94%) identity at the amino acid sequence level with the purple acid phosphatase (PAP) from bovine spleen and with pig uteroferrin. These iron-containing purple enzymes contain a binuclear iron centre, with a tyrosinate-to-Fe(III) charge-transfer transition responsible for the purple colour. In the present study, production of rat osteoclast TRAP could be achieved at a level of 4.3 mg/litre of medium using a baculovirus expression system. The enzyme was purified to apparent homogeneity using a combination of cation-exchange, hydrophobic-interaction, lectin-affinity and gel-permeation chromatography steps. The protein as isolated had a purple colour, a specific activity of 428 units/mg of protein and consisted of the single-chain form of molecular mass 34 kDa, with only trace amounts of proteolytically derived subunits. The recombinant enzyme had the ability to dephosphorylate bone matrix phosphoproteins, as previously shown for bone TRAP. Light absorption spectroscopy of the isolated purple enzyme showed a lambda max at 544 nm, which upon reduction with ascorbic acid changed to 515 nm, concomitant with the transition to a pink colour. EPR spectroscopic analysis of the reduced enzyme at 3.6 K revealed a typical mu-hydr(oxo)-bridged mixed-valent Fe(II)Fe(III) signal with g-values at 1.96, 1.74 and 1.60, proving that recombinant rat TRAP belongs to the family of PAPs. To validate the use of recombinant PAP in substituting for the rat bone counterpart in functional studies, various comparative studies were carried out. The enzyme isolated from bone exhibited a lower K(m) for p-nitrophenyl phosphate and was slightly more sensitive to PAP inhibitors such as molybdate, tungstate, arsenate and phosphate. In contrast with the recombinant enzyme, TRAP from bone was isolated predominantly as the proteolytically cleaved, two-subunit, form. Both the recombinant enzyme and rat bone TRAP were shown to be substituted with N-linked oligosaccharides. A slightly higher apparent molecular mass of the monomeric form and N-terminal chain of bone TRAP compared with the recombinant enzyme could not be accounted for by differential N-glycosylation. Despite differences in specific post-translational modifications, the recombinant PAP should be useful in future studies on the properties and regulation of the mammalian PAP enzyme. PMID:9020859

Expression of three gonadotropin subunits and gonadotropin receptor mRNA during male-to-female sex change in the cinnamon clownfish, Amphiprion melanopus.

PubMed

An, Kwang Wook; Lee, Jehee; Choi, Cheol Young

2010-08-01

To quantify the sex-change progression from male to female in the cinnamon clownfish, Amphiprion melanopus, we divided gonadal development into three stages (I, mature male; II, male at 90 days after removal of the female; and III, mature female), and the expression of GTH subunits and GTH receptors during each of these stages was investigated. The mRNA of the three GTH subunits and their receptors increased with progression from male to female. To understand the effect of gonadotropin-releasing hormone (GnRH) on this progression, we examined expression of genes encoding the GTH subunit mRNA in the pituitary and the GTH-receptor mRNA in the gonads in addition to investigating changes in plasma E(2) levels after GnRH analogue (GnRHa) injection. GnRHa treatment increased mRNA expression levels of these genes, as well as plasma E(2) levels, indicating that GnRH plays an important regulatory role in the brain-pituitary-gonad axis of immature cinnamon clownfish. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Molecular contacts in the transmembrane c-subunit oligomer of F-ATPases identified by tryptophan substitution mutagenesis.

PubMed

Schnick, C; Forrest, L R; Sansom, M S; Groth, G

2000-07-20

When isolated in its monomeric form, subunit c of the proton transporting ATP synthase of Escherichia coli was shown to fold in a hairpin-like structure consisting of two hydrophobic membrane spanning helices and a short connecting hydrophilic loop. In the plasma membrane of Escherichia coli, however, about 9-12 c-subunit monomers form an oligomeric complex that functions in transmembrane proton conduction and in energy transduction to the catalytic F1 domain. The arrangement of the monomers and the molecular architecture of the complex were studied by tryptophan scanning mutagenesis and restrained MD simulations. Residues 12-24 of the N-terminal transmembrane segment of subunit c were individually substituted by the large and moderately hydrophobic tryptophan side chain. Effects on the activity of the mutant proteins were studied in selective growth experiments and various ATP synthase specific activity assays. The results identify potential intersubunit contacts and structurally non-distorted, accessible residues in the c-oligomer and add constraints to the arrangement of monomers in the oligomeric complex. Results from our mutagenesis experiments were interpreted in structural models of the c-oligomer that have been obtained by restrained MD simulations. Different stoichiometries and monomer orientations were applied in these calculations. A cylindrical complex consisting of 10 monomers that are arranged in two concentric rings with the N-terminal helices of the monomers located at the periphery shows the best match with the experimental data.

Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei

PubMed Central

Lopez-Zavala, Alonso A.; Carrasco-Miranda, Jesus S.; Ramirez-Aguirre, Claudia D.; López-Hidalgo, Marisol; Benitez-Cardoza, Claudia G.; Ochoa-Leyva, Adrian; Cardona-Felix, Cesar S.; Diaz-Quezada, Corina; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.; Brieba, Luis G.

2016-01-01

Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7 Å resolution. LvTIM assembles as a homodimer with residues 166-176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a "ball and socket" interaction. LvTIM presents a Cys47 at the "ball" inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation. PMID:27614148

Identification of the Catalytic Ubiquinone-binding Site of Vibrio cholerae Sodium-dependent NADH Dehydrogenase

PubMed Central

Tuz, Karina; Li, Chen; Fang, Xuan; Raba, Daniel A.; Liang, Pingdong; Minh, David D. L.; Juárez, Oscar

2017-01-01

The sodium-dependent NADH dehydrogenase (Na+-NQR) is a key component of the respiratory chain of diverse prokaryotic species, including pathogenic bacteria. Na+-NQR uses the energy released by electron transfer between NADH and ubiquinone (UQ) to pump sodium, producing a gradient that sustains many essential homeostatic processes as well as virulence factor secretion and the elimination of drugs. The location of the UQ binding site has been controversial, with two main hypotheses that suggest that this site could be located in the cytosolic subunit A or in the membrane-bound subunit B. In this work, we performed alanine scanning mutagenesis of aromatic residues located in transmembrane helices II, IV, and V of subunit B, near glycine residues 140 and 141. These two critical glycine residues form part of the structures that regulate the site's accessibility. Our results indicate that the elimination of phenylalanine residue 211 or 213 abolishes the UQ-dependent activity, produces a leak of electrons to oxygen, and completely blocks the binding of UQ and the inhibitor HQNO. Molecular docking calculations predict that UQ interacts with phenylalanine 211 and pinpoints the location of the binding site in the interface of subunits B and D. The mutagenesis and structural analysis allow us to propose a novel UQ-binding motif, which is completely different compared with the sites of other respiratory photosynthetic complexes. These results are essential to understanding the electron transfer pathways and mechanism of Na+-NQR catalysis. PMID:28053088

Loss of the smallest subunit of cytochrome c oxidase, COX8A, causes Leigh-like syndrome and epilepsy.

PubMed

Hallmann, Kerstin; Kudin, Alexei P; Zsurka, Gábor; Kornblum, Cornelia; Reimann, Jens; Stüve, Burkhard; Waltz, Stephan; Hattingen, Elke; Thiele, Holger; Nürnberg, Peter; Rüb, Cornelia; Voos, Wolfgang; Kopatz, Jens; Neumann, Harald; Kunz, Wolfram S

2016-02-01

Isolated cytochrome c oxidase (complex IV) deficiency is one of the most frequent respiratory chain defects in humans and is usually caused by mutations in proteins required for assembly of the complex. Mutations in nuclear-encoded structural subunits are very rare. In a patient with Leigh-like syndrome presenting with leukodystrophy and severe epilepsy, we identified a homozygous splice site mutation in COX8A, which codes for the ubiquitously expressed isoform of subunit VIII, the smallest nuclear-encoded subunit of complex IV. The mutation, affecting the last nucleotide of intron 1, leads to aberrant splicing, a frame-shift in the highly conserved exon 2, and decreased amount of the COX8A transcript. The loss of the wild-type COX8A protein severely impairs the stability of the entire cytochrome c oxidase enzyme complex and manifests in isolated complex IV deficiency in skeletal muscle and fibroblasts, similar to the frequent c.845_846delCT mutation in the assembly factor SURF1 gene. Stability and activity of complex IV could be rescued in the patient's fibroblasts by lentiviral expression of wild-type COX8A. Our findings demonstrate that COX8A is indispensable for function of human complex IV and its mutation causes human disease. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Interaction between glycosaminoglycans and immunoglobulin light chains.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, X.; Myatt, E.; Lykos, P.

1997-01-01

Amyloidosis is a pathological process in which normally soluble proteins polymerize to form insoluble fibrils (amyloid). Amyloid formation is found in a number of diseases, including Alzheimer's disease, adult-onset diabetes, and light-chain-associated amyloidosis. No pharmaceutical methods currently exist to prevent this process or to remove the fibrils from tissue. The search for treatment and prevention methods is hampered by a limited understanding of the biophysical basis of amyloid formation. Glycosaminoglycans (GAGs) are long, unbranched heteropolysaccharides composed of repeating disaccharide subunits and are known to associate with amyloid fibrils. The interaction of amyloid-associated free light chains with GAGs was tested bymore » both size-exclusion high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments. The results indicated that heparin 16 000 and chondroitin sulfate B and C precipitated both human intact light chains and recombinant light chain variable domains. Although all light chains interacted with heparin, the strongest interactions were obtained with proteins that had formed amyloid. Molecular modeling indicated the possibility of interaction between heparin and the conserved saddle like surface of the light chain dimer opposite the complementarity-determining segments that form part of the antigen-binding site of a functional antibody. This suggestion might offer a new path to block the aggregation of amyloid-associated light chain proteins, by design of antagonists based on properties of GAG binding. A hexasaccharide was modeled as the basis for a possible antagonist.« less

Polyphosphate kinase: demonstration that short chain polyphosphate serves as a primer for the enzymatic synthesis of polyphosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robinson, N.A.; Wood, H.G.

1986-05-01

Polyphosphate (poly(P)) kinase, isolated from Propionibacterium shermanii, catalyzes the following reaction: poly(P/sub n/) + ATPin equilibriumpoly(P/sub n+1/) + ADP. The authors have purified this enzyme to 90% homogeneity and have shown it to be composed of 2-3 identical subunits of M/sub r/ 80,000. Investigation of the reaction mechanism by product analysis has revealed that the elongation phase is processive whereby successive elongation occurs without release of intermediate sizes until very long chains are formed. The initiation phase of synthesis has been investigated using (/sup 32/P) poly(P) primer of chain length 11-60. It is incorporated into long chain poly(P) and themore » /sup 32/P has been shown, by use of poly(P) glucokinase, to be localized at the end of the molecule. Calculation of average chain length based upon the incorporation of /sup 32/P, however, yields a value approx.3 fold higher than the value calculated by another method using poly(P) glucokinase. This result indicates that initiation of poly(P) synthesis occurs by at least one other route which does not involve short chain poly(P) primers. The effect of temperature and concentration of poly(P) primer upon the average chain length of poly(P) synthesized was also investigated. A general trend was observed in which the chain length of the synthesized poly(P) decreased as either temperature or concentration or primer was increased.« less

The Role of Glycine Residues 140 and 141 of Subunit B in the Functional Ubiquinone Binding Site of the Na+-pumping NADH:quinone Oxidoreductase from Vibrio cholerae*

PubMed Central

Juárez, Oscar; Neehaul, Yashvin; Turk, Erin; Chahboun, Najat; DeMicco, Jessica M.; Hellwig, Petra; Barquera, Blanca

2012-01-01

The Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) is the main entrance for electrons into the respiratory chain of many marine and pathogenic bacteria. The enzyme accepts electrons from NADH and donates them to ubiquinone, and the free energy released by this redox reaction is used to create an electrochemical gradient of sodium across the cell membrane. Here we report the role of glycine 140 and glycine 141 of the NqrB subunit in the functional binding of ubiquinone. Mutations at these residues altered the affinity of the enzyme for ubiquinol. Moreover, mutations in residue NqrB-G140 almost completely abolished the electron transfer to ubiquinone. Thus, NqrB-G140 and -G141 are critical for the binding and reaction of Na+-NQR with its electron acceptor, ubiquinone. PMID:22645140

Ribosome rearrangements at the onset of translational bypassing

PubMed Central

Agirrezabala, Xabier; Samatova, Ekaterina; Klimova, Mariia; Zamora, Miguel; Gil-Carton, David; Rodnina, Marina V.; Valle, Mikel

2017-01-01

Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNAGly to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap. PMID:28630923

Association of Mitochondrial DNA 10398 Polymorphism in Invasive Breast Cancer in Malay Population of Peninsular Malaysia

PubMed Central

Tengku Baharudin, Nadiah; Jaafar, Hasnan; Zainuddin, Zafarina

2012-01-01

Background: The mitochondrial DNA (mtDNA) 10398 polymorphism is hypothesised to alter a mitochondrial subunit of the electron transfer chain and is associated with several neurodegenerative disorders and cancers. Methods: In this study, an mtDNA polymorphism at nucleotide position 10398 was screened in 101 Malay female patients with invasive breast cancer and 90 age-matched healthy female controls using minisequencing analysis. Results: The Malay women with the 10398G variant showed a significantly increased risk of invasive breast cancer (OR = 2.29, 95% CI 1.25–4.20, P = 0.007). Immunohistochemistry analysis was conducted to investigate the effect of this polymorphism on the levels of apoptosis in breast cancer cells. The level of Bax (a pro-apoptotic protein) expression was significantly higher than that of Bcl-2 (an anti-apoptotic protein) in patients carrying the G allele (P = 0.016) but not in those carrying the A allele (P = 0.48). Conclusion: Based on these findings, we propose that the mtDNA 10398 polymorphism may be a potential risk marker for breast cancer susceptibility in the Malay population. PMID:22977373

High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing α4β3δ subunits.

PubMed

Zhou, Xiaojuan; Desai, Rooma; Zhang, Yinghui; Stec, Wojciech J; Miller, Keith W; Jounaidi, Youssef

2018-01-01

The inhibitory γ-aminobutyric acid type A receptors are implicated in numerous physiological processes, including cognition and inhibition of neurotransmission, rendering them important molecular targets for many classes of drugs. Functionally, the entire GABAAR family of receptors can be subdivided into phasic, fast acting synaptic receptors, composed of α-, β- and γ-subunits, and tonic extrasynaptic receptors, many of which contain the δ-subunit in addition to α- and β-subunits. Whereas the subunit arrangement of the former group is agreed upon, that of the αβδ GABAARs remains unresolved by electrophysiological and pharmacological research. To resolve such issues will require biophysical techniques that demand quantities of receptor that have been previously unavailable. Therefore, we have engineered a stable cell line with tetracycline inducible expression of human α4-, β3- and N-terminally Flag-tagged δ-subunits. This cell line achieved a specific activity between 15 and 20 pmol [3H]muscimol sites/mg of membrane protein, making it possible to obtain 1 nmole of purified α4β3δ GABAAR from sixty 15-cm culture dishes. When induced, these cells exhibited agonist-induced currents with characteristics comparable to those previously reported for this receptor and a pharmacology that included strong modulation by etomidate and the δ-subunit-specific ligand, DS2. Immunoaffinity purification and reconstitution in CHAPS/asolectin micelles resulted in the retention of equilibrium allosteric interactions between the separate agonist, anesthetic and DS2 sites. Moreover, all three subunits retained glycosylation. The establishment of this well-characterized cell line will allow molecular level studies of tonic receptors to be undertaken.

Downregulation of GABA[Subscript A] Receptor Protein Subunits a6, ß2, d, e, ?2, ?, and ?2 in Superior Frontal Cortex of Subjects with Autism

ERIC Educational Resources Information Center

Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rustan, Oyvind G.; Rooney, Robert J.; Thuras, Paul D.

2014-01-01

We measured protein and mRNA levels for nine gamma-aminobutyric acid A (GABA[subscript A]) receptor subunits in three brain regions (cerebellum, superior frontal cortex, and parietal cortex) in subjects with autism versus matched controls. We observed changes in mRNA for a number of GABA[subscript A] and GABA[subscript B] subunits and overall…

Fasciola hepatica Infection in an Indigenous Community of the Peruvian Jungle.

PubMed

Cabada, Miguel M; Castellanos-Gonzalez, Alejandro; Lopez, Martha; Caravedo, María Alejandra; Arque, Eulogia; White, Arthur Clinton

2016-06-01

Fasciola hepatica is a zoonotic infection with a worldwide distribution. Autochthonous cases have not been reported in the Amazon region of Peru. Operculated eggs resembling F. hepatica were identified in the stools of five out of 215 subjects in a remote indigenous community of the Peruvian jungle. Polymerase chain reaction targeting Fasciola hepatica cytochrome oxidase subunit 1 (COI) gene and sequencing of the products confirmed Fasciola infection. © The American Society of Tropical Medicine and Hygiene.

Naphthalenesulphonamides block neutrophil superoxide production by intact cells and in a cell-free system: is myosin light chain kinase responsible for these effects?

PubMed Central

Heyworth, P G; Erickson, R W; Ding, J; Curnutte, J T; Badwey, J A

1995-01-01

Selective antagonists of myosin light chain kinase (MLCK) [e.g. ML-7; 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride] were found to inhibit superoxide (O2-) release from stimulated neutrophils. The concentrations of ML-7 that were inhibitory were substantially lower than those reported for a selective antagonist of protein kinase C [i.e. H-7; 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride]. ML-7 also reduced the phosphorylation of the 47 kDa subunit of the NADPH-oxidase system (p47-phox) and blocked translocation of this protein to the Triton X-100-insoluble fraction in stimulated cells. Interestingly, ML-7 also inhibited O2- production in a cell-free system derived from neutrophils at concentrations similar to those that were effective in vivo. This cell-free system does not require ATP and is insensitive to all other inhibitors of protein kinases tested, including some highly effective against MLCK (i.e. staurosporine). Thus, the data suggest that ML-7 does not block O2- release by inhibiting a protein kinase but instead may interact directly with a subunit of the oxidase. The binding site for ML-7 may provide a valuable target for inhibiting the inflammatory properties of phagocytic leucocytes by naphthalenesulphonamides designed to lack activity against protein kinases. Images Figure 3 Figure 4 PMID:7575484

A Conserved Region in the F2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation▿

PubMed Central

Gardner, Amanda E.; Dutch, Rebecca E.

2007-01-01

Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F1 and F2. Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F1 (CBF1) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F2 subunit (CBF2). To analyze the functions of CBF2, alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF2 mutations resulted in folding and expression defects. However, the CBF2 mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF2 Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF2 I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF2 in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion. PMID:17507474

Molecular cloning, phylogenetic analysis, and expression profiling of endoplasmic reticulum molecular chaperone BiP genes from bread wheat (Triticum aestivum L.).

PubMed

Zhu, Jiantang; Hao, Pengchao; Chen, Guanxing; Han, Caixia; Li, Xiaohui; Zeller, Friedrich J; Hsam, Sai L K; Hu, Yingkao; Yan, Yueming

2014-10-01

The endoplasmic reticulum chaperone binding protein (BiP) is an important functional protein, which is involved in protein synthesis, folding assembly, and secretion. In order to study the role of BiP in the process of wheat seed development, we cloned three BiP homologous cDNA sequences in bread wheat (Triticum aestivum), completed by rapid amplification of cDNA ends (RACE), and examined the expression of wheat BiP in wheat tissues, particularly the relationship between BiP expression and the subunit types of HMW-GS using near-isogenic lines (NILs) of HMW-GS silencing, and under abiotic stress. Sequence analysis demonstrated that all BiPs contained three highly conserved domains present in plants, animals, and microorganisms, indicating their evolutionary conservation among different biological species. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that TaBiP (Triticum aestivum BiP) expression was not organ-specific, but was predominantly localized to seed endosperm. Furthermore, immunolocalization confirmed that TaBiP was primarily located within the protein bodies (PBs) in wheat endosperm. Three TaBiP genes exhibited significantly down-regulated expression following high molecular weight-glutenin subunit (HMW-GS) silencing. Drought stress induced significantly up-regulated expression of TaBiPs in wheat roots, leaves, and developing grains. The high conservation of BiP sequences suggests that BiP plays the same role, or has common mechanisms, in the folding and assembly of nascent polypeptides and protein synthesis across species. The expression of TaBiPs in different wheat tissue and under abiotic stress indicated that TaBiP is most abundant in tissues with high secretory activity and with high proportions of cells undergoing division, and that the expression level of BiP is associated with the subunit types of HMW-GS and synthesis. The expression of TaBiPs is developmentally regulated during seed development and early seedling growth, and under various abiotic stresses.

Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.

PubMed

Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

2016-01-01

Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels.

Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

PubMed Central

Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

2016-01-01

Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels. PMID:26966698

β-Subunits Promote the Expression of CaV2.2 Channels by Reducing Their Proteasomal Degradation*

PubMed Central

Waithe, Dominic; Ferron, Laurent; Page, Karen M.; Chaggar, Kanchan; Dolphin, Annette C.

2011-01-01

The β-subunits of voltage-gated calcium channels regulate their functional expression and properties. Two mechanisms have been proposed for this, an effect on gating and an enhancement of expression. With respect to the effect on expression, β-subunits have been suggested to enhance trafficking by masking an unidentified endoplasmic reticulum (ER) retention signal. Here we have investigated whether, and how, β-subunits affect the level of CaV2.2 channels within somata and neurites of cultured sympathetic neurons. We have used YFP-CaV2.2 containing a mutation (W391A), that prevents binding of β-subunits to its I-II linker and found that expression of this channel was much reduced compared with WT CFP-CaV2.2 when both were expressed in the same neuron. This effect was particularly evident in neurites and growth cones. The difference between the levels of YFP-CaV2.2(W391A) and CFP-CaV2.2(WT) was lost in the absence of co-expressed β-subunits. Furthermore, the relative reduction of expression of CaV2.2(W391A) compared with the WT channel was reversed by exposure to two proteasome inhibitors, MG132 and lactacystin, particularly in the somata. In further experiments in tsA-201 cells, we found that proteasome inhibition did not augment the cell surface CaV2.2(W391A) level but resulted in the observation of increased ubiquitination, particularly of mutant channels. In contrast, we found no evidence for selective retention of CaV2.2(W391A) in the ER, in either the soma or growth cones. In conclusion, there is a marked effect of β-subunits on CaV2.2 expression, particularly in neurites, but our results point to protection from proteasomal degradation rather than masking of an ER retention signal. PMID:21233207

Absence of Complex I Is Associated with Diminished Respiratory Chain Function in European Mistletoe.

PubMed

Maclean, Andrew E; Hertle, Alexander P; Ligas, Joanna; Bock, Ralph; Balk, Janneke; Meyer, Etienne H

2018-05-21

Parasitism is a life history strategy found across all domains of life whereby nutrition is obtained from a host. It is often associated with reductive evolution of the genome, including loss of genes from the organellar genomes [1, 2]. In some unicellular parasites, the mitochondrial genome (mitogenome) has been lost entirely, with far-reaching consequences for the physiology of the organism [3, 4]. Recently, mitogenome sequences of several species of the hemiparasitic plant mistletoe (Viscum sp.) have been reported [5, 6], revealing a striking loss of genes not seen in any other multicellular eukaryotes. In particular, the nad genes encoding subunits of respiratory complex I are all absent and other protein-coding genes are also lost or highly diverged in sequence, raising the question what remains of the respiratory complexes and mitochondrial functions. Here we show that oxidative phosphorylation (OXPHOS) in European mistletoe, Viscum album, is highly diminished. Complex I activity and protein subunits of complex I could not be detected. The levels of complex IV and ATP synthase were at least 5-fold lower than in the non-parasitic model plant Arabidopsis thaliana, whereas alternative dehydrogenases and oxidases were higher in abundance. Carbon flux analysis indicates that cytosolic reactions including glycolysis are greater contributors to ATP synthesis than the mitochondrial tricarboxylic acid (TCA) cycle. Our results describe the extreme adjustments in mitochondrial functions of the first reported multicellular eukaryote without complex I. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy.

PubMed

Tsai, Y-S; Shiau, A-L; Chen, Y-F; Tsai, H-T; Tzai, T-S; Wu, C-L

2010-01-01

The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

Selection of reference genes for expression analyses of red-fleshed sweet orange (Citrus sinensis).

PubMed

Pinheiro, T T; Nishimura, D S; De Nadai, F B; Figueira, A; Latado, R R

2015-12-28

Red-fleshed oranges (Citrus sinensis) contain high levels of carotenoids and lycopene. The growing consumer demand for products with health benefits has increased interest in these types of Citrus cultivars as a potential source of nutraceuticals. However, little is known about the physiology of these cultivars under Brazilian conditions. Transcriptome and gene expression analyses are important tools in the breeding and management of red-fleshed sweet orange cultivars. Reverse transcription quantitative polymerase chain reaction is a method of quantifying gene expression, but various standardizations are required to obtain precise, accurate, and specific results. Among the standardizations required, the choice of suitable stable reference genes is fundamental. The objective of this study was to evaluate the stability of 11 candidate genes using various tissue and organ samples from healthy plants or leaves from citrus greening disease (Huanglongbing)-symptomatic plants of a Brazilian red-fleshed cultivar ('Sanguínea de Mombuca'), in order to select the most suitable reference gene for investigating gene expression under these conditions. geNorm and NormFinder identified genes that encoded translation initiation factor 3, ribosomal protein L35, and translation initiation factor 5A as the most stable genes under the biological conditions tested, and genes coding actin (ACT) and the subunit of the PSI reaction center subunit III were the least stable. Phosphatase, malate dehydrogenase, and ACT were the most stable genes in the leaf samples of infected plants.

Leigh disease presenting in utero due to a novel missense mutation in the mitochondrial DNA-ND3.

PubMed

Leshinsky-Silver, Esther; Lev, Dorit; Malinger, Gustavo; Shapira, Daniel; Cohen, Sarit; Lerman-Sagie, Tally; Saada, Ann

2010-05-01

Leigh syndrome can be caused by defects in both nuclear and mitochondrial genes involved in energy metabolism. Recently, an increasing number of mutations in mitochondrial DNA encoding regions, especially in NADH dehydrogenase (respiratory chain complex I) subunits, have been reported as causative of early onset Leigh syndrome. We describe a patient whose fetal brain ultrasound demonstrated periventricular pseudocyst suggestive of a possible mitochondrial disorder who presented postnatally with Leigh syndrome. A muscle biopsy demonstrated a partial decrease in complex I and pyruvate dehydrogenase (PDH-E1 alpha) activity. Sequencing of the PDH-E1 alpha gene did not reveal any mutation. Sequencing of the mtDNA revealed a novel heteroplasmic G10254A (D66N) mutation in the ND3 gene. This change results in a substitution of aspartic acid to asparagine in a highly conserved domain of the ND3 subunit. The mutation could not be detected in the mother's blood or urine sediment. Blue native gel electrophoresis of muscle mitochondria revealed a normal size, albeit a decreased level of complex I. The G10254A substitution in the mtDNA-ND3 gene is another cause of maternally inherited Leigh syndrome. This case demonstrates that periventricular pseudocysts may be the initial in utero presentation in patients with mitochondrial disorders. We emphasize the importance of screening the mtDNA in pediatric patients as the first step in molecular diagnosis of Leigh syndrome. (c) 2010 Elsevier Inc. All rights reserved.

Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes.

PubMed

Li, Hao; van der Linden, Wouter A; Verdoes, Martijn; Florea, Bogdan I; McAllister, Fiona E; Govindaswamy, Kavitha; Elias, Joshua E; Bhanot, Purnima; Overkleeft, Herman S; Bogyo, Matthew

2014-08-15

The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

Contributions of arginines-43 and -94 of human choriogonadotropin. beta. to receptor binding and activation as determined by oligonucleotide-based mutagenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang Chen; Puett, D.

1991-10-22

Members of the glycoprotein hormone family contain a common {alpha} subunit and a hormone-specific {beta} subunit. Human choriogonadotropin (hCG) {beta} is a 145 amino acid residue protein glycosylated at 6 positions (2 N-linked and 4 O-linked oligosaccharides). In an effort to elucidate receptor determinants on hCG{beta}, the authors have used site-directed mutagenesis to prepare and express several mutant cDNAs with replacements at arginines-43 and -94. Arg-43 is invariant in all known mammalian CG/lutropin {beta} amino acid sequences, and Arg-94 is conserved in 10 of the 12 sequences. Moreover, various studies involving synthetic peptides and enzymatic digestions of intact {beta} chainsmore » suggest that these residues may be important in hCG receptor binding. Point mutants were made in which these two arginines were replaced with the corresponding residues in human follitropin {beta}, Leu-43 and Asp-94. The wild-type and mutant {beta} chains were expressed in CHO cells containing a stably integrated gene for bovine {alpha}, and heterodimer formation occurred. These heterologous gonadotropins were active in assays using transformed Leydig cells, competitive binding with standard {sup 125}I-hCG, and cAMP and progesterone production, but the potency was considerably less than that associated with the hCG{beta} wild-type-containing gonadotropin. The double-mutant protein Arg-43 to Leu/Arg-94 to Asp also associated with bovine {alpha}, but the resultant heterodimer exhibited only low activity. Replacement but the Lys-43-containing {beta} chain appeared to exhibit a low degree of subunit association or reduced stability relative to the expressed hCG{beta} wild type. These results demonstrate that arginines-43 and -94 contribute to receptor binding through a positive charge.« less

Phosphorylation-dependent autoinhibition of myosin light chain phosphatase accounts for Ca2+ sensitization force of smooth muscle contraction.

PubMed

Khromov, Alexander; Choudhury, Nandini; Stevenson, Andra S; Somlyo, Avril V; Eto, Masumi

2009-08-07

The reversible regulation of myosin light chain phosphatase (MLCP) in response to agonist stimulation and cAMP/cGMP signals plays an important role in the regulation of smooth muscle (SM) tone. Here, we investigated the mechanism underlying the inhibition of MLCP induced by the phosphorylation of myosin phosphatase targeting subunit (MYPT1), a regulatory subunit of MLCP, at Thr-696 and Thr-853 using glutathione S-transferase (GST)-MYPT1 fragments having the inhibitory phosphorylation sites. GST-MYPT1 fragments, including only Thr-696 and only Thr-853, inhibited purified MLCP (IC(50) = 1.6 and 60 nm, respectively) when they were phosphorylated with RhoA-dependent kinase (ROCK). The activities of isolated catalytic subunits of type 1 and type 2A phosphatases (PP1 and PP2A) were insensitive to either fragment. Phospho-GST-MYPT1 fragments docked directly at the active site of MLCP, and this was blocked by a PP1/PP2A inhibitor microcystin (MC)-LR or by mutation of the active sites in PP1. GST-MYPT1 fragments induced a contraction of beta-escin-permeabilized ileum SM at constant pCa 6.3 (EC(50) = 2 microm), which was eliminated by Ala substitution of the fragment at Thr-696 or by ROCK inhibitors or 8Br-cGMP. GST-MYPT1-(697-880) was 5-times less potent than fragments including Thr-696. Relaxation induced by 8Br-cGMP was not affected by Ala substitution at Ser-695, a known phosphorylation site for protein kinase A/G. Thus, GST-MYPT1 fragments are phosphorylated by ROCK in permeabilized SM and mimic agonist-induced inhibition and cGMP-induced activation of MLCP. We propose a model in which MYPT1 phosphorylation at Thr-696 and Thr-853 causes an autoinhibition of MLCP that accounts for Ca(2+) sensitization of smooth muscle force.

Another cat and mouse game: Deciphering the evolution of the SCGB superfamily and exploring the molecular similarity of major cat allergen Fel d 1 and mouse ABP using computational approaches

PubMed Central

Pageat, Patrick; Bienboire-Frosini, Cécile

2018-01-01

The mammalian secretoglobin (SCGB) superfamily contains functionally diverse members, among which the major cat allergen Fel d 1 and mouse salivary androgen-binding protein (ABP) display similar subunits. We searched for molecular similarities between Fel d 1 and ABP to examine the possibility that they play similar roles. We aimed to i) cluster the evolutionary relationships of the SCGB superfamily; ii) identify divergence patterns, structural overlap, and protein-protein docking between Fel d 1 and ABP dimers; and iii) explore the residual interaction between ABP dimers and steroid binding in chemical communication using computational approaches. We also report that the evolutionary tree of the SCGB superfamily comprises seven unique palm-like clusters, showing the evolutionary pattern and divergence time tree of Fel d 1 with 28 ABP paralogs. Three ABP subunits (A27, BG27, and BG26) share phylogenetic relationships with Fel d 1 chains. The Fel d 1 and ABP subunits show similarities in terms of sequence conservation, identical motifs and binding site clefts. Topologically equivalent positions were visualized through superimposition of ABP A27:BG27 (AB) and ABP A27:BG26 (AG) dimers on a heterodimeric Fel d 1 model. In docking, Fel d 1-ABP dimers exhibit the maximum surface binding ability of AG compared with that of AB dimers and the several polar interactions between ABP dimers with steroids. Hence, cat Fel d 1 is an ABP-like molecule in which monomeric chains 1 and 2 are the equivalent of the ABPA and ABPBG monomers, respectively. These findings suggest that the biological and molecular function of Fel d 1 is similar to that of ABP in chemical communication, possibly via pheromone and/or steroid binding. PMID:29771985

Another cat and mouse game: Deciphering the evolution of the SCGB superfamily and exploring the molecular similarity of major cat allergen Fel d 1 and mouse ABP using computational approaches.

PubMed

Durairaj, Rajesh; Pageat, Patrick; Bienboire-Frosini, Cécile

2018-01-01

The mammalian secretoglobin (SCGB) superfamily contains functionally diverse members, among which the major cat allergen Fel d 1 and mouse salivary androgen-binding protein (ABP) display similar subunits. We searched for molecular similarities between Fel d 1 and ABP to examine the possibility that they play similar roles. We aimed to i) cluster the evolutionary relationships of the SCGB superfamily; ii) identify divergence patterns, structural overlap, and protein-protein docking between Fel d 1 and ABP dimers; and iii) explore the residual interaction between ABP dimers and steroid binding in chemical communication using computational approaches. We also report that the evolutionary tree of the SCGB superfamily comprises seven unique palm-like clusters, showing the evolutionary pattern and divergence time tree of Fel d 1 with 28 ABP paralogs. Three ABP subunits (A27, BG27, and BG26) share phylogenetic relationships with Fel d 1 chains. The Fel d 1 and ABP subunits show similarities in terms of sequence conservation, identical motifs and binding site clefts. Topologically equivalent positions were visualized through superimposition of ABP A27:BG27 (AB) and ABP A27:BG26 (AG) dimers on a heterodimeric Fel d 1 model. In docking, Fel d 1-ABP dimers exhibit the maximum surface binding ability of AG compared with that of AB dimers and the several polar interactions between ABP dimers with steroids. Hence, cat Fel d 1 is an ABP-like molecule in which monomeric chains 1 and 2 are the equivalent of the ABPA and ABPBG monomers, respectively. These findings suggest that the biological and molecular function of Fel d 1 is similar to that of ABP in chemical communication, possibly via pheromone and/or steroid binding.

Assembly and Architecture of Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1)*

PubMed Central

Lee, Hyung Ho; Nemecek, Daniel; Schindler, Christina; Smith, William J.; Ghirlando, Rodolfo; Steven, Alasdair C.; Bonifacino, Juan S.; Hurley, James H.

2012-01-01

BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. We show that BLOC-1 is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted, BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, ∼300 Å long and ∼30 Å in diameter. The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45°. Two stable subcomplexes were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course of tubular endosome biogenesis and sorting. PMID:22203680

Respiratory processes in non-photosynthetic plastids

PubMed Central

Renato, Marta; Boronat, Albert; Azcón-Bieto, Joaquín

2015-01-01

Chlororespiration is a respiratory process located in chloroplast thylakoids which consists in an electron transport chain from NAD(P)H to oxygen. This respiratory chain involves the NAD(P)H dehydrogenase complex, the plastoquinone pool and the plastid terminal oxidase (PTOX), and it probably acts as a safety valve to prevent the over-reduction of the photosynthetic machinery in stress conditions. The existence of a similar respiratory activity in non-photosynthetic plastids has been less studied. Recently, it has been reported that tomato fruit chromoplasts present an oxygen consumption activity linked to ATP synthesis. Etioplasts and amyloplasts contain several electron carriers and some subunits of the ATP synthase, so they could harbor a similar respiratory process. This review provides an update on the study about respiratory processes in chromoplasts, identifying the major gaps that need to be addressed in future research. It also reviews the proteomic data of etioplasts and amyloplasts, which suggest the presence of a respiratory electron transport chain in these plastids. PMID:26236317

Neonatal finasteride administration alters hippocampal α4 and δ GABAAR subunits expression and behavioural responses to progesterone in adult rats.

PubMed

Modol, Laura; Casas, Caty; Navarro, Xavier; Llidó, Anna; Vallée, Monique; Pallarès, Marc; Darbra, Sònia

2014-02-01

Allopregnanolone is a neurosteroid that has been reported to fluctuate during early developmental stages. Previous experiments reported the importance of neonatal endogenous allopregnanolone levels for the maturation of the central nervous system and particularly for the hippocampus. Changes in neonatal allopregnanolone levels have been related to altered adult behaviour and with psychopathological susceptibility, including anxiety disorders, schizophrenia and drug abuse. However, the mechanism underlying these changes remains to be elucidated. In the present study we assessed changes in hippocampal expression of α4 and δ GABAA receptor (GABAAR) subunits as a consequence of neonatal finasteride (a 5-α reductase inhibitor) administration during early development (PD6 to PD15) in male rats. We observed that the treatment altered the temporal window of the natural peak in the expression of these subunits during development. Additionally, the level of these subunits were higher than in non-handled and control animals in the adult hippocampus. We observed that in adulthood, neonatal finasteride-treated animals presented an anxiogenic-like profile in response to progesterone administration which was absent in the rest of the groups. In conclusion, these results corroborate the relevance of neonatal maintenance of neurosteroid levels for behavioural anxiety responses in the adult, and point to some of the mechanisms involved in this alterations.

Alcohol Withdrawal-Induced Seizure Susceptibility is Associated with an Upregulation of CaV1.3 Channels in the Rat Inferior Colliculus

PubMed Central

Akinfiresoye, Luli R.; Allard, Joanne S.; Lovinger, David M.

2015-01-01

Background: We previously reported increased current density through L-type voltage-gated Ca2+ (CaV1) channels in inferior colliculus (IC) neurons during alcohol withdrawal. However, the molecular correlate of this increased CaV1 current is currently unknown. Methods: Rats received three daily doses of ethanol every 8 hours for 4 consecutive days; control rats received vehicle. The IC was dissected at various time intervals following alcohol withdrawal, and the mRNA and protein levels of the CaV1.3 and CaV1.2 α1 subunits were measured. In separate experiments, rats were tested for their susceptibility to alcohol withdrawal–induced seizures (AWS) 3, 24, and 48 hours after alcohol withdrawal. Results: In the alcohol-treated group, AWS were observed 24 hours after withdrawal; no seizures were observed at 3 or 48 hours. No seizures were observed at any time in the control-treated rats. Compared to control-treated rats, the mRNA level of the CaV1.3 α1 subunit was increased 1.4-fold, 1.9-fold, and 1.3-fold at 3, 24, and 48 hours, respectively. In contrast, the mRNA level of the CaV1.2 α1 subunit increased 1.5-fold and 1.4-fold at 24 and 48 hours, respectively. At 24 hours, Western blot analyses revealed that the levels of the CaV1.3 and CaV1.2 α1 subunits increased by 52% and 32%, respectively, 24 hours after alcohol withdrawal. In contrast, the CaV1.2 and CaV1.3 α1 subunits were not altered at either 3 or 48 hours during alcohol withdrawal. Conclusions: Expression of the CaV1.3 α1 subunit increased in parallel with AWS development, suggesting that altered L-type CaV1.3 channel expression is an important feature of AWS pathogenesis. PMID:25556199

Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

PubMed Central

Mayer-Jaekel, R E; Baumgartner, S; Bilbe, G; Ohkura, H; Glover, D M; Hemmings, B A

1992-01-01

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner. Images PMID:1320961

The Compact and Biologically Relevant Structure of Inter-α-inhibitor Is Maintained by the Chondroitin Sulfate Chain and Divalent Cations.

PubMed

Scavenius, Carsten; Nikolajsen, Camilla Lund; Stenvang, Marcel; Thøgersen, Ida B; Wyrożemski, Łukasz; Wisniewski, Hans-Georg; Otzen, Daniel E; Sanggaard, Kristian W; Enghild, Jan J

2016-02-26

Inter-α-inhibitor is a proteoglycan of unique structure. The protein consists of three subunits, heavy chain 1, heavy chain 2, and bikunin covalently joined by a chondroitin sulfate chain originating at Ser-10 of bikunin. Inter-α-inhibitor interacts with an inflammation-associated protein, tumor necrosis factor-inducible gene 6 protein, in the extracellular matrix. This interaction leads to transfer of the heavy chains from the chondroitin sulfate of inter-α-inhibitor to hyaluronan and consequently to matrix stabilization. Divalent cations and heavy chain 2 are essential co-factors in this transfer reaction. In the present study, we have investigated how divalent cations in concert with the chondroitin sulfate chain influence the structure and stability of inter-α-inhibitor. The results showed that Mg(2+) or Mn(2+), but not Ca(2+), induced a conformational change in inter-α-inhibitor as evidenced by a decrease in the Stokes radius and a bikunin chondroitin sulfate-dependent increase of the thermodynamic stability. This structure was shown to be essential for the ability of inter-α-inhibitor to participate in extracellular matrix stabilization. In addition, the data revealed that bikunin was positioned adjacent to both heavy chains and that the two heavy chains also were in close proximity. The chondroitin sulfate chain interacted with all protein components and inter-α-inhibitor dissociated when it was degraded. Conventional purification protocols result in the removal of the Mg(2+) found in plasma and because divalent cations influence the conformation and affect function it is important to consider this when characterizing the biological activity of inter-α-inhibitor. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Dynamics of Human Telomerase Holoenzyme Assembly and Subunit Exchange across the Cell Cycle*

PubMed Central

Vogan, Jacob M.; Collins, Kathleen

2015-01-01

Human telomerase acts on telomeres during the genome synthesis phase of the cell cycle, accompanied by its concentration in Cajal bodies and transient colocalization with telomeres. Whether the regulation of human telomerase holoenzyme assembly contributes to the cell cycle restriction of telomerase function is unknown. We investigated the steady-state levels, assembly, and exchange dynamics of human telomerase subunits with quantitative in vivo cross-linking and other methods. We determined the physical association of telomerase subunits in cells blocked or progressing through the cell cycle as synchronized by multiple protocols. The total level of human telomerase RNA (hTR) was invariant across the cell cycle. In vivo snapshots of telomerase holoenzyme composition established that hTR remains bound to human telomerase reverse transcriptase (hTERT) throughout all phases of the cell cycle, and subunit competition assays suggested that hTERT-hTR interaction is not readily exchangeable. In contrast, the telomerase holoenzyme Cajal body-associated protein, TCAB1, was released from hTR in mitotic cells coincident with TCAB1 delocalization from Cajal bodies. This telomerase holoenzyme disassembly was reversible with cell cycle progression without any change in total TCAB1 protein level. Consistent with differential cell cycle regulation of hTERT-hTR and TCAB1-hTR protein-RNA interactions, overexpression of hTERT or TCAB1 had limited if any influence on hTR assembly of the other subunit. Overall, these findings revealed a cell cycle regulation that disables human telomerase association with telomeres while preserving the co-folded hTERT-hTR ribonucleoprotein catalytic core. Studies here, integrated with previous work, led to a unifying model for telomerase subunit assembly and trafficking in human cells. PMID:26170453

The Goat (Capra hircus) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis.

PubMed

Cugno, Graziano; Parreira, José R; Ferlizza, Enea; Hernández-Castellano, Lorenzo E; Carneiro, Mariana; Renaut, Jenny; Castro, Noemí; Arguello, Anastasio; Capote, Juan; Campos, Alexandre M O; Almeida, André M

2016-01-01

Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer's incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15-20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss.

The Goat (Capra hircus) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis

PubMed Central

Cugno, Graziano; Parreira, José R.; Ferlizza, Enea; Hernández-Castellano, Lorenzo E.; Carneiro, Mariana; Renaut, Jenny; Castro, Noemí; Arguello, Anastasio; Capote, Juan

2016-01-01

Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer’s incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15–20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss. PMID:27031334

Fluorescence detection of the movement of single KcsA subunits reveals cooperativity

PubMed Central

Blunck, Rikard; McGuire, Hugo; Hyde, H. Clark; Bezanilla, Francisco

2008-01-01

The prokaryotic KcsA channel is gated at the helical bundle crossing by intracellular protons and inactivates at the extracellular selectivity filter. The C-terminal transmembrane helix has to undergo a conformational change for potassium ions to access the central cavity. Whereas a partial opening of the tetrameric channel is suggested to be responsible for subconductance levels of ion channels, including KcsA, a cooperative opening of the 4 subunits is postulated as the final opening step. In this study, we used single-channel fluorescence spectroscopy of KcsA to directly observe the movement of each subunit and the temporal correlation between subunits. Purified KcsA channels labeled at the C terminus near the bundle crossing have been inserted into supported lipid bilayer, and the fluorescence traces analyzed by means of a cooperative or independent Markov model. The analysis revealed that the 4 subunits do not move fully independently but instead showed a certain degree of cooperativity. However, the 4 subunits do not simply open in 1 concerted step. PMID:19074286

Na+, K+-ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos.

PubMed

Hu, Yau-Chung; Chu, Keng-Fu; Yang, Wen-Kai; Lee, Tsung-Han

2017-10-01

The euryhaline milkfish (Chanos chanos) is a popular aquaculture species that can be cultured in fresh water, brackish water, or seawater in Southeast Asia. In gills of the milkfish, Na + , K + -ATPase (i.e., NKA; sodium pump) responds to salinity challenges including changes in mRNA abundance, protein amount, and activity. The functional pump is composed of a heterodimeric protein complex composed of α- and β-subunits. Among the NKA genes, α1-β1 isozyme comprises the major form of NKA subunits in mammalian osmoregulatory organs; however, most studies on fish gills have focused on the α1 subunit and did not verify the α1-β1 isozyme. Based on the sequenced milkfish transcriptome, an NKA β1 subunit gene was identified that had the highest amino acid homology to β233, a NKA β1 subunit paralog originally identified in the eel. Despite this high level of homology to β233, phylogenetic analysis and the fact that only a single NKA β1 subunit gene exists in the milkfish suggest that the milkfish gene should be referred to as the NKA β1 subunit gene. The results of accurate domain prediction of the β1 subunit, co-localization of α1 and β1 subunits in epithelial ionocytes, and co-immunoprecipitation of α1 and β1 subunits, indicated the formation of a α1-β1 complex in milkfish gills. Moreover, when transferred to hyposmotic media (fresh water) from seawater, parallel increases in branchial mRNA and protein expression of NKA α1 and β1 subunits suggested their roles in hypo-osmoregulation of euryhaline milkfish. This study molecularly characterized the NKA β1 subunit and provided the first evidence for an NKA α1-β1 association in gill ionocytes of euryhaline teleosts.

Expression of accessory colonization factor subunit A (ACFA) of Vibrio cholerae and ACFA fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

PubMed

Sharma, Manoj Kumar; Jani, Dewal; Thungapathra, M; Gautam, J K; Meena, L S; Singh, Yogendra; Ghosh, Amit; Tyagi, Akhilesh Kumar; Sharma, Arun Kumar

2008-05-20

In earlier study from our group, cholera toxin B subunit had been expressed in tomato for developing a plant-based vaccine against cholera. In the present investigation, gene for accessory colonization factor (acf) subunit A, earlier reported to be essential for efficient colonization in the intestine, has been expressed in Escherichia coli as well as tomato plants. Gene encoding for a chimeric protein having a fusion of cholera toxin B subunit and accessory colonization factor A was also expressed in tomato to generate more potent combinatorial antigen. CaMV35S promoter with a duplicated enhancer sequence was used for expression of these genes in tomato. Integration of transgenes into tomato genome was confirmed by PCR and Southern hybridization. Expression of the genes was confirmed at transcript and protein levels. Accessory colonization factor A and cholera toxin B subunit fused to this protein accumulated up to 0.25% and 0.08% of total soluble protein, respectively, in the fruits of transgenic plants. Whereas protein purified from E. coli, in combination with cholera toxin B subunit can be used for development of conventional subunit vaccine, tomato fruits expressing these proteins can be used together with tomato plants expressing cholera toxin B subunit for development of oral vaccine against cholera.

Maple Syrup Urine Disease: Identification and Carrier-Frequency Determination of a Novel Founder Mutation in the Ashkenazi Jewish Population

PubMed Central

Edelmann, Lisa; Wasserstein, Melissa P.; Kornreich, Ruth; Sansaricq, Claude; Snyderman, Selma E.; Diaz, George A.

2001-01-01

Maple syrup urine disease (MSUD) is a rare, autosomal recessive disorder of branched-chain amino acid metabolism. We noted that a large proportion (10 of 34) of families with MSUD that were followed in our clinic were of Ashkenazi Jewish (AJ) descent, leading us to search for a common mutation within this group. On the basis of genotyping data suggestive of a conserved haplotype at tightly linked markers on chromosome 6q14, the BCKDHB gene encoding the E1β subunit was sequenced. Three novel mutations were identified in seven unrelated AJ patients with MSUD. The locations of the affected residues in the crystal structure of the E1β subunit suggested possible mechanisms for the deleterious effects of these mutations. Large-scale population screening of AJ individuals for R183P, the mutation present in six of seven patients, revealed that the carrier frequency of the mutant allele was ∼1/113; the patient not carrying R183P had a previously described homozygous mutation in the gene encoding the E2 subunit. These findings suggested that a limited number of mutations might underlie MSUD in the AJ population, potentially facilitating prenatal diagnosis and carrier detection of MSUD in this group. PMID:11509994

Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases.

PubMed

Xu, Yuquan; Zhou, Tong; Zhang, Shuwei; Espinosa-Artiles, Patricia; Wang, Luoyi; Zhang, Wei; Lin, Min; Gunatilaka, A A Leslie; Zhan, Jixun; Molnár, István

2014-08-26

Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into "build-couple-pair" combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.

Maple syrup urine disease: identification and carrier-frequency determination of a novel founder mutation in the Ashkenazi Jewish population.

PubMed

Edelmann, L; Wasserstein, M P; Kornreich, R; Sansaricq, C; Snyderman, S E; Diaz, G A

2001-10-01

Maple syrup urine disease (MSUD) is a rare, autosomal recessive disorder of branched-chain amino acid metabolism. We noted that a large proportion (10 of 34) of families with MSUD that were followed in our clinic were of Ashkenazi Jewish (AJ) descent, leading us to search for a common mutation within this group. On the basis of genotyping data suggestive of a conserved haplotype at tightly linked markers on chromosome 6q14, the BCKDHB gene encoding the E1beta subunit was sequenced. Three novel mutations were identified in seven unrelated AJ patients with MSUD. The locations of the affected residues in the crystal structure of the E1beta subunit suggested possible mechanisms for the deleterious effects of these mutations. Large-scale population screening of AJ individuals for R183P, the mutation present in six of seven patients, revealed that the carrier frequency of the mutant allele was approximately 1/113; the patient not carrying R183P had a previously described homozygous mutation in the gene encoding the E2 subunit. These findings suggested that a limited number of mutations might underlie MSUD in the AJ population, potentially facilitating prenatal diagnosis and carrier detection of MSUD in this group.

The mitochondrial electron transfer flavoprotein complex is essential for survival of Arabidopsis in extended darkness.

PubMed

Ishizaki, Kimitsune; Schauer, Nicolas; Larson, Tony R; Graham, Ian A; Fernie, Alisdair R; Leaver, Christopher J

2006-09-01

In mammals, the electron transfer flavoprotein (ETF) is a heterodimeric protein composed of two subunits, alpha and beta, that is responsible for the oxidation of at least nine mitochondrial matrix flavoprotein dehydrogenases. Electrons accepted by ETF are further transferred to the main respiratory chain via the ETF ubiquinone oxide reductase (ETFQO). Sequence analysis of the unique Arabidopsis homologues of two subunits of ETF revealed their high similarity to both subunits of the mammalian ETF. Yeast two-hybrid experiments showed that the Arabidopsis ETFalpha and ETFbeta can form a heteromeric protein. Isolation and characterization of two independent T-DNA insertional Arabidopsis mutants of the ETFbeta gene revealed accelerated senescence and early death compared to wild-type during extended darkness. Furthermore in contrast to wild-type, the etfb mutants demonstrated a significant accumulation of several amino acids, isovaleryl CoA and phytanoyl CoA during dark-induced carbohydrate deprivation. These phenotypic characteristics of etfb mutants are broadly similar to those that we observed previously in Arabidopsis etfqo mutants, suggesting functional association between ETF and ETFQO in Arabidopsis, and confirming the essential roles of the ETF/ETFQO electron transfer complex in the catabolism of leucine and involvement in the chlorophyll degradation pathway activated during dark-induced carbohydrate deprivation.

Localization of Ubiquinone-8 in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae*

PubMed Central

Casutt, Marco S.; Nedielkov, Ruslan; Wendelspiess, Severin; Vossler, Sara; Gerken, Uwe; Murai, Masatoshi; Miyoshi, Hideto; Möller, Heiko M.; Steuber, Julia

2011-01-01

Na+ is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) as the first complex in its respiratory chain. The Na+-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na+ translocation by the Na+-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na+-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na+-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA. PMID:21885438

Structural Basis of Resistance to Anti-Cytochrome bc1 Complex Inhibitors: Implication for Drug Improvement

PubMed Central

Esser, Lothar; Yu, Chang-An; Xia, Di

2016-01-01

The emergence of drug resistance has devastating economic and social consequences, a testimonial of which is the rise and fall of inhibitors against the respiratory component cytochrome bc1 complex, a time tested and highly effective target for disease control. Unfortunately, the mechanism of resistance is a multivariate problem, including primarily mutations in the gene of the cytochrome b subunit but also activation of alternative pathways of ubiquinol oxidation and pharmacokinetic effects. There is a considerable interest in designing new bc1 inhibitors with novel modes of binding and lower propensity to induce the development of resistance. The accumulation of crystallographic data of bc1 complexes with and without inhibitors bound provides the structural basis for rational drug design. In particular, the cytochrome b subunit offers two distinct active sites that can be targeted for inhibition - the quinol oxidation site and the quinone reduction site. This review brings together available structural information of inhibited bc1 by various quinol oxidation- and reduction-site inhibitors, the inhibitor binding modes, conformational changes upon inhibitor binding of side chains in the active site and large scale domain movements of the iron-sulfur protein subunit. Structural data analysis provides a clear understanding of where and why existing inhibitors fail and points towards promising alternatives. PMID:23688079

Short-term sleep deprivation impairs spatial working memory and modulates expression levels of ionotropic glutamate receptor subunits in hippocampus.

PubMed

Xie, Meilan; Yan, Jie; He, Chao; Yang, Li; Tan, Gang; Li, Chao; Hu, Zhian; Wang, Jiali

2015-06-01

Hippocampus-dependent learning memory is sensitive to sleep deprivation (SD). Although the ionotropic glutamate receptors play a vital role in synaptic plasticity and learning and memory, however, whether the expression of these receptor subunits is modulated by sleep loss remains unclear. In the present study, western blotting was performed by probing with specific antibodies against the ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1, GluA2, GluA3, and against the N-methyl-d-aspartate (NMDA) glutamate receptor subunits GluN1, GluN2A, GluN2B. In hippocampus, down regulation of surface GluA1 and GluN2A surface expression were observed in both SD groups. However, surface expression level of GluA2, GluA3, GluN1 and GluN2B was significantly up-regulated in 8h-SD rats when compared to the 4h-SD rats. In parallel with the complex changes in AMPA and NMDA receptor subunit expressions, we found the 8h-SD impaired rat spatial working memory in 30-s-delay T-maze task, whereas no impairment of spatial learning was observed in 4h-SD rats. These results indicate that sleep loss alters the relative expression levels of the AMPA and NMDA receptors, thus affects the synaptic strength and capacity for plasticity and partially contributes to spatial memory impairment. Copyright © 2015. Published by Elsevier B.V.

L-type Calcium Channel Blockers Enhance Trafficking and Function of Epilepsy-associated α1(D219N) Subunits of GABA(A) Receptors.

PubMed

Han, Dong-Yun; Guan, Bo-Jhih; Wang, Ya-Juan; Hatzoglou, Maria; Mu, Ting-Wei

2015-09-18

Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory ion channels in the mammalian central nervous system and play an essential role in regulating inhibition-excitation balance in neural circuits. The α1 subunit harboring the D219N mutation of GABAA receptors was reported to be retained in the endoplasmic reticulum (ER) and traffic inefficiently to the plasma membrane, leading to a loss of function of α1(D219N) subunits and thus idiopathic generalized epilepsy (IGE). We present the use of small molecule proteostasis regulators to enhance the forward trafficking of α1(D219N) subunits to restore their function. We showed that treatment with verapamil (4 μM, 24 h), an L-type calcium channel blocker, substantially increases the α1(D219N) subunit cell surface level in both HEK293 cells and neuronal SH-SY5Y cells and remarkably restores the GABA-induced maximal chloride current in HEK293 cells expressing α1(D219N)β2γ2 receptors to a level that is comparable to wild type receptors. Our drug mechanism study revealed that verapamil treatment promotes the ER to Golgi trafficking of the α1(D219N) subunits post-translationally. To achieve that, verapamil treatment enhances the interaction between the α1(D219N) subunit and β2 subunit and prevents the aggregation of the mutant protein by shifting the protein from the detergent-insoluble fractions to detergent-soluble fractions. By combining (35)S pulse-chase labeling and MG-132 inhibition experiments, we demonstrated that verapamil treatment does not inhibit the ER-associated degradation of the α1(D219N) subunit. In addition, its effect does not involve a dynamin-1 dependent endocytosis. To gain further mechanistic insight, we showed that verapamil increases the interaction between the mutant protein and calnexin and calreticulin, two major lectin chaperones in the ER. Moreover, calnexin binding promotes the forward trafficking of the mutant subunit. Taken together, our data indicate that verapamil treatment enhances the calnexin-assisted forward trafficking and subunit assembly, which leads to substantially enhanced functional surface expression of the mutant receptors. Since verapamil is an FDA-approved drug that crosses blood-brain barrier and has been used as an additional medication for some epilepsies, our findings suggest that verapamil holds great promise to be developed to ameliorate IGE resulting from α1(D219N) subunit trafficking deficiency.

Archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2B alpha-beta-delta subunit families

NASA Technical Reports Server (NTRS)

Kyrpides, N. C.; Woese, C. R.

1998-01-01

As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits alpha, beta, and delta, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.

The Crystallographic Structure of Panicum Mosaic Virus (PMV)

PubMed Central

Makino, Debora L.; Larson, Steven B.; McPherson, Alexander

2012-01-01

The structure of Panicum Mosaic Virus (PMV) was determined by X-ray diffraction analysis to 2.9 Å resolution. The crystals were of pseudo symmetry F23; the true crystallographic unit cell was of space group P21 with a=411.7 Å, b=403.9 Å and c=412.5 Å, with β=89.7°. The asymmetric unit was two entire T=3 virus particles, or 360 protein subunits. The structure was solved by conventional molecular replacement from two distant homologues, Cocksfoot Mottle Virus (CfMV) and Tobacco Necrosis Virus (TNV), of ~20% sequence identity followed by phase extension. The model was initially refined with exact icosahedral constraints and then with icosahedral restraints. The virus has Ca++ ions octahedrally coordinated by six aspartic acid residues on quasi threefold axes, which is completely different than for either CfMV or TNV. Amino terminal residues 1–53, 1–49 and 1-21 of the A, B and C subunits, respectively, and the four C-terminal residues (239-242) are not visible in electron density maps. The additional ordered residues of the C chain form a prominent “arm” that intertwines with symmetry equivalent “arms” at icosahedral threefold axes, as was seen in both CfMV and TNV. A 17 nucleotide hairpin segment of genomic RNA is icosahedrally ordered and bound at 60 equivalent sites at quasi twofold A–B subunit interfaces at the interior surface of the capsid. This segment of RNA may serve as a conformational switch for coat protein subunits, as has been proposed for similar RNA segments in other viruses. PMID:23123270

Structural investigation of nonpolar sulfur cross-linked macromolecules in petroleum

NASA Astrophysics Data System (ADS)

Adam, P.; Schmid, J. C.; Mycke, B.; Strazielle, C.; Connan, J.; Huc, A.; Riva, A.; Albrecht, P.

1993-07-01

A novel hexane-soluble nonpolar macromolecular fraction (NPMF) has been found to occur in substantial amounts (up to 32%) in sulfur-rich crude oils and a rock extract. It is highly aliphatic and has a molecular weight culminating at several thousand mass units, as proven by spectroscopic and molecular weight studies. C-S bond hydrogenolysis of NPMF with Raney nickel as a catalyst yields high proportions of aliphatic hydrocarbons in which long linear, acyclic polyisoprenoid and carotenoid chains usually predominate (except in one case) over polycyclic structures, such as steroids and hopanoids. Hence, NPMF consists mainly of macromolecules composed of low molecular weight hydrocarbon subunits cross-linked with sulfide bridges. Use of deuterated Raney nickel indicated in one case (Rozel Point oil) that the long chains and some hopanoids are multiattached to the macromolecular network, whereas other structural subunits, such as steroids or gammacerane, are essentially monoattached. Detailed structural determinations of the hydrocarbon "building blocks" of NPMF give information on their origin and the mode of formation of these macromolecules in the subsurface. Indeed, most of the building blocks can be related to algal (e.g., long linear chains, steroids, β-carotene, and related carotenoids) or bacterial (e.g., acyclic and monocyclic carotenoids, long-chain acyclic isoprenoids) precursors which essentially exist in living organisms as monounsaturated or polyunsaturated species or are easily transformed into such species by diagenetic processes (e.g., steroids). It appears that these alkenes or polyenes become selectively trapped into a macromolecular network by reaction with inorganic sulfur species produced by bacteria in a kind of natural, low-temperature, vulcanization process. This process could start at early diagenesis already in the water column or eventually continue in the bottom sediment. Although its exact nature is yet unknown, it seems likely that the cross-linking reaction can be initiated by the cleavage of sulfur species in a radical type mechanism. The alkanes formed upon desulfurization of NPMF usually represent much higher amounts than the free alkanes of the samples and show a dramatically different composition. They may deliver very useful, complementary information in studies related to source and palaeoenvironment.

Differential regulation of luteinizing hormone and follicle-stimulating hormone expression during ovarian development and under sexual steroid feedback in the European eel.

PubMed

Schmitz, Monika; Aroua, Salima; Vidal, Bernadette; Le Belle, Nadine; Elie, Pierre; Dufour, Sylvie

2005-01-01

Pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are, in teleosts as in mammals, under the control of hypothalamic factors and steroid feedbacks. In teleosts, feedback regulations largely vary depending on species and physiological stage. In the present study the regulation of FSH and LH expression was investigated in the European eel, a fish of biological and phylogenetical interest as a representative of an early group of teleosts. The eel FSHbeta subunit was cloned, sequenced and together with earlier isolated eel LHbeta and glycoprotein hormone alpha (GPalpha) subunits used to study the differential regulation of LH and FSH. In situ hybridization indicated that FSHbeta and LHbeta are expressed by separate cells of the proximal pars distalis of the adenohypophysis, differently from the situation in mammals. The profiles of LHbeta and FSHbeta subunit expression were compared during experimental ovarian maturation, using dot-blot assays. Expression levels for LHbeta and GPalpha increased throughout ovarian development with a positive correlation between these two subunits. Conversely, FSHbeta mRNA levels decreased. To understand the role of sex steroids in these opposite variations, immature eels were treated with estradiol (E2)and testosterone (T), both steroids being produced in eel ovaries during gonadal development. E2 treatment induced increases in both LHbeta and GPalpha mRNA levels, without any significant effect on FSHbeta. In contrast, T treatment induced a decrease in FSHbeta mRNA levels, without any significant effect on the other subunits. These data demonstrate that steroids exert a differential feedback on eel gonadotropin expression, with an E2-specific positive feedback on LH and a T-specific negative feedback on FSH, leading to an opposite regulation of LH and FSH during ovarian development. Copyright 2005 S. Karger AG, Basel

Identification and Characterization of an Alternatively Spliced Isoform of the Human Protein Phosphatase 2Aα Catalytic Subunit*

PubMed Central

Migueleti, Deivid L. S.; Smetana, Juliana H. C.; Nunes, Hugo F.; Kobarg, Jörg; Zanchin, Nilson I. T.

2012-01-01

PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Acα2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, α4 out-competes PME-1 and LCMT-1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation. PMID:22167190

Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Machaalani, R., E-mail: rita.machaalani@sydney.edu.au; Bosch Institute, The University of Sydney, NSW 2006; The Children's Hospital at Westmead, NSW 2145

Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and comparedmore » mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta.« less

Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics.

PubMed

Jin, Zhe; Bhandage, Amol K; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R; Birnir, Bryndis

2014-01-01

The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence.

Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics

PubMed Central

Jin, Zhe; Bhandage, Amol K.; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R.; Birnir, Bryndis

2014-01-01

The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence. PMID:25278838

[Molecular cloning of the DNA sequence of activin beta A subunit gene mature peptides from panda and related species and its application in the research of phylogeny and taxonomy].

PubMed

Wang, Xiao-Jing; Wang, Xiao-Xing; Wang, Ya-Jun; Wang, Xi-Zhong; He, Guang-Xin; Chen, Hong-Wei; Fei, Li-Song

2002-09-01

Activin, which is included in the transforming growth factor-beta (TGF beta) superfamily of proteins and receptors, is known to have broad-ranging effects in the creatures. The mature peptide of beta A subunit of this gene, one of the most highly conserved sequence, can elevate the basal secretion of follicle-stimulating hormone (FSH) in the pituitary and FSH is pivotal to organism's reproduction. Reproduction block is one of the main reasons which cause giant panda to extinct. The sequence of Activin beta A subunit gene mature peptides has been successfully amplified from giant panda, red panda and malayan sun bear's genomic DNA by using polymerase chain reaction (PCR) with a pair of degenerate primers. The PCR products were cloned into the vector pBlueScript+ of Esherichia coli. Sequence analysis of Activin beta A subunit gene mature peptides shows that the length of this gene segment is the same (359 bp) and there is no intron in all three species. The sequence encodes a peptide of 119 amino acid residues. The homology comparison demonstrates 93.9% DNA homology and 99% homology in amino acid among these three species. Both GenBank blast search result and restriction enzyme map reveal that the sequences of Activin beta A subunit gene mature peptides of different species are highly conserved during the evolution process. Phylogeny analysis is performed with PHYLIP software package. A consistent phylogeny tree has been drawn with three different methods. The software analysis outcome accords with the academic view that giant panda has a closer relationship to the malayan sun bear than the red panda. Giant panda should be grouped into the bear family (Uersidae) with the malayan sun bear. As to the red panda, it would be better that this animal be grouped into the unique family (red panda family) because of great difference between the red panda and the bears (Uersidae).

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.

PubMed

Mundus, D; Wollenzien, P

1998-11-01

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.

Ionotropic glutamate receptor mRNA editing in the prefrontal cortex: no alterations in schizophrenia or bipolar disorder.

PubMed

Lyddon, Rebecca; Navarrett, Scott; Dracheva, Stella

2012-07-01

Dysfunction of glutamate neurotransmission has been implicated in the pathology of schizophrenia and bipolar disorder, and one mechanism by which glutamate signalling can be altered is through RNA editing of ionotropic glutamate receptors (iGluRs). The objectives of the present study were to evaluate the editing status of iGluRs in the human prefrontal cortex, determine whether iGluR editing is associated with psychiatric disease or suicide and evaluate a potential association between editing and alternative splicing in the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) iGluR subunits' pre-mRNA. We studied specimens derived from patients with antemortem diagnoses of bipolar disorder (n = 31) or schizophrenia (n = 34) who died by suicide or other causes, and from psychiatrically healthy controls (n = 34) who died from causes other than suicide. The RNA editing at all 8 editing sites within AMPA (GluA2-4 subunits) and kainate (GluK1-2 subunits) iGluRs was analyzed using a novel real-time quantitative polymerase chain reaction assay. No differences in editing were detected among schizophrenia, bipolar or control groups or between suicide completers and patients who died from causes other than suicide. The editing efficiency was significantly higher in the flop than in the flip splicoforms of GluA3-4 AMPA subunits (all p < 0.001). The study is limited by the near absence of specimens from medicationnaive psychiatric patients and considerable variation in medication regimens among individuals, both of which introduce considerable uncertainty into the analysis of potential medication effects. We found that iGluR RNA editing status was not associated with bipolar disorder, schizophrenia or suicide. Differences in editing between flip and flop splicoforms suggest that glutamate sensitivity of receptors containing GluA3 and/or GluA4 flop subunits is moderated as a result of increased editing.

The role of geochemistry and energetics in the evolution of modern respiratory complexes from a proton-reducing ancestor.

PubMed

Schut, Gerrit J; Zadvornyy, Oleg; Wu, Chang-Hao; Peters, John W; Boyd, Eric S; Adams, Michael W W

2016-07-01

Complex I or NADH quinone oxidoreductase (NUO) is an integral component of modern day respiratory chains and has a close evolutionary relationship with energy-conserving [NiFe]-hydrogenases of anaerobic microorganisms. Specifically, in all of biology, the quinone-binding subunit of Complex I, NuoD, is most closely related to the proton-reducing, H2-evolving [NiFe]-containing catalytic subunit, MbhL, of membrane-bound hydrogenase (MBH), to the methanophenzine-reducing subunit of a methanogenic respiratory complex (FPO) and to the catalytic subunit of an archaeal respiratory complex (MBX) involved in reducing elemental sulfur (S°). These complexes also pump ions and have at least 10 homologous subunits in common. As electron donors, MBH and MBX use ferredoxin (Fd), FPO uses either Fd or cofactor F420, and NUO uses either Fd or NADH. In this review, we examine the evolutionary trajectory of these oxidoreductases from a proton-reducing ancestral respiratory complex (ARC). We hypothesize that the diversification of ARC to MBH, MBX, FPO and eventually NUO was driven by the larger energy yields associated with coupling Fd oxidation to the reduction of oxidants with increasing electrochemical potential, including protons, S° and membrane soluble organic compounds such as phenazines and quinone derivatives. Importantly, throughout Earth's history, the availability of these oxidants increased as the redox state of the atmosphere and oceans became progressively more oxidized as a result of the origin and ecological expansion of oxygenic photosynthesis. ARC-derived complexes are therefore remarkably stable respiratory systems with little diversity in core structure but whose general function appears to have co-evolved with the redox state of the biosphere. This article is part of a Special Issue entitled Respiratory Complex I, edited by Volker Zickermann and Ulrich Brandt. Copyright © 2016 Elsevier B.V. All rights reserved.

Interleukin-9 enhances interleukin-5 receptor expression, differentiation, and survival of human eosinophils.

PubMed

Gounni, A S; Gregory, B; Nutku, E; Aris, F; Latifa, K; Minshall, E; North, J; Tavernier, J; Levit, R; Nicolaides, N; Robinson, D; Hamid, Q

2000-09-15

Interleukin-9 (IL-9) has been implicated in the pathogenesis of allergic disorders. To examine the interaction between IL-9 and eosinophils, we evaluated mature peripheral blood eosinophils for their expression of the specific alpha-subunit of the IL-9 receptor (IL-9R-alpha). The expression of IL-9R-alpha by human eosinophils was detected at the messenger RNA (mRNA) and protein levels by reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, and immunocytochemical analysis, respectively. Functional analyses demonstrated that recombinant human (rh)IL-9 inhibited in vitro peripheral blood human eosinophil apoptosis in a concentration-dependent manner. We then examined the role of IL-9 in eosinophil differentiation using the human cord blood CD34(+) cells and human promyelocytic leukemia cells (HL-60). The addition of IL-9 to CD34(+) cells cultured in IL-3 and IL-5 enhanced eosinophil development, and IL-9 alone induced the expression of IL-5R-alpha. IL-9 also up-regulated the IL-5R-alpha chain cell surface expression during terminal eosinophil differentiation of the HL-60 cell line. Our findings suggest that IL-9 may potentiate in vivo eosinophil function by increasing their survival and IL-5-mediated differentiation and maturation. Taken together, these results suggest a mechanism by which IL-9 potentiates airway and tissue eosinophilia.

Invariant Chain Complexes and Clusters as Platforms for MIF Signaling

PubMed Central

Lindner, Robert

2017-01-01

Invariant chain (Ii/CD74) has been identified as a surface receptor for migration inhibitory factor (MIF). Most cells that express Ii also synthesize major histocompatibility complex class II (MHC II) molecules, which depend on Ii as a chaperone and a targeting factor. The assembly of nonameric complexes consisting of one Ii trimer and three MHC II molecules (each of which is a heterodimer) has been regarded as a prerequisite for efficient delivery to the cell surface. Due to rapid endocytosis, however, only low levels of Ii-MHC II complexes are displayed on the cell surface of professional antigen presenting cells and very little free Ii trimers. The association of Ii and MHC II has been reported to block the interaction with MIF, thus questioning the role of surface Ii as a receptor for MIF on MHC II-expressing cells. Recent work offers a potential solution to this conundrum: Many Ii-complexes at the cell surface appear to be under-saturated with MHC II, leaving unoccupied Ii subunits as potential binding sites for MIF. Some of this work also sheds light on novel aspects of signal transduction by Ii-bound MIF in B-lymphocytes: membrane raft association of Ii-MHC II complexes enables MIF to target Ii-MHC II to antigen-clustered B-cell-receptors (BCR) and to foster BCR-driven signaling and intracellular trafficking. PMID:28208600

A Regulatory Hierarchy of the Arabidopsis Branched-Chain Amino Acid Metabolic Network.

PubMed

Xing, Anqi; Last, Robert L

2017-06-01

The branched-chain amino acids (BCAAs) Ile, Val, and Leu are essential nutrients that humans and other animals obtain from plants. However, total and relative amounts of plant BCAAs rarely match animal nutritional needs, and improvement requires a better understanding of the mechanistic basis for BCAA homeostasis. We present an in vivo regulatory model of BCAA homeostasis derived from analysis of feedback-resistant Arabidopsis thaliana mutants for the three allosteric committed enzymes in the biosynthetic network: threonine deaminase (also named l- O -methylthreonine resistant 1 [OMR1]), acetohydroxyacid synthase small subunit 2 (AHASS2), and isopropylmalate synthase 1 (IPMS1). In this model, OMR1 exerts primary control on Ile accumulation and functions independently of AHAS and IPMS AHAS and IPMS regulate Val and Leu homeostasis, where AHAS affects total Val+Leu and IPMS controls partitioning between these amino acids. In addition, analysis of feedback-resistant and loss-of-function single and double mutants revealed that each AHAS and IPMS isoenzyme contributes to homeostasis rather than being functionally redundant. The characterized feedback resistance mutations caused increased free BCAA levels in both seedlings and seeds. These results add to our understanding of the basis of in vivo BCAA homeostasis and inform approaches to improve the amount and balance of these essential nutrients in crops. © 2017 American Society of Plant Biologists. All rights reserved.

Cardiomyocyte hypertrophy induced by Endonuclease G deficiency requires reactive oxygen radicals accumulation and is inhibitable by the micropeptide humanin.

PubMed

Blasco, Natividad; Cámara, Yolanda; Núñez, Estefanía; Beà, Aida; Barés, Gisel; Forné, Carles; Ruíz-Meana, Marisol; Girón, Cristina; Barba, Ignasi; García-Arumí, Elena; García-Dorado, David; Vázquez, Jesús; Martí, Ramon; Llovera, Marta; Sanchis, Daniel

2018-06-01

The endonuclease G gene (Endog), which codes for a mitochondrial nuclease, was identified as a determinant of cardiac hypertrophy. How ENDOG controls cardiomyocyte growth is still unknown. Thus, we aimed at finding the link between ENDOG activity and cardiomyocyte growth. Endog deficiency induced reactive oxygen species (ROS) accumulation and abnormal growth in neonatal rodent cardiomyocytes, altering the AKT-GSK3β and Class-II histone deacethylases (HDAC) signal transduction pathways. These effects were blocked by ROS scavengers. Lack of ENDOG reduced mitochondrial DNA (mtDNA) replication independently of ROS accumulation. Because mtDNA encodes several subunits of the mitochondrial electron transport chain, whose activity is an important source of cellular ROS, we investigated whether Endog deficiency compromised the expression and activity of the respiratory chain complexes but found no changes in these parameters nor in ATP content. MtDNA also codes for humanin, a micropeptide with possible metabolic functions. Nanomolar concentrations of synthetic humanin restored normal ROS levels and cell size in Endog-deficient cardiomyocytes. These results support the involvement of redox signaling in the control of cardiomyocyte growth by ENDOG and suggest a pathway relating mtDNA content to the regulation of cell growth probably involving humanin, which prevents reactive oxygen radicals accumulation and hypertrophy induced by Endog deficiency. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: Is resting calcium responsible?

PubMed Central

Smith, Ian C.; Gittings, William; Huang, Jian; McMillan, Elliott M.; Quadrilatero, Joe; Tupling, A. Russell

2013-01-01

The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca2+ concentration ([Ca2+]i) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca2+-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca2+ levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P < 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P < 0.01) during potentiation, indicating a significant increase in resting [Ca2+]i. Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca2+ transients (ICTs) that triggered potentiated twitches during this time (P < 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting [Ca2+]i, in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation. PMID:23401574

Diminished superoxide generation is associated with respiratory chain dysfunction and changes in the mitochondrial proteome of sensory neurons from diabetic rats.

PubMed

Akude, Eli; Zherebitskaya, Elena; Chowdhury, Subir K Roy; Smith, Darrell R; Dobrowsky, Rick T; Fernyhough, Paul

2011-01-01

Impairments in mitochondrial function have been proposed to play a role in the etiology of diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in axons of sensory neurons in type 1 diabetes is due to abnormal activity of the respiratory chain and an altered mitochondrial proteome. Proteomic analysis using stable isotope labeling with amino acids in cell culture (SILAC) determined expression of proteins in mitochondria from dorsal root ganglia (DRG) of control, 22-week-old streptozotocin (STZ)-diabetic rats, and diabetic rats treated with insulin. Rates of oxygen consumption and complex activities in mitochondria from DRG were measured. Fluorescence imaging of axons of cultured sensory neurons determined the effect of diabetes on mitochondrial polarization status, oxidative stress, and mitochondrial matrix-specific reactive oxygen species (ROS). Proteins associated with mitochondrial dysfunction, oxidative phosphorylation, ubiquinone biosynthesis, and the citric acid cycle were downregulated in diabetic samples. For example, cytochrome c oxidase subunit IV (COX IV; a complex IV protein) and NADH dehydrogenase Fe-S protein 3 (NDUFS3; a complex I protein) were reduced by 29 and 36% (P < 0.05), respectively, in diabetes and confirmed previous Western blot studies. Respiration and mitochondrial complex activity was significantly decreased by 15 to 32% compared with control. The axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria, an aberrant adaption to oligomycin-induced mitochondrial membrane hyperpolarization, but reduced levels of intramitochondrial superoxide compared with control. Abnormal mitochondrial function correlated with a downregulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar DRG in diabetes. The reduced activity of the respiratory chain was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons. Alternative pathways involving polyol pathway activity appear to contribute to raised ROS in axons of diabetic neurons under high glucose concentration.

Developmental regulation of N-methyl-D-aspartate- and kainate-type glutamate receptor expression in the rat spinal cord

NASA Technical Reports Server (NTRS)

Stegenga, S. L.; Kalb, R. G.

2001-01-01

Spinal motor neurons undergo experience-dependent development during a critical period in early postnatal life. It has been suggested that the repertoire of glutamate receptor subunits differs between young and mature motor neurons and contributes to this activity-dependent development. In the present study we examined the expression patterns of N-methyl-D-aspartate- and kainate-type glutamate receptor subunits during the postnatal maturation of the spinal cord. Young motor neurons express much higher levels of the N-methyl-D-aspartate receptor subunit NR1 than do adult motor neurons. Although there are eight potential splice variants of NR1, only a subgroup is expressed by motor neurons. With respect to NR2 receptor subunits, young motor neurons express NR2A and C, while adult motor neurons express only NR2A. Young motor neurons express kainate receptor subunits GluR5, 6 and KA2 but we are unable to detect these or any other kainate receptor subunits in the adult spinal cord. Other spinal cord regions display a distinct pattern of developmental regulation of N-methyl-D-aspartate and kainate receptor subunit expression in comparison to motor neurons. Our findings indicate a precise spatio-temporal regulation of individual subunit expression in the developing spinal cord. Specific combinations of subunits in developing neurons influence their excitable properties and could participate in the emergence of adult neuronal form and function.

How does a scanning ribosomal particle move along the 5'-untranslated region of eukaryotic mRNA? Brownian Ratchet model.

PubMed

Spirin, Alexander S

2009-11-17

A model of the ATP-dependent unidirectional movement of the 43S ribosomal initiation complex (=40S ribosomal subunit + eIF1 + eIF1A + eIF2.GTP.Met-tRNA(i) + eIF3) during scanning of the 5'-untranslated region of eukaryotic mRNA is proposed. The model is based on the principles of molecular Brownian ratchet machines and explains several enigmatic data concerning the scanning complex. In this model, the one-dimensional diffusion of the ribosomal initiation complex along the mRNA chain is rectified into the net-unidirectional 5'-to-3' movement by the Feynman ratchet-and-pawl mechanism. The proposed mechanism is organized by the heterotrimeric protein eIF4F (=eIF4A + eIF4E + eIF4G), attached to the scanning ribosomal particle via eIF3, and the RNA-binding protein eIF4B that is postulated to play the role of the pawl. The energy for the useful work of the ratchet-and-pawl mechanism is supplied from ATP hydrolysis induced by the eIF4A subunit: ATP binding and its hydrolysis alternately change the affinities of eIF4A for eIF4B and for mRNA, resulting in the restriction of backward diffusional sliding of the 43S ribosomal complex along the mRNA chain, while stochastic movements ahead are allowed.

l-Arginine Attenuates Cardiac Dysfunction, But Further Down-Regulates α-Myosin Heavy Chain Expression in Isoproterenol-Induced Cardiomyopathy.

PubMed

Kralova, Eva; Doka, Gabriel; Pivackova, Lenka; Srankova, Jasna; Kuracinova, Kristina; Janega, Pavol; Babal, Pavel; Klimas, Jan; Krenek, Peter

2015-10-01

In view of previously reported increased capacity for nitric oxide production, we suggested that l-arginine (ARG), the nitric oxide synthase (NOS) substrate, supplementation would improve cardiac function in isoproterenol (ISO)-induced heart failure. Male Wistar rats were treated with ISO for 8 days (5 mg/kg/day, i.p.) or vehicle. ARG was given to control (ARG) and ISO-treated (ISO+ARG) rats in water (0.4 g/kg/day). ISO administration was associated with 40% mortality, ventricular hypertrophy, decreased heart rate, left ventricular dysfunction, fibrosis and ECG signs of ischaemia. RT-PCR showed increased mRNA levels of cardiac hypertrophy marker atrial natriuretic peptide, but not BNP, decreased expression of myosin heavy chain isoform MYH6 and unaltered expression of pathological MYH7. ISO increased the protein levels of endothelial nitric oxide synthase, but at the same time it markedly up-regulated mRNA and protein levels of gp91phox, a catalytical subunit of superoxide-producing NADPH oxidase. Fibrosis was markedly increased by ISO. ARG treatment moderately ameliorated left ventricular dysfunction, but was without effect on cardiac hypertrophy and fibrosis. Combination of ISO and ARG led to a decrease in cav-1 expression, a further increase in MYH7 expression and a down-regulation of MYH6 that inversely correlated with gp91phox mRNA levels. Although ARG, at least partially, improved ISO-impaired basal left ventricular systolic function, it failed to reduce cardiac hypertrophy, fibrosis, oxidative stress and mortality. The protection of contractile performance might be related to increased capacity for nitric oxide production and the up-regulation of MYH7 which may compensate for the marked down-regulation of the major MYH6 isoform. © 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Method for altering antibody light chain interactions

DOEpatents

Stevens, Fred J.; Stevens, Priscilla Wilkins; Raffen, Rosemarie; Schiffer, Marianne

2002-01-01

A method for recombinant antibody subunit dimerization including modifying at least one codon of a nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in the interface segment of the light polypeptide variable region, the charged amino acid having a first polarity; and modifying at least one codon of the nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in an interface segment of the heavy polypeptide variable region corresponding to a position in the light polypeptide variable region, the charged amino acid having a second polarity opposite the first polarity. Nucleic acid sequences which code for novel light chain proteins, the latter of which are used in conjunction with the inventive method, are also provided.

Allosteric mechanism controls traffic in the chaperone/usher pathway.

PubMed

Di Yu, Xiao; Dubnovitsky, Anatoly; Pudney, Alex F; Macintyre, Sheila; Knight, Stefan D; Zavialov, Anton V

2012-11-07

Many virulence organelles of Gram-negative bacterial pathogens are assembled via the chaperone/usher pathway. The chaperone transports organelle subunits across the periplasm to the outer membrane usher, where they are released and incorporated into growing fibers. Here, we elucidate the mechanism of the usher-targeting step in assembly of the Yersinia pestis F1 capsule at the atomic level. The usher interacts almost exclusively with the chaperone in the chaperone:subunit complex. In free chaperone, a pair of conserved proline residues at the beginning of the subunit-binding loop form a "proline lock" that occludes the usher-binding surface and blocks usher binding. Binding of the subunit to the chaperone rotates the proline lock away from the usher-binding surface, allowing the chaperone-subunit complex to bind to the usher. We show that the proline lock exists in other chaperone/usher systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of the free chaperone. Copyright © 2012 Elsevier Ltd. All rights reserved.

Arylphorin from Manduca sexta: carbohydrate structure and immunological studies.

PubMed

Ryan, R O; Anderson, D R; Grimes, W J; Law, J H

1985-11-15

The major hemolymph protein in the last larval stage of Manduca sexta is a hexameric glycoprotein, arylphorin (Mr = 450,000). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified arylphorin reveals the presence of two subunits, A1 and A2. Both subunits are glycosylated and have apparent Mr = 77,000 and 72,000, respectively. Pronase digestion of arylphorin yielded a single major glycopeptide. 250 MHz NMR spectroscopy of arylphorin glycopeptide revealed a Man9GlcNAc2 oligosaccharide structure similar to that observed in mammalian glycoproteins. Endoglycosidase-H treatment of arylphorin was employed to remove covalently linked carbohydrate residues. The carbohydrate removal lowered the apparent Mr of subunits A1 and A2 to 72,000 and 69,000, respectively, indicating that the difference in arylphorin subunit size is not due to levels of glycosylation. Immunoblotting experiments with anti-arylphorin antiserum and Bombyx mori storage proteins indicated cross reactivity with the corresponding arylphorin of this insect. Preparation of subunit A2 monospecific antibodies, followed by immunoblotting of arylphorin showed a close immunological relationship between subunits A1 and A2.

G-protein gamma subunit 1 is required for sugar reception in Drosophila

PubMed Central

Ishimoto, Hiroshi; Takahashi, Kuniaki; Ueda, Ryu; Tanimura, Teiichi

2005-01-01

Though G-proteins have been implicated in the primary step of taste signal transduction, no direct demonstration has been done in insects. We show here that a G-protein gamma subunit, Gγ1, is required for the signal transduction of sugar taste reception in Drosophila. The Gγ1 gene is expressed mainly in one of the gustatory receptor neurons. Behavioral responses of the flies to sucrose were reduced by the targeted suppression of neural functions of Gγ1-expressing cells using neural modulator genes such as the modified Shaker K+ channel (EKO), the tetanus toxin light chain or the shibire (shits1) gene. RNA interference targeting to the Gγ1 gene reduced the amount of Gγ1 mRNA and suppressed electrophysiological response of the sugar receptor neuron. We also demonstrated that responses to sugars were lowered in Gγ1 null mutant, Gγ1N159. These results are consistent with the hypothesis that Gγ1 participates in the signal transduction of sugar taste reception. PMID:16121192

Structure and function of archaeal prefoldin, a co-chaperone of group II chaperonin.

PubMed

Ohtaki, Akashi; Noguchi, Keiichi; Yohda, Masafumi

2010-01-01

Molecular chaperones are key cellular components involved in the maintenance of protein homeostasis and other unrelated functions. Prefoldin is a chaperone that acts as a co-factor of group II chaperonins in eukaryotes and archaea. It assists proper folding of protein by capturing nonnative proteins and delivering it to the group II chaperonin. Eukaryotic prefoldin is a multiple subunit complex composed of six different polypeptide chains. Archaeal prefoldin, on the other hand, is a heterohexameric complex composed of two alpha and four beta subunits, and forms a double beta barrel assembly with six long coiled coils protruding from it like a jellyfish with six tentacles. Based on the structural information of the archaeal prefoldin, substrate recognition and prefoldin-chaperonin binding mechanisms have been investigated. In this paper, we review a series of studies on the molecular mechanisms of archaeal PFD function. Particular emphasis will be placed on the molecular structures revealed by X-ray crystallography and molecular dynamics induced by binding to nonnative protein substrates.

Modelling toxin effects on protein biosynthesis in eukaryotic cells.

PubMed

Skakauskas, Vladas; Katauskis, Pranas

2017-08-01

We present a rather generic model for toxin (ricin) inhibition of protein biosynthesis in eukaryotic cells. We also study reduction of the ricin toxic effects with application of antibodies against the RTB subunit of ricin molecules. Both species initially are delivered extracellularly. The model accounts for the pinocytotic and receptor-mediated toxin endocytosis and the intact toxin exocytotic removal out of the cell. The model also includes the lysosomal toxin destruction, the intact toxin motion to the endoplasmic reticulum (ER) for separation of its molecules into the RTA and RTB subunits, and the RTA chain translocation into the cytosol. In the cytosol, one portion of the RTA undergoes degradation via the ERAD. The other its portion can inactivate ribosomes at a large rate. The model is based on a system of deterministic ODEs. The influence of the kinetic parameters on the protein concentration and antibody protection factor is studied in detail. Copyright © 2017 Elsevier Ltd. All rights reserved.

Building a flagellum in biological outer space.

PubMed

Evans, Lewis D B; Hughes, Colin; Fraser, Gillian M

2014-02-01

Flagella, the rotary propellers on the surface of bacteria, present a paradigm for how cells build and operate complex molecular 'nanomachines'. Flagella grow at a constant rate to extend several times the length of the cell, and this is achieved by thousands of secreted structural subunits transiting through a central channel in the lengthening flagellum to incorporate into the nascent structure at the distant extending tip. A great mystery has been how flagella can assemble far outside the cell where there is no conventional energy supply to fuel their growth. Recent work published by Evans et al. [ Nature (2013) 504: 287-290], has gone some way towards solving this puzzle, presenting a simple and elegant transit mechanism in which growth is powered by the subunits them selves as they link head-to-tail in a chain that is pulled through the length of the growing structure to the tip. This new mechanism answers an old question and may have resonance in other assembly processes.

Novel Carvedilol Analogs that Suppress Store Overload Induced Ca2+ Release

PubMed Central

Smith, Chris D.; Wang, Aixia; Vembaiyan, Kannan; Zhang, Jingqun; Xie, Cuihong; Zhou, Qiang; Wu, Guogen; Wayne Chen, S. R.; Back, Thomas G.

2013-01-01

Carvedilol is a uniquely effective drug for the treatment of cardiac arrhythmias in patients with heart failure. This activity is in part due to its ability to inhibit store overload-induced calcium release (SOICR) through the RyR2 channel. We describe the synthesis, characterization and bioassay of ca. 100 compounds based on the carvedilol motif in order to identify features that correlate with and optimize SOICR inhibition. A single cell bioassay was employed based on the RyR2-R4496C mutant HEK-293 cell line, in which calcium release from the endoplasmic reticulum through the defective channel was measured. IC50 values for SOICR inhibition were thus obtained. The compounds investigated contained modifications to the three principal subunits of carvedilol, including the carbazole and catechol moieties, as well as the linker chain containing the β-amino alcohol functionality. The SAR results indicate that significant alterations are tolerated in each of the three subunits. PMID:24124794

Direct Force Measurements on Neurofilaments: Gel Expanded to Gel Condensed Transition

NASA Astrophysics Data System (ADS)

Beck, R.; Deek, J.; Jones, J. B.; Safinya, C. R.

2010-03-01

Neurofilaments (NFs)--the major cytoskeletal constituent of axons in vertebrates, consist of three subunit proteins assembled to form filaments with protruding unstructured C-terminus sidearms. Liquid crystal gel networks of sidearm-mediated NF assemblies play a key role in the mechanical while disruptions of this network, due to over-accumulation or incorrect sidearm interactions, are a hallmark of motor neuron diseases. Using synchrotron SAXS [1,2] and microscopy techniques [1,3] we report a direct force measurement of reconstituted NF-gels under osmotic pressure (P), which revealed the role of subunit sidearms on structure and interaction of NFs. With increasing P, near physiological condition, the gels undergo an abrupt nonreversible gel expanded to gel condensed transition that indicates sidearm-mediated attractions between NFs. This attraction is consistent with an electrostatic model of interpenetrating chains.[4pt] [1] J.B. Jones, C.R. Safinya, Biophys. J. 95, 823 (2008);[0pt] [2] R. Beck et al., Nature Mat. (2009) in press;[0pt] [3] H. Hess et al. Langmuir 24, 8397 (2008)

Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor.

PubMed

Jouvin, M H; Adamczewski, M; Numerof, R; Letourneur, O; Vallé, A; Kinet, J P

1994-02-25

Nonreceptor tyrosine kinases such as the newly described 70-kDa (ZAP-70/Syk) and Src-related tyrosine kinases are coupled to a variety of receptors, including the antigen receptors on B- and T-cells and the Fc receptors for IgE (Fc epsilon RI) and IgG (Fc gamma RI, Fc gamma RIII/CD16). Various subunits of these receptors contain homologous activation motifs which appear capable of autonomously triggering cell activation. Two forms of this motif are present in the Fc epsilon RI multimeric complex: one in the beta chain and one in the gamma chain. Here we show that each of the two tyrosine kinases known to be involved in Fc epsilon RI signaling is controlled by a distinct motif-containing chain. Lyn associates with the nonactivated beta chain, whereas gamma promotes the activation of Syk. We also show that neither the beta nor the gamma motif alone can account for the full signaling capacity of the entire receptor. We propose that, upon triggering of the tetrameric receptor, Lyn already bound to beta becomes activated and phosphorylates beta and gamma; the phosphorylation of gamma induces the association of Syk with gamma and also the activation of Syk, resulting in the phosphorylation and activation of phospholipase C gamma 1. Cooperative recruitment of specific kinases by the various signaling chains found in this family of antigen receptors could represent a way to achieve the full signaling capacity of the multimeric complexes.

Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

PubMed

Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

2016-10-01

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana

PubMed Central

Bhoo, Seong Hee; Lai, Huafang; Ma, Julian; Arntzen, Charles J.; Chen, Qiang; Mason, Hugh S.

2014-01-01

Summary Filoviruses (Ebola and Marburg viruses) cause severe and often fatal hemorrhagic fever in humans and non-human primates. The US Centers for Disease Control identify Ebola and Marburg viruses as “category A” pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost-effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C-terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co-expression of the GP1-heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of Nicotiana benthamiana produced assembled immunoglobulin, which was purified by ammonium sulfate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti-Ebola virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. These results show excellent potential for a plant-expressed EIC as a human vaccine. PMID:21281425

Biological responses to PDGF-BB versus PDGF-DD in human mesangial cells.

PubMed

van Roeyen, C R C; Ostendorf, T; Denecke, B; Bokemeyer, D; Behrmann, I; Strutz, F; Lichenstein, H S; LaRochelle, W J; Pena, C E; Chaudhuri, A; Floege, J

2006-04-01

Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more selectively to the beta-chain, suggesting potential differences in the biological activities. Signal transduction and regulation of gene expression induced by PDGF-BB and -DD were compared in primary human mesangial cells (HMCs), which expressed PDGF alpha- and beta-receptor subunits. The growth factor concentrations used were chosen based on their equipotency in inducing HMCs proliferation and binding to the betabeta-receptor. Both growth factors, albeit at different concentrations induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. In addition, PDGFs led to the phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1) and STAT3. HMCs proliferation induced by either PDGF-BB or -DD could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase-, Janus kinase (JAK)/STAT-, or phosphatidyl-inositol 3-kinase pathways. Using a gene chip array and subsequent verification by real-time reverse transcriptase (RT)-polymerase chain reaction, we found that in HMC genes for matrix metalloproteinase 13 (MMP-13) and MMP-14 and, to a low extent, cytochrome B5 and cathepsin L were exclusively regulated by PDGF-BB, whereas no exclusive gene regulation was detected by PDGF-DD. However, at the protein level, both MMP-13 and -14 were equally induced by PDGF-BB and -DD. PDGF-BB and -DD effect similar biological responses in HMCs albeit at different potencies. Rare apparently differential gene regulation did not result in different protein expression, suggesting that in HMCs both PDGFs exert their biological activity almost exclusively via the PDGF beta-receptor.

Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana.

PubMed

Phoolcharoen, Waranyoo; Bhoo, Seong H; Lai, Huafang; Ma, Julian; Arntzen, Charles J; Chen, Qiang; Mason, Hugh S

2011-09-01

Filoviruses (Ebola and Marburg viruses) cause severe and often fatal haemorrhagic fever in humans and non-human primates. The US Centers for Disease Control identifies Ebola and Marburg viruses as 'category A' pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost-effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C-terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co-expression of the GP1-heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of N. benthamiana produced assembled immunoglobulin, which was purified by ammonium sulphate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size-exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti-Ebola virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. These results show excellent potential for a plant-expressed EIC as a human vaccine. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Tripartite motif ligases catalyze polyubiquitin chain formation through a cooperative allosteric mechanism.

PubMed

Streich, Frederick C; Ronchi, Virginia P; Connick, J Patrick; Haas, Arthur L

2013-03-22

Ligation of polyubiquitin chains to proteins is a fundamental post-translational modification, often resulting in targeted degradation of conjugated proteins. Attachment of polyubiquitin chains requires the activities of an E1 activating enzyme, an E2 carrier protein, and an E3 ligase. The mechanism by which polyubiquitin chains are formed remains largely speculative, especially for RING-based ligases. The tripartite motif (TRIM) superfamily of ligases functions in many cellular processes including innate immunity, cellular localization, development and differentiation, signaling, and cancer progression. The present results show that TRIM ligases catalyze polyubiquitin chain formation in the absence of substrate, the rates of which can be used as a functional readout of enzyme function. Initial rate studies under biochemically defined conditions show that TRIM32 and TRIM25 are specific for the Ubc5 family of E2-conjugating proteins and, along with TRIM5α, exhibit cooperative kinetics with respect to Ubc5 concentration, with submicromolar [S]0.5 and Hill coefficients of 3-5, suggesting they possess multiple binding sites for their cognate E2-ubiquitin thioester. Mutation studies reveal a second, non-canonical binding site encompassing the C-terminal Ubc5α-helix. Polyubiquitin chain formation requires TRIM subunit oligomerization through the conserved coiled-coil domain, but can be partially replaced by fusing the catalytic domain to GST to promote dimerization. Other results suggest that TRIM32 assembles polyubiquitin chains as a Ubc5-linked thioester intermediate. These results represent the first detailed mechanistic study of TRIM ligase activity and provide a functional context for oligomerization observed in the superfamily.

Phylogeny, divergence time and historical biogeography of Laetiporus (Basidiomycota, Polyporales).

PubMed

Song, Jie; Cui, Bao-Kai

2017-04-20

The aim of this study was to characterize the molecular relationship, origin and historical biogeography of the species in important brown rot fungal genus Laetiporus from East Asia, Europe, Pan-America, Hawaii and South Africa. We used six genetic markers to estimate a genus-level phylogeny including (1) the internal transcribed spacer (ITS), (2) nuclear large subunit rDNA (nrLSU), (3) nuclear small subunit rDNA (nrSSU), (4) translation elongation factor 1-α (EF-1α), (5) DNA-directed RNA polymerase II subunit 2 (RPB2), and (6) mitochondrial small subunit rDNA (mtSSU). Results of multi-locus phylogenetic analyses show clade support for at least seventeen species-level lineages including two new Laetiporus in China. Molecular dating using BEAST estimated the present crown group diverged approximately 20.16 million years ago (Mya) in the early Miocene. Biogeographic analyses using RASP indicated that Laetiporus most likely originated in temperate zones with East Asia and North America having the highest probability (48%) of being the ancestral area. Four intercontinental dispersal routes and a possible concealed dispersal route were established for the first time.

Regulation of Na(+)/K(+)-ATPase by nuclear respiratory factor 1: implication in the tight coupling of neuronal activity, energy generation, and energy consumption.

PubMed

Johar, Kaid; Priya, Anusha; Wong-Riley, Margaret T T

2012-11-23

NRF-1 regulates mediators of neuronal activity and energy generation. NRF-1 transcriptionally regulates Na(+)/K(+)-ATPase subunits α1 and β1. NRF-1 functionally regulates mediators of energy consumption in neurons. NRF-1 mediates the tight coupling of neuronal activity, energy generation, and energy consumption at the molecular level. Energy generation and energy consumption are tightly coupled to neuronal activity at the cellular level. Na(+)/K(+)-ATPase, a major energy-consuming enzyme, is well expressed in neurons rich in cytochrome c oxidase, an important enzyme of the energy-generating machinery, and glutamatergic receptors that are mediators of neuronal activity. The present study sought to test our hypothesis that the coupling extends to the molecular level, whereby Na(+)/K(+)-ATPase subunits are regulated by the same transcription factor, nuclear respiratory factor 1 (NRF-1), found recently by our laboratory to regulate all cytochrome c oxidase subunit genes and some NMDA and AMPA receptor subunit genes. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation, promoter mutational analysis, and real-time quantitative PCR, NRF-1 was found to functionally bind to the promoters of Atp1a1 and Atp1b1 genes but not of the Atp1a3 gene in neurons. The transcripts of Atp1a1 and Atp1b1 subunit genes were up-regulated by KCl and down-regulated by tetrodotoxin. Atp1b1 is positively regulated by NRF-1, and silencing of NRF-1 with small interference RNA blocked the up-regulation of Atp1b1 induced by KCl, whereas overexpression of NRF-1 rescued these transcripts from being suppressed by tetrodotoxin. On the other hand, Atp1a1 is negatively regulated by NRF-1. The binding sites of NRF-1 on Atp1a1 and Atp1b1 are conserved among mice, rats, and humans. Thus, NRF-1 regulates key Na(+)/K(+)-ATPase subunits and plays an important role in mediating the tight coupling between energy consumption, energy generation, and neuronal activity at the molecular level.

Revealing the functionality of hypothetical protein KPN00728 from Klebsiella pneumoniae MGH78578: molecular dynamics simulation approaches

PubMed Central

2011-01-01

Background Previously, the hypothetical protein, KPN00728 from Klebsiella pneumoniae MGH78578 was the Succinate dehydrogenase (SDH) chain C subunit via structural prediction and molecular docking simulation studies. However, due to limitation in docking simulation, an in-depth understanding of how SDH interaction occurs across the transmembrane of mitochondria could not be provided. Results In this present study, molecular dynamics (MD) simulation of KPN00728 and SDH chain D in a membrane was performed in order to gain a deeper insight into its molecular role as SDH. Structural stability was successfully obtained in the calculation for area per lipid, tail order parameter, thickness of lipid and secondary structural properties. Interestingly, water molecules were found to be highly possible in mediating the interaction between Ubiquinone (UQ) and SDH chain C via interaction with Ser27 and Arg31 residues as compared with earlier docking study. Polar residues such as Asp95 and Glu101 (KPN00728), Asp15 and Glu78 (SDH chain D) might have contributed in the creation of a polar environment which is essential for electron transport chain in Krebs cycle. Conclusions As a conclusion, a part from the structural stability comparability, the dynamic of the interacting residues and hydrogen bonding analysis had further proved that the interaction of KPN00728 as SDH is preserved and well agreed with our postulation earlier. PMID:22372825

Na, K-ATPase subunits as markers for epithelial-mesenchymal transition in cancer and fibrosis

PubMed Central

Rajasekaran, Sigrid A.; Huynh, Thu P.; Wolle, Daniel G.; Espineda, Cromwell E.; Inge, Landon J.; Skay, Anna; Lassman, Charles; Nicholas, Susanne B.; Harper, Jeffrey F.; Reeves, Anna E.; Ahmed, Mansoor M.; Leatherman, James M; Mullin, James M.; Rajasekaran, Ayyappan K.

2010-01-01

Epithelial-to-mesenchymal transition (EMT) is an important developmental process, participates in tissue repair and occurs during pathological processes of tumor invasiveness, metastasis and tissue fibrosis. The molecular mechanisms leading to EMT are poorly understood. While it is well documented that transforming growth factor (TGF)-β plays a central role in the induction of EMT, the targets of TGF-β signaling are poorly defined. We have shown earlier that Na,K-ATPase β1-subunit levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients’ tumor samples. In this study, we provide evidence that Na,K-ATPase is a new target of TGF-β1-mediated EMT in renal epithelial cells, a model system used in studies of both cancer progression and fibrosis. We show that following treatment with TGF-β1 the surface expression of the β1-subunit of Na,K-ATPase is reduced, prior to well-characterized EMT markers and is associated with the acquisition of a mesenchymal phenotype. RNAi mediated knockdown confirmed the specific involvement of the Na,K-ATPase β1-subunit in the loss of the epithelial phenotype and exogenous over-expression of the Na,K-ATPase β1-subunit attenuated TGF-β1-mediated EMT. We further show that both Na,K-ATPase α- and β-subunit levels are highly reduced in renal fibrotic tissues. These findings for the first time reveal that Na,K-ATPase is a target of TGF-β1-mediated EMT and is associated with the progression of EMT in both cancer and fibrosis. PMID:20501797

Regulatory Subunit B′γ of Protein Phosphatase 2A Prevents Unnecessary Defense Reactions under Low Light in Arabidopsis1[W][OA

PubMed Central

Trotta, Andrea; Wrzaczek, Michael; Scharte, Judith; Tikkanen, Mikko; Konert, Grzegorz; Rahikainen, Moona; Holmström, Maija; Hiltunen, Hanna-Maija; Rips, Stephan; Sipari, Nina; Mulo, Paula; Weis, Engelbert; von Schaewen, Antje; Aro, Eva-Mari; Kangasjärvi, Saijaliisa

2011-01-01

Light is an important environmental factor that modulates acclimation strategies and defense responses in plants. We explored the functional role of the regulatory subunit B′γ (B′γ) of protein phosphatase 2A (PP2A) in light-dependent stress responses of Arabidopsis (Arabidopsis thaliana). The predominant form of PP2A consists of catalytic subunit C, scaffold subunit A, and highly variable regulatory subunit B, which determines the substrate specificity of PP2A holoenzymes. Mutant leaves of knockdown pp2a-b′γ plants show disintegration of chloroplasts and premature yellowing conditionally under moderate light intensity. The cell-death phenotype is accompanied by the accumulation of hydrogen peroxide through a pathway that requires CONSTITUTIVE EXPRESSION OF PR GENES5 (CPR5). Moreover, the pp2a-b′γ cpr5 double mutant additionally displays growth suppression and malformed trichomes. Similar to cpr5, the pp2a-b′γ mutant shows constitutive activation of both salicylic acid- and jasmonic acid-dependent defense pathways. In contrast to cpr5, however, pp2a-b′γ leaves do not contain increased levels of salicylic acid or jasmonic acid. Rather, the constitutive defense response associates with hypomethylation of DNA and increased levels of methionine-salvage pathway components in pp2a-b′γ leaves. We suggest that the specific B′γ subunit of PP2A is functionally connected to CPR5 and operates in the basal repression of defense responses under low irradiance. PMID:21571669

Protein farnesyltransferase in plants: molecular characterization and involvement in cell cycle control.

PubMed Central

Qian, D; Zhou, D; Ju, R; Cramer, C L; Yang, Z

1996-01-01

Farnesylation is required for membrane targeting, protein-protein interactions, and the biological activity of key regulatory proteins, such as Ras small GTPases and protein kinases in a wide range of eukaryotes. In this report, we describe the molecular identification of a plant protein farnesyltransferase (FTase) and evidence for its role in the control of the cell cycle in plants. A pea gene encoding a homolog of the FTase beta subunit was previously cloned using a polymerase chain reaction-based strategy. A similar approach was used to clone a pea gene encoding a homolog of the FTase alpha subunit. The biochemical function of the pea FTase homologs was demonstrated by the reconstitution of FTase enzyme activity using FTase fusion proteins coexpressed in Escherichia coll. RNA gel blot analyses showed that levels of FTase mRNAs are generally higher in tissues, such as those of nodules, that are active in cell division. The relationship of FTase to cell division was further analyzed during the growth of suspension-cultured tobacco BY-2 cells. A biphasic fluctuation of FTase enzyme activity preceded corresponding changes in mitotic activity at the early log phase of cell growth. Moreover, manumycin, a specific inhibitor of FTase, was effective in inhibiting mitosis and growth in these cells. Using synchronized BY-2 cells, manumycin completely blocked mitosis when added at the early S phase but not when added at the G2 phase. These data suggest that FTase is required for the plant cell cycle, perhaps by modulating the progression through the S phase and the transition from G1 to the S phase. PMID:8989889

Identification and Modulation of the Key Amino Acid Residue Responsible for the pH Sensitivity of Neoculin, a Taste-Modifying Protein

PubMed Central

Nakajima, Ken-ichiro; Yokoyama, Kanako; Koizumi, Taichi; Koizumi, Ayako; Asakura, Tomiko; Terada, Tohru; Masuda, Katsuyoshi; Ito, Keisuke; Shimizu-Ibuka, Akiko; Misaka, Takumi; Abe, Keiko

2011-01-01

Neoculin occurring in the tropical fruit of Curculigo latifolia is currently the only protein that possesses both a sweet taste and a taste-modifying activity of converting sourness into sweetness. Structurally, this protein is a heterodimer consisting of a neoculin acidic subunit (NAS) and a neoculin basic subunit (NBS). Recently, we found that a neoculin variant in which all five histidine residues are replaced with alanine elicits intense sweetness at both neutral and acidic pH but has no taste-modifying activity. To identify the critical histidine residue(s) responsible for this activity, we produced a series of His-to-Ala neoculin variants and evaluated their sweetness levels using cell-based calcium imaging and a human sensory test. Our results suggest that NBS His11 functions as a primary pH sensor for neoculin to elicit taste modification. Neoculin variants with substitutions other than His-to-Ala were further analyzed to clarify the role of the NBS position 11 in the taste-modifying activity. We found that the aromatic character of the amino acid side chain is necessary to elicit the pH-dependent sweetness. Interestingly, since the His-to-Tyr variant is a novel taste-modifying protein with alternative pH sensitivity, the position 11 in NBS can be critical to modulate the pH-dependent activity of neoculin. These findings are important for understanding the pH-sensitive functional changes in proteinaceous ligands in general and the interaction of taste receptor–taste substance in particular. PMID:21559382

Effects of recombinant gonadotropin hormones on the expression of vitellogenin, gonadotropin subunits and gonadotropin receptors in cinnamon clownfish, Amphiprion melanopus.

PubMed

Kim, Na Na; Habibi, Hamid R; Lee, Jehee; Choi, Cheol Young

2012-08-01

Gonadotropins (GTHs) are the key regulators of reproduction in vertebrates. The present study investigated autoregulatory effects of gonadotropins, using recombinant FSH (rFSH) and LH (rLH) in cinnamon clownfish (Amphiprion melanopus). Experiments were carried out to investigate the actions of cinnamon clownfish rFSH and rLH on expression of GTH subunits, GTH receptors, and vitellogenin (Vtg) mRNA in vivo and in vitro. Plasma estradiol-17β (E(2)) level was also measured in immature fish following treatments with rFSH and rLH. The results demonstrate increasing levels of GTH subunits, GTH-receptors, Vtg mRNA levels, as well as plasma E(2) levels following injection with rFSH and rLH. The findings support the hypothesis that LH and FSH stimulate reproduction, in part, by autoregulatory mechanisms leading to upregulation of GTH receptors and GTH hormone production in cinnamon clownfish. The results provide a framework for better understanding of the mechanisms of GTH-mediated control of reproduction in cinnamon clownfish and other vertebrates. Copyright © 2012 Elsevier Inc. All rights reserved.

Central cavity of fructose-1,6-bisphosphatase and the evolution of AMP/fructose 2,6-bisphosphate synergism in eukaryotic organisms.

PubMed

Gao, Yang; Shen, Lu; Honzatko, Richard B

2014-03-21

The effects of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) on porcine fructose-1,6-bisphosphatase (pFBPase) and Escherichia coli FBPase (eFBPase) differ in three respects. AMP/Fru-2,6-P2 synergism in pFBPase is absent in eFBPase. Fru-2,6-P2 induces a 13° subunit pair rotation in pFBPase but no rotation in eFBPase. Hydrophilic side chains in eFBPase occupy what otherwise would be a central aqueous cavity observed in pFBPase. Explored here is the linkage of AMP/Fru-2,6-P2 synergism to the central cavity and the evolution of synergism in FBPases. The single mutation Ser(45) → His substantially fills the central cavity of pFBPase, and the triple mutation Ser(45) → His, Thr(46) → Arg, and Leu(186) → Tyr replaces porcine with E. coli type side chains. Both single and triple mutations significantly reduce synergism while retaining other wild-type kinetic properties. Similar to the effect of Fru-2,6-P2 on eFBPase, the triple mutant of pFBPase with bound Fru-2,6-P2 exhibits only a 2° subunit pair rotation as opposed to the 13° rotation exhibited by the Fru-2,6-P2 complex of wild-type pFBPase. The side chain at position 45 is small in all available eukaryotic FBPases but large and hydrophilic in bacterial FBPases, similar to eFBPase. Sequence information indicates the likelihood of synergism in the FBPase from Leptospira interrogans (lFBPase), and indeed recombinant lFBPase exhibits AMP/Fru-2,6-P2 synergism. Unexpectedly, however, AMP also enhances Fru-6-P binding to lFBPase. Taken together, these observations suggest the evolution of AMP/Fru-2,6-P2 synergism in eukaryotic FBPases from an ancestral FBPase having a central aqueous cavity and exhibiting synergistic feedback inhibition by AMP and Fru-6-P.

Cross-bridge kinetics of fast and slow fibres of cat jaw and limb muscles: correlations with myosin subunit composition.

PubMed

Hoh, Joseph F Y; Li, Zhao-Bo; Qin, Han; Hsu, Michael K H; Rossmanith, Gunther H

2007-01-01

Mechanical properties of the jaw-closing muscles of the cat are poorly understood. These muscles are known to differ in myosin and fibre type compositions from limb muscles. This work aims to correlate mechanical properties of single fibres in cat jaw and limb muscles with their myosin subunit compositions. The stiffness minimum frequency, f(min), which reflects isometric cross-bridge kinetics, was measured in Ca(2+)-activated glycerinated fast and slow fibres from cat jaw and limb muscles for temperatures ranging between 15 and 30 degrees C by mechanical perturbation analysis. At 15 degrees C, f(min) was 0.5 Hz for limb-slow fibres, 4-6 Hz for jaw-slow fibres, and 10-13 Hz for limb-fast and jaw-fast fibres. The activation energy for f(min) obtained from the slope of the Arrhenius plot for limb-slow fibres was 30-40% higher than values for the other three types of fibres. SDS-PAGE and western blotting using highly specific antibodies verified that limb-fast fibres contained IIA or IIX myosin heavy chain (MyHC). Jaw-fast fibres expressed masticatory MyHC while both jaw-fast and jaw-slow fibres expressed masticatory myosin light chains (MLCs). The nucleotide sequences of the 3' ends of the slow MyHC cDNAs isolated from cat masseter and soleus cDNA libraries showed identical coding and 3'-untranslated regions, suggesting that jaw-slow and limb-slow fibres express the same slow MyHC gene. We conclude that the isometric cross-bridge cycling kinetics of jaw-fast and limb-fast fibres detected by f(min) are indistinguishable in spite of differences in MyHC and light chain compositions. However, jaw-slow fibres, in which the same slow MyHCs are found in combination with MLCs of the jaw type, show enhanced cross-bridge cycling kinetics and reduced activation energy for cross-bridge detachment.

New metal-organic complexes based on bis(tetrazole) ligands: Synthesis, structures and properties

NASA Astrophysics Data System (ADS)

Du, Ceng-Ceng; Fan, Jian-Zhong; Wang, Xin-Fang; Zhou, Sheng-Bin; Wang, Duo-Zhi

2017-04-01

In this paper, a series of new complexes, [Zn2(HL1)2(H2O)4]·H2O (1), [Co2(HL1)2]·TEA (2), [Co3(HL1)2(H2L1)2(H2O)4]n (3), [Cu(HL1)(H2O)2]n (4), {[Cu5(HL2)2(OH)4(ClO4)2]·4H2O}n (5) and [Cu2(L3)]n (6) were successfully prepared by utilizing three bis(tetrazole) ligands [bis-(1H-tetrazol-5-ylmethyl)-amine (H3L1), bis-(1H-tetrazol-5-ylethyl)-amine (H3L2) and 1,5-bis(5-tetrazolo)-3-thiapentane (H2L3)], all of which have been characterized by elemental analyses, FT-IR spectroscopy, powder X-ray diffraction (PXRD), thermogravimetric analyses as well as single-crystal X-ray diffraction analyses showing different dimensionalities (0D, 1D and 3D). Complexes 1 and 2 are 0D structures, 1 shows a dinuclear structure, 2 displays two crystallographically different mononuclear structures, 1 and 2 are further assembled to form 3D supramolecular framework and 2D supramolecular network by hydrogen-bonding interactions, respectively. Complexes 3, 4 and 5 are 1D structures, 3 features a mononuclear unit and a 1D chain, which are arranged into 3D supramolecular architecture by hydrogen-bonding interactions, 4 presents a zigzag chain, 5 shows an infinite chain structure constructed from pentanuclear Cu(II) subunits and ClO4- anions. Complex 6 exhibits a 3D coordination framework based on cyclic [Cu4(L3)2] dimmer subunits as nodes possessing an 8-connected network topology with the point symbol {424·64}. Further, semiconductor behaviors, the solid-state luminescent properties of the complexes 1-3 and 6 were measured and studied seriously at room temperature.

Dissection of the Caffeate Respiratory Chain in the Acetogen Acetobacterium woodii: Identification of an Rnf-Type NADH Dehydrogenase as a Potential Coupling Site▿

PubMed Central

Imkamp, Frank; Biegel, Eva; Jayamani, Elamparithi; Buckel, Wolfgang; Müller, Volker

2007-01-01

The anaerobic acetogenic bacterium Acetobacterium woodii couples caffeate reduction with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions, a process referred to as caffeate respiration. We addressed the nature of the hitherto unknown enzymatic activities involved in this process and their cellular localization. Cell extract of A. woodii catalyzes H2-dependent caffeate reduction. This reaction is strictly ATP dependent but can be activated also by acetyl coenzyme A (CoA), indicating that there is formation of caffeyl-CoA prior to reduction. Two-dimensional gel electrophoresis revealed proteins present only in caffeate-grown cells. Two proteins were identified by electrospray ionization-mass spectrometry/mass spectrometry, and the encoding genes were cloned. These proteins are very similar to subunits α (EtfA) and β (EtfB) of electron transfer flavoproteins present in various anaerobic bacteria. Western blot analysis demonstrated that they are induced by caffeate and localized in the cytoplasm. Etf proteins are known electron carriers that shuttle electrons from NADH to different acceptors. Indeed, NADH was used as an electron donor for cytosolic caffeate reduction. Since the hydrogenase was soluble and used ferredoxin as an electron acceptor, the missing link was a ferredoxin:NAD+ oxidoreductase. This activity could be determined and, interestingly, was membrane bound. A search for genes that could encode this activity revealed DNA fragments encoding subunits C and D of a membrane-bound Rnf-type NADH dehydrogenase that is a potential Na+ pump. These data suggest the following electron transport chain: H2 → ferredoxin → NAD+ → Etf → caffeyl-CoA reductase. They also imply that the sodium motive step in the chain is the ferredoxin-dependent NAD+ reduction catalyzed by Rnf. PMID:17873051

Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs

PubMed Central

Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

2016-01-01

In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process. PMID:27099964

Effects of sulfate concentrations on the expression of a soybean seed storage protein gene and its reversibility in transgenic Arabidopsis thaliana.

PubMed

Hirai, M Y; Fujiwara, T; Chino, M; Naito, S

1995-10-01

Transgenic expression of genes encoding the alpha' and beta subunits of beta-conglycinin, one of the major seed storage proteins of soybean (Glycine max [L.] Merr.), was analyzed in Arabidopsis thaliana (L.) Heynh. under conditions of sulfate deficiency. Temporal patterns of expression of both the intact beta subunit gene and the beta subunit gene promoter fused to the beta-glucuronidase (GUS) gene are similar in soil-less cultures using rockwool, suggesting that the response to sulfate deficiency is regulated mainly at the level of transcription. In hydroponic cultures with various concentrations of sulfate, expression of both the intact beta subunit gene and the beta subunit gene promoter-GUS fusion gene were negatively correlated to increased sulfate concentrations in the culture medium. Transfer of transgenic A. thaliana plants carrying the beta subunit gene promoter-GUS fusion from sulfate-deficient to sulfate-sufficient control medium caused GUS activity in developing siliques to be repressed within two days. A reverse shift, where the plants were transferred from the control to sulfate-deficient medium, caused GUS activity to become higher than that in seeds of the control plants within two days. These results indicate that the expression of the beta subunit gene promoter responds rapidly to changes of sulfate availability.

Conformational Flexibility and Subunit Arrangement of the Modular Yeast Spt-Ada-Gcn5 Acetyltransferase Complex*

PubMed Central

Setiaputra, Dheva; Ross, James D.; Lu, Shan; Cheng, Derrick T.; Dong, Meng-Qiu; Yip, Calvin K.

2015-01-01

The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility. PMID:25713136