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Sample records for acid protein ligases

  1. Multisite clickable modification of proteins using lipoic acid ligase.

    PubMed

    Plaks, Joseph G; Falatach, Rebecca; Kastantin, Mark; Berberich, Jason A; Kaar, Joel L

    2015-06-17

    Approaches that allow bioorthogonal and, in turn, site-specific chemical modification of proteins present considerable opportunities for modulating protein activity and stability. However, the development of such approaches that enable site-selective modification of proteins at multiple positions, including internal sites within a protein, has remained elusive. To overcome this void, we have developed an enzymatic approach for multisite clickable modification based on the incorporation of azide moieties in proteins using lipoic acid ligase (LplA). The ligation of azide moieties to the model protein, green fluorescent protein (GFP), at the N-terminus and two internal sites using lipoic acid ligase was shown to proceed efficiently with near-complete conversion. Modification of the ligated azide groups with poly(ethylene glycol) (PEG), α-d-mannopyranoside, and palmitic acid resulted in highly homogeneous populations of protein-polymer, protein-sugar, and protein-fatty acid conjugates. The homogeneity of the conjugates was confirmed by mass spectrometry (MALDI-TOF) and SDS-PAGE electrophoresis. In the case of PEG attachment, which involved the use of strain-promoted azide-alkyne click chemistry, the conjugation reaction resulted in highly homogeneous PEG-GFP conjugates in less than 30 min. As further demonstration of the utility of this approach, ligated GFP was also covalently immobilized on alkyne-terminated self-assembled monolayers. These results underscore the potential of this approach for, among other applications, site-specific multipoint protein PEGylation, glycosylation, fatty acid modification, and protein immobilization.

  2. Control of Amino Acid Homeostasis by a Ubiquitin Ligase-Coactivator Protein Complex.

    PubMed

    Guerra, Damian; Chapiro, Sonia M; Pratelli, Réjane; Yu, Shi; Jia, Weitao; Leary, Julie; Pilot, Guillaume; Callis, Judy

    2017-03-03

    Intercellular amino acid transport is essential for the growth of all multicellular organisms, and its dysregulation is implicated in developmental disorders. By an unknown mechanism, amino acid efflux is stimulated in plants by overexpression of a membrane-localized protein (GLUTAMINE DUMPER 1 (GDU1)) that requires a ubiquitin ligase (LOSS OF GDU 2 (LOG2). Here we further explore the physiological consequences of the interaction between these two proteins. LOG2 ubiquitin ligase activity is necessary for GDU1-dependent tolerance to exogenous amino acids, and LOG2 self-ubiquitination was markedly stimulated by the GDU1 cytosolic domain, suggesting that GDU1 functions as an adaptor or coactivator of amino acid exporter(s). However, other consequences more typical of a ligase-substrate relationship are observed: disruption of the LOG2 gene increased the in vivo half-life of GDU1, mass spectrometry confirmed that LOG2 ubiquitinates GDU1 at cytosolic lysines, and GDU1 protein levels decreased upon co-expression with active, but not enzymatically inactive LOG2. Altogether these data indicate LOG2 negatively regulates GDU1 protein accumulation by a mechanism dependent upon cytosolic GDU1 lysines. Although GDU1-lysine substituted protein exhibited diminished in vivo ubiquitination, overexpression of GDU1 lysine mutants still conferred amino acid tolerance in a LOG2-dependent manner, consistent with GDU1 being both a substrate and facilitator of LOG2 function. From these data, we offer a model in which GDU1 activates LOG2 to stimulate amino acid export, a process that could be negatively regulated by GDU1 ubiquitination and LOG2 self-ubiquitination.

  3. Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology

    PubMed Central

    Chambers, Cecilia R.; Patrick, Wayne M.

    2015-01-01

    With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and biotechnology. Currently, the nucleic acid ligases from bacteriophage T4 are used extensively in these protocols. In this review, we argue that the nucleic acid ligases from Archaea represent a largely untapped pool of enzymes with diverse and potentially favourable properties for new and emerging biotechnological applications. We summarise the current state of knowledge on archaeal DNA and RNA ligases, which makes apparent the relative scarcity of information on in vitro activities that are of most relevance to biotechnologists (such as the ability to join blunt- or cohesive-ended, double-stranded DNA fragments). We highlight the existing biotechnological applications of archaeal DNA ligases and RNA ligases. Finally, we draw attention to recent experiments in which protein engineering was used to modify the activities of the DNA ligase from Pyrococcus furiosus and the RNA ligase from Methanothermobacter thermautotrophicus, thus demonstrating the potential for further work in this area. PMID:26494982

  4. Arabidopsis BPM proteins function as substrate adaptors to a cullin3-based E3 ligase to affect fatty acid metabolism in plants.

    PubMed

    Chen, Liyuan; Lee, Joo Hyun; Weber, Henriette; Tohge, Takayuki; Witt, Sandra; Roje, Sanja; Fernie, Alisdair R; Hellmann, Hanjo

    2013-06-01

    Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity regulation because they play a critical role as key mediators assuring specific expression of target genes. In this work, we show that cullin3-based E3 ligases have the potential to interact with a broad range of ethylene response factor (ERF)/APETALA2 (AP2) transcription factors, mediated by Math-BTB/POZ (for Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog)-Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the wrinkled1 ERF/AP2 protein. Furthermore, loss of Math-BTB/POZ proteins widely affects plant development and causes altered fatty acid contents in mutant seeds. Overall, this work demonstrates a link between fatty acid metabolism and E3 ligase activities in plants and establishes CUL3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members.

  5. Is there a protein ligase?

    PubMed

    Erhan, S

    1976-01-01

    Results obtained from experiments dealing with mammalian, bacterial, phage and mitochondrial protein biosynthesis as well as certain enzymatically performed amino acid replacement studies on Kunitz trypsin inhibitor strongly suggest that protein ligation may be occuring in vivo. Amino acid substitution experiments prove the reversibility of endopeptidase reactions, and protein ligation is the reverse of endopeptidase reaction. These experiments are discussed in detail and the suggestion is made that ligation may also be useful in the repair of certain essential proteins which may become damaged.

  6. Protein Neddylation: Beyond Cullin-RING Ligases

    PubMed Central

    Enchev, Radoslav I.; Schulman, Brenda A.; Peter, Matthias

    2016-01-01

    NEDD8 is a ubiquitin-like protein that activates the largest ubiquitin E3 ligase family, the cullin RING ligases. Many non-cullin neddylation targets have been proposed in recent years. However, overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery, which makes validating potential NEDD8 targets challenging. Here we re-evaluate these studies in light of the current understanding of the neddylation pathway, and suggest criteria for the identification of genuine neddylation substrates under homeostatic conditions. We describe the biological processes that might be regulated by non-cullin neddylation, and the utility of neddylation inhibitors for research and as potential therapies. Understanding the biological significance of non-cullin neddylation is an exciting research prospect primed to reveal fundamental insights. PMID:25531226

  7. Systems Biology of Lignin Biosynthesis in Populus trichocarpa: Heteromeric 4-Coumaric Acid:Coenzyme A Ligase Protein Complex Formation, Regulation, and Numerical Modeling[W

    PubMed Central

    Chen, Hsi-Chuan; Song, Jina; Wang, Jack P.; Lin, Ying-Chung; Ducoste, Joel; Shuford, Christopher M.; Liu, Jie; Li, Quanzi; Shi, Rui; Nepomuceno, Angelito; Isik, Fikret; Muddiman, David C.; Williams, Cranos; Sederoff, Ronald R.; Chiang, Vincent L.

    2014-01-01

    As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein–protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation. PMID:24619612

  8. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    SciTech Connect

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

  9. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    DOE PAGES

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; ...

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of themore » intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.« less

  10. Endocytosis of the Aspartic Acid/Glutamic Acid Transporter Dip5 Is Triggered by Substrate-Dependent Recruitment of the Rsp5 Ubiquitin Ligase via the Arrestin-Like Protein Aly2 ▿

    PubMed Central

    Hatakeyama, Riko; Kamiya, Masao; Takahara, Terunao; Maeda, Tatsuya

    2010-01-01

    Endocytosis of nutrient transporters is stimulated under various conditions, such as elevated nutrient availability. In Saccharomyces cerevisiae, endocytosis is triggered by ubiquitination of transporters catalyzed by the E3 ubiquitin ligase Rsp5. However, how the ubiquitination is accelerated under certain conditions remains obscure. Here we demonstrate that closely related proteins Aly2/Art3 and Aly1/Art6, which are poorly characterized members of the arrestin-like protein family, mediate endocytosis of the aspartic acid/glutamic acid transporter Dip5. In aly2Δ cells, Dip5 is stabilized at the plasma membrane and is not endocytosed efficiently. Efficient ubiquitination of Dip5 is dependent on Aly2. aly1Δ cells also show deficiency in Dip5 endocytosis, although less remarkably than aly2Δ cells. Aly2 physically interacts in vivo with Rsp5 at its PY motif and also with Dip5, thus serving as an adaptor linking Rsp5 with Dip5 to achieve Dip5 ubiquitination. Importantly, the interaction between Aly2 and Dip5 is accelerated in response to elevated aspartic acid availability. This result indicates that the regulation of Dip5 endocytosis is accomplished by dynamic recruitment of Rsp5 via Aly2. PMID:20956561

  11. TRIM proteins as RING finger E3 ubiquitin ligases.

    PubMed

    Ikeda, Kazuhiro; Inoue, Satoshi

    2012-01-01

    The tripartite motif(TRIM) proteins harboring the RING finger, B-box and coiled-coil (RBCC) domain motifs form a large protein family. The members of this family are involved in various biological processes, including growth, differentiation, apoptosis and transcription and also in diseases and oncogenesis. Recent studies have revealed that TRIM proteins play key roles in innate antiviral immunity. An accumulating body of evidence has demonstrated that some TRIM proteins function as E3 ubiquitin ligases in specific ubiquitin-mediated protein degradation pathways; however, the precise mechanisms underlying this function have not been fully elucidated. In this chapter, we focus on the TRIM family of proteins specially with regard to E3 ligase.

  12. Involvement of fatty acid-CoA ligase 4 in hepatocellular carcinoma growth: Roles of cyclic AMP and p38 mitogen-activated protein kinase

    PubMed Central

    Liang, Yu-Chih; Wu, Chih-Hsiung; Chu, Jan-Show; Wang, Chung-Kwe; Hung, Ling-Fang; Wang, Ying-Jan; Ho, Yuan-Soon; Chang, Jan-Gowth; Lin, Shyr-Yi

    2005-01-01

    AIM: Fatty acid-CoA ligase 4 (FACL4) is an arachidonate-preferring enzyme which has been shown to be up-regulated in human colon cancer tissues and implicated in the colon tumorigenesis. The purpose of this study was to investigate the role of FACL4 in the human hepatocellular carcinoma (HCC) tumorigenesis and the specific signal pathways involved in this process. METHODS: We investigated the expression and regulation of FACL4 in HCC, adjacent non-tumorous liver tissues, and cell lines. RESULTS: In HCC patients, we demonstrated that FACL4 gene expression was markedly elevated in the cancerous tissues than in the adjacent non-cancerous liver tissues. In addition, several human hepatoma cell lines, including Hep3B and HepG2, expressed high levels of FACL4. Stable overex-pression of FACL4 knockdown plasmids (small interfering RNA, siRNA) to Hep3B cells significantly decreased FACL4 expression and subsequently limited the cell proliferation. Treatment of Hep3B cells with 8-bromo-cAMP and SB203508 (p38 MAPK inhibitor) significantly suppressed the FACL4 expression. CONCLUSION: FACL4 is involved in the HCC tumorigenesis and both cAMP and p38 MAPK pathways are associated with the regulation of FACL4 in HCC. PMID:15849811

  13. A network of ubiquitin ligases is important for the dynamics of misfolded protein aggregates in yeast.

    PubMed

    Theodoraki, Maria A; Nillegoda, Nadinath B; Saini, Jagdeep; Caplan, Avrom J

    2012-07-06

    Quality control ubiquitin ligases promote degradation of misfolded proteins by the proteasome. If the capacity of the ubiquitin/proteasome system is exceeded, then misfolded proteins accumulate in aggregates that are cleared by the autophagic system. To identify components of the ubiquitin/proteasome system that protect against aggregation, we analyzed a GFP-tagged protein kinase, Ste11ΔN(K444R)-GFP, in yeast strains deleted for 14 different ubiquitin ligases. We show that deletion of almost all of these ligases affected the proteostatic balance in untreated cells such that Ste11ΔN(K444R)-GFP aggregation was changed significantly compared with the levels found in wild type cells. By contrast, aggregation was increased significantly in only six E3 deletion strains when Ste11ΔN(K444R)-GFP folding was impaired due to inhibition of the molecular chaperone Hsp90 with geldanamycin. The increase in aggregation of Ste11ΔN(K444R)-GFP due to deletion of UBR1 and UFD4 was partially suppressed by deletion of UBR2 due to up-regulation of Rpn4, which controls proteasome activity. Deletion of UBR1 in combination with LTN1, UFD4, or DOA10 led to a marked hypersensitivity to azetidine 2-carboxylic acid, suggesting some redundancy in the networks of quality control ubiquitin ligases. Finally, we show that Ubr1 promotes clearance of protein aggregates when the autophagic system is inactivated. These results provide insight into the mechanics by which ubiquitin ligases cooperate and provide feedback regulation in the clearance of misfolded proteins.

  14. SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.

    PubMed Central

    Willems, A R; Goh, T; Taylor, L; Chernushevich, I; Shevchenko, A; Tyers, M

    1999-01-01

    Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis. PMID:10582239

  15. SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.

    PubMed

    Willems, A R; Goh, T; Taylor, L; Chernushevich, I; Shevchenko, A; Tyers, M

    1999-09-29

    Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.

  16. Biotin protein ligase from Corynebacterium glutamicum: role for growth and L: -lysine production.

    PubMed

    Peters-Wendisch, P; Stansen, K C; Götker, S; Wendisch, V F

    2012-03-01

    Corynebacterium glutamicum is a biotin auxotrophic Gram-positive bacterium that is used for large-scale production of amino acids, especially of L-glutamate and L-lysine. It is known that biotin limitation triggers L-glutamate production and that L-lysine production can be increased by enhancing the activity of pyruvate carboxylase, one of two biotin-dependent proteins of C. glutamicum. The gene cg0814 (accession number YP_225000) has been annotated to code for putative biotin protein ligase BirA, but the protein has not yet been characterized. A discontinuous enzyme assay of biotin protein ligase activity was established using a 105aa peptide corresponding to the carboxyterminus of the biotin carboxylase/biotin carboxyl carrier protein subunit AccBC of the acetyl CoA carboxylase from C. glutamicum as acceptor substrate. Biotinylation of this biotin acceptor peptide was revealed with crude extracts of a strain overexpressing the birA gene and was shown to be ATP dependent. Thus, birA from C. glutamicum codes for a functional biotin protein ligase (EC 6.3.4.15). The gene birA from C. glutamicum was overexpressed and the transcriptome was compared with the control strain revealing no significant gene expression changes of the bio-genes. However, biotin protein ligase overproduction increased the level of the biotin-containing protein pyruvate carboxylase and entailed a significant growth advantage in glucose minimal medium. Moreover, birA overexpression resulted in a twofold higher L-lysine yield on glucose as compared with the control strain.

  17. Reconciling Ligase Ribozyme Activity with Fatty Acid Vesicle Stability

    PubMed Central

    Anella, Fabrizio; Danelon, Christophe

    2014-01-01

    The “RNA world” and the “Lipid world” theories for the origin of cellular life are often considered incompatible due to the differences in the environmental conditions at which they can emerge. One obstacle resides in the conflicting requirements for divalent metal ions, in particular Mg2+, with respect to optimal ribozyme activity, fatty acid vesicle stability and protection against RNA strand cleavage. Here, we report on the activity of a short L1 ligase ribozyme in the presence of myristoleic acid (MA) vesicles at varying concentrations of Mg2+. The ligation rate is significantly lower at low-Mg2+ conditions. However, the loss of activity is overcompensated by the increased stability of RNA leading to a larger amount of intact ligated substrate after long reaction periods. Combining RNA ligation assays with fatty acid vesicles we found that MA vesicles made of 5 mM amphiphile are stable and do not impair ligase ribozyme activity in the presence of approximately 2 mM Mg2+. These results provide a scenario in which catalytic RNA and primordial membrane assembly can coexist in the same environment. PMID:25513761

  18. The Streptomyces coelicolor lipoate-protein ligase is a circularly permuted version of the Escherichia coli enzyme composed of discrete interacting domains.

    PubMed

    Cao, Xinyun; Cronan, John E

    2015-03-13

    Lipoate-protein ligases are used to scavenge lipoic acid from the environment and attach the coenzyme to its cognate proteins, which are generally the E2 components of the 2-oxoacid dehydrogenases. The enzymes use ATP to activate lipoate to its adenylate, lipoyl-AMP, which remains tightly bound in the active site. This mixed anhydride is attacked by the ϵ-amino group of a specific lysine present on a highly conserved acceptor protein domain, resulting in the amide-linked coenzyme. The Streptomyces coelicolor genome encodes only a single putative lipoate ligase. However, this protein had only low sequence identity (<25%) to the lipoate ligases of demonstrated activity and appears to be a circularly permuted version of the known lipoate ligase proteins in that the canonical C-terminal domain seems to have been transposed to the N terminus. We tested the activity of this protein both by in vivo complementation of an Escherichia coli ligase-deficient strain and by in vitro assays. Moreover, when the domains were rearranged into a protein that mimicked the arrangement found in the canonical lipoate ligases, the enzyme retained complementation activity. Finally, when the two domains were separated into two proteins, both domain-containing proteins were required for complementation and catalysis of the overall ligase reaction in vitro. However, only the large domain-containing protein was required for transfer of lipoate from the lipoyl-AMP intermediate to the acceptor proteins, whereas both domain-containing proteins were required to form lipoyl-AMP.

  19. Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase

    PubMed Central

    Puthenveetil, Sujiet; Liu, Daniel S.; White, Katharine A.; Thompson, Samuel; Ting, Alice Y.

    2009-01-01

    E. coli lipoic acid ligase (LplA) catalyzes ATP-dependent covalent ligation of lipoic acid onto specific lysine sidechains of three acceptor proteins involved in oxidative metabolism. Our lab has shown that LplA and engineered mutants can ligate useful small-molecule probes such as alkyl azides (Nat. Biotechnol. 2007, 25, 1483–1487) and photocrosslinkers (Angew. Chem Int. Ed Engl. 2008, 47, 7018–7021) in place of lipoic acid, facilitating imaging and proteomic studies. Both to further our understanding of lipoic acid metabolism, and to improve LplA’s utility as a biotechnological platform, we have engineered a novel 13-amino acid peptide substrate for LplA. LplA’s natural protein substrates have a conserved β-hairpin structure, a conformation that is difficult to recapitulate in a peptide, and thus we performed in vitro evolution to engineer the LplA peptide substrate, called “LplA Acceptor Peptide” (LAP). A ~107 library of LAP variants was displayed on the surface of yeast cells, labeled by LplA with either lipoic acid or bromoalkanoic acid, and the most efficiently labeled LAP clones were isolated by fluorescence activated cell sorting. Four rounds of evolution followed by additional rational mutagenesis produced a “LAP2” sequence with a kcat/Km of 0.99 μM−1min−1, >70-fold better than our previous rationally-designed 22-amino acid LAP1 sequence (Nat. Biotechnol. 2007, 25, 1483–1487), and only 8-fold worse than the kcat/Km values of natural lipoate and biotin acceptor proteins. The kinetic improvement over LAP1 allowed us to rapidly label cell surface peptide-fused receptors with quantum dots. PMID:19863063

  20. DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories.

    PubMed

    Vago, Riccardo; Leva, Valentina; Biamonti, Giuseppe; Montecucco, Alessandra

    2009-08-15

    DNA ligase I is the main DNA ligase activity involved in eukaryotic DNA replication acting in the joining of Okazaki fragments. This enzyme is also implicated in nucleotide excision repair and in the long-patch base excision repair while its role in the recombinational repair pathways is poorly understood. DNA ligase I is phosphorylated during cell cycle at several serine and threonine residues that regulate its participation in different DNA transactions by modulating the interaction with different protein partners. Here we use an antibody-based array method to identify novel DNA ligase-interacting partners. We show that DNA ligase I participates in several multiprotein complexes with proteins involved in DNA replication and repair, cell cycle control, and protein modification. In particular we demonstrate that DNA ligase I complexes with Nbs1, a core component of the MRN complex critical for detection, processing and repair of double-stranded DNA breaks. The analysis of epitope tagged DNA ligase I mutants demonstrates that the association is mediated by the catalytic fragment of the enzyme. DNA ligase I and Nbs1 colocalize at replication factories during unperturbed replication and after treatment with DNA damaging agents. Since MRN complex is involved in the repair of double-stranded DNA breaks by homologous recombination at stalled replication forks our data support the notion that DNA ligase I participates in homology dependent pathways that deal with replication-associated lesions generated when replication fork encounters DNA damage.

  1. DNA ligases.

    PubMed

    Tabor, S

    2001-05-01

    DNA ligases catalyze the formation of phosphodiester bonds between juxtaposed 5' phosphate and a 3'-hydroxyl terminus in duplex DNA. This activity can repair single-stranded nicks in duplex DNA and join duplex DNA restriction fragments having either blunt ends or homologous cohesive ends. Two ligases are used for nucleic acid research and their reaction conditions and applications are described in this unit: E. coli ligase and T4 ligase. These enzymes differ in two important properties. One is the source of energy: T4 ligase uses ATP, while E. coli ligase uses NAD. Another important difference is their ability to ligate blunt ends; under normal reaction conditions, only T4 DNA ligase will ligate blunt ends.

  2. Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

    NASA Technical Reports Server (NTRS)

    Lecker, Stewart H.; Goldberg, A. L. (Principal Investigator)

    2003-01-01

    PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

  3. SUMO E3 ligase activity of TRIM proteins.

    PubMed

    Chu, Y; Yang, X

    2011-03-03

    SUMOylation governs numerous cellular processes and is essential to most eukaryotic life. Despite increasing recognition of the importance of this process, an extremely limited number of small ubiquitin-like modifier (SUMO) protein ligases (E3s) have been identified. Here we show that at least some members of the functionally diverse tripartite motif (TRIM) superfamily are SUMO E3s. These TRIM proteins bind both the SUMO-conjugating enzyme Ubc9 and substrates and strongly enhance transfer of SUMOs from Ubc9 to these substrates. Among the substrates of TRIM SUMO E3s are the tumor suppressor p53 and its principal antagonist Mdm2. The E3 activity depends on the TRIM motif, suggesting it to be the first widespread SUMO E3 motif. Given the large number of TRIM proteins, our results may greatly expand the identified SUMO E3s. Furthermore, TRIM E3 activity may be an important contributor to SUMOylation specificity and the versatile functions of TRIM proteins.

  4. Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci.

    PubMed Central

    Greene, M; Firshein, W

    1976-01-01

    Deoxyribonucleic acid (DNA) ligase has been detected in a DNA membrane fraction extracted from Pneumococcus. The specific activity of the enzyme in this fraction is 10-fold greater than in the remaining cell extract. It remains firmly bound (with other enzymes) to the complex after a purification procedure in which a considerable percentage of the macromolecules are dissociated. The ligase acts in two ways in the DNA membrane fraction in vitro. One, it catalyzes the linkage of small-molecular-weight pieces of newly synthesized DNA into heavier-molecular-weight DNA strands as shown by others (M Gellert, 1976; R. Okazaki, A. Sugino, S. Hirose, T. Okazaki, Y. Imae, R. Kainuma-Kuroda, T. Ogawa, M. Arisawa, and Y. Kurosowa, 1973; B. Olivera and I. Lehman, 14; and A. Sugino, S. Hirose, and R. Okazaki, 1972) and, two, it protects DNA from degradation by deoxyribonucleases. This latter effect is due to a competition between the ability of the nucleases to degrade DNA and the ability of DNA ligase to seal the nicks produced by these degradative enzymes. The ligase acts cooperatively with other enzymes in the DNA membrane fraction to synthesize DNA. PMID:4433

  5. From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase.

    PubMed

    Tanabe, Maiko; Ishino, Yoshizumi; Nishida, Hirokazu

    2015-01-01

    DNA ligases are indispensable in all living cells and ubiquitous in all organs. DNA ligases are broadly utilized in molecular biology research fields, such as genetic engineering and DNA sequencing technologies. Here we review the utilization of DNA ligases in a variety of in vitro gene manipulations, developed over the past several decades. During this period, fewer protein engineering attempts for DNA ligases have been made, as compared to those for DNA polymerases. We summarize the recent progress in the elucidation of the DNA ligation mechanisms obtained from the tertiary structures solved thus far, in each step of the ligation reaction scheme. We also present some examples of engineered DNA ligases, developed from the viewpoint of their three-dimensional structures.

  6. From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase

    PubMed Central

    Tanabe, Maiko; Ishino, Yoshizumi; Nishida, Hirokazu

    2015-01-01

    DNA ligases are indispensable in all living cells and ubiquitous in all organs. DNA ligases are broadly utilized in molecular biology research fields, such as genetic engineering and DNA sequencing technologies. Here we review the utilization of DNA ligases in a variety of in vitro gene manipulations, developed over the past several decades. During this period, fewer protein engineering attempts for DNA ligases have been made, as compared to those for DNA polymerases. We summarize the recent progress in the elucidation of the DNA ligation mechanisms obtained from the tertiary structures solved thus far, in each step of the ligation reaction scheme. We also present some examples of engineered DNA ligases, developed from the viewpoint of their three-dimensional structures. PMID:26508902

  7. Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin

    PubMed Central

    Howarth, Mark; Ting, Alice Y

    2009-01-01

    This protocol describes a simple and efficient way to label specific cell surface proteins with biophysical probes on mammalian cells. Cell surface proteins tagged with a 15-amino acid peptide are biotinylated by Escherichia coli biotin ligase (BirA), whereas endogenous proteins are not modified. The biotin group then allows sensitive and stable binding by streptavidin conjugates. This protocol describes the optimal use of BirA and streptavidin for site-specific labeling and also how to produce BirA and monovalent streptavidin. Streptavidin is tetravalent and the cross-linking of biotinylated targets disrupts many of streptavidin’s applications. Monovalent streptavidin has only a single functional biotin-binding site, but retains the femtomolar affinity, low off-rate and high thermostability of wild-type streptavidin. Site-specific biotinylation and streptavidin staining take only a few minutes, while expression of BirA takes 4 d and expression of monovalent streptavidin takes 8 d. PMID:18323822

  8. Successful Conversion of the Bacillus subtilis BirA Group II Biotin Protein Ligase into a Group I Ligase

    PubMed Central

    Henke, Sarah K.; Cronan, John E.

    2014-01-01

    Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that allows transcriptional regulation of biotin biosynthetic and transport genes whereas Group I BPLs lack this N-terminal domain. The Bacillus subtilis BPL, BirA, is classified as a Group II BPL based on sequence predictions of an N-terminal helix-turn-helix motif and mutational alteration of its regulatory properties. We report evidence that B. subtilis BirA is a Group II BPL that regulates transcription at three genomic sites: bioWAFDBI, yuiG and yhfUTS. Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function. This is the first example of successful conversion of a Group II BPL to a Group I BPL with retention of full ligase activity. PMID:24816803

  9. Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase.

    PubMed

    Lamas-Maceiras, Mónica; Vaca, Inmaculada; Rodríguez, Esther; Casqueiro, Javier; Martín, Juan F

    2006-04-01

    A gene, phl, encoding a phenylacetyl-CoA ligase was cloned from a phage library of Penicillium chrysogenum AS-P-78. The presence of five introns in the phl gene was confirmed by reverse transcriptase-PCR. The phl gene encoded an aryl-CoA ligase closely related to Arabidopsis thaliana 4-coumaroyl-CoA ligase. The Phl protein contained most of the amino acids defining the aryl-CoA (4-coumaroyl-CoA) ligase substrate-specificity code and differed from acetyl-CoA ligase and other acyl-CoA ligases. The phl gene was not linked to the penicillin gene cluster. Amplification of phl in an autonomous replicating plasmid led to an 8-fold increase in phenylacetyl-CoA ligase activity and a 35% increase in penicillin production. Transformants containing the amplified phl gene were resistant to high concentrations of phenylacetic acid (more than 2.5 g/l). Disruption of the phl gene resulted in a 40% decrease in penicillin production and a similar reduction of phenylacetyl-CoA ligase activity. The disrupted mutants were highly susceptible to phenylacetic acid. Complementation of the disrupted mutants with the phl gene restored normal levels of penicillin production and resistance to phenylacetic acid. The phenylacetyl-CoA ligase encoded by the phl gene is therefore involved in penicillin production, although a second aryl-CoA ligase appears to contribute partially to phenylacetic acid activation. The Phl protein lacks a peptide-carrier-protein domain and behaves as an aryl-capping enzyme that activates phenylacetic acid and transfers it to the isopenicillin N acyltransferase. The Phl protein contains the peroxisome-targeting sequence that is also present in the isopenicillin N acyltransferase. The peroxisomal co-localization of these two proteins indicates that the last two enzymes of the penicillin pathway form a peroxisomal functional complex.

  10. E3 ubiquitin ligases and abscisic acid signaling

    PubMed Central

    Liu, Hongxia

    2011-01-01

    The ubiquitin proteasome system is involved in the regulation of nearly every aspect of plant growth and development. Protein ubiquitination involves the covalent attachment of ubiquitin to target proteins through a cascade catalyzed by three enzymes known as E1, E2 and E3. E3s are of particular interest as they confer substrate specificity during ubiquitination through their diverse substrate recognition domains. Recently, a number of E3s have been identified that actively participate in abscisic acid hormone biology, including regulation of biosynthesis, de-repression or activation of abscisic acid response and degradation of signaling components. In this review, we summarize recent exciting studies of the different types of E3s that target specific mediators of abscisic acid signaling or affect the plants response to the hormone. PMID:21364320

  11. Phage display evolution of a peptide substrate for yeast biotin ligase and application to two-color quantum dot labeling of cell surface proteins.

    PubMed

    Chen, Irwin; Choi, Yoon-Aa; Ting, Alice Y

    2007-05-23

    Site-specific protein labeling with Escherichia coli biotin ligase (BirA) has been used to introduce fluorophores, quantum dots (QDs), and photocross-linkers onto recombinant proteins fused to a 15-amino acid acceptor peptide (AP) substrate for BirA and expressed on the surface of living mammalian cells. Here, we used phage display to engineer a new and orthogonal biotin ligase-AP pair for site-specific protein labeling. Yeast biotin ligase (yBL) does not recognize the AP, but we discovered a new 15-amino acid substrate for yBL called the yeast acceptor peptide (yAP), using two generations of phage display selection from 15-mer peptide libraries. The yAP is not recognized by BirA, and thus, we were able to specifically label AP and yAP fusion proteins coexpressed in the same cell with differently colored QDs. We fused the yAP to a variety of recombinant proteins and demonstrated biotinylation by yBL at the N-terminus, C-terminus, and within a flexible internal region. yBL is extremely sequence-specific, as endogenous proteins on the surface of yeast and HeLa cells are not biotinylated. This new methodology expands the scope of biotin ligase labeling to two-color imaging and yeast-based applications.

  12. Deubiquitinase FAM/USP9X Interacts with the E3 Ubiquitin Ligase SMURF1 Protein and Protects It from Ligase Activity-dependent Self-degradation

    PubMed Central

    Xie, Yang; Avello, Monika; Schirle, Markus; McWhinnie, Elizabeth; Feng, Yan; Bric-Furlong, Eva; Wilson, Christopher; Nathans, Robin; Zhang, Jing; Kirschner, Marc W.; Huang, Shih-Min A.; Cong, Feng

    2013-01-01

    Ubiquitination is an essential post-translational modification that mediates diverse cellular functions. SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) belongs to the Nedd4 family of HECT ubiquitin ligases that directly catalyzes ubiquitin conjugation onto diverse substrates. As a result, SMURF1 regulates a great variety of cellular physiologies including bone morphogenetic protein (BMP) signaling, cell migration, and planar cell polarity. Structurally, SMURF1 consists of a C2 domain, two WW domain repeats, and a catalytic HECT domain essential for its E3 ubiquitin ligase activity. This modular architecture allows for interactions with other proteins, which are either substrates or adaptors of SMURF1. Despite the increasing number of SMURF1 substrates identified, current knowledge regarding regulatory proteins and their modes of action on controlling SMURF1 activity is still limited. In this study, we employed quantitative mass spectrometry to analyze SMURF1-associated cellular complexes, and identified the deubiquitinase FAM/USP9X as a novel interacting protein for SMURF1. Through domain mapping study, we found the second WW domain of SMURF1 and the carboxyl terminus of USP9X critical for this interaction. SMURF1 is autoubiquitinated through its intrinsic HECT E3 ligase activity, and is degraded by the proteasome. USP9X association antagonizes this activity, resulting in deubiquitination and stabilization of SMURF1. In MDA-MB-231 breast cancer cells, SMURF1 expression is elevated and is required for cellular motility. USP9X stabilizes endogenous SMURF1 in MDA-MB-231 cells. Depletion of USP9X led to down-regulation of SMURF1 and significantly impaired cellular migration. Taken together, our data reveal USP9X as an important regulatory protein of SMURF1 and suggest that the association between deubiquitinase and E3 ligase may serve as a common strategy to control the cellular protein dynamics through modulating E3 ligase stability. PMID:23184937

  13. Cullin-RING ubiquitin ligases in salicylic acid-mediated plant immune signaling

    PubMed Central

    Furniss, James J.; Spoel, Steven H.

    2015-01-01

    Plant immune responses against biotrophic pathogens are regulated by the signaling hormone salicylic acid (SA). SA establishes immunity by regulating a variety of cellular processes, including programmed cell death (PCD) to isolate and kill invading pathogens, and development of systemic acquired resistance (SAR) which provides long-lasting, broad-spectrum resistance throughout the plant. Central to these processes is post-translational modification of SA-regulated signaling proteins by ubiquitination, i.e., the covalent addition of small ubiquitin proteins. Emerging evidence indicates SA-induced protein ubiquitination is largely orchestrated by Cullin-RING ligases (CRLs), which recruit specific substrates for ubiquitination using interchangeable adaptors. Ligation of ubiquitin chains interlinked at lysine 48 leads to substrate degradation by the 26S proteasome. Here we discuss how CRL-mediated degradation of both nucleotide-binding/leucine-rich repeat domain containing immune receptors and SA-induced transcription regulators are critical for functional PCD and SAR responses, respectively. By placing these recent findings in context of knowledge gained in other eukaryotic model species, we highlight potential alternative roles for processive ubiquitination in regulating the activity of SA-mediated immune responses. PMID:25821454

  14. Protein-protein interactions involved in the recognition of p27 by E3 ubiquitin ligase.

    PubMed Central

    Xu, Kui; Belunis, Charles; Chu, Wei; Weber, David; Podlaski, Frank; Huang, Kuo-Sen; Reed, Steven I; Vassilev, Lyubomir T

    2003-01-01

    The p27(Kip1) protein is a potent cyclin-dependent kinase inhibitor, the level of which is decreased in many common human cancers as a result of enhanced ubiquitin-dependent degradation. The multiprotein complex SCF(Skp2) has been identified as the ubiquitin ligase that targets p27, but the functional interactions within this complex are not well understood. One component, the F-box protein Skp2, binds p27 when the latter is phosphorylated on Thr(187), thus providing substrate specificity for the ligase. Recently, we and others have shown that the small cell cycle regulatory protein Cks1 plays a critical role in p27 ubiquitination by increasing the binding affinity of Skp2 for p27. Here we report the development of a homogeneous time-resolved fluorescence assay that allows the quantification of the molecular interactions between human recombinant Skp2, Cks1 and a p27-derived peptide phosphorylated on Thr(187). Using this assay, we have determined the dissociation constant of the Skp2-Cks1 complex (K(d) 140 +/- 14 nM) and have shown that Skp2 binds phosphorylated p27 peptide with high affinity only in the presence of Cks1 (K(d) 37 +/- 2 nM). Cks1 does not bind directly to the p27 phosphopeptide or to Skp1, which confirms its suggested role as an allosteric effector of Skp2. PMID:12529174

  15. Effective interaction studies for inhibition of DNA ligase protein from Staphylococcus aureus.

    PubMed

    Vijayalakshmi, Periyasamy; Daisy, Pitchai

    2015-02-01

    Staphylococcus aureus has been recognized as an important human pathogen for more than 100 years. It is among the most important causative agent of human infections in the twenty-first century. DNA ligase is the main protein responsible for the replication of S. aureus. In order to control the replication mechanism, DNA ligase is a successive drug target, hence we have chosen this protein for this study. We performed virtual screening using ZINC database for identification of potent inhibitor against DNA ligase. Based on the scoring methods, we have selected best five compounds from the ZINC database. In order to improve the accuracy, selected compounds were subjected into Quantum Polarized Ligand Docking (QPLD) docking, for which the results showed high docking score, compared to glide docking score. QPLD is more accurate as it includes charges in the scoring function, which was not available in the glide docking. Binding energy calculation results also indicated that selected compounds have good binding capacity with the target protein. In addition, these compounds on screening have good absorption, distribution, metabolism, excretion and toxicity property. In this study, we identified few compounds that particularly work against DNA ligase protein, having better interaction phenomenon and it would help further the experimental analysis.

  16. Genetic evidence for an additional function of phage T4 gene 32 protein: interaction with ligase.

    PubMed

    Mosig, G; Breschkin, A M

    1975-04-01

    Gene 32 of bacteriophage T4 is essential for DNA replication, recombination, and repair. In an attempt to clarify the role of the corresponding gene product, we have looked for mutations that specifically inactivate one but not all of its functions and for compensating suppressor mutations in other genes. Here we describe a gene 32 ts mutant that does not produce progeny, but in contrast to an am mutant investigated by others, is capable of some primary and secondary DNA replication and of forming "joint" recombinational intermediates after infection of Escherichia coli B at the restrictive temperature. However, parental and progeny DNA strands are not ligated to covalently linked "recombinant" molecules, and single strands of vegetative DNA do not exceed unit length. Progeny production as well as capacity for covalent linkage in this gene 32 ts mutant are partially restored by additional rII mutations. Suppression by rII depends on functioning host ligase [EC 6.5.1.2; poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming, NMN-forming)]. This gene 32 ts mutation (unlike some others) in turn suppresses the characteristic plaque morphology of rII mutants. We conclude that gene 32 protein, in addition to its role in DNA replication and in the formation of "joint" recombinational intermediates, interacts with T4 ligase [EC 6.5.1.1; poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming)] when recombining DNA strands are covalently linked. The protein of the mutant that we describe here is mainly defective in this interaction, thus inactivating T4 ligase in recombination. Suppressing rII mutations facilitate substitution of host ligase. There is suggestive evidence that these interactions occur at the membrane.

  17. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells

    PubMed Central

    Kim, Dae In; Raida, Manfred; Burke, Brian

    2012-01-01

    We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment. PMID:22412018

  18. Inactivation of SAG E3 ubiquitin ligase blocks embryonic stem cell differentiation and sensitizes leukemia cells to retinoid acid.

    PubMed

    Tan, Mingjia; Li, Yun; Yang, Ruiguo; Xi, Ning; Sun, Yi

    2011-01-01

    Sensitive to Apoptosis Gene (SAG), also known as RBX2 (RING box protein-2), is the RING component of SCF (SKP1, Cullin, and F-box protein) E3 ubiquitin ligase. Our previous studies have demonstrated that SAG is an anti-apoptotic protein and an attractive anti-cancer target. We also found recently that Sag knockout sensitized mouse embryonic stem cells (mES) to radiation and blocked mES cells to undergo endothelial differentiation. Here, we reported that compared to wild-type mES cells, the Sag(-/-) mES cells were much more sensitive to all-trans retinoic acid (RA)-induced suppression of cell proliferation and survival. While wild-type mES cells underwent differentiation upon exposure to RA, Sag(-/-) mES cells were induced to death via apoptosis instead. The cell fate change, reflected by cellular stiffness, can be detected as early as 12 hrs post RA exposure by AFM (Atomic Force Microscopy). We then extended this novel finding to RA differentiation therapy of leukemia, in which the resistance often develops, by testing our hypothesis that SAG inhibition would sensitize leukemia to RA. Indeed, we found a direct correlation between SAG overexpression and RA resistance in multiple leukemia lines. By using MLN4924, a small molecule inhibitor of NEDD8-Activating Enzyme (NAE), that inactivates SAG-SCF E3 ligase by blocking cullin neddylation, we were able to sensitize two otherwise resistant leukemia cell lines, HL-60 and KG-1 to RA. Mechanistically, RA sensitization by MLN4924 was mediated via enhanced apoptosis, likely through accumulation of pro-apoptotic proteins NOXA and c-JUN, two well-known substrates of SAG-SCF E3 ligase. Taken together, our study provides the proof-of-concept evidence for effective treatment of leukemia patients by RA-MLN4924 combination.

  19. Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins.

    PubMed

    Crowder, Justin J; Geigges, Marco; Gibson, Ryan T; Fults, Eric S; Buchanan, Bryce W; Sachs, Nadine; Schink, Andrea; Kreft, Stefan G; Rubenstein, Eric M

    2015-07-24

    Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3'-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane.

  20. Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins*

    PubMed Central

    Crowder, Justin J.; Geigges, Marco; Gibson, Ryan T.; Fults, Eric S.; Buchanan, Bryce W.; Sachs, Nadine; Schink, Andrea; Kreft, Stefan G.; Rubenstein, Eric M.

    2015-01-01

    Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3′-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane. PMID:26055716

  1. Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

    PubMed Central

    Xie, Chuan-Ming; Wei, Wenyi; Sun, Yi

    2013-01-01

    Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainly controlled by the ubiquitin—proteasome system (UPS), which consists of two distinct steps: (1) ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and (2) subsequent degradation by the 26S proteasome. Among all E3 ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such as cancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore, altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis. PMID:23522382

  2. RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

    SciTech Connect

    Sheren, Jamie E.; Kassenbrock, C. Kenneth

    2013-11-01

    Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

  3. Ubiquitin protein ligase Nedd4 binds to connexin43 by a phosphorylation-modulated process.

    PubMed

    Leykauf, Kerstin; Salek, Mojibrahman; Bomke, Jörg; Frech, Matthias; Lehmann, Wolf-Dieter; Dürst, Matthias; Alonso, Angel

    2006-09-01

    Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization.

  4. The Arabidopsis MIEL1 E3 ligase negatively regulates ABA signalling by promoting protein turnover of MYB96

    PubMed Central

    Lee, Hong Gil; Seo, Pil Joon

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant responses to various environmental challenges. Controlled protein turnover is an important component of ABA signalling. Here we show that the RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (MIEL1) regulates ABA sensitivity by promoting MYB96 turnover in Arabidopsis. Germination of MIEL1-deficient mutant seeds is hypersensitive to ABA, whereas MIEL1-overexpressing transgenic seeds are less sensitive. MIEL1 can interact with MYB96, a regulator of ABA signalling, and stimulate its ubiquitination and degradation. Genetic analysis shows that MYB96 is epistatic to MIEL1 in the control of ABA sensitivity in seeds. While MIEL1 acts primarily via MYB96 in seed germination, MIEL1 regulates protein turnover of both MYB96 and MYB30 in vegetative tissues. We find that ABA regulates the expression of MYB30-responsive genes during pathogen infection and this regulation is partly dependent on MIEL1. These results suggest that MIEL1 may facilitate crosstalk between ABA and biotic stress signalling. PMID:27615387

  5. The ETS protein MEF is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCFSkp2.

    PubMed

    Liu, Yan; Hedvat, Cyrus V; Mao, Shifeng; Zhu, Xin-Hua; Yao, Jinjuan; Nguyen, Hoang; Koff, Andrew; Nimer, Stephen D

    2006-04-01

    MEF is an ETS-related transcription factor with strong transcriptional activating activity that affects hematopoietic stem cell behavior and is required for normal NK cell and NK T-cell development. The MEF (also known as ELF4) gene is repressed by several leukemia-associated fusion transcription factor proteins (PML-retinoic acid receptor alpha and AML1-ETO), but it is also activated by retroviral insertion in several cancer models. We have previously shown that cyclin A-dependent phosphorylation of MEF largely restricts its activity to the G(1) phase of the cell cycle; we now show that MEF is a short-lived protein whose expression level also peaks during late G(1) phase. Mutagenesis studies show that the rapid turnover of MEF in S phase is dependent on the specific phosphorylation of threonine 643 and serine 648 at the C terminus of MEF by cdk2 and on the Skp1/Cul1/F-box (SCF) E3 ubiquitin ligase complex SCF(Skp2), which targets MEF for ubiquitination and proteolysis. Overexpression of MEF drives cells through the G(1)/S transition, thereby promoting cell proliferation. The tight regulation of MEF levels during the cell cycle contributes to its effects on regulating cell cycle entry and cell proliferation.

  6. Functional analysis of the mammalian RNA ligase for IRE1 in the unfolded protein response.

    PubMed

    Poothong, Juthakorn; Tirasophon, Witoon; Kaufman, Randal J

    2017-04-30

    The unfolded protein response (UPR) is a conserved signalling pathway activated on the accumulation of unfolded proteins within the endoplasmic reticulum (ER), termed ER stress. Upon ER stress, HAC1/XBP1 undergoes exon/intron-specific excision by inositol requiring enzyme 1 (IRE1) to remove an intron and liberate the 5' and 3' exons. In yeast, the 5' and 3' HAC1 exons are subsequently ligated by tRNA ligase (Rlg1p), whereas XBP1 ligation in mammalian cells is catalysed by a recently identified ligase, RtcB. In the present study, RNA ligase activity of the human RtcB (hRtcB) involved in the unconventional splicing of XBP1/HAC1 mRNA was explored in an rlg1-100 mutant yeast strain. Distinct from Escherichia coli RtcB and Rlg1p, expression of hRtcB alone inefficiently complemented HAC1/XBP1 splicing and the hRtcB cofactor (archease) was required to promote enzymatic activity of hRtcB to catalyse RNA ligation.

  7. Structure and Function of the First Full-Length Murein Peptide Ligase (Mpl) Cell Wall Recycling Protein

    PubMed Central

    Das, Debanu; Hervé, Mireille; Feuerhelm, Julie; Farr, Carol L.; Chiu, Hsiu-Ju; Elsliger, Marc-André; Knuth, Mark W.; Klock, Heath E.; Miller, Mitchell D.; Godzik, Adam; Lesley, Scott A.; Deacon, Ashley M.; Mengin-Lecreulx, Dominique; Wilson, Ian A.

    2011-01-01

    Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In Gram-negative bacteria, ∼30–60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships. PMID:21445265

  8. RING finger ubiquitin-protein isopeptide ligase Nrdp1/FLRF regulates parkin stability and activity.

    PubMed

    Zhong, Ling; Tan, Ying; Zhou, An; Yu, Qingming; Zhou, Jianhua

    2005-03-11

    Parkin is a ubiquitin-protein isopeptide ligase. It has been suggested that loss of function in parkin causes accumulation and aggregation of its substrates, leading to death of dopaminergic neurons in Parkinson disease. Using the yeast two-hybrid screen, we isolated a RING finger protein that interacted with the N terminus of parkin in a Drosophila cDNA library. Interaction between human parkin and the mammalian RING finger protein homologue Nrdp1/FLRF, a ubiquitin-protein isopeptide ligase that ubiquitinates ErbB3 and ErbB4, was validated by in vitro binding assay, co-immunoprecipitation, and immunofluorescence co-localization. Significantly, pulse-chase experiments showed that cotransfection of Nrdp1 and parkin reduced the half-life of parkin from 5 to 2.5 h. Consistent with these findings, we further observed that degradation of CDCrel-1, a parkin substrate, was facilitated by overexpression of parkin protein. However, co-transfection of Nrdp1 with parkin reversed the effects of parkin on CDCrel-1 degradation. We conclude that Nrdp1 is a parkin modifier that accelerates degradation of parkin, resulting in a reduction of parkin activity.

  9. Chlorovirus Skp1-Binding Ankyrin Repeat Protein Interplay and Mimicry of Cellular Ubiquitin Ligase Machinery

    PubMed Central

    Noel, Eric A.; Kang, Ming; Adamec, Jiri; Oyler, George A.

    2014-01-01

    ABSTRACT The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins. IMPORTANCE Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their

  10. 4-Coumaroyl and caffeoyl shikimic acids inhibit 4-coumaric acid:coenzyme A ligases and modulate metabolic flux for 3-hydroxylation in monolignol biosynthesis of Populus trichocarpa.

    PubMed

    Lin, Chien-Yuan; Wang, Jack P; Li, Quanzi; Chen, Hsi-Chuan; Liu, Jie; Loziuk, Philip; Song, Jina; Williams, Cranos; Muddiman, David C; Sederoff, Ronald R; Chiang, Vincent L

    2015-01-01

    Downregulation of 4-coumaric acid:coenzyme A ligase (4CL) can reduce lignin content in a number of plant species. In lignin precursor (monolignol) biosynthesis during stem wood formation in Populus trichocarpa, two enzymes, Ptr4CL3 and Ptr4CL5, catalyze the coenzyme A (CoA) ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. CoA ligation of 4-coumaric acid is essential for the 3-hydroxylation of 4-coumaroyl shikimic acid. This hydroxylation results from sequential reactions of 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) and 4-coumaric acid 3-hydroxylase 3 (PtrC3H3). Alternatively, 3-hydroxylation of 4-coumaric acid to caffeic acid may occur through an enzyme complex of cinnamic acid 4-hydroxylase 1 and 2 (PtrC4H1 and PtrC4H2) and PtrC3H3. We found that 4-coumaroyl and caffeoyl shikimic acids are inhibitors of Ptr4CL3 and Ptr4CL5. 4-Coumaroyl shikimic acid strongly inhibits the formation of 4-coumaroyl-CoA and caffeoyl-CoA. Caffeoyl shikimic acid inhibits only the formation of 4-coumaroyl-CoA. 4-Coumaroyl and caffeoyl shikimic acids both act as competitive and uncompetitive inhibitors. Metabolic flux in wild-type and PtrC3H3 downregulated P. trichocarpa transgenics has been estimated by absolute protein and metabolite quantification based on liquid chromatography-tandem mass spectrometry, mass action kinetics, and inhibition equations. Inhibition by 4-coumaroyl and caffeoyl shikimic acids may play significant regulatory roles when these inhibitors accumulate.

  11. Distinct and overlapping functions of the cullin E3 ligase scaffolding proteins CUL4A and CUL4B

    PubMed Central

    Hannah, Jeffrey

    2016-01-01

    The cullin 4 subfamily of genes includes CUL4A and CUL4B, which share a mostly identical amino acid sequence aside from the elongated N-terminal region in CUL4B. Both act as scaffolding proteins for modular cullin RING ligase 4 (CRL4) complexes which promote the ubiquitination of a variety of substrates. CRL4 function is vital to cells as loss of both genes or their shared substrate adaptor protein DDB1 halts proliferation and eventually leads to cell death. Due to their high structural similarity, CUL4A and CUL4B share a substantial overlap in function. However, in some cases, differences in subcellular localization, spatiotemporal expression patterns and stress-inducibility preclude functional compensation. In this review, we highlight the most essential functions of the CUL4 genes in: DNA repair and replication, chromatin-remodeling, cell cycle regulation, embryogenesis, hematopoiesis and spermatogenesis. CUL4 genes are also clinically relevant as dysregulation can contribute to the onset of cancer and CRL4 complexes are often hijacked by certain viruses to promote viral replication and survival. Also, mutations in CUL4B have been implicated in a subset of patients suffering from syndromic X-linked intellectual disability (AKA mental retardation). Interestingly, the antitumor effects of immunomodulatory drugs are caused by their binding to the CRL4CRBN complex and re-directing the E3 ligase towards the Ikaros transcription factors IKZF1 and IKZF3. Because of their influence over key cellular functions and relevance to human disease, CRL4s are considered promising targets for therapeutic intervention. PMID:26344709

  12. Properties of bacteriophage T4 mutants defective in gene 30 (deoxyribonucleic acid ligase) and the rII gene.

    PubMed

    Karam, J D; Barker, B

    1971-02-01

    In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rII double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or inhibited by continued DNA synthesis. Mutations of the rII gene decreased but did not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivity to ultraviolet irradiation.

  13. The Ubiquitin E3 Ligase NOSIP Modulates Protein Phosphatase 2A Activity in Craniofacial Development

    PubMed Central

    Hoffmeister, Meike; Prelle, Carola; Küchler, Philipp; Kovacevic, Igor; Moser, Markus; Müller-Esterl, Werner; Oess, Stefanie

    2014-01-01

    Holoprosencephaly is a common developmental disorder in humans characterised by incomplete brain hemisphere separation and midface anomalies. The etiology of holoprosencephaly is heterogeneous with environmental and genetic causes, but for a majority of holoprosencephaly cases the genes associated with the pathogenesis could not be identified so far. Here we report the generation of knockout mice for the ubiquitin E3 ligase NOSIP. The loss of NOSIP in mice causes holoprosencephaly and facial anomalies including cleft lip/palate, cyclopia and facial midline clefting. By a mass spectrometry based protein interaction screen we identified NOSIP as a novel interaction partner of protein phosphatase PP2A. NOSIP mediates the monoubiquitination of the PP2A catalytic subunit and the loss of NOSIP results in an increase in PP2A activity in craniofacial tissue in NOSIP knockout mice. We conclude, that NOSIP is a critical modulator of brain and craniofacial development in mice and a candidate gene for holoprosencephaly in humans. PMID:25546391

  14. Role of a ribosome-associated E3 ubiquitin ligase in protein quality control.

    PubMed

    Bengtson, Mario H; Joazeiro, Claudio A P

    2010-09-23

    Messenger RNA lacking stop codons ('non-stop mRNA') can arise from errors in gene expression, and encode aberrant proteins whose accumulation could be deleterious to cellular function. In bacteria, these 'non-stop proteins' become co-translationally tagged with a peptide encoded by ssrA/tmRNA (transfer-messenger RNA), which signals their degradation by energy-dependent proteases. How eukaryotic cells eliminate non-stop proteins has remained unknown. Here we show that the Saccharomyces cerevisiae Ltn1 RING-domain-type E3 ubiquitin ligase acts in the quality control of non-stop proteins, in a process that is mechanistically distinct but conceptually analogous to that performed by ssrA: Ltn1 is predominantly associated with ribosomes, and it marks nascent non-stop proteins with ubiquitin to signal their proteasomal degradation. Ltn1-mediated ubiquitylation of non-stop proteins seems to be triggered by their stalling in ribosomes on translation through the poly(A) tail. The biological relevance of this process is underscored by the finding that loss of Ltn1 function confers sensitivity to stress caused by increased non-stop protein production. We speculate that defective protein quality control may underlie the neurodegenerative phenotype that results from mutation of the mouse Ltn1 homologue Listerin.

  15. Global conformational change associated with the two-step reaction catalyzed by Escherichia coli lipoate-protein ligase A.

    PubMed

    Fujiwara, Kazuko; Maita, Nobuo; Hosaka, Harumi; Okamura-Ikeda, Kazuko; Nakagawa, Atsushi; Taniguchi, Hisaaki

    2010-03-26

    Lipoate-protein ligase A (LplA) catalyzes the attachment of lipoic acid to lipoate-dependent enzymes by a two-step reaction: first the lipoate adenylation reaction and, second, the lipoate transfer reaction. We previously determined the crystal structure of Escherichia coli LplA in its unliganded form and a binary complex with lipoic acid (Fujiwara, K., Toma, S., Okamura-Ikeda, K., Motokawa, Y., Nakagawa, A., and Taniguchi, H. (2005) J Biol. Chem. 280, 33645-33651). Here, we report two new LplA structures, LplA.lipoyl-5'-AMP and LplA.octyl-5'-AMP.apoH-protein complexes, which represent the post-lipoate adenylation intermediate state and the pre-lipoate transfer intermediate state, respectively. These structures demonstrate three large scale conformational changes upon completion of the lipoate adenylation reaction: movements of the adenylate-binding and lipoate-binding loops to maintain the lipoyl-5'-AMP reaction intermediate and rotation of the C-terminal domain by about 180 degrees . These changes are prerequisites for LplA to accommodate apoprotein for the second reaction. The Lys(133) residue plays essential roles in both lipoate adenylation and lipoate transfer reactions. Based on structural and kinetic data, we propose a reaction mechanism driven by conformational changes.

  16. The E3 Ubiquitin Ligase TMEM129 Is a Tri-Spanning Transmembrane Protein

    PubMed Central

    van de Weijer, Michael L.; van Muijlwijk, Guus H.; Visser, Linda J.; Costa, Ana I.; Wiertz, Emmanuel J. H. J.; Lebbink, Robert Jan

    2016-01-01

    Misfolded proteins from the endoplasmic reticulum (ER) are transported back into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 hijacks this ER-associated protein degradation (ERAD) pathway to downregulate human leukocyte antigen (HLA) class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. Recently, we identified the E3 ubiquitin ligase transmembrane protein 129 (TMEM129) as a key player in this process, where interference with TMEM129 activity in human cells completely abrogates US11-mediated class I degradation. Here, we set out to further characterize TMEM129. We show that TMEM129 is a non-glycosylated protein containing a non-cleaved signal anchor sequence. By glycosylation scanning mutagenesis, we show that TMEM129 is a tri-spanning ER-membrane protein that adopts an Nexo–Ccyto orientation. This insertion in the ER membrane positions the C-terminal really interesting new gene (RING) domain of TMEM129 in the cytosol, making it available to catalyze ubiquitination reactions that are required for cytosolic degradation of secretory proteins. PMID:27854284

  17. Mitochondrial E3 Ubiquitin Protein Ligase 1 Mediates Cigarette Smoke-Induced Endothelial Cell Death and Dysfunction.

    PubMed

    Kim, Sun-Yong; Kim, Hyo Jeong; Park, Mi Kyeong; Huh, Jin Won; Park, Hye Yun; Ha, Sang Yun; Shin, Joo-Ho; Lee, Yun-Song

    2016-02-01

    By virtue of the critical roles of Akt in vascular endothelial cell (EC) survival and function, cigarette smoke-induced Akt reduction may contribute to EC death and dysfunction in smokers' lungs. One of the negative Akt regulatory mechanisms is K48-linked Akt ubiquitination and subsequent proteasomal degradation. Here, we assessed the involvement of mitochondrial E3 ubiquitin protein ligase 1 (MUL1), recently revealed as a novel Akt ubiquitin E3 ligase, in cigarette smoke-induced Akt ubiquitination and its contribution to pulmonary EC death and dysfunction. In human lung microvascular ECs (HLMVECs), cigarette smoke extract (CSE) noticeably elevated MUL1 expression and K48-linked Akt ubiquitination, whereas Akt, p-Akt, eNOS, and p-eNOS levels were decreased. MUL1 knockdown suppressed CSE-induced Akt ubiquitination/degradation and cytoplasmic reductions of Akt and p-Akt. Furthermore, MUL1 knockdown attenuated reductions of eNOS and p-eNOS and alleviated EC survival, migration, and tube formation in the presence of CSE exposure. In addition, overexpression of K284R Akt, a mutant for a MUL1-ubiquitination site, produced similar effects. In HLMVECs exposed to CSE, Akt-MUL1 interaction was increased in coimmunoprecipitation and in situ proximity ligation assays. Similarly, the proximity ligation assay signals were elevated in rat lungs exposed to cigarette smoke for 3 months, during which Mul1 levels were noticeably increased. Finally, we found that CSE-mediated MUL1 induction in HLMVECs is mediated by retinoic acid receptor-related orphan receptor α. Taken together, these data suggest that cigarette smoke-induced MUL1 elevation mediates Akt ubiquitination/degradation, potentially leading to pulmonary EC death and functional impairment.

  18. Purification, crystallization and preliminary crystallographic analysis of the biotin–protein ligase from Pyrococcus horikoshii OT3

    SciTech Connect

    Bagautdinov, Bagautdin; Kuroishi, Chizu; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-02-01

    The biotin–protein ligase from P. horikoshii OT3 was overexpressed, purified, crystallized and cocrystallized with biotin, ADP and biotinyl-5′-AMP. The crystals belong to space group P2{sub 1} and diffract to beyond 1.6 Å resolution.

  19. Ubiquitin Ligase Substrate Identification through Quantitative Proteomics at Both the Protein and Peptide Levels

    PubMed Central

    Lee, Kimberly A.; Hammerle, Lisa P.; Andrews, Paul S.; Stokes, Matthew P.; Mustelin, Tomas; Silva, Jeffrey C.; Black, Roy A.; Doedens, John R.

    2011-01-01

    Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His6-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology. PMID:21987572

  20. The E3 ubiquitin ligase Itch controls the protein stability of p63.

    PubMed

    Rossi, Mario; Aqeilan, Rami I; Neale, Michael; Candi, Eleonora; Salomoni, Paolo; Knight, Richard A; Croce, Carlo M; Melino, Gerry

    2006-08-22

    p63, a member of the p53 family of transcription factors, plays an important role in epithelial development, regulating both cell cycle and apoptosis. Even though p63 activity is regulated mainly at the posttranslational level, the control of p63 protein stability is far from being fully understood. Here, we show that the Hect (homologous to the E6-associated protein C terminus)-containing Nedd4-like ubiquitin protein ligase Itch binds, ubiquitylates, and promotes the degradation of p63. The physical interaction occurs at the border between the PY and the SAM (sterile alpha motif) domains; a single Y504F mutation significantly affects p63 degradation. Itch and p63 are coexpressed in the epidermis and in primary keratinocytes where Itch controls the p63 protein steady-state level. Accordingly, p63 protein levels are significantly increased in Itch knockout keratinocytes. These data suggest that Itch has a fundamental role in the mechanism that controls endogenous p63 protein levels and therefore contributes to regulation of p63 in physiological conditions.

  1. Dual roles of F123 in protein homodimerization and inhibitor binding to biotin protein ligase from Staphylococcus aureus.

    PubMed

    Soares da Costa, Tatiana P; Yap, Min Y; Perugini, Matthew A; Wallace, John C; Abell, Andrew D; Wilce, Matthew C J; Polyak, Steven W; Booker, Grant W

    2014-01-01

    Protein biotinylation is catalysed by biotin protein ligase (BPL). The most characterized BPL is from Escherichia coli where it functions as both a biotin ligase and a homodimeric transcriptional repressor. Here we investigated another bifunctional BPL from the clinically important Staphylococcus aureus (SaBPL). Unliganded SaBPL (apo) exists in a dimer-monomer equilibrium at low micromolar concentrations - a stark contrast to E. coli BPL (EcBPL) that is monomeric under the same conditions. EMSA and SAXS analysis demonstrated that dimeric apo SaBPL adopted a conformation that was competent to bind DNA and necessary for it to function as a transcription factor. The SaBPL dimer-monomer dissociation constant was 5.8-fold tighter when binding the inhibitor biotin acetylene, but unchanged with biotin. F123, located in the dimer interface, was critical for homodimerization. Inhibition studies together with surface plasmon resonance analyses revealed a strong correlation between inhibitor potency and slow dissociation kinetics. A 24-fold difference in Ki values for these two enzymes was explained by differences in enzyme:inhibitor dissociation rates. Substitution of F123 in SaBPL and its equivalent in EcBPL altered both inhibitor potency and dissociation. Hence, F123 in SaBPL has novel roles in both protein dimerization and ligand-binding that have not been reported in EcBPL.

  2. E6-AP/UBE3A protein acts as a ubiquitin ligase toward SOX9 protein.

    PubMed

    Hattori, Takako; Kishino, Tetsuya; Stephen, Shelley; Eberspaecher, Heidi; Maki, Sayumi; Takigawa, Masaharu; de Crombrugghe, Benoit; Yasuda, Hideyo

    2013-12-06

    SOX9 is a transcription factor that acts as a key regulator at various stages of cartilage differentiation. There is ample evidence that intracellular SOX9 protein levels are tightly regulated both by sumoylation and by degradation through the ubiquitin-proteasome pathway. Using a proteomics approach, here we report the identification of a SOX9-binding protein, E6-AP/UBE3A, that may act as a ubiquitin ligase toward Sox9. E6-AP bound SOX9 through the region consisting mostly of its high mobility group domain in vitro. In nuclear lysates, FLAG-tagged E6-AP coprecipitated with Sox9 and its high mobility group domain. This finding was estimated using nuclear lysates from a chondrocytic cell line that endogenously expresses E6-AP and SOX9. Accordingly, ectopically expressed E6-AP and SOX9 colocalized in the nucleus. We show that E6-AP ubiquitinates SOX9 in vitro and in vivo and that SOX9 levels are enhanced after addition of the proteasome inhibitor bortezomib. Similar, siRNA knockdown of E6-AP and the E2 ligase Ubc9 increased cellular SOX9 amounts, supporting the notion that SOX9 may be ubiquitinated in hypertrophic chondrocytes by E6-AP and degraded by proteasomes. This is in accordance with the distribution of SOX9 levels, which are high in proliferating and prehypertrophic chondrocytes but low in hypertrophic chondrocytes, whereas E6-AP levels are high in hypertrophic chondrocytes and low in prehypertrophic chondrocytes. Furthermore, E6-AP-deficient mice showed SOX9 accumulation in chondrocytes and the brain. These findings support the concept that E6-AP regulates SOX9 levels in developing cartilage by acting as a ubiquitin ligase.

  3. DNA Ligase I is an In Vivo Substrate of DNA-Dependent Protein Kinase and is Activated by Phosphorylation in Response to DNA Double-Strand Breaks

    DTIC Science & Technology

    2006-01-01

    anlysis. to the procedure described by Malanga and Althaus (8). Gel Electrophoresis and A utoradiography. Immunopre- DNA Ligase and Protein Assays. DNA...by casein kinase 11, EMBO J. 11, 2925-2933. In conclusion, we have demonstrated that DNA ligase I 8. Malanga , M., and Althaus, F. R. (1994) Poly (ADP

  4. The von Hippel-Lindau tumor suppressor protein is a component of an E3 ubiquitin-protein ligase activity.

    PubMed

    Lisztwan, J; Imbert, G; Wirbelauer, C; Gstaiger, M; Krek, W

    1999-07-15

    pVHL, the product of the VHL tumor suppressor gene, plays an important role in the regulation of cell growth and differentiation of human kidney cells, and inactivation of the VHL gene is the most frequent genetic event in human kidney cancer. The biochemical function of pVHL is unknown. Here we report that pVHL exists in vivo in a complex that displays ubiquitination-promoting activity in conjunction with the universally required components E1, E2, and ubiquitin. pVHL-associated ubiquitination activity requires, at a minimum, pVHL to bind elongin C and Cul-2, relatives of core components of SCF (Skp1-Cdc53/Cul-1-F-box protein) E3 ligase complexes. Notably, certain tumor-derived mutants of pVHL demonstrate loss of associated ubiquitination promoting activity. These results identify pVHL as a component of a potential SCF-like E3 ubiquitin-protein ligase complex and suggest a direct link between pVHL tumor suppressor and the process of ubiquitination.

  5. The SCF ubiquitin ligase protein slimb regulates centrosome duplication in Drosophila.

    PubMed

    Wojcik, E J; Glover, D M; Hays, T S

    2000-09-21

    The duplication of the centrosome is a key event in the cell-division cycle. Although defects in centrosome duplication are thought to contribute to genomic instability [1-3] and are a hallmark of certain transformed cells and human cancer [4-6], the mechanism responsible for centrosome duplication is not understood. Recent experiments have established that centrosome duplication requires the activity of cyclin-dependent kinase 2 (Cdk2) and cyclins E and A [7-9]. The stability of cyclin E is regulated by the ubiquitin ligase SCF, which is a protein complex composed of Skp1, Cdc53 (Cullin) and F-box proteins [10-12]. The Skp1 and Cullin components have been detected on mammalian centrosomes, and shown to be essential for centrosome duplication and separation in Xenopus [13]. Here, we report that Slimb, an F-box protein that targets proteins to the SCFcomplex [14,15], plays a role in limiting centrosome replication. We found that, in the fruit fly Drosophila, the hypomorphic mutation slimb(crd) causes the appearance of additional centrosomes and mitotic defects in mutant larval neuroblasts.

  6. Ataxia and hypogonadism caused by the loss of ubiquitin ligase activity of the U box protein CHIP

    PubMed Central

    Shi, Chang-He; Schisler, Jonathan C.; Rubel, Carrie E.; Tan, Song; Song, Bo; McDonough, Holly; Xu, Lei; Portbury, Andrea L.; Mao, Cheng-Yuan; True, Cadence; Wang, Rui-Hao; Wang, Qing-Zhi; Sun, Shi-Lei; Seminara, Stephanie B.; Patterson, Cam; Xu, Yu-Ming

    2014-01-01

    Gordon Holmes syndrome (GHS) is a rare Mendelian neurodegenerative disorder characterized by ataxia and hypogonadism. Recently, it was suggested that disordered ubiquitination underlies GHS though the discovery of exome mutations in the E3 ligase RNF216 and deubiquitinase OTUD4. We performed exome sequencing in a family with two of three siblings afflicted with ataxia and hypogonadism and identified a homozygous mutation in STUB1 (NM_005861) c.737C→T, p.Thr246Met, a gene that encodes the protein CHIP (C-terminus of HSC70-interacting protein). CHIP plays a central role in regulating protein quality control, in part through its ability to function as an E3 ligase. Loss of CHIP function has long been associated with protein misfolding and aggregation in several genetic mouse models of neurodegenerative disorders; however, a role for CHIP in human neurological disease has yet to be identified. Introduction of the Thr246Met mutation into CHIP results in a loss of ubiquitin ligase activity measured directly using recombinant proteins as well as in cell culture models. Loss of CHIP function in mice resulted in behavioral and reproductive impairments that mimic human ataxia and hypogonadism. We conclude that GHS can be caused by a loss-of-function mutation in CHIP. Our findings further highlight the role of disordered ubiquitination and protein quality control in the pathogenesis of neurodegenerative disease and demonstrate the utility of combining whole-exome sequencing with molecular analyses and animal models to define causal disease polymorphisms. PMID:24113144

  7. Functional characterization of SAG/RBX2/ROC2/RNF7, an antioxidant protein and an E3 ubiquitin ligase.

    PubMed

    Sun, Yi; Li, Hua

    2013-02-01

    SAG (Sensitive to Apoptosis Gene), also known as RBX2 (RING box protein 2), ROC2 (Regulator of Cullins 2), or RNF7 (RING Finger Protein 7), was originally cloned in our laboratory as a redox inducible antioxidant protein and later characterized as the second member of the RBX/ROC RING component of the SCF (SKP1-CUL-F-box Proteins) E3 ubiquitin ligase. When acting alone, SAG scavenges oxygen radicals by forming inter- and intra-molecular disulfide bonds, whereas by forming a complex with other components of the SCF E3 ligase, SAG promotes ubiquitination and degradation of a number of protein substrates, including c-JUN, DEPTOR, HIF-1α, IκBα, NF1, NOXA, p27, and procaspase-3, thus regulating various signaling pathways and biological processes. Specifically, SAG protects cells from apoptosis, confers radioresistance, and plays an essential and non-redundant role in mouse embryogenesis and vasculogenesis. Furthermore, stress-inducible SAG is overexpressed in a number of human cancers and SAG overexpression correlates with poor patient prognosis. Finally, SAG transgenic expression in epidermis causes an early stage inhibition, but later stage promotion, of skin tumorigenesis triggered by DMBA/TPA. Given its major role in promoting targeted degradation of tumor suppressive proteins, leading to apoptosis suppression and accelerated tumorigenesis, SAG E3 ligase appears to be an attractive anticancer target.

  8. Salicylic acid-mediated innate immunity in Arabidopsis is regulated by SIZ1 SUMO E3 ligase.

    PubMed

    Lee, Jiyoung; Nam, Jaesung; Park, Hyeong Cheol; Na, Gunnam; Miura, Kenji; Jin, Jing Bo; Yoo, Chan Yul; Baek, Dongwon; Kim, Doh Hoon; Jeong, Jae Cheol; Kim, Donggiun; Lee, Sang Yeol; Salt, David E; Mengiste, Tesfaye; Gong, Qingqiu; Ma, Shisong; Bohnert, Hans J; Kwak, Sang-Soo; Bressan, Ray A; Hasegawa, Paul M; Yun, Dae-Jin

    2007-01-01

    Reversible modifications of target proteins by small ubiquitin-like modifier (SUMO) proteins are involved in many cellular processes in yeast and animals. Yet little is known about the function of sumoylation in plants. Here, we show that the SIZ1 gene, which encodes an Arabidopsis SUMO E3 ligase, regulates innate immunity. Mutant siz1 plants exhibit constitutive systemic-acquired resistance (SAR) characterized by elevated accumulation of salicylic acid (SA), increased expression of pathogenesis-related (PR) genes, and increased resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Transfer of the NahG gene to siz1 plants results in reversal of these phenotypes back to wild-type. Analyses of the double mutants, npr1 siz1, pad4 siz1 and ndr1 siz1 revealed that SIZ1 controls SA signalling. SIZ1 interacts epistatically with PAD4 to regulate PR expression and disease resistance. Consistent with these observations, siz1 plants exhibited enhanced resistance to Pst DC3000 expressing avrRps4, a bacterial avirulence determinant that responds to the EDS1/PAD4-dependent TIR-NBS-type R gene. In contrast, siz1 plants were not resistant to Pst DC3000 expressing avrRpm1, a bacterial avirulence determinant that responds to the NDR1-dependent CC-NBS-type R gene. Jasmonic acid (JA)-induced PDF1.2 expression and susceptibility to Botrytis cinerea were unaltered in siz1 plants. Taken together, these results demonstrate that SIZ1 is required for SA and PAD4-mediated R gene signalling, which in turn confers innate immunity in Arabidopsis.

  9. Regulation of neddylation and deneddylation of cullin1 in SCFSkp2 ubiquitin ligase by F-box protein and substrate

    PubMed Central

    Bornstein, Gil; Ganoth, Dvora; Hershko, Avram

    2006-01-01

    The activity of cullin-containing ubiquitin protein ligase complexes is stimulated by linkage to cullin of the ubiquitin-like protein Nedd8 (“neddylation”). Neddylation is inhibited by the tight binding of cullins to CAND1 (cullin-associated and neddylation-dissociated 1) protein, and Nedd8 is removed from cullins by specific isopeptidase activity of the COP9/signalosome (CSN) complex. The mechanisms that regulate neddylation and deneddylation of cullins were unknown. We examined this problem for the case of SCFSkp2, a cullin1 (Cul1)-containing ubiquitin ligase complex that contains the S phase-associated protein Skp2 as the substrate-binding F-box protein subunit. SCFSkp2 targets for degradation the cyclin-dependent kinase (cdk) inhibitor p27 in the G1-to-S phase transition, a process that requires its phosphorylation and binding to cdk2-cyclin E. Because levels of Skp2, cyclin E, and the accessory protein Cks1 (cyclin kinase subunit 1) all rise at the end of G1 phase, it seemed possible that the neddylation of Cul1 in SCFSkp2 is regulated by the availability of the F-box protein and/or the substrate. We found that the supplementation of Skp2–Skp1 and substrate (along with further components necessary for substrate presentation to the ubiquitin ligase) to extracts of HeLa cells synergistically increased levels of neddylated Cul1. Skp2–Skp1 abrogates the inhibitory influence of CAND1 on the neddylation of Cul1 by promoting the dissociation of the cullin–CAND1 complex, whereas substrate, together with substrate-presenting components, prevents the action of CSN to deneddylate cullin. We propose a sequence of events in which the increased availability of Skp2 and substrate in the transition of cells to S phase promotes the neddylation and assembly of the SCFSkp2 ubiquitin ligase complex. PMID:16861300

  10. Direct recognition of the C-terminal polylysine residues of nonstop protein by Ltn1, an E3 ubiquitin ligase.

    PubMed

    Sung, Kwang Hoon; Song, Hyun Kyu

    2014-10-24

    When mRNAs lack stop codons, errors in gene expression and coding of aberrant proteins that are harmful in cells can result. In Saccharomyces cerevisiae, a 180-kDa E3-ubiquitin ligase, Ltn1 has been known to associate with ribosomes and marks translationally-arrested aberrant nascent polypeptides for proteasomal degradation. Here, we demonstrate the Ltn1 E3-ubiquitin ligase directly binds to the nonstop proteins and efficiently ubiquitylates them. The middle domain of Ltn1 is responsible for recognizing the polylysine residues of the nonstop protein with an affinity of 2-3μM. This biochemical characterization of Ltn1 expands our knowledge regarding the fundamental process that removes aberrant nascent polypeptides in eukaryotes.

  11. Expression and biochemical characterization of Plasmodium falciparum DNA ligase I.

    PubMed

    Buguliskis, Jeffrey S; Casta, Louis J; Butz, Charles E; Matsumoto, Yoshihiro; Taraschi, Theodore F

    2007-10-01

    We report that Plasmodium falciparum (Pf) encodes a 912 amino acid ATP-dependent DNA ligase. Protein sequence analysis of Pf DNA ligase I indicates a strong sequence similarity, particularly in the C-terminal region, to DNA ligase I homologues. The activity of recombinant Pf DNA ligase I (PfLigI) was investigated using protein expressed in HEK293 cells. The PfLigI gene product is approximately 94kDa and catalyzes phosphodiester bond formation on a singly nicked DNA substrate. The enzyme is most active at alkaline pH (8.5) and with Mg(2+) or Mn(2+) and ATP as cofactors. Kinetic studies of PfLigI revealed that the enzyme has similar substrate affinity (K(m) 2.6nM) as compared to human DNA ligase I and k(cat) (2.3x10(-3)s(-1)) and k(cat)/K(m) (8.8x10(5)M(-1)s(-1)) which are similar to other ATP-dependent DNA ligases. PfLigI was able to join RNA-DNA substrates only when the RNA sequence was upstream of the nick, confirming that it is DNA ligase I and has no associated DNA ligase III like activity.

  12. Types of Ubiquitin Ligases.

    PubMed

    Morreale, Francesca Ester; Walden, Helen

    2016-03-24

    Ubiquitination is a post-translational modification of proteins involved in a variety of cellular processes. Ubiquitination requires the sequential action of three enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin ligases). This SnapShot highlights the main types of E3 ubiquitin ligases, which can be classified in three families depending on the presence of characteristic domains and on the mechanism of ubiquitin transfer to the substrate protein.

  13. Regulating ehrlich and demethiolation pathways for alcohols production by the expression of ubiquitin-protein ligase gene HUWE1

    PubMed Central

    Zhang, Quan; Jia, Kai-Zhi; Xia, Shi-Tao; Xu, Yang-Hua; Liu, Rui-Sang; Li, Hong-Mei; Tang, Ya-Jie

    2016-01-01

    Ehrlich and demethiolation pathways as two competing branches converted amino acid into alcohols. Controlling both pathways offers considerable potential for industrial applications including alcohols overproduction, flavor-quality control and developing new flavors. While how to regulate ehrlich and demethiolation pathways is still not applicable. Taking the conversion of methionine into methionol and methanethiol for example, we constructed two suppression subtractive cDNA libraries of Clonostachys rosea by using suppression subtractive hybridization (SSH) technology for screening regulators controlling the conversion. E3 ubiquitin-protein ligase gene HUWE1 screened from forward SSH library was validated to be related with the biosynthesis of end products. Overexpressing HUWE1 in C. rosea and S. cerevisiae significantly increased the biosynthesis of methanethiol and its derivatives in demethiolation pathway, while suppressed the biosynthesis of methional and methionol in ehrlich pathway. These results attained the directional regulation of both pathways by overexpressing HUWE1. Thus, HUWE1 has potential to be a key target for controlling and enhancing alcohols production by metabolic engineering. PMID:26860895

  14. Genome-wide identification and gene expression profiling of ubiquitin ligases for endoplasmic reticulum protein degradation

    PubMed Central

    Kaneko, Masayuki; Iwase, Ikuko; Yamasaki, Yuki; Takai, Tomoko; Wu, Yan; Kanemoto, Soshi; Matsuhisa, Koji; Asada, Rie; Okuma, Yasunobu; Watanabe, Takeshi; Imaizumi, Kazunori; Nomura, Yausyuki

    2016-01-01

    Endoplasmic reticulum (ER)-associated degradation (ERAD) is a mechanism by which unfolded proteins that accumulate in the ER are transported to the cytosol for ubiquitin–proteasome-mediated degradation. Ubiquitin ligases (E3s) are a group of enzymes responsible for substrate selectivity and ubiquitin chain formation. The purpose of this study was to identify novel E3s involved in ERAD. Thirty-seven candidate genes were selected by searches for proteins with RING-finger motifs and transmembrane regions, which are the major features of ERAD E3s. We performed gene expression profiling for the identified E3s in human and mouse tissues. Several genes were specifically or selectively expressed in both tissues; the expression of four genes (RNFT1, RNF185, CGRRF1 and RNF19B) was significantly upregulated by ER stress. To determine the involvement of the ER stress-responsive genes in ERAD, we investigated their ER localisation, in vitro autoubiquitination activity and ER stress resistance. All were partially localised to the ER, whereas CGRRF1 did not possess E3 activity. RNFT1 and RNF185, but not CGRRF1 and RNF19B, exhibited significant resistance to ER stressor in an E3 activity-dependent manner. Thus, these genes are possible candidates for ERAD E3s. PMID:27485036

  15. The Highwire Ubiquitin Ligase Promotes Axonal Degeneration by Tuning Levels of Nmnat Protein

    PubMed Central

    Xiong, Xin; Hao, Yan; Sun, Kan; Li, Jiaxing; Li, Xia; Mishra, Bibhudatta; Soppina, Pushpanjali; Wu, Chunlai; Hume, Richard I.; Collins, Catherine A.

    2012-01-01

    Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury. PMID:23226106

  16. Lysine 63-Linked TANK-Binding Kinase 1 Ubiquitination by Mindbomb E3 Ubiquitin Protein Ligase 2 Is Mediated by the Mitochondrial Antiviral Signaling Protein

    PubMed Central

    Ye, Jung Sook; Kim, Nari; Lee, Kyoung Jin; Nam, Young Ran; Lee, Uk

    2014-01-01

    ABSTRACT Beta interferon (IFN-β) is involved in a wide range of cellular functions, and its secretion must be tightly controlled to inhibit viral spreading while minimizing cellular damage. Intracellular viral replication triggers cellular signaling cascades leading to the activation of the transcription factors NF-κB and interferon regulatory factor 3 (IRF3) and IRF7 (IRF3/7), which synergistically bind to the IFN-β gene promoter to induce its expression. The mitochondrial antiviral signaling protein (MAVS) is a governing adaptor protein that mediates signaling communications between virus-sensing proteins and transcription factors. The activity of MAVS in the regulation of IFN-β secretion is affected by many cellular factors. However, the mechanism of MAVS-mediated IRF3/7 activation is not completely understood. Here, we identified a highly conserved DLAIS motif at amino acid positions 438 to 442 of MAVS that is indispensable for IRF3/7 activation. Specifically, the L439S and A440R mutations suppress IRF3/7 activation. Pulldown experiments using wild-type and mutant MAVS showed that mindbomb E3 ubiquitin protein ligase 2 (MIB2) binds to the DLAIS motif. Furthermore, the DLAIS motif was found to be critical for MIB2 binding, the ligation of K63-linked ubiquitin to TANK-binding kinase 1, and phosphorylation-mediated IRF3/7 activation. Our results suggest that MIB2 plays a putative role in MAVS-mediated interferon signaling. IMPORTANCE Mitochondrial antiviral signaling protein (MAVS) mediates signaling from virus-sensing proteins to transcription factors for the induction of beta interferon. However, the mechanism underlying activation of MAVS-mediated interferon regulatory factors 3 and 7 (IRF3/7) is not completely understood. We found a highly conserved DLAIS motif in MAVS that is indispensable for IRF3/7 activation through TANK-binding kinase 1 (TBK1) and identified it as the binding site for mindbomb E3 ubiquitin protein ligase 2 (MIB2). The mutations that

  17. The Glomuvenous Malformation Protein Glomulin Binds Rbx1 and Regulates Cullin RING Ligase-Mediated Turnover of Fbw7

    PubMed Central

    Tron, Adriana E.; Arai, Takehiro; Duda, David M.; Kuwabara, Hiroshi; Olszewski, Jennifer L.; Fujiwara, Yuko; Bahamon, Brittany N.; Signoretti, Sabina; Schulman, Brenda A.; DeCaprio, James A.

    2012-01-01

    SUMMARY Fbw7, a substrate receptor for Cul1-RING-ligase (CRL1), facilitates the ubiquitination and degradation of several proteins including Cyclin E and c-Myc. In spite of much effort, the mechanisms underlying Fbw7 regulation are mostly unknown. Here we show that Glomulin (Glmn), a protein found mutated in the vascular disorder Glomuvenous Malformation (GVM), binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity. Loss of Glmn in a variety of cells, tissues and GVM lesions results in decreased levels of Fbw7 and increased levels of Cyclin E and c-Myc. The increased turnover of Fbw7 is dependent on CRL and proteasome activity indicating that Glmn modulates the E3 activity of CRL1Fbw7. These data reveal an unexpected functional connection between Glmn and Rbx1 and demonstrate that defective regulation of Fbw7 levels contributes to GVM. PMID:22405651

  18. The glomuvenous malformation protein Glomulin binds Rbx1 and regulates cullin RING ligase-mediated turnover of Fbw7.

    PubMed

    Tron, Adriana E; Arai, Takehiro; Duda, David M; Kuwabara, Hiroshi; Olszewski, Jennifer L; Fujiwara, Yuko; Bahamon, Brittany N; Signoretti, Sabina; Schulman, Brenda A; DeCaprio, James A

    2012-04-13

    Fbw7, a substrate receptor for Cul1-RING-ligase (CRL1), facilitates the ubiquitination and degradation of several proteins, including Cyclin E and c-Myc. In spite of much effort, the mechanisms underlying Fbw7 regulation are mostly unknown. Here, we show that Glomulin (Glmn), a protein found mutated in the vascular disorder glomuvenous malformation (GVM), binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity. Loss of Glmn in a variety of cells, tissues, and GVM lesions results in decreased levels of Fbw7 and increased levels of Cyclin E and c-Myc. The increased turnover of Fbw7 is dependent on CRL and proteasome activity, indicating that Glmn modulates the E3 activity of CRL1(Fbw7). These data reveal an unexpected functional connection between Glmn and Rbx1 and demonstrate that defective regulation of Fbw7 levels contributes to GVM.

  19. Targeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL) with Nucleoside-Based Bisubstrate Adenylation Inhibitors

    PubMed Central

    Petrelli, Riccardo; De la Mora-Rey, Teresa; Tiwari, Divya; Liu, Feng; Dawadi, Surrendra; Nandakumar, Madhumitha; Rhee, Kyu Y.; Schnappinger, Dirk; Finzel, Barry C.; Aldrich, Courtney C.

    2015-01-01

    Mycobacterium tuberculosis (Mtb) responsible for both latent and symptomatic tuberculosis (TB) remains the second leading cause of mortality among infectious diseases worldwide. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. We report the synthesis and evaluation of a systematic series of potent nucleoside-based inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with KD's below 2 nM. Additionally, we obtained high-resolution co-crystal structures for a majority of the compounds. Despite fairly uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MIC) ranging from 0.78 to >100 μM. Cellular accumulation studies showed a nearly 10-fold enhanced accumulation of a C-2′-α analog over the corresponding C-2′-β analog, consistent with their differential whole-cell activity. PMID:26299766

  20. Regulation of EGFR protein stability by the HECT-type ubiquitin ligase SMURF2.

    PubMed

    Ray, Dipankar; Ahsan, Aarif; Helman, Abigail; Chen, Guoan; Hegde, Ashok; Gurjar, Susmita Ramanand; Zhao, Lili; Kiyokawa, Hiroaki; Beer, David G; Lawrence, Theodore S; Nyati, Mukesh K

    2011-07-01

    Epidermal growth factor receptor (EGFR) is overexpressed in a variety of epithelial tumors and is considered to be an important therapeutic target. Although gene amplification is responsible for EGFR overexpression in certain human malignancies including lung and head and neck cancers, additional molecular mechanisms are likely. Here, we report a novel interaction of EGFR with an HECT-type ubiquitin ligase SMURF2, which can ubiquitinate, but stabilize EGFR by protecting it from c-Cbl-mediated degradation. Conversely, small interfering RNA (siRNA)-mediated knockdown of SMURF2 destabilized EGFR, induced an autophagic response and reduced the clonogenic survival of EGFR-expressing cancer cell lines, with minimal effects on EGFR-negative cancer cells, normal fibroblasts, and normal epithelial cells. UMSCC74B head and neck squamous cancer cells, which form aggressive tumors in nude mice, significantly lost in vivo tumor-forming ability on siRNA-mediated SMURF2 knockdown. Gene expression microarray data from 443 lung adenocarcinoma patients, and tissue microarray data from 67 such patients, showed a strong correlation of expression between EGFR and SMURF2 at the messenger RNA and protein levels, respectively. Our findings suggest that SMURF2-mediated protective ubiquitination of EGFR may be responsible for EGFR overexpression in certain tumors and support targeting SMURF2-EGFR interaction as a novel therapeutic approach in treating EGFR-addicted tumors.

  1. The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein-coupled receptor CXCR4.

    PubMed

    Holleman, Justine; Marchese, Adriano

    2014-06-15

    G protein-coupled receptor (GPCR) sorting into the degradative pathway is important for limiting the duration and magnitude of signaling. Agonist activation of the GPCR CXCR4 induces its rapid ubiquitination and sorting to lysosomes via the endosomal sorting complex required for transport (ESCRT) pathway. We recently reported that ESCRT-0 ubiquitination is linked to the efficiency with which CXCR4 is sorted for lysosomal degradation; however mechanistic insight is lacking. Here we define a novel role for the really interesting new gene-domain E3 ubiquitin ligase deltex-3-like (DTX3L) in regulating CXCR4 sorting from endosomes to lysosomes. We show that DTX3L localizes to early endosomes upon CXCR4 activation and interacts directly with and inhibits the activity of the E3 ubiquitin ligase atrophin-1 interacting protein 4. This serves to limit the extent to which ESCRT-0 is ubiquitinated and is able to sort CXCR4 for lysosomal degradation. Therefore we define a novel role for DTX3L in GPCR endosomal sorting and reveal an unprecedented link between two distinct E3 ubiquitin ligases to control the activity of the ESCRT machinery.

  2. The ubiquitin E3 ligase LOSS OF GDU2 is required for GLUTAMINE DUMPER1-induced amino acid secretion in Arabidopsis.

    PubMed

    Pratelli, Réjane; Guerra, Damian D; Yu, Shi; Wogulis, Mark; Kraft, Edward; Frommer, Wolf B; Callis, Judy; Pilot, Guillaume

    2012-04-01

    Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.

  3. Structural and functional interaction between the human DNA repair proteins DNA ligase IV and XRCC4.

    PubMed

    Wu, Peï-Yu; Frit, Philippe; Meesala, SriLakshmi; Dauvillier, Stéphanie; Modesti, Mauro; Andres, Sara N; Huang, Ying; Sekiguchi, JoAnn; Calsou, Patrick; Salles, Bernard; Junop, Murray S

    2009-06-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  4. Subcellular relocalization of a long-chain fatty acid CoA ligase by a suppressor mutation alleviates a respiration deficiency in Saccharomyces cerevisiae.

    PubMed Central

    Harington, A; Schwarz, E; Slonimski, P P; Herbert, C J

    1994-01-01

    We have isolated an extragenic suppressor, FAM1-1, which is able to restore respiratory growth to a deletion of the CEM1 gene (mitochondrial beta-keto-acyl synthase). The sequence of the suppressor strongly suggests that it encodes a long-chain fatty acid CoA ligase (fatty-acyl-CoA synthetase). We have also cloned and sequenced the wild-type FAM1 gene, which is devoid of suppressor activity. The comparison of the two sequences shows that the suppressor mutation is an A-->T transversion, which creates a new initiation codon and adds 18 amino acids to the N-terminus of the protein. This extension has all the characteristics of a mitochondrial targeting sequence, whilst the N-terminus of the wild-type protein has none of these characteristics. In vitro mitochondrial import experiments show that the N-terminal half of the suppressor protein, but not of the wild-type, is transported into mitochondria. Thus, we hypothesize that the suppressor acts by changing the subcellular localization of the protein and relocating at least some of the enzyme from the cytosol to the mitochondria. These results support the hypothesis that some form of fatty acid synthesis, specific for the mitochondria, is essential for the function of the organelle. Images PMID:7988550

  5. The Pepper E3 Ubiquitin Ligase RING1 Gene, CaRING1, Is Required for Cell Death and the Salicylic Acid-Dependent Defense Response1[C][W][OA

    PubMed Central

    Lee, Dong Hyuk; Choi, Hyong Woo; Hwang, Byung Kook

    2011-01-01

    Ubiquitination is essential for ubiquitin/proteasome-mediated protein degradation in plant development and defense. Here, we identified a novel E3 ubiquitin ligase RING1 gene, CaRING1, from pepper (Capsicum annuum). In pepper, CaRING1 expression is induced by avirulent Xanthomonas campestris pv vesicatoria infection. CaRING1 contains an amino-terminal transmembrane domain and a carboxyl-terminal RING domain. In addition, it displays in vitro E3 ubiquitin ligase activity, and the RING domain is essential for E3 ubiquitin ligase activity in CaRING1. CaRING1 also localizes to the plasma membrane. In pepper plants, virus-induced gene silencing of CaRING1 confers enhanced susceptibility to avirulent X. campestris pv vesicatoria infection, which is accompanied by compromised hypersensitive cell death, reduced expression of PATHOGENESIS-RELATED1, and lowered salicylic acid levels in leaves. Transient expression of CaRING1 in pepper leaves induces cell death and the defense response that requires the E3 ubiquitin ligase activity of CaRING1. By contrast, overexpression of CaRING1 in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to hemibiotrophic Pseudomonas syringae pv tomato and biotrophic Hyaloperonospora arabidopsidis infections. Taken together, these results suggest that CaRING1 is involved in the induction of cell death and the regulation of ubiquitination during the defense response to microbial pathogens. PMID:21628629

  6. Structure of a Putative Lipoate Protein Ligase from Thermoplasma acidophilum and the Mechanism of Target Selection for Post-Translational Modification

    SciTech Connect

    McManus,E.; Luisi, B.; Perham, R.

    2006-01-01

    Lipoyl-lysine swinging arms are crucial to the reactions catalysed by the 2-oxo acid dehydrogenase multienzyme complexes. A gene encoding a putative lipoate protein ligase (LplA) of Thermoplasma acidophilum was cloned and expressed in Escherichia coli. The recombinant protein, a monomer of molecular mass 29 kDa, was catalytically inactive. Crystal structures in the absence and presence of bound lipoic acid were solved at 2.1 Angstroms resolution. The protein was found to fall into the a/{beta} class and to be structurally homologous to the catalytic domains of class II aminoacyl-tRNA synthases and biotin protein ligase, BirA. Lipoic acid in LplA was bound in the same position as biotin in BirA. The structure of the T. acidophilum LplA and limited proteolysis of E. coli LplA together highlighted some key features of the post-translational modification. A loop comprising residues 71-79 in the T. acidophilumligase is proposed as interacting with the dithiolane ring of lipoic acid and discriminating against the entry of biotin. A second loop comprising residues 179-193 was disordered in the T. acidophilum structure; tryptic cleavage of the corresponding loop in the E. coli LplA under non-denaturing conditions rendered the enzyme catalytically inactive, emphasizing its importance. The putative LplA of T. acidophilum lacks a C-terminal domain found in its counterparts in E. coli (Gram-negative) or Streptococcus pneumoniae (Gram-positive). A gene encoding a protein that appears to have structural homology to the additional domain in the E. coli and S. pneumoniae enzymes was detected alongside the structural gene encoding the putative LplA in the T. acidophilum genome. It is likely that this protein is required to confer activity on the LplA as currently purified, one protein perhaps catalysing the formation of the obligatory lipoyl-AMP intermediate, and the other transferring the lipoyl group from it to the specific lysine residue in the target protein.

  7. Electron microscopy visualization of DNA-protein complexes formed by Ku and DNA ligase IV.

    PubMed

    Grob, Patricia; Zhang, Teri T; Hannah, Ryan; Yang, Hui; Hefferin, Melissa L; Tomkinson, Alan E; Nogales, Eva

    2012-01-02

    The repair of DNA double-stranded breaks (DSBs) is essential for cell viability and genome stability. Aberrant repair of DSBs has been linked with cancer predisposition and aging. During the repair of DSBs by non-homologous end joining (NHEJ), DNA ends are brought together, processed and then joined. In eukaryotes, this repair pathway is initiated by the binding of the ring-shaped Ku heterodimer and completed by DNA ligase IV. The DNA ligase IV complex, DNA ligase IV/XRRC4 in humans and Dnl4/Lif1 in yeast, is recruited to DNA ends in vitro and in vivo by an interaction with Ku and, in yeast, Dnl4/Lif1 stabilizes the binding of yKu to in vivo DSBs. Here we have analyzed the interactions of these functionally conserved eukaryotic NHEJ factors with DNA by electron microscopy. As expected, the ring-shaped Ku complex bound stably and specifically to DNA ends at physiological salt concentrations. At a ratio of 1 Ku molecule per DNA end, the majority of DNA ends were occupied by a single Ku complex with no significant formation of linear DNA multimers or circular loops. Both Dnl4/Lif1 and DNA ligase IV/XRCC4 formed complexes with Ku-bound DNA ends, resulting in intra- and intermolecular DNA end bridging, even with non-ligatable DNA ends. Together, these studies, which provide the first visualization of the conserved complex formed by Ku and DNA ligase IV at juxtaposed DNA ends by electron microscopy, suggest that the DNA ligase IV complex mediates end-bridging by engaging two Ku-bound DNA ends.

  8. Genome-wide identification and characterization of the apple (Malus domestica) HECT ubiquitin-protein ligase family and expression analysis of their responsiveness to abiotic stresses.

    PubMed

    Xu, Jianing; Xing, Shanshan; Cui, Haoran; Chen, Xuesen; Wang, Xiaoyun

    2016-04-01

    The ubiquitin-protein ligases (E3s) directly participate in ubiquitin (Ub) transferring to the target proteins in the ubiquitination pathway. The HECT ubiquitin-protein ligase (UPL), one type of E3s, is characterized as containing a conserved HECT domain of approximately 350 amino acids in the C terminus. Some UPLs were found to be involved in trichome development and leaf senescence in Arabidopsis. However, studies on plant UPLs, such as characteristics of the protein structure, predicted functional motifs of the HECT domain, and the regulatory expression of UPLs have all been limited. Here, we present genome-wide identification of the genes encoding UPLs (HECT gene) in apple. The 13 genes (named as MdUPL1-MdUPL13) from ten different chromosomes were divided into four groups by phylogenetic analysis. Among these groups, the encoding genes in the intron-exon structure and the included additional functional domains were quite different. Notably, the F-box domain was first found in MdUPL7 in plant UPLs. The HECT domain in different MdUPL groups also presented different spatial features and three types of conservative motifs were identified. The promoters of each MdUPL member carried multiple stress-response related elements by cis-acting element analysis. Experimental results demonstrated that the expressions of several MdUPLs were quite sensitive to cold-, drought-, and salt-stresses by qRT-PCR assay. The results of this study helped to elucidate the functions of HECT proteins, especially in Rosaceae plants.

  9. The Atypical Occurrence of Two Biotin Protein Ligases in Francisella novicida Is Due to Distinct Roles in Virulence and Biotin Metabolism

    PubMed Central

    Feng, Youjun; Chin, Chui-Yoke; Chakravartty, Vandana; Gao, Rongsui; Crispell, Emily K.

    2015-01-01

    ABSTRACT The physiological function of biotin requires biotin protein ligase activity in order to attach the coenzyme to its cognate proteins, which are enzymes involved in central metabolism. The model intracellular pathogen Francisella novicida is unusual in that it encodes two putative biotin protein ligases rather than the usual single enzyme. F. novicida BirA has a ligase domain as well as an N-terminal DNA-binding regulatory domain, similar to the prototypical BirA protein in E. coli. However, the second ligase, which we name BplA, lacks the N-terminal DNA binding motif. It has been unclear why a bacterium would encode these two disparate biotin protein ligases, since F. novicida contains only a single biotinylated protein. In vivo complementation and enzyme assays demonstrated that BirA and BplA are both functional biotin protein ligases, but BplA is a much more efficient enzyme. BirA, but not BplA, regulated transcription of the biotin synthetic operon. Expression of bplA (but not birA) increased significantly during F. novicida infection of macrophages. BplA (but not BirA) was required for bacterial replication within macrophages as well as in mice. These data demonstrate that F. novicida has evolved two distinct enzymes with specific roles; BplA possesses the major ligase activity, whereas BirA acts to regulate and thereby likely prevent wasteful synthesis of biotin. During infection BplA seems primarily employed to maximize the efficiency of biotin utilization without limiting the expression of biotin biosynthetic genes, representing a novel adaptation strategy that may also be used by other intracellular pathogens. PMID:26060274

  10. E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors

    PubMed Central

    Zhang, Yan; Yokoyama, Shigetoshi; Herriges, John C.; Zhang, Zhen; Young, Randee E.; Verheyden, Jamie M.; Sun, Xin

    2016-01-01

    The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis. PMID:27335464

  11. Prokaryotic DNA ligases unwind superhelical DNA.

    PubMed

    Ivanchenko, M; van Holde, K; Zlatanova, J

    1996-09-13

    We have studied the effect on DNA topology of binding of prokaryotic DNA ligases (T4 and E. coli) to superhelical or nicked circular DNA. Performing topoisomerase I-mediated relaxation in the presence of increasing amounts of T4 ligase led to a shift in the topoisomer distribution to increasingly more negative values. This result suggested that T4 ligase unwound the DNA and was further substantiated by ligation of nicked circular molecules by E. coli DNA ligase in the presence of increasing amounts of T4 ligase. Such an experiment was possible since the two DNA ligases require different cofactors for enzymatic activity. Performing a similar experiment with reverse partners, using E. coli DNA ligase as ligand, and T4 ligase as sealing agent, we observed that the E. coli enzyme also unwound the DNA. Thus, prokaryotic DNA ligases can be added to an ever-growing list of DNA-binding proteins that unwind the DNA upon binding.

  12. DNA ligase I, the replicative DNA ligase.

    PubMed

    Howes, Timothy R L; Tomkinson, Alan E

    2012-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication.

  13. The Membrane Associated RING-CH Proteins: A Family of E3 Ligases with Diverse Roles through the Cell

    PubMed Central

    Means, Robert E.

    2014-01-01

    Since the discovery that conjugation of ubiquitin to proteins can drive proteolytic degradation, ubiquitination has been shown to perform a diverse range of functions in the cell. It plays an important role in endocytosis, signal transduction, trafficking of vesicles inside the cell, and even DNA repair. The process of ubiquitination-mediated control has turned out to be remarkably complex, involving a diverse array of proteins and many levels of control. This review focuses on a family of structurally related E3 ligases termed the membrane-associated RING-CH (MARCH) ubiquitin ligases, which were originally discovered as structural homologs to the virals E3s, K3, and K5 from Kaposi's sarcoma-associated herpesvirus (KSHV). These proteins contain a catalytic RING-CH finger and are typically membrane-bound, with some having up to 14 putative transmembrane domains. Despite several lines of evidence showing that the MARCH proteins play a complex and essential role in several cellular processes, this family remains understudied. PMID:27419207

  14. The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

    PubMed Central

    Hoxhaj, Gerta; Caddye, Edward; Najafov, Ayaz; Houde, Vanessa P; Johnson, Catherine; Dissanayake, Kumara; Toth, Rachel; Campbell, David G; Prescott, Alan R; MacKintosh, Carol

    2016-01-01

    The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1. DOI: http://dx.doi.org/10.7554/eLife.12278.001 PMID:27244671

  15. D-Amino acid dipeptide production utilizing D-alanine-D-alanine ligases with novel substrate specificity.

    PubMed

    Sato, Masaru; Kirimura, Kohtaro; Kino, Kuniki

    2005-06-01

    D-Alanine-D-alanine ligase (Ddl) is an important enzyme in the synthesis of bacterial peptidoglycan. The genes encoding Ddls from Escherichia coli K12 (EcDdlB), Oceanobacillus iheyensis JCM 11309 (OiDdl), Synechocystis sp. PCC 6803 (SsDdl) and Thermotoga maritima ATCC 43589 (TmDdl), the genomic DNA sequences of which have been determined, were cloned and the substrate specificities of these recombinant Ddls were investigated. Although OiDdl had a high substrate specificity for D-alanine; EcDdlB, SsDdl and TmDdl showed broad substrate specificities for D-serine, D-threonine, D-cysteine and glycine, in addition to D-alanine. Four D-amino acid dipeptides were produced using EcDdlB, and D-amino acid homo-dipeptides were successfully produced at high yields except for D-threonyl-D-threonine.

  16. The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy.

    PubMed

    Sun, Aiqin; Wei, Jing; Childress, Chandra; Shaw Iv, John H; Peng, Ke; Shao, Genbao; Yang, Wannian; Lin, Qiong

    2017-01-13

    The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.

  17. Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2.

    PubMed

    Rosario, Fredrick J; Dimasuay, Kris Genelyn; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

    2016-04-01

    Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

  18. Genetically engineered mouse models for functional studies of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases

    PubMed Central

    Zhou, Weihua; Wei, Wenyi; Sun, Yi

    2013-01-01

    The SCF (SKP1 (S-phase-kinase-associated protein 1), Cullin-1, F-box protein) E3 ubiquitin ligases, the founding member of Cullin-RING ligases (CRLs), are the largest family of E3 ubiquitin ligases in mammals. Each individual SCF E3 ligase consists of one adaptor protein SKP1, one scaffold protein cullin-1 (the first family member of the eight cullins), one F-box protein out of 69 family members, and one out of two RING (Really Interesting New Gene) family proteins RBX1/ROC1 or RBX2/ROC2/SAG/RNF7. Various combinations of these four components construct a large number of SCF E3s that promote the degradation of many key regulatory proteins in cell-context, temporally, and spatially dependent manners, thus controlling precisely numerous important cellular processes, including cell cycle progression, apoptosis, gene transcription, signal transduction, DNA replication, maintenance of genome integrity, and tumorigenesis. To understand how the SCF E3 ligases regulate these cellular processes and embryonic development under in vivo physiological conditions, a number of mouse models with transgenic (Tg) expression or targeted deletion of components of SCF have been established and characterized. In this review, we will provide a brief introduction to the ubiquitin-proteasome system (UPS) and the SCF E3 ubiquitin ligases, followed by a comprehensive overview on the existing Tg and knockout (KO) mouse models of the SCF E3s, and discuss the role of each component in mouse embryogenesis, cell proliferation, apoptosis, carcinogenesis, as well as other pathogenic processes associated with human diseases. We will end with a brief discussion on the future directions of this research area and the potential applications of the knowledge gained to more effective therapeutic interventions of human diseases. PMID:23528706

  19. Subunit architecture of the Golgi Dsc E3 ligase required for sterol regulatory element-binding protein (SREBP) cleavage in fission yeast.

    PubMed

    Lloyd, S Julie-Ann; Raychaudhuri, Sumana; Espenshade, Peter J

    2013-07-19

    The membrane-bound sterol regulatory element-binding protein (SREBP) transcription factors regulate lipogenesis in mammalian cells and are activated through sequential cleavage by the Golgi-localized Site-1 and Site-2 proteases. The mechanism of fission yeast SREBP cleavage is less well defined and, in contrast, requires the Golgi-localized Dsc E3 ligase complex. The Dsc E3 ligase consists of five integral membrane subunits, Dsc1 through Dsc5, and resembles membrane E3 ligases that function in endoplasmic reticulum-associated degradation. Using immunoprecipitation assays and blue native electrophoresis, we determined the subunit architecture for the complex of Dsc1 through Dsc5, showing that the Dsc proteins form subcomplexes and display defined connectivity. Dsc2 is a rhomboid pseudoprotease family member homologous to mammalian UBAC2 and a central component of the Dsc E3 ligase. We identified conservation in the architecture of the Dsc E3 ligase and the multisubunit E3 ligase gp78 in mammals. Specifically, Dsc1-Dsc2-Dsc5 forms a complex resembling gp78-UBAC2-UBXD8. Further characterization of Dsc2 revealed that its C-terminal UBA domain can bind to ubiquitin chains but that the Dsc2 UBA domain is not essential for yeast SREBP cleavage. Based on the ability of rhomboid superfamily members to bind transmembrane proteins, we speculate that Dsc2 functions in SREBP recognition and binding. Homologs of Dsc1 through Dsc4 are required for SREBP cleavage and virulence in the human opportunistic pathogen Aspergillus fumigatus. Thus, these studies advance our organizational understanding of multisubunit E3 ligases involved in endoplasmic reticulum-associated degradation and fungal pathogenesis.

  20. Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1

    PubMed Central

    Heck, Jarrod W.; Cheung, Samantha K.; Hampton, Randolph Y.

    2009-01-01

    Eukaryotic cells maintain proteostasis by quality control (QC) degradation. These pathways can specifically target a wide variety of distinct misfolded proteins, and so are important for management of cellular stress. Although a number of conserved QC pathways have been described in yeast, the E3 ligases responsible for cytoplasmic QC are unknown. We now show that Ubr1 and San1 mediate chaperone-dependent ubiquitination of numerous misfolded cytoplasmic proteins. This action of Ubr1 is distinct from its role in the “N-end rule.” In this capacity, Ubr1 functions to protect cells from proteotoxic stresses. Our phenotypic and biochemical studies of Ubr1 and San1 indicate that two strategies are employed for cytoplasmic QC: chaperone-assisted ubiquitination by Ubr1 and chaperone-dependent delivery to nuclear San1. The broad conservation of Ubr ligases and the relevant chaperones indicates that these mechanisms will be important in understanding both basic and biomedical aspects of cellular proteostasis. PMID:20080635

  1. A Large Complement of the Predicted Arabidopsis ARM Repeat Proteins Are Members of the U-Box E3 Ubiquitin Ligase Family1[w

    PubMed Central

    Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L.; Salt, Jennifer N.; Goring, Daphne R.

    2004-01-01

    The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis. PMID:14657406

  2. Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

    PubMed Central

    Mudhasani, Rajini; Tran, Julie P.; Retterer, Cary; Kota, Krishna P.; Whitehouse, Chris A.; Bavari, Sina

    2016-01-01

    Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCFFBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCFFBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection. PMID

  3. A cancer-associated RING finger protein, RNF43, is a ubiquitin ligase that interacts with a nuclear protein, HAP95

    SciTech Connect

    Sugiura, Takeyuki Yamaguchi, Aya; Miyamoto, Kentaro

    2008-04-15

    RNF43 is a recently discovered RING finger protein that is implicated in colon cancer pathogenesis. This protein possesses growth-promoting activity but its mechanism remains unknown. In this study, to gain insight into the biological action of RNF43 we characterized it biochemically and intracellularly. A combination of indirect immunofluorescence analysis and biochemical fractionation experiments suggests that RNF43 resides in the endoplasmic reticulum (ER) as well as in the nuclear envelope. Sucrose density gradient fractionation demonstrates that RNF43 co-exists with emerin, a representative inner nuclear membrane protein in the nuclear subcompartment. The cell-free system with pure components reveals that recombinant RNF43 fused with maltose-binding protein has autoubiquitylation activity. By the yeast two-hybrid screening we identified HAP95, a chromatin-associated protein interfacing the nuclear envelope, as an RNF43-interacting protein and substantiated this interaction in intact cells by the co-immunoprecipitation experiments. HAP95 is ubiquitylated and subjected to a proteasome-dependent degradation pathway, however, the experiments in which 293 cells expressing both RNF43 and HAP95 were treated with a proteasome inhibitor, MG132, show that HAP95 is unlikely to serve as a substrate of RNF43 ubiquitin ligase. These results infer that RNF43 is a resident protein of the ER and, at least partially, the nuclear membrane, with ubiquitin ligase activity and may be involved in cell growth control potentially through the interaction with HAP95.

  4. A HECT Ubiquitin-Protein Ligase as a Novel Candidate Gene for Altered Quinine and Quinidine Responses in Plasmodium falciparum

    PubMed Central

    Sanchez, Cecilia P.; Cyrklaff, Marek; Mu, Jianbing; Ferdig, Michael T.; Stein, Wilfred D.; Lanzer, Michael

    2014-01-01

    The emerging resistance to quinine jeopardizes the efficacy of a drug that has been used in the treatment of malaria for several centuries. To identify factors contributing to differential quinine responses in the human malaria parasite Plasmodium falciparum, we have conducted comparative quantitative trait locus analyses on the susceptibility to quinine and also its stereoisomer quinidine, and on the initial and steady-state intracellular drug accumulation levels in the F1 progeny of a genetic cross. These data, together with genetic screens of field isolates and laboratory strains associated differential quinine and quinidine responses with mutated pfcrt, a segment on chromosome 13, and a novel candidate gene, termed MAL7P1.19 (encoding a HECT ubiquitin ligase). Despite a strong likelihood of association, episomal transfections demonstrated a role for the HECT ubiquitin-protein ligase in quinine and quinidine sensitivity in only a subset of genetic backgrounds, and here the changes in IC50 values were moderate (approximately 2-fold). These data show that quinine responsiveness is a complex genetic trait with multiple alleles playing a role and that more experiments are needed to unravel the role of the contributing factors. PMID:24830312

  5. Balancing protein stability and activity in cancer: a new approach for identifying driver mutations affecting CBL ubiquitin ligase activation

    PubMed Central

    Li, Minghui; Kales, Stephen C.; Ma, Ke; Shoemaker, Benjamin A.; Crespo-Barreto, Juan; Cangelosi, Andrew L.; Lipkowitz, Stanley; Panchenko, Anna R.

    2015-01-01

    Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved depicting the protein at different stages of its activation cycle and thus provide mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than did random non-cancer mutations. We further tested the ability of these computational models assessing the changes in CBL stability and its binding to ubiquitin conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two-thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL. PMID:26676746

  6. Ectromelia Virus BTB/kelch Proteins, EVM150 and EVM167, Interact with Cullin-3 Based Ubiquitin Ligases

    PubMed Central

    Wilton, Brianne A.; Campbell, Stephanie; Van Buuren, Nicholas; Garneau, Robyn; Furukawa, Manabu; Xiong, Yue; Barry., Michele

    2008-01-01

    Cellular proteins containing BTB and kelch domains have been shown to function as adapters for the recruitment of substrates to cullin-3-based ubiquitin ligases. Poxviruses are the only family of viruses known to encode multiple BTB/kelch proteins, suggesting that poxviruses may modulate the ubiquitin pathway through interaction with cullin-3. Ectromelia virus encodes four BTB/kelch proteins and one BTB-only protein. Here we demonstrate that two of the ectromelia virus encoded BTB/kelch proteins, EVM150 and EVM167, interacted with cullin-3. Similar to cellular BTB proteins, the BTB domain of EVM150 and EVM167 was necessary and sufficient for cullin-3 interaction. During infection, EVM150 and EVM167 localized to discrete cytoplasmic regions, which co-localized with cullin-3. Furthermore, EVM150 and EVM167 co-localized and interacted with conjugated ubiquitin, as demonstrated by confocal microscopy and co-immunoprecipitation. Our findings suggest that the ectromelia virus encoded BTB/kelch proteins, EVM150 and EVM167, interact with cullin-3 potentially functioning to recruit unidentified substrates for ubiquitination. PMID:18221766

  7. Ectromelia virus BTB/kelch proteins, EVM150 and EVM167, interact with cullin-3-based ubiquitin ligases.

    PubMed

    Wilton, Brianne A; Campbell, Stephanie; Van Buuren, Nicholas; Garneau, Robyn; Furukawa, Manabu; Xiong, Yue; Barry, Michele

    2008-04-25

    Cellular proteins containing BTB and kelch domains have been shown to function as adapters for the recruitment of substrates to cullin-3-based ubiquitin ligases. Poxviruses are the only family of viruses known to encode multiple BTB/kelch proteins, suggesting that poxviruses may modulate the ubiquitin pathway through interaction with cullin-3. Ectromelia virus encodes four BTB/kelch proteins and one BTB-only protein. Here we demonstrate that two of the ectromelia virus-encoded BTB/kelch proteins, EVM150 and EVM167, interacted with cullin-3. Similar to cellular BTB proteins, the BTB domain of EVM150 and EVM167 was necessary and sufficient for cullin-3 interaction. During infection, EVM150 and EVM167 localized to discrete cytoplasmic regions, which co-localized with cullin-3. Furthermore, EVM150 and EVM167 co-localized and interacted with conjugated ubiquitin, as demonstrated by confocal microscopy and co-immunoprecipitation. Our findings suggest that the ectromelia virus-encoded BTB/kelch proteins, EVM150 and EVM167, interact with cullin-3 potentially functioning to recruit unidentified substrates for ubiquitination.

  8. Human Mre11/human Rad50/Nbs1 and DNA ligase IIIalpha/XRCC1 protein complexes act together in an alternative nonhomologous end joining pathway.

    PubMed

    Della-Maria, Julie; Zhou, Yi; Tsai, Miaw-Sheue; Kuhnlein, Jeff; Carney, James P; Paull, Tanya T; Tomkinson, Alan E

    2011-09-30

    Recent studies have implicated a poorly defined alternative pathway of nonhomologous end joining (alt-NHEJ) in the generation of large deletions and chromosomal translocations that are frequently observed in cancer cells. Here, we describe an interaction between two factors, hMre11/hRad50/Nbs1 (MRN) and DNA ligase IIIα/XRCC1, that have been linked with alt-NHEJ. Expression of DNA ligase IIIα and the association between MRN and DNA ligase IIIα/XRCC1 are altered in cell lines defective in the major NHEJ pathway. Most notably, DNA damage induced the association of these factors in DNA ligase IV-deficient cells. MRN interacts with DNA ligase IIIα/XRCC1, stimulating intermolecular ligation, and together these proteins join incompatible DNA ends in a reaction that mimics alt-NHEJ. Thus, our results provide novel mechanistic insights into the alt-NHEJ pathway that not only contributes to genome instability in cancer cells but may also be a therapeutic target.

  9. Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins

    PubMed Central

    Takahashi, Hirotaka; Uematsu, Atsushi; Yamanaka, Satoshi; Imamura, Mei; Nakajima, Tatsuro; Doi, Kousuke; Yasuoka, Saki; Takahashi, Chikako; Takeda, Hiroyuki; Sawasaki, Tatsuya

    2016-01-01

    Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3). Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1) targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3—which there have been no report to bind p53—were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein. PMID:27249653

  10. Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene.

    PubMed

    Barany, F; Gelfand, D H

    1991-12-20

    Thermostable DNA ligase has been harnessed for the detection of single-base genetic diseases using the ligase chain reaction [Barany, Proc. Natl. Acad. Sci. USA 88 (1991) 189-193]. The Thermus thermophilus (Tth) DNA ligase-encoding gene (ligT) was cloned in Escherichia coli by genetic complementation of a ligts 7 defect in an E. coli host. Nucleotide sequence analysis of the gene revealed a single chain of 676 amino acid residues with 47% identity to the E. coli ligase. Under phoA promoter control, Tth ligase was overproduced to greater than 10% of E. coli cellular proteins. Adenylated and deadenylated forms of the purified enzyme were distinguished by apparent molecular weights of 81 kDa and 78 kDa, respectively, after separation via sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.

  11. NIRF, a Novel Ubiquitin Ligase, Inhibits Hepatitis B Virus Replication Through Effect on HBV Core Protein and H3 Histones.

    PubMed

    Qian, Guanhua; Hu, Bin; Zhou, Danlin; Xuan, Yanyan; Bai, Lu; Duan, Changzhu

    2015-05-01

    Np95/ICBP90-like RING finger protein (NIRF), a novel E3 ubiquitin ligase, has been shown to interact with HBc and promote its degradation. This study investigated the effects of NIRF on replication of hepatitis B virus (HBV) and the mechanisms. We have shown that NIRF inhibits replication of HBV DNA and secretion of HBsAg and HBeAg in HepG2 cells transfected with pAAV-HBV1.3. NIRF also inhibits the replication and secretion of HBV in a mouse model that expressed HBV. NIRF reduces acetylation of HBV cccDNA-bound H3 histones. These results showed that NIRF is involved in the HBV replication cycle not only through direct interaction with HBc but also reduces acetylation of HBV cccDNA-bound H3 histones.

  12. Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection.

    PubMed

    Ferretti, Lorenza P; Himmels, Sarah-Felicitas; Trenner, Anika; Walker, Christina; von Aesch, Christine; Eggenschwiler, Aline; Murina, Olga; Enchev, Radoslav I; Peter, Matthias; Freire, Raimundo; Porro, Antonio; Sartori, Alessandro A

    2016-08-26

    Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein-protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity.

  13. Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase

    SciTech Connect

    Wang, Yechun; Yi, Hankuil; Wang, Melissa; Yu, Oliver; Jez, Joseph M.

    2012-10-24

    To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the first molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 {angstrom} of each other provides the basis for enhanced in vivo synthesis of resveratrol.

  14. An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination.

    PubMed Central

    Mackey, Z B; Ramos, W; Levin, D S; Walter, C A; McCarrey, J R; Tomkinson, A E

    1997-01-01

    Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase. PMID:9001252

  15. Peroxisomal ubiquitin-protein ligases peroxin2 and peroxin10 have distinct but synergistic roles in matrix protein import and peroxin5 retrotranslocation in Arabidopsis.

    PubMed

    Burkhart, Sarah E; Kao, Yun-Ting; Bartel, Bonnie

    2014-11-01

    Peroxisomal matrix proteins carry peroxisomal targeting signals (PTSs), PTS1 or PTS2, and are imported into the organelle with the assistance of peroxin (PEX) proteins. From a microscopy-based screen to identify Arabidopsis (Arabidopsis thaliana) mutants defective in matrix protein degradation, we isolated unique mutations in PEX2 and PEX10, which encode ubiquitin-protein ligases anchored in the peroxisomal membrane. In yeast (Saccharomyces cerevisiae), PEX2, PEX10, and a third ligase, PEX12, ubiquitinate a peroxisome matrix protein receptor, PEX5, allowing the PEX1 and PEX6 ATP-hydrolyzing enzymes to retrotranslocate PEX5 out of the membrane after cargo delivery. We found that the pex2-1 and pex10-2 Arabidopsis mutants exhibited defects in peroxisomal physiology and matrix protein import. Moreover, the pex2-1 pex10-2 double mutant exhibited severely impaired growth and synergistic physiological defects, suggesting that PEX2 and PEX10 function cooperatively in the wild type. The pex2-1 lesion restored the unusually low PEX5 levels in the pex6-1 mutant, implicating PEX2 in PEX5 degradation when retrotranslocation is impaired. PEX5 overexpression altered pex10-2 but not pex2-1 defects, suggesting that PEX10 facilitates PEX5 retrotranslocation from the peroxisomal membrane. Although the pex2-1 pex10-2 double mutant displayed severe import defects of both PTS1 and PTS2 proteins into peroxisomes, both pex2-1 and pex10-2 single mutants exhibited clear import defects of PTS1 proteins but apparently normal PTS2 import. A similar PTS1-specific pattern was observed in the pex4-1 ubiquitin-conjugating enzyme mutant. Our results indicate that Arabidopsis PEX2 and PEX10 cooperate to support import of matrix proteins into plant peroxisomes and suggest that some PTS2 import can still occur when PEX5 retrotranslocation is slowed.

  16. Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization

    NASA Astrophysics Data System (ADS)

    Perdih, Andrej; Hrast, Martina; Pureber, Kaja; Barreteau, Hélène; Grdadolnik, Simona Golič; Kocjan, Darko; Gobec, Stanislav; Solmajer, Tom; Wolber, Gerhard

    2015-06-01

    Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/ d-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/ d-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8- 11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.

  17. Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection

    PubMed Central

    Ferretti, Lorenza P.; Himmels, Sarah-Felicitas; Trenner, Anika; Walker, Christina; von Aesch, Christine; Eggenschwiler, Aline; Murina, Olga; Enchev, Radoslav I.; Peter, Matthias; Freire, Raimundo; Porro, Antonio; Sartori, Alessandro A.

    2016-01-01

    Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein–protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity. PMID:27561354

  18. Clomipramine causes osteoporosis by promoting osteoclastogenesis via E3 ligase Itch, which is prevented by Zoledronic acid

    PubMed Central

    Li, Xing; Sun, Wen; Li, Jinbo; Wang, Mengmeng; Zhang, Hengwei; Pei, Lingpeng; Boyce, Brendan F.; Wang, Zhiyu; Xing, Lianping

    2017-01-01

    Patients taking antidepressants, including Clomipramine (CLP), have an increased risk of osteoporotic fracture. However, the effects of CLP on bone metabolism are unknown. Here, we demonstrate that WT mice treated with CLP for 2 weeks had significantly reduced trabecular bone volume and cortical bone thickness, associated with increased osteoclast (OC) numbers, but had no change in osteoblast numbers or bone formation rate. Bone marrow cells from CLP-treated mice had normal OC precursor frequency, but formed significantly more OCs when they were cultured with RANKL and M-CSF. CLP promoted OC formation and bone resorption and expression of OC-associated genes. CLP-induced bone loss was prevented by Zoledronic acid. At the molecular level, CLP inhibited the activity of the ubiquitin E3 ligase Itch. CLP did not promote OC formation from bone marrow cells of Itch−/− mice in vitro nor induce bone loss in Itch−/− mice. Our findings indicate that CLP causes bone loss by enhancing Itch-mediated osteoclastogenesis, which was prevented by Zoledronic acid. Thus, anti-resorptive therapy could be used to prevent bone loss in patients taking antidepressants, such as CLP. PMID:28145497

  19. Multiple interactions drive adaptor-mediated recruitment of the ubiquitin ligase rsp5 to membrane proteins in vivo and in vitro.

    PubMed

    Sullivan, James A; Lewis, Michael J; Nikko, Elina; Pelham, Hugh R B

    2007-07-01

    Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain "PY" motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY-WW interactions is required for the ubiquitination of Smf1.

  20. Inhibition of the ubiquitin ligase activity improves the production of biologically active fusion protein HSA-HGF in Chinese hamster ovary cells.

    PubMed

    Xu, Dongsheng; Wan, Aini; Zhang, Jingjing; Peng, Lin; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian

    2016-10-18

    Hepatocyte growth factor (HGF) is a potent multi-functional protein that stimulates proliferation, survival, motility, scattering and differentiation during growth and development, and has been considered to be a potential therapeutic agent for the treatment of a number of intractable diseases. The aim of this study was to enhance the expression of recombinant fusion protein HSA-HGF (R494E) in CHO cells by inhibiting the intracellular ubiquitin ligase activity. The high stable expression sub-clones with different signal peptides were selected by western blot (WB) analysis and used for suspension culture. We found that the expression of fusion protein HSA-HGF (R494E) on day 3 achieved 50 mg/L during the 8 day culture process, a large number of fusion proteins were intracellular degradated by ubiquitination pathway during day 4 to day 8. Furthermore, ubiquitin ligase inhibitor, thalidomide, was added in culture process, and resulted in efficient and stable secretion of HSA-HGF (R494E) in CHO cells. According to biological activity assays, HSA-HGF (R494E) possessed various biological activities similar to native HGF. In conclusion, innhibition of intracellular ubiquitin ligase activity was successfully improve the expression of biologically active fusion protein HSA-HGF (R494E) in CHO cells. Our data may be beneficial to enhance the production of other therapeutic proteins in fed-batch culture.

  1. A CUL-2 ubiquitin ligase containing three FEM proteins degrades TRA-1 to regulate C. elegans sex determination

    PubMed Central

    Starostina, Natalia G.; Lim, Jae-min; Schvarzstein, Mara; Wells, Lance; Spence, Andrew M.; Kipreos, Edward T.

    2007-01-01

    Summary In Caenorhabditis elegans, the Gli-family transcription factor TRA-1 is the terminal effector of the sex determination pathway. TRA-1 activity inhibits male development and allows female fates. Genetic studies have indicated that TRA-1 is negatively regulated by the fem-1, fem-2, and fem-3 genes. However, the mechanism of this regulation has not been understood. Here, we present data that TRA-1 is regulated by degradation mediated by a CUL-2-based ubiquitin ligase complex that contains FEM-1 as the substrate-recognition subunit, and FEM-2 and FEM-3 as cofactors. CUL-2 physically associates with both FEM-1 and TRA-1 in vivo, and cul-2 mutant males share feminization phenotypes with fem mutants. CUL-2 and the FEM proteins negatively regulate TRA-1 protein levels in C. elegans. When expressed in human cells, the FEM proteins interact with human CUL2 and induce the proteasome-dependent degradation of TRA-1. This work demonstrates that the terminal step in C. elegans sex determination is controlled by ubiquitin-mediated proteolysis. PMID:17609115

  2. Protein–Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP)*

    PubMed Central

    Narayan, Vikram; Landré, Vivien; Ning, Jia; Hernychova, Lenka; Muller, Petr; Verma, Chandra; Walkinshaw, Malcolm D.; Blackburn, Elizabeth A.; Ball, Kathryn L.

    2015-01-01

    CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. PMID:26330542

  3. Ubiquitin ligase gene neurl3 plays a role in spermatogenesis of half-smooth tongue sole (Cynoglossus semilaevis) by regulating testis protein ubiquitination.

    PubMed

    Xu, Wenteng; Li, Hailong; Dong, Zhongdian; Cui, Zhongkai; Zhang, Ning; Meng, Liang; Zhu, Ying; Liu, Yang; Li, Yangzhen; Guo, Hua; Ma, Jialu; Wei, Zhanfei; Zhang, Nianwei; Yang, Yingming; Chen, Songlin

    2016-10-30

    E3 ubiquitin ligases are a large gene family that plays a diversity of roles in spermatogenesis. In this study, the functional characterization of a neuralized E3 ubiquitin protein ligase 3 (neurl3) revealed its potential participation in spermatogenesis. Firstly, we found that neurl3 exhibited male-biased transcription and that its translation was predominant in testis germ cells. The knockdown of neurl3 by RNA interference caused increased transcription of spermatogenesis-related genes. These results corroborate previous studies indicating a role for neurl3 in spermatogenesis. Moreover, the levels of neurl3 transcription and testis protein ubiquitination were closely correlated. Based on these findings, we speculate that neurl3 modulates testis protein ubiquitination in a dosage-dependent manner and that this influences spermatogenesis.

  4. The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic acid, a side-chain precursor for cephem antibiotics.

    PubMed

    Koetsier, Martijn J; Gombert, Andreas K; Fekken, Susan; Bovenberg, Roel A L; van den Berg, Marco A; Kiel, Jan A K W; Jekel, Peter A; Janssen, Dick B; Pronk, Jack T; van der Klei, Ida J; Daran, Jean-Marc

    2010-01-01

    Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the beta-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-aminopenicillanic acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins.

  5. DNA and RNA ligases: structural variations and shared mechanisms.

    PubMed

    Pascal, John M

    2008-02-01

    DNA and RNA ligases join 3' OH and 5' PO4 ends in polynucleotide substrates using a three-step reaction mechanism that involves covalent modification of both the ligase enzyme and the polynucleotide substrate with AMP. In the past three years, several polynucleotide ligases have been crystallized in complex with nucleic acid, providing the introductory views of ligase enzymes engaging their substrates. Crystal structures for two ATP-dependent DNA ligases, an NAD+-dependent DNA ligase, and an ATP-dependent RNA ligase demonstrate how ligases utilize the AMP group and their multi-domain architectures to manipulate nucleic acid structure and catalyze the end-joining reaction. Together with unliganded crystal structures of DNA and RNA ligases, a more comprehensive and dynamic understanding of the multi-step ligation reaction mechanism has emerged.

  6. E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells

    PubMed Central

    Samant, Rahul S.; Clarke, Paul A.; Workman, Paul

    2014-01-01

    The molecular chaperone heat shock protein 90 (HSP90) is required for the activity and stability of its client proteins. Pharmacologic inhibition of HSP90 leads to the ubiquitin-mediated degradation of clients, particularly activated or mutant oncogenic protein kinases. Client ubiquitination occurs via the action of one or more E3 ubiquitin ligases. We sought to identify the role of Cullin-RING family E3 ubiquitin ligases in the cellular response to HSP90 inhibition. Through a focused siRNA screen of 28 Cullin-RING ligase family members, we found that CUL5 and RBX2 were required for degradation of several HSP90 clients upon treatment of human cancer cells with the clinical HSP90 inhibitor 17-AAG. Surprisingly, silencing Cullin-5 (CUL5) also delayed the earlier loss of HSP90 client protein activity at the same time as delaying cochaperone dissociation from inhibited HSP90–client complexes. Expression of a dominant-negative CUL5 showed that NEDD8 conjugation of CUL5 is required for client degradation but not for loss of client activity or recruitment of clients and HSP90 to CUL5. Silencing CUL5 reduced cellular sensitivity to three distinct HSP90 inhibitors, across four cancer types driven by different protein kinases. Our results reveal the importance of CUL5 in multiple aspects of the cellular response to HSP90 inhibition. PMID:24760825

  7. Ubiquitin ligase RNF20/40 facilitates spindle assembly and promotes breast carcinogenesis through stabilizing motor protein Eg5

    PubMed Central

    Duan, Yang; Huo, Dawei; Gao, Jie; Wu, Heng; Ye, Zheng; Liu, Zhe; Zhang, Kai; Shan, Lin; Zhou, Xing; Wang, Yue; Su, Dongxue; Ding, Xiang; Shi, Lei; Wang, Yan; Shang, Yongfeng; Xuan, Chenghao

    2016-01-01

    Whether transcriptional regulators are functionally involved in mitosis is a fundamental question in cell biology. Here we report that the RNF20/40 complex, a major ubiquitin ligase catalysing histone H2B monoubiquitination, interacts with the motor protein Eg5 during mitosis and participates in spindle assembly. We show that the RNF20/40 complex monoubiquitinates and stabilizes Eg5. Loss of RNF20/40 results in spindle assembly defects, cell cycle arrest and apoptosis. Consistently, depletion of either RNF20/40 or Eg5 suppresses breast cancer in vivo. Significantly, RNF20/40 and Eg5 are concurrently upregulated in human breast carcinomas and high Eg5 expression is associated with poorer overall survival of patients with luminal A, or B, breast cancer. Our study uncovers an important spindle assembly role of the RNF20/40 complex, and implicates the RNF20/40-Eg5 axis in breast carcinogenesis, supporting the pursuit of these proteins as potential targets for breast cancer therapeutic interventions. PMID:27557628

  8. The E3 ligase axotrophin/MARCH-7: protein expression profiling of human tissues reveals links to adult stem cells.

    PubMed

    Szigyarto, Cristina A; Sibbons, Paul; Williams, Gill; Uhlen, Mathias; Metcalfe, Su M

    2010-04-01

    Axotrophin/MARCH-7 was first identified in mouse embryonic stem cells as a neural stem cell gene. Using the axotrophin/MARCH-7 null mouse, we discovered profound effects on T lymphocyte responses, including 8-fold hyperproliferation and 5-fold excess release of the stem cell cytokine leukemia inhibitory factor (LIF). Our further discovery that axotrophin/MARCH-7 is required for targeted degradation of the LIF receptor subunit gp190 implies a direct role in the regulation of LIF signaling. Bioinformatics studies revealed a highly conserved RING-CH domain in common with the MARCH family of E3-ubiquitin ligases, and accordingly, axotrophin was renamed "MARCH-7." To probe protein expression of human axotrophin/MARCH-7, we prepared antibodies against different domains of the protein. Each antibody bound its specific target epitope with high affinity, and immunohistochemistry cross-validated target specificity. Forty-eight human tissue types were screened. Epithelial cells stained strongly, with trophoblasts having the greatest staining. In certain tissues, specific cell types were selectively positive, including neurons and neuronal progenitor cells in the hippocampus and cerebellum, endothelial sinusoids of the spleen, megakaryocytes in the bone marrow, crypt stem cells of the small intestine, and alveolar macrophages in the lung. Approximately 20% of central nervous system neuropils were positive. Notably, axotrophin/MARCH-7 has an expression profile that is distinct from that of other MARCH family members. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

  9. Structural and biochemical basis for ubiquitin ligase recruitment by arrestin-related domain-containing protein-3 (ARRDC3).

    PubMed

    Qi, Shiqian; O'Hayre, Morgan; Gutkind, J Silvio; Hurley, James H

    2014-02-21

    After protracted stimulation, the β2-adrenergic receptor and many other G-protein-coupled receptors are ubiquitinated and down-regulated. Arrestin-related domain-containing protein-3 (ARRDC3) has been proposed to recruit the ubiquitin ligase Nedd4 to the β2-adrenergic receptor. ARRDC3 contains two PPXY motifs that could potentially interact with any of the four WW domains of Nedd4. Here we dissect the interaction determinants. ARRDC3 PPXY-Nedd4 WW dissociation constants vary from unmeasurable to Kd = 3 μM for the third WW domain of Nedd4 binding to the first PPXY motif of ARRDC3. Structures of the uncomplexed and PPXY1-bound WW3 domain were determined at 1.1 and 1.7 Å resolution. The structures revealed conformational changes upon binding and the hydrogen bonding network in exquisite detail. Tight packing of ARRDC3 Val-352', part of a 310 helix at the C terminus of PPXY1, is important for high affinity binding to WW3. Although no single WW domain is strictly essential for the binding of Nedd4 and ARRDC3 expressed in HEK293 cells, high affinity binding of full-length ARRDC3 and Nedd4 is driven by the avid interaction of both PPXY motifs with either the WW2-WW3 or WW3-WW4 combinations, with Kd values as low as 300 nM.

  10. Pub1 acts as an E6-AP-like protein ubiquitiin ligase in the degradation of cdc25.

    PubMed Central

    Nefsky, B; Beach, D

    1996-01-01

    The level of the mitotic activating tyrosine phosphatase cdc25 is regulated by both transcriptional and post-transcriptional mechanisms in the fission yeast Schizosaccharomyces pombe. We have found that cdc25 is ubiquitinated and have cloned pub1, a gene which regulates this event. Pub1 contains a region highly homologous to the putative catalytic domain of the human protein ubiquitin ligase E6-AP. Disruption of pub1 elevates the level of cdc25 protein in vivo rendering cells relatively resistant to the cdc25-opposing tyrosine kinases wee1 and mik1. In addition, loss of wee1 activity in a pub1-disruption background results in a lethal premature entry into mitosis which can be rescued by loss of cdc25 function. A ubiquitin-thioester adduct of pub1 was isolated from fission yeast and disruption of pub1 dramatically reduced ubiquitination of cdc25 in vivo. These results suggest that pub1 directly ubiquitinates cdc25 in vivo. Images PMID:8635463

  11. SCF ubiquitin ligase targeted therapies

    PubMed Central

    Skaar, Jeffrey R.; Pagan, Julia K.; Pagano, Michele

    2015-01-01

    Summary The recent clinical successes of inhibitors of the proteasome for the treatment of cancer have highlighted the therapeutic potential of this protein degradation system. Proteasome inhibitors prevent the degradation of numerous proteins, so increased specificity could be achieved by inhibiting the components of the ubiquitin-proteasome system that target specific subsets of proteins for degradation. F-box proteins are the substrate-targeting subunits of SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complexes. Through the degradation of a plethora of diverse substrates, SCF ubiquitin ligases control a large number of processes at the cellular and organismal levels, and their misregulation is implicated in many pathologies. SCF ligases are characterized by a high specificity for their substrates, so they represent promising drug targets. However, the potential for therapeutic manipulation of SCF complexes remains an underdeveloped area. This review will explore and discuss potential strategies to target SCF-mediated biology to treat human diseases. PMID:25394868

  12. Identification of a Protein Network Interacting with TdRF1, a Wheat RING Ubiquitin Ligase with a Protective Role against Cellular Dehydration1[C][W

    PubMed Central

    Guerra, Davide; Mastrangelo, Anna Maria; Lopez-Torrejon, Gema; Marzin, Stephan; Schweizer, Patrick; Stanca, Antonio Michele; del Pozo, Juan Carlos; Cattivelli, Luigi; Mazzucotelli, Elisabetta

    2012-01-01

    Plants exploit ubiquitination to modulate the proteome with the final aim to ensure environmental adaptation and developmental plasticity. Ubiquitination targets are specifically driven to degradation through the action of E3 ubiquitin ligases. Genetic analyses have indicated wide functions of ubiquitination in plant life; nevertheless, despite the large number of predicted E3s, only a few of them have been characterized so far, and only a few ubiquitination targets are known. In this work, we characterized durum wheat (Triticum durum) RING Finger1 (TdRF1) as a durum wheat nuclear ubiquitin ligase. Moreover, its barley (Hordeum vulgare) homolog was shown to protect cells from dehydration stress. A protein network interacting with TdRF1 has been defined. The transcription factor WHEAT BEL1-TYPE HOMEODOMAIN1 (WBLH1) was degraded in a TdRF1-dependent manner through the 26S proteasome in vivo, the mitogen-activated protein kinase TdWNK5 [for Triticum durum WITH NO LYSINE (K)5] was able to phosphorylate TdRF1 in vitro, and the RING-finger protein WHEAT VIVIPAROUS-INTERACTING PROTEIN2 (WVIP2) was shown to have a strong E3 ligase activity. The genes coding for the TdRF1 interactors were all responsive to cold and/or dehydration stress, and a negative regulative function in dehydration tolerance was observed for the barley homolog of WVIP2. A role in the control of plant development was previously known, or predictable based on homology, for wheat BEL1-type homeodomain1(WBLH1). Thus, TdRF1 E3 ligase might act regulating the response to abiotic stress and remodeling plant development in response to environmental constraints. PMID:22167118

  13. The ubiquitin-protein ligase Nedd4 targets Notch1 in skeletal muscle and distinguishes the subset of atrophies caused by reduced muscle tension.

    PubMed

    Koncarevic, Alan; Jackman, Robert W; Kandarian, Susan C

    2007-02-01

    Ubiquitination-dependent proteolysis is a fundamental process underlying skeletal muscle atrophy. Thus, the role of ubiquitin ligases is of great interest. There are no focused studies in muscle on the ubiquitin ligase Nedd4. We first confirmed increased mRNA expression in rat soleus muscles due to 1-14 days of hind limb unloading. Nedd4 protein localized to the sarcolemmal region of muscle fibers. Hind limb unloading, sciatic nerve denervation, starvation, and diabetes led to atrophy of soleus, plantaris, and gastrocnemius muscles, but only unloaded and denervated muscles showed a marked increase in Nedd4 protein expression. This increase was strongly correlated with decreased Notch1 expression, a known target of Nedd4 in other cell types. Overexpression of dominant negative Nedd4 in soleus muscles completely reversed the unloading-induced decrease of Notch1 expression, indicating that Nedd4 is required for Notch1 inactivation. Overexpression of wild-type Nedd4 in soleus muscles of weight bearing rats caused a decrease in Notch1 protein, indicating that Nedd4 is sufficient for Notch1 down-regulation. To further show that Notch1 is a Nedd4 substrate in muscle, conditional overexpression of Nedd4 in C2C12 myotubes induced ubiquitination of Notch1. This is the first finding of a Nedd4 substrate in muscle and of an ubiquitin ligase, the activity of which distinguishes disuse from cachexia atrophy.

  14. CRL4-DCAF1 ubiquitin E3 ligase directs protein phosphatase 2A degradation to control oocyte meiotic maturation.

    PubMed

    Yu, Chao; Ji, Shu-Yan; Sha, Qian-Qian; Sun, Qing-Yuan; Fan, Heng-Yu

    2015-08-18

    Oocyte meiosis is a specialized cell cycle that gives rise to fertilizable haploid gametes and is precisely controlled in various dimensions. We recently found that E3 ubiquitin ligase CRL4 is required for female fertility by regulating DNA hydroxymethylation to maintain oocyte survival and to promote zygotic genome reprogramming. However, not all phenotypes of CRL4-deleted oocytes could be explained by this mechanism. Here we show that CRL4 controls oocyte meiotic maturation by proteasomal degradation of protein phosphatase 2A scaffold subunit, PP2A-A. Oocyte-specific deletion of DDB1 or DCAF1 (also called VPRBP) results in delayed meiotic resumption and failure to complete meiosis I along with PP2A-A accumulation. DCAF1 directly binds to and results in the poly-ubiquitination of PP2A-A. Moreover, combined deletion of Ppp2r1a rescues the meiotic defects caused by DDB1/DCAF1 deficiency. These results provide in vivo evidence that CRL4-directed PP2A-A degradation is physiologically essential for regulating oocyte meiosis and female fertility.

  15. The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch.

    PubMed

    Oberst, Andrew; Malatesta, Martina; Aqeilan, Rami I; Rossi, Mario; Salomoni, Paolo; Murillas, Rodolfo; Sharma, Prashant; Kuehn, Michael R; Oren, Moshe; Croce, Carlo M; Bernassola, Francesca; Melino, Gerry

    2007-07-03

    Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin-protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73 alpha, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73 alpha for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73 alpha and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1(-/-) cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73 alpha and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy.

  16. Protein and amino acid nutrition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dairy cow protein and amino acid nutrition have a significant role in sustainable dairying. Protein, amino acids, and nitrogen are inextricably linked through effects in the rumen, metabolism of the cow, and environmental nutrient management. Feeding systems have been making progress toward emphasiz...

  17. ATL9, a RING Zinc Finger Protein with E3 Ubiquitin Ligase Activity Implicated in Chitin- and NADPH Oxidase-Mediated Defense Responses

    PubMed Central

    Berrocal-Lobo, Marta; Stone, Sophia; Yang, Xin; Antico, Jay; Callis, Judy; Ramonell, Katrina M.; Somerville, Shauna

    2010-01-01

    Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 ( = ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst. PMID:21203445

  18. Quality Control of Plasma Membrane Proteins by Saccharomyces cerevisiae Nedd4-Like Ubiquitin Ligase Rsp5p under Environmental Stress Conditions

    PubMed Central

    Shiga, Takeki; Yoshida, Nobuyuki; Shimizu, Yuko; Suzuki, Etsuko; Sasaki, Toshiya; Watanabe, Daisuke

    2014-01-01

    In Saccharomyces cerevisiae, when a rich nitrogen source such as ammonium is added to the culture medium, the general amino acid permease Gap1p is ubiquitinated by the yeast Nedd4-like ubiquitin ligase Rsp5p, followed by its endocytosis to the vacuole. The arrestin-like Bul1/2p adaptors for Rsp5p specifically mediate this process. In this study, to investigate the downregulation of Gap1p in response to environmental stresses, we determined the intracellular trafficking of Gap1p under various stress conditions. An increase in the extracellular ethanol concentration induced ubiquitination and trafficking of Gap1p from the plasma membrane to the vacuole in wild-type cells, whereas Gap1p remained stable on the plasma membrane under the same conditions in rsp5A401E and Δend3 cells. A 14C-labeled citrulline uptake assay using a nonubiquitinated form of Gap1p (Gap1pK9R/K16R) revealed that ethanol stress caused a dramatic decrease of Gap1p activity. These results suggest that Gap1p is inactivated and ubiquitinated by Rsp5p for endocytosis when S. cerevisiae cells are exposed to a high concentration of ethanol. It is noteworthy that this endocytosis occurs in a Bul1/2p-independent manner, whereas ammonium-triggered downregulation of Gap1p was almost completely inhibited in Δbul1/2 cells. We also found that other environmental stresses, such as high temperature, H2O2, and LiCl, also promoted endocytosis of Gap1p. Similar intracellular trafficking caused by ethanol occurred in other plasma membrane proteins (Agp1p, Tat2p, and Gnp1p). Our findings suggest that stress-induced quality control is a common process requiring Rsp5p for plasma membrane proteins in yeast. PMID:25001409

  19. Cyclin F/FBXO1 Interacts with HIV-1 Viral Infectivity Factor (Vif) and Restricts Progeny Virion Infectivity by Ubiquitination and Proteasomal Degradation of Vif Protein through SCF(cyclin F) E3 Ligase Machinery.

    PubMed

    Augustine, Tracy; Chaudhary, Priyanka; Gupta, Kailash; Islam, Sehbanul; Ghosh, Payel; Santra, Manas Kumar; Mitra, Debashis

    2017-03-31

    Cyclin F protein, also known as FBXO1, is the largest among all cyclins and oscillates in the cell cycle like other cyclins. Apart from being a G2/M cyclin, cyclin F functions as the substrate-binding subunit of SCF(cyclin F) E3 ubiquitin ligase. In a gene expression analysis performed to identify novel gene modulations associated with cell cycle dysregulation during HIV-1 infection in CD4(+) T cells, we observed down-regulation of the cyclin F gene (CCNF). Later, using gene overexpression and knockdown studies, we identified cyclin F as negatively influencing HIV-1 viral infectivity without any significant impact on virus production. Subsequently, we found that cyclin F negatively regulates the expression of viral protein Vif (viral infectivity factor) at the protein level. We also identified a novel host-pathogen interaction between cyclin F and Vif protein in T cells during HIV-1 infection. Mutational analysis of a cyclin F-specific amino acid motif in the C-terminal region of Vif indicated rescue of the protein from cyclin F-mediated down-regulation. Subsequently, we showed that Vif is a novel substrate of the SCF(cyclin F) E3 ligase, where cyclin F mediates the ubiquitination and proteasomal degradation of Vif through physical interaction. Finally, we showed that cyclin F augments APOBEC3G expression through degradation of Vif to regulate infectivity of progeny virions. Taken together, our results demonstrate that cyclin F is a novel F-box protein that functions as an intrinsic cellular regulator of HIV-1 Vif and has a negative regulatory effect on the maintenance of viral infectivity by restoring APOBEC3G expression.

  20. E3 ligase FLRF (Rnf41) regulates differentiation of hematopoietic progenitors by governing steady-state levels of cytokine and retinoic acid receptors

    PubMed Central

    Jing, Xin; Infante, Jorge; Nachtman, Ronald G.; Jurecic, Roland

    2008-01-01

    Objective FLRF (Rnf41) gene was identified through screening of subtracted cDNA libraries form murine hematopoietic stem cells and progenitors. Subsequent work has revealed that FLRF acts as E3 ubiquitin ligase, and that it regulates steady-state levels of neuregulin receptor ErbB3, and participates in degradation of IAP protein BRUCE and parkin. The objective of this study was to start exploring the role of FLRF during hematopoiesis. Methods FLRF was over-expressed in a murine multipotent hematopoietic progenitor cell line EML, which can differentiate into almost all blood cell lineages, and in pro-B progenitor cell line BaF3. The impact of FLRF over-expression on EML cell differentiation into myelo-erythroid lineages was studied using hematopoietic colony-forming assays. The interaction of FLRF with cytokine receptors and receptor levels in control cells and EML and BaF3 cells over-expressing FLRF were examined with Western and immunoprecipitation. Results Remarkably, over-expression of FLRF significantly attenuated erythroid and myeloid differentiation of EML cells in response to cytokines Epo and IL-3, and retinoic acid (RA), and resulted in significant and constitutive decrease of steady-state levels of IL-3, Epo and RA receptor RARα in EML and BaF3 cells. Immunoprecipitation has revealed that FLRF interacts with IL-3, Epo and RARα receptors in EML and BaF3 cells, and that FLRF-mediated down-regulation of these receptors is ligand binding-independent. Conclusions The results of this study have revealed new FLRF-mediated pathway for ligand-independent receptor level regulation, and support the notion that through maintaining basal levels of cytokine receptors, FLRF is involved in the control of hematopoietic progenitor cell differentiation into myelo-erythroid lineages. PMID:18495327

  1. Characterization of a long-chain fatty acid-CoA ligase 1 gene and association between its SNPs and growth traits in the clam Meretrix meretrix.

    PubMed

    Dai, Ping; Huan, Pin; Wang, Hongxia; Lu, Xia; Liu, Baozhong

    2015-07-25

    Long-chain fatty acid-CoA ligases (ACSLs) play crucial roles in fatty acid (FA) metabolism. They convert free long-chain FA into acyl-CoAs, which are key intermediates in both anabolic and catabolic pathways. A long-chain fatty acid-CoA ligase gene was cloned in the clam Meretrix meretrix (MmeACSL1), with a full-length cDNA of 1865 bp encoding 475 amino acids. Its expression was only detected in hepatopancreas by semi-quantitative reverse transcription PCR. Expression level of MmeACSL1 exhibited a significant increase in a starvation experiment (P<0.05). This indicates that MmeACSL1 plays an important role in normal metabolism of M. meretrix and may be involved in energy supply and storage. Two exon SNPs and six intron SNPs were developed in this gene by direct sequencing. A marker-trait association analysis showed that five of these SNPs were significantly associated with growth traits (P<0.05). Also, haplotypes comprised of the five SNPs were revealed to be significantly growth-related (P<0.05), which further corroborates the relationship of this gene to growth traits of M. meretrix.

  2. Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3

    SciTech Connect

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2007-04-01

    A truncated form of biotin carboxyl carrier protein containing the C-terminal half fragment (BCCPΔN76) and the biotin protein ligase (BPL) with the mutation R48A (BPL*) or the double mutation R48A K111A (BPL**) were successfully cocrystallized in the presence of ATP and biotin. The BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals belong to space group P2{sub 1} and diffract X-rays to 2.7 and 2.0 Å resolution, respectively. Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein–protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPΔN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*–BCCPΔN76 and BPL**–BCCPΔN76 complexes as well as crystals of BPL*, BPL** and BCCPΔN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals were collected at 100 K to 2.7 and 2.0 Å resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2{sub 1}, with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 Å, β = 95.9°. Assuming two subunits of the complex per asymmetric unit gives a V{sub M} value of 2.45 Å{sup 3} Da{sup −1} and a solvent content of 50%.

  3. p53 E3 ubiquitin protein ligase homolog regulates p53 in vivo in the adult mouse eye lens

    PubMed Central

    Jaramillo-Rangel, Gilberto; Ortega-Martínez, Marta; Sepúlveda-Saavedra, Julio; Saucedo-Cárdenas, Odila; Montes-de-Oca-Luna, Roberto

    2013-01-01

    Purpose p53 is a transcription factor that plays an important role in preventing cancer development. p53 participates in relevant aspects of cell biology, including apoptosis and cell cycle control and must be strictly regulated to maintain normal tissue homeostasis. p53 E3 ubiquitin protein ligase homolog (Mdm2) is an important negative regulator of p53. The purpose of this study was to determine if Mdm2 regulates p53 in vivo in the adult lens. Methods We analyzed mice expressing human p53 transgene (Tgp53) selectively in the lens in the presence or absence of Mdm2. Mice with the required genotypes were obtained by crossing transgenic, mdm2+/−, and p53−/− mice. Eye phenotype and lens histology and ultrastructure were analyzed in adult mice. Results In a wild-type genetic background (mdm2+/+), lens damage and microphthalmia were observed only in mice homozygous for Tgp53 (t/t). However, in an mdm2 null background, just one allele of Tgp53 (mdm2−/−/Tgp53t/0 mice) was sufficient to cause lens damage and microphthalmia. Furthermore, Mdm2 in only one allele was sufficient to rescue these deleterious effects, since the mdm2+/−/Tgp53t/0 mice had eye size and lens morphology similar to the control mice. Conclusions Mdm2 regulates p53 in the adult lens in vivo. This information may have relevance for analyzing normal and pathological conditions of the lens, and designing cancer therapies targeting Mdm2–p53 interaction. PMID:24339722

  4. A Novel Retinoblastoma Protein (RB) E3 Ubiquitin Ligase (NRBE3) Promotes RB Degradation and Is Transcriptionally Regulated by E2F1 Transcription Factor.

    PubMed

    Wang, Yingshuang; Zheng, Zongfang; Zhang, Jingyi; Wang, You; Kong, Ruirui; Liu, Jiangying; Zhang, Ying; Deng, Hongkui; Du, Xiaojuan; Ke, Yang

    2015-11-20

    Retinoblastoma protein (RB) plays critical roles in tumor suppression and is degraded through the proteasomal pathway. However, E3 ubiquitin ligases responsible for proteasome-mediated degradation of RB are largely unknown. Here we characterize a novel RB E3 ubiquitin ligase (NRBE3) that binds RB and promotes RB degradation. NRBE3 contains an LXCXE motif and bound RB in vitro. NRBE3 interacted with RB in cells when proteasome activity was inhibited. NRBE3 promoted RB ubiquitination and degradation via the ubiquitin-proteasome pathway. Importantly, purified NRBE3 ubiquitinated recombinant RB in vitro, and a U-box was identified as essential for its E3 activity. Surprisingly, NRBE3 was transcriptionally activated by E2F1/DP1. Consequently, NRBE3 affected the cell cycle by promoting G1/S transition. Moreover, NRBE3 was up-regulated in breast cancer tissues. Taken together, we identified NRBE3 as a novel ubiquitin E3 ligase for RB that might play a role as a potential oncoprotein in human cancers.

  5. PARAQUAT TOLERANCE3 Is an E3 Ligase That Switches off Activated Oxidative Response by Targeting Histone-Modifying PROTEIN METHYLTRANSFERASE4b

    PubMed Central

    Du, Jin; Zhao, Tao-Lan; Wang, Peng-Fei; Zhao, Ping-Xia; Xie, Qi; Cao, Xiao-Feng; Xiang, Cheng-Bin

    2016-01-01

    Oxidative stress is unavoidable for aerobic organisms. When abiotic and biotic stresses are encountered, oxidative damage could occur in cells. To avoid this damage, defense mechanisms must be timely and efficiently modulated. While the response to oxidative stress has been extensively studied in plants, little is known about how the activated response is switched off when oxidative stress is diminished. By studying Arabidopsis mutant paraquat tolerance3, we identified the genetic locus PARAQUAT TOLERANCE3 (PQT3) as a major negative regulator of oxidative stress tolerance. PQT3, encoding an E3 ubiquitin ligase, is rapidly down-regulated by oxidative stress. PQT3 has E3 ubiquitin ligase activity in ubiquitination assay. Subsequently, we identified PRMT4b as a PQT3-interacting protein. By histone methylation, PRMT4b upregulates the expression of APX1 and GPX1, encoding two key enzymes against oxidative stress. On the other hand, PRMT4b is recognized by PQT3 for targeted degradation via 26S proteasome. Therefore, we have identified PQT3 as an E3 ligase that acts as a negative regulator of activated response to oxidative stress and found that histone modification by PRMT4b at APX1 and GPX1 loci plays an important role in oxidative stress tolerance. PMID:27676073

  6. DNA-ligase IV and DNA-protein kinase play a critical role in deficient caspases activation in apoptosis-resistant cancer cells by using doxorubicin.

    PubMed

    Friesen, Claudia; Uhl, Miriam; Pannicke, Ulrich; Schwarz, Klaus; Miltner, Erich; Debatin, Klaus-Michael

    2008-08-01

    Resistance toward cytotoxic drugs is one of the primary causes for therapeutic failure in cancer therapy. DNA repair mechanisms as well as deficient caspases activation play a critical role in apoptosis resistance of tumor cells toward anticancer drug treatment. Here, we discovered that deficient caspases activation in apoptosis-resistant cancer cells depends on DNA-ligase IV and DNA-protein kinase (DNA-PK), playing crucial roles in the nonhomologous end joining (NHEJ) pathway, which is the predominant pathway for DNA double-strand break repair (DNA-DSB-repair) in mammalian cells. DNA-PK(+/+) as well as DNA-ligase IV (+/+) cancer cells were apoptosis resistant and deficient in activation of caspase-3, caspase-9, and caspase-8 and in cleavage of poly(ADP-ribose) polymerase after doxorubicin treatment. Inhibition of NHEJ by knocking out DNA-PK or DNA-ligase IV restored caspases activation and apoptosis sensitivity after doxorubicin treatment. In addition, inhibition of caspases activation prevented doxorubicin-induced apoptosis but could not prevent doxorubicin-induced DNA damage, indicating that induction of DNA damage is independent of caspases activation. However, caspases activation depends on induction of DNA damage left unrepaired by NHEJ-DNA-DSB-repair. We conclude that DNA damage left unrepaired by DNA-ligase IV or DNA-PK might be the initiator for caspases activation by doxorubicin in cancer cells. Failure in caspases activation using doxorubicin depends on loss of DNA damage and is due to higher rates of NHEJ-DNA-DBS-repair.

  7. The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development*♦

    PubMed Central

    Rahman, Kazi; Zhao, Peng; Mandalasi, Msano; van der Wel, Hanke; Wells, Lance; Blader, Ira J.; West, Christopher M.

    2016-01-01

    Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts. PMID:26719340

  8. The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development.

    PubMed

    Rahman, Kazi; Zhao, Peng; Mandalasi, Msano; van der Wel, Hanke; Wells, Lance; Blader, Ira J; West, Christopher M

    2016-02-26

    Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.

  9. Ube3a, the E3 ubiquitin ligase causing Angelman syndrome and linked to autism, regulates protein homeostasis through the proteasomal shuttle Rpn10.

    PubMed

    Lee, So Young; Ramirez, Juanma; Franco, Maribel; Lectez, Benoît; Gonzalez, Monika; Barrio, Rosa; Mayor, Ugo

    2014-07-01

    Ubiquitination, the covalent attachment of ubiquitin to a target protein, regulates most cellular processes and is involved in several neurological disorders. In particular, Angelman syndrome and one of the most common genomic forms of autism, dup15q, are caused respectively by lack of or excess of UBE3A, a ubiquitin E3 ligase. Its Drosophila orthologue, Ube3a, is also active during brain development. We have now devised a protocol to screen for substrates of this particular ubiquitin ligase. In a neuronal cell system, we find direct ubiquitination by Ube3a of three proteasome-related proteins Rpn10, Uch-L5, and CG8209, as well as of the ribosomal protein Rps10b. Only one of these, Rpn10, is targeted for degradation upon ubiquitination by Ube3a, indicating that degradation might not be the only effect of Ube3a on its substrates. Furthermore, we report the genetic interaction in vivo between Ube3a and the C-terminal part of Rpn10. Overexpression of these proteins leads to an enhanced accumulation of ubiquitinated proteins, further supporting the biochemical evidence of interaction obtained in neuronal cells.

  10. BTB-BACK Domain Protein POB1 Suppresses Immune Cell Death by Targeting Ubiquitin E3 ligase PUB17 for Degradation

    PubMed Central

    Mesmar, Joelle; McLellan, Hazel; Yang, Chengwei; Craig, Adam; Zhang, Cunjin; Moore, Jonathan David; Tian, Zhendong; Birch, Paul R. J.; Sadanandom, Ari

    2017-01-01

    Hypersensitive response programmed cell death (HR-PCD) is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses. PMID:28056034

  11. Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration.

    PubMed

    Del Prete, Dolores; Rice, Richard C; Rajadhyaksha, Anjali M; D'Adamio, Luciano

    2016-08-12

    The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the NH2 terminus of the ACR, and CRL4(CRBN), which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the COOH terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4(CRBN), pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome analysis indicates that these E3 ligases are enzymatically active and ubiquitinate the ACR residues Lys(649/650/651/676/688) Deletion of Crbn reduces ubiquitination of Lys(676) suggesting that Lys(676) is physiologically ubiquitinated by CRL4(CRBN) The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible.

  12. Ectopic expression of PtaRHE1, encoding a poplar RING-H2 protein with E3 ligase activity, alters plant development and induces defence-related responses

    PubMed Central

    Mukoko Bopopi, Johnny; Vandeputte, Olivier M.; Himanen, Kristiina; Mol, Adeline; Vaessen, Quentin; El Jaziri, Mondher; Baucher, Marie

    2010-01-01

    RING (really interesting new gene)-H2 domain-containing proteins are widely represented in plants and play important roles in the regulation of many developmental processes as well as in plant–environment interactions. In the present report, experiments were performed to unravel the role of the poplar gene PtaRHE1, coding for a RING-H2 protein. In vitro ubiquitination assays indicate a functional E3 ligase activity for PtaRHE1 with the specific E2 ubiquitin-conjugating enzyme UbcH5a. The overexpression of PtaRHE1 in tobacco resulted in a pleiotropic phenotype characterized by a curling of the leaves, the formation of necrotic lesions on leaf blades, growth retardation, and a delay in floral transition. The plant gene expression response to PtaRHE1 overexpression provided evidence for the up-regulation of defence- and/or programmed cell death-related genes. Moreover, genes coding for WRKY transcription factors as well as for mitogen-activated protein kinases, such as wound-induced protein kinase (WIPK), were also found to be induced in the transgenic lines as compared with the wild type. In addition, histochemical β-glucuronidase staining showed that the PtaRHE1 promoter is induced by plant pathogens and by elicitors such as salicylic acid and cellulase. Taken together, these results suggest that the E3 ligase PtaRHE1 plays a role in the ubiquitination-mediated regulation of defence response, possibly by acting upstream of WIPK and/or in the activation of WRKY factors. PMID:19892745

  13. Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 Is Critical for Plant Carbon/Nitrogen Nutrient Balance Response and Controls the Stability of 14-3-3 Proteins*

    PubMed Central

    Yasuda, Shigetaka; Sato, Takeo; Maekawa, Shugo; Aoyama, Shoki; Fukao, Yoichiro; Yamaguchi, Junji

    2014-01-01

    Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr209, Ser247, Ser270, and Ser303 as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr209 and Ser247 on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr209 peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response. PMID:24722992

  14. Hey bHLH Proteins Interact with a FBXO45 Containing SCF Ubiquitin Ligase Complex and Induce Its Translocation into the Nucleus.

    PubMed

    Salat, Daniela; Winkler, Anja; Urlaub, Henning; Gessler, Manfred

    2015-01-01

    The Hey protein family, comprising Hey1, Hey2 and HeyL in mammals, conveys Notch signals in many cell types. The helix-loop-helix (HLH) domain as well as the Orange domain, mediate homo- and heterodimerization of these transcription factors. Although distinct interaction partners have been identified so far, their physiological relevance for Hey functions is still largely unclear. Using a tandem affinity purification approach and mass spectrometry analysis we identified members of an ubiquitin E3-ligase complex consisting of FBXO45, PAM and SKP1 as novel Hey1 associated proteins. There is a direct interaction between Hey1 and FBXO45, whereas FBXO45 is needed to mediate indirect Hey1 binding to SKP1. Expression of Hey1 induces translocation of FBXO45 and PAM into the nucleus. Hey1 is a short-lived protein that is degraded by the proteasome, but there is no evidence for FBXO45-dependent ubiquitination of Hey1. On the contrary, Hey1 mediated nuclear translocation of FBXO45 and its associated ubiquitin ligase complex may extend its spectrum to additional nuclear targets triggering their ubiquitination. This suggests a novel mechanism of action for Hey bHLH factors.

  15. SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus

    PubMed Central

    Jöhnk, Bastian; Bayram, Özgür; Heinekamp, Thorsten; Mattern, Derek J.; Brakhage, Axel A.; Jacobsen, Ilse D.; Valerius, Oliver; Braus, Gerhard H.

    2016-01-01

    F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus. PMID:27649508

  16. Cellular DNA ligase I is recruited to cytoplasmic vaccinia virus factories and masks the role of the vaccinia ligase in viral DNA replication.

    PubMed

    Paran, Nir; De Silva, Frank S; Senkevich, Tatiana G; Moss, Bernard

    2009-12-17

    Vaccinia virus (VACV) encodes DNA polymerase and additional proteins that enable cytoplasmic replication. We confirmed the ability of VACV DNA ligase mutants to replicate and tested the hypothesis that cellular ligases compensate for loss of viral gene expression. RNA silencing of human DNA ligase I expression and a small molecule inhibitor of human DNA ligase I [corrected] severely reduced replication of viral DNA in cells infected with VACV ligase-deficient mutants, indicating that the cellular enzyme plays a complementary role. Replication of ligase-deficient VACV was greatly reduced and delayed in resting primary cells, correlating with initial low levels of ligase I and subsequent viral induction and localization of ligase I in virus factories. These studies indicate that DNA ligation is essential for poxvirus replication and explain the ability of ligase deletion mutants to replicate in dividing cells but exhibit decreased pathogenicity in mice. Encoding its own ligase might allow VACV to "jump-start" DNA synthesis.

  17. Production of resveratrol from p-coumaric acid in recombinant Saccharomyces cerevisiae expressing 4-coumarate:coenzyme A ligase and stilbene synthase genes.

    PubMed

    Shin, So-Yeon; Han, Nam Soo; Park, Yong-Cheol; Kim, Myoung-Dong; Seo, Jin-Ho

    2011-01-05

    Resveratrol is a well-known polyphenol present in red wine and exerts antioxidative and anti-carcinogenic effects on the human body. To produce resveratrol in a food-grade yeast, the 4-coumarate:coenzyme A ligase gene (4CL1) from Arabidopsis thaliana and stilbene synthase gene (STS) from Arachis hypogaea were cloned and transformed into Saccharomyces cerevisiae W303-1A. The resveratrol produced was unglycosylated and secreted into the culture medium. A batch culture with 15.3mg/l p-coumaric acid used as precursor resulted in the production of 3.1mg/l resveratrol with 14.4 mol% yield. Deletion of the putative phenyl acrylic acid decarboxylase gene (PAD1) did not enhance resveratrol production.

  18. Molecular Basis of Interactions Between SH3 Domain-Containing Proteins and the Proline-Rich Region of the Ubiquitin Ligase Itch.

    PubMed

    Desrochers, Guillaume; Cappadocia, Laurent; Lussier-Price, Mathieu; Ton, Anh-Tien; Ayoubi, Riham; Serohijos, Adrian; Omichinski, James G; Angers, Annie

    2017-02-24

    The ligase Itch plays major roles in signalling pathways by inducing ubiquitylation-dependent degradation of several substrates. Substrate recognition and binding is critical for the regulation of this reaction. Like closely related ligases, Itch can interact with proteins containing a PPxY motif via its WW domains. In addition to these WW domains, Itch possesses a proline-rich region (PRR) that has been shown to interact with several Src Homology 3 (SH3) domain-containing proteins. We have previously established that despite the apparent surface uniformity and conserved fold of SH3 domains, they display different binding mechanisms and affinities for their interaction with the PRR of Itch. Here, we attempt to determine the molecular bases underlying the wide range of binding properties of the Itch PRR. Using pull-down assays combined with mass spectrometry analysis, we show that the Itch PRR preferentially forms complexes with Endophilins, Amphyphisins and Pacsins, but can also target a variety of other SH3 domain-containing proteins. In addition, we map the binding sites of these proteins using a combination of PRR sub-sequences and mutants. We find that different SH3 domains target distinct proline-rich sequences overlapping significantly. We also structurally analyze these protein complexes using crystallography and molecular modelling. These structures depict the position of Itch PRR engaged in a 1:2 protein complex with β-PIX and a 1:1 complex with the other SH3 domain-containing proteins. Taken together, these results reveal the binding preferences of the Itch PRR towards its most common SH3 domain-containing partners, and demonstrate that the PRR region is sufficient for binding.

  19. Partial suppression of bacteriophage T4 ligase mutations by T4 endonuclease II deficiency: role of host ligase.

    PubMed

    Warner, H R

    1971-04-01

    Endonuclease II-deficient, ligase-deficient double mutants of phage T4 induce considerably more deoxyribonucleic acid (DNA) synthesis after infection of Escherichia coli B than does the ligase-deficient single mutant. Furthermore, the double mutant can replicate 10 to 15% as well as wild-type T4, whereas the single mutant fails to replicate. When the E. coli host is also deficient in ligase, the double mutant resembles the single mutant. The results indicate that host ligase can substitute for phage ligase when the host DNA is not attacked by the phage-induced endonuclease II.

  20. The SUMO E3 Ligase-Like Proteins PIAL1 and PIAL2 Interact with MOM1 and Form a Novel Complex Required for Transcriptional Silencing.

    PubMed

    Han, Yong-Feng; Zhao, Qiu-Yuan; Dang, Liang-Liang; Luo, Yu-Xi; Chen, Shan-Shan; Shao, Chang-Rong; Huang, Huan-Wei; Li, Yong-Qiang; Li, Lin; Cai, Tao; Chen, She; He, Xin-Jian

    2016-05-01

    The mechanism by which MORPHEUS' MOLECULE1 (MOM1) contributes to transcriptional gene silencing has remained elusive since the gene was first identified and characterized. Here, we report that two Arabidopsis thaliana PIAS (PROTEIN INHIBITOR OF ACTIVATED STAT)-type SUMO E3 ligase-like proteins, PIAL1 and PIAL2, function redundantly to mediate transcriptional silencing at MOM1 target loci. PIAL1 and PIAL2 physically interact with each other and with MOM1 to form a high molecular mass complex. In the absence of either PIAL2 or MOM1, the formation of the high molecular mass complex is disrupted. We identified a previously uncharacterized IND (interacting domain) in PIAL1 and PIAL2 and demonstrated that IND directly interacts with MOM1. The CMM2 (conserved MOM1 motif 2) domain of MOM1 was previously shown to be required for the dimerization of MOM1. We demonstrated that the CMM2 domain is also required for the interaction of MOM1 with PIAL1 and PIAL2. We found that although PIAL2 has SUMO E3 ligase activity, the activity is dispensable for PIAL2's function in transcriptional silencing. This study suggests that PIAL1 and PIAl2 act as components of the MOM1-containing complex to mediate transcriptional silencing at heterochromatin regions.

  1. Yeast sterol regulatory element-binding protein (SREBP) cleavage requires Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing subunit of the Golgi Dsc E3 ligase.

    PubMed

    Stewart, Emerson V; Lloyd, S Julie-Ann; Burg, John S; Nwosu, Christine C; Lintner, Robert E; Daza, Riza; Russ, Carsten; Ponchner, Karen; Nusbaum, Chad; Espenshade, Peter J

    2012-01-02

    Schizosaccharomyces pombe Sre1 is a membrane-bound transcription factor that controls adaptation to hypoxia. Like its mammalian homolog, sterol regulatory element-binding protein (SREBP), Sre1 activation requires release from the membrane. However, in fission yeast, this release occurs through a strikingly different mechanism that requires the Golgi Dsc E3 ubiquitin ligase complex and the proteasome. The mechanistic details of Sre1 cleavage, including the link between the Dsc E3 ligase complex and proteasome, are not well understood. Here, we present results of a genetic selection designed to identify additional components required for Sre1 cleavage. From the selection, we identified two new components of the fission yeast SREBP pathway: Dsc5 and Cdc48. The AAA (ATPase associated with diverse cellular activities) ATPase Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing protein, interact with known Dsc complex components and are required for SREBP cleavage. These findings provide a mechanistic link between the Dsc E3 ligase complex and the proteasome in SREBP cleavage and add to a growing list of similarities between the Dsc E3 ligase and membrane E3 ligases involved in endoplasmic reticulum-associated degradation.

  2. The tomato DWD motif-containing protein DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase and plays a pivotal role in abiotic stress responses

    SciTech Connect

    Miao, Min; Zhu, Yunye; Qiao, Maiju; Tang, Xiaofeng; Zhao, Wei; Xiao, Fangming; Liu, Yongsheng

    2014-08-08

    Highlights: • We identify DDI1 as a DAMAGED DNA BINDING PROTEIN1 (DDB1)-interacting protein. • DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase in the nucleus. • DDI1 plays a positive role in regulating abiotic stress response in tomato. - Abstract: CULLIN4(CUL4)–DAMAGED DNA BINDING PROTEIN1 (DDB1)-based ubiquitin ligase plays significant roles in multiple physiological processes via ubiquitination-mediated degradation of relevant target proteins. The DDB1–CUL4-associated factor (DCAF) acts as substrate receptor in the CUL4–DDB1 ubiquitin ligase complex and determines substrate specificity. In this study, we identified a tomato (Solanum lycopersicum) DDB1-interacting (DDI1) protein as a DCAF protein involved in response to abiotic stresses, including UV radiation, high salinity and osmotic stress. Co-immunoprecipitation and bimolecular fluorescence complementation assay indicated that DDI1 associates with CUL4–DDB1 in the nucleus. Quantitative RT-PCR analysis indicated the DDI1 gene is induced by salt, mannitol and UV-C treatment. Moreover, transgenic tomato plants with overexpression or knockdown of the DDI1 gene exhibited enhanced or attenuated tolerance to salt/mannitol/UV-C, respectively. Thus, our data suggest that DDI1 functions as a substrate receptor of the CUL4–DDB1 ubiquitin ligase, positively regulating abiotic stress response in tomato.

  3. Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis

    PubMed Central

    Dong, Hui; Dumenil, Jack; Lu, Fu-Hao; Na, Li; Vanhaeren, Hannes; Naumann, Christin; Klecker, Maria; Prior, Rachel; Smith, Caroline; McKenzie, Neil; Saalbach, Gerhard; Chen, Liangliang; Xia, Tian; Gonzalez, Nathalie; Seguela, Mathilde; Inzé, Dirk; Dissmeyer, Nico; Li, Yunhai; Bevan, Michael W.

    2017-01-01

    The characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana. The peptidase is activated by two RING E3 ligases, Big Brother (BB) and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PROTEOLYSIS 1 (PRT1) of the N-end rule pathway. DA1 peptidase activity also cleaves the deubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TEOSINTE BRANCED 1/CYCLOIDEA/PCF 15 (TCP15) and TCP22, which promote cell proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins. PMID:28167503

  4. Quantifying protein by bicinchoninic Acid.

    PubMed

    Simpson, Richard J

    2008-08-01

    INTRODUCTIONThis protocol describes a method of quantifying protein that is a variation of the Lowry assay. It uses bicinchoninic acid (BCA) to enhance the detection of Cu(+) generated under alkaline conditions at sites of complexes between Cu(2+) and protein. The resulting chromophore absorbs at 562 nm. This technique is divided into three parts: Standard Procedure, Microprocedure, and 96-Well Microtiter Plate Procedure. For each procedure, test samples are assayed in parallel with protein standards that are used to generate a calibration curve, and the exact concentration of protein in the test samples is interpolated. The standard BCA assay uses large volumes of both reagents and samples and cannot easily be automated. If these issues are important, the Microprocedure is recommended. This in turn can be adapted for use with a microplate reader in the 96-Well Microtiter Plate Procedure. If the microplate reader is interfaced with a computer, more than 1000 samples can be read per hour.

  5. Nucleic acid amplification tests (polymerase chain reaction, ligase chain reaction) for the diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae in pediatric emergency medicine.

    PubMed

    Corneli, Howard M

    2005-04-01

    Nucleic acid amplification tests, such as ligase chain reaction and polymerase chain reaction, offer potential advantages of speed, simplicity, and accuracy in the detection of genitourinary tract infection with Neisseria gonorrhoeae and Chlamydia trachomatis. Their appropriate use in pediatric emergency medicine depends on an understanding of their strengths and weaknesses. Problems arise in defining the sensitivity and, especially, specificity of these tests. The clinical scenario, the site of infection, the age and sex of the patient, and especially the presence or absence of medicolegal concerns strongly affect the applicability of these tests. The risk of false positives may be significant even when legal concerns do not arise and even if a highly specific test is used. This article reviews the uses and limitations of such tests in pediatric emergency medicine. Discussion is directed to both technical and practical considerations.

  6. SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1

    PubMed Central

    Brown, James R.; Conn, Kristen L.; Wasson, Peter; Charman, Matthew; Tong, Lily; Grant, Kyle; McFarlane, Steven

    2016-01-01

    ABSTRACT Aspects of intrinsic antiviral immunity are mediated by promyelocytic leukemia nuclear body (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is the small ubiquitin-like modifier (SUMO) signaling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligase protein inhibitor of activated STAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in a SUMO interaction motif (SIM)-dependent manner that requires SUMOylated or SUMOylation-competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well-characterized PML-NB proteins. In contrast to that of Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation. IMPORTANCE Adaptive, innate, and intrinsic immunity cooperatively and efficiently restrict the propagation of viral pathogens. Intrinsic immunity mediated by constitutively expressed cellular proteins represents the first line of intracellular defense against

  7. Structure-Guided Design and Optimization of Small Molecules Targeting the Protein–Protein Interaction between the von Hippel–Lindau (VHL) E3 Ubiquitin Ligase and the Hypoxia Inducible Factor (HIF) Alpha Subunit with in Vitro Nanomolar Affinities

    PubMed Central

    2014-01-01

    E3 ubiquitin ligases are attractive targets in the ubiquitin–proteasome system, however, the development of small-molecule ligands has been rewarded with limited success. The von Hippel–Lindau protein (pVHL) is the substrate recognition subunit of the VHL E3 ligase that targets HIF-1α for degradation. We recently reported inhibitors of the pVHL:HIF-1α interaction, however they exhibited moderate potency. Herein, we report the design and optimization, guided by X-ray crystal structures, of a ligand series with nanomolar binding affinities. PMID:25166285

  8. Differential response between the p53 ubiquitin-protein ligases Pirh2 and MdM2 following DNA damage in human cancer cells

    SciTech Connect

    Duan Wenrui; Gao, Li; Wu Xin; Zhang Yang; Otterson, Gregory A.; Villalona-Calero, Miguel A. . E-mail: Miguel.villalona@osumc.edu

    2006-10-15

    Pirh2, a recently identified ubiquitin-protein ligase, has been reported to promote p53 degradation. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of MDM2. Like MDM2, Pirh2 is thought to participate in an autoregulatory feedback loop that controls p53 function. We have previously reported that Pirh2 was overexpressed in human and murine lung cancers as compared to uninvolved lung tissue. Pirh2 increase could potentially cause degradation of wildtype p53 and reduce its tumor suppression function in the lung tumor cells. Since Pirh2 has been reported to be transactivated by p53, however, the mechanisms by which a high level of Pirh2 expression is maintained in tumor cells despite low level of wildtype p53 protein are unclear. In order to evaluate p53 involvement in the transactivation of Pirh2, we evaluated Pirh2, MDM2, p53 and p21 expression with Western blot analysis and real time PCR after {gamma} irradiation or cisplatin DNA damage treatment using human cancer cell lines containing wildtype (A549, MCF-7), mutant (H719) and null (H1299) p53. Surprisingly, Pirh2 expression was not affected by the presence of wildtype p53 in the cancer cells. In contrast, MDM2 was upregulated by wildtype p53 in A549 and MCF-7 cells and was absent from the H1299 and the H719 cells. We conclude that Pirh2 operates in a distinct manner from MDM2 in response to DNA damage in cancer cells. Pirh2 elevation in p53 null cells indicates the existence of additional molecular mechanisms for Pirh2 upregulation and suggests that p53 is not the sole target of Pirh2 ubiquitin ligase activity.

  9. Cardiac Nav 1.5 is modulated by ubiquitin protein ligase E3 component n-recognin UBR3 and 6.

    PubMed

    Zhao, Chunxia; Wang, Lijie; Ma, Xiue; Zhu, Weidong; Yao, Lei; Cui, Yingyu; Liu, Yi; Li, Jun; Liang, Xingqun; Sun, Yunfu; Li, Li; Chen, Yi-Han

    2015-09-01

    The voltage-gated Na(+) channel Nav 1.5 is essential for action potential (AP) formation and electrophysiological homoeostasis in the heart. The ubiquitin-proteasome system (UPS) is a major degradative system for intracellular proteins including ion channels. The ubiquitin protein ligase E3 component N-recognin (UBR) family is a part of the UPS; however, their roles in regulating cardiac Nav 1.5 channels remain elusive. Here, we found that all of the UBR members were expressed in cardiomyocytes. Individual knockdown of UBR3 or UBR6, but not of other UBR members, significantly increased Nav 1.5 protein levels in neonatal rat ventricular myocytes, and this effect was verified in HEK293T cells expressing Nav 1.5 channels. The UBR3/6-dependent regulation of Nav 1.5 channels was not transcriptionally mediated, and pharmacological inhibition of protein biosynthesis failed to counteract the increase in Nav 1.5 protein caused by UBR3/6 reduction, suggesting a degradative modulation of UBR3/6 on Nav 1.5. Furthermore, the effects of UBR3/6 knockdown on Nav 1.5 proteins were abolished under the inhibition of proteasome activity, and UBR3/6 knockdown reduced Nav 1.5 ubiquitylation. The double UBR3-UBR6 knockdown resulted in comparable increases in Nav 1.5 proteins to that observed for single knockdown of either UBR3 or UBR6. Electrophysiological recordings showed that UBR3/6 reduction-mediated increase in Nav 1.5 protein enhanced the opening of Nav 1.5 channels and thereby the amplitude of the AP. Thus, our findings indicate that UBR3/6 regulate cardiomyocyte Nav 1.5 channel protein levels via the ubiquitin-proteasome pathway. It is likely that UBR3/6 have the potential to be a therapeutic target for cardiac arrhythmias.

  10. Reduced association of anti-apoptotic protein Mcl-1 with E3 ligase Mule increases the stability of Mcl-1 in breast cancer cells

    PubMed Central

    Pervin, S; Tran, A; Tran, L; Urman, R; Braga, M; Chaudhuri, G; Singh, R

    2011-01-01

    Background: Mechanisms that increase resistance to apoptosis help promote cellular transformation. Cancer cells have deregulated apoptotic pathways, where increased expression and stability of anti-apoptotic proteins Mcl-1 and Bcl-2 increases resistance to apoptosis. Pathways that increase the stability of proteins in cancer cells remain poorly understood. Methods: Using human mammary epithelial and established breast cancer cell lines, we assessed the mechanisms that increase the stability of anti-apoptotic proteins in breast cancer cells by caspase assay, western blot, small-inhibitory RNA treatment and immunoprecipitation. Results: While breast cancer cells were resistant to de novo inhibition of protein synthesis, a rapid proteosome-mediated degradation of Mcl-1 and Bcl-2 induced apoptosis in mammary epithelial cells. Although Mule, an E3 ligase that targets Mcl-1 for degradation was expressed in mammary epithelial and breast cancer cell lines, rapid increase of polyubiquitinated Mcl-1 and Bcl-2 was detected only in mammary epithelial cells. Only transient formation of the Mule–Mcl-1 complex was detected in breast cancer cells. Downregulation of pERK1/2 in breast cancer cells reduced Mcl-1 levels and increased Mcl-1/Mule complex. Conclusion: Our findings suggest that reduced Mule/Mcl-1 complex has a significant role in increasing the stability of Mcl-1 in breast cancer cells and increased resistance to apoptosis. PMID:21730980

  11. Peptidic degron in EID1 is recognized by an SCF E3 ligase complex containing the orphan F-box protein FBXO21

    PubMed Central

    Zhang, Cuiyan; Li, Xiaotong; Adelmant, Guillaume; Dobbins, Jessica; Geisen, Christoph; Oser, Matthew G.; Wucherpfenning, Kai W.; Marto, Jarrod A.; Kaelin, William G.

    2015-01-01

    EP300-interacting inhibitor of differentiation 1 (EID1) belongs to a protein family implicated in the control of transcription, differentiation, DNA repair, and chromosomal maintenance. EID1 has a very short half-life, especially in G0 cells. We discovered that EID1 contains a peptidic, modular degron that is necessary and sufficient for its polyubiquitylation and proteasomal degradation. We found that this degron is recognized by an Skp1, Cullin, and F-box (SCF)-containing ubiquitin ligase complex that uses the F-box Only Protein 21 (FBXO21) as its substrate recognition subunit. SCFFBXO21 polyubiquitylates EID1 both in vitro and in vivo and is required for the efficient degradation of EID1 in both cycling and quiescent cells. The EID1 degron partially overlaps with its retinoblastoma tumor suppressor protein-binding domain and is congruent with a previously defined melanoma-associated antigen-binding motif shared by EID family members, suggesting that binding to retinoblastoma tumor suppressor and melanoma-associated antigen family proteins could affect the polyubiquitylation and turnover of EID family members in cells. PMID:26631746

  12. The E3 ligase Ubr3 regulates Usher syndrome and MYH9 disorder proteins in the auditory organs of Drosophila and mammals

    PubMed Central

    Li, Tongchao; Giagtzoglou, Nikolaos; Eberl, Daniel F; Jaiswal, Sonal Nagarkar; Cai, Tiantian; Godt, Dorothea; Groves, Andrew K; Bellen, Hugo J

    2016-01-01

    Myosins play essential roles in the development and function of auditory organs and multiple myosin genes are associated with hereditary forms of deafness. Using a forward genetic screen in Drosophila, we identified an E3 ligase, Ubr3, as an essential gene for auditory organ development. Ubr3 negatively regulates the mono-ubiquitination of non-muscle Myosin II, a protein associated with hearing loss in humans. The mono-ubiquitination of Myosin II promotes its physical interaction with Myosin VIIa, a protein responsible for Usher syndrome type IB. We show that ubr3 mutants phenocopy pathogenic variants of Myosin II and that Ubr3 interacts genetically and physically with three Usher syndrome proteins. The interactions between Myosin VIIa and Myosin IIa are conserved in the mammalian cochlea and in human retinal pigment epithelium cells. Our work reveals a novel mechanism that regulates protein complexes affected in two forms of syndromic deafness and suggests a molecular function for Myosin IIa in auditory organs. DOI: http://dx.doi.org/10.7554/eLife.15258.001 PMID:27331610

  13. Haploinsufficiency of the E3 ubiquitin ligase C-terminus of heat shock cognate 70 interacting protein (CHIP) produces specific behavioral impairments.

    PubMed

    McLaughlin, Bethann; Buendia, Matthew A; Saborido, Tommy P; Palubinsky, Amy M; Stankowski, Jeannette N; Stanwood, Gregg D

    2012-01-01

    The multifunctional E3 ubiquitin ligase CHIP is an essential interacting partner of HSP70, which together promote the proteasomal degradation of client proteins. Acute CHIP overexpression provides neuroprotection against neurotoxic mitochondrial stress, glucocorticoids, and accumulation of toxic amyloid fragments, as well as genetic mutations in other E3 ligases, which have been shown to result in familial Parkinson's disease. These studies have created a great deal of interest in understanding CHIP activity, expression and modulation. While CHIP knockout mice have the potential to provide essential insights into the molecular control of cell fate and survival, the animals have been difficult to characterize in vivo due to severe phenotypic and behavioral dysfunction, which have thus far been poorly characterized. Therefore, in the present study we conducted a battery of neurobehavioral and physiological assays of adult CHIP heterozygotic (HET) mutant mice to provide a better understanding of the functional consequence of CHIP deficiency. We found that CHIP HET mice had normal body and brain weight, body temperature, muscle tone and breathing patterns, but do have a significant elevation in baseline heart rate. Meanwhile basic behavioral screens of sensory, motor, emotional and cognitive functions were normative. We observed no alterations in performance in the elevated plus maze, light-dark preference and tail suspension assays, or two simple cognitive tasks: novel object recognition and spontaneous alternation in a Y maze. Significant deficits were found, however, when CHIP HET mice performed wire hang, inverted screen, wire maneuver, and open field tasks. Taken together, our data indicate a clear subset of behaviors that are altered at baseline in CHIP deficient animals, which will further guide whole animal studies of the effects of CHIP dysregulation on cardiac function, brain circuitry and function, and responsiveness to environmental and cellular stress.

  14. Overexpression of a Soybean Ariadne-Like Ubiquitin Ligase Gene GmARI1 Enhances Aluminum Tolerance in Arabidopsis

    PubMed Central

    Zhang, Xiaolian; Wang, Ning; Chen, Pei; Gao, Mengmeng; Liu, Juge; Wang, Yufeng; Zhao, Tuanjie; Li, Yan; Gai, Junyi

    2014-01-01

    Ariadne (ARI) subfamily of RBR (Ring Between Ring fingers) proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene) finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L.) Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2–4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress. PMID:25364908

  15. Combined walking exercise and alkali therapy in patients with CKD4-5 regulates intramuscular free amino acid pools and ubiquitin E3 ligase expression.

    PubMed

    Watson, Emma L; Kosmadakis, George C; Smith, Alice C; Viana, Joao L; Brown, Jeremy R; Molyneux, Karen; Pawluczyk, Izabella Z A; Mulheran, Michael; Bishop, Nicolette C; Shirreffs, Susan; Maughan, Ronald J; Owen, Paul J; John, Stephen G; McIntyre, Christopher W; Feehally, John; Bevington, Alan

    2013-08-01

    Muscle-wasting in chronic kidney disease (CKD) arises from several factors including sedentary behaviour and metabolic acidosis. Exercise is potentially beneficial but might worsen acidosis through exercise-induced lactic acidosis. We studied the chronic effects of exercise in CKD stage 4-5 patients (brisk walking, 30 min, 5 times/week), and non-exercising controls; each group receiving standard oral bicarbonate (STD), or additional bicarbonate (XS) (Total n = 26; Exercising + STD n = 9; Exercising +XS n = 6; Control + STD n = 8; Control + XS n = 3). Blood and vastus lateralis biopsies were drawn at baseline and 6 months. The rise in blood lactate in submaximal treadmill tests was suppressed in the Exercising + XS group. After 6 months, intramuscular free amino acids (including the branched chain amino acids) in the Exercising + STD group showed a striking chronic depletion. This did not occur in the Exercising + XS group. The effect in Exercising + XS patients was accompanied by reduced transcription of ubiquitin E3-ligase MuRF1 which activates proteolysis via the ubiquitin-proteasome pathway. Other anabolic indicators (Akt activation and suppression of the 14 kDa actin catabolic marker) were unaffected in Exercising + XS patients. Possibly because of this, overall suppression of myofibrillar proteolysis (3-methylhistidine output) was not observed. It is suggested that alkali effects in exercisers arose by countering exercise-induced acidosis. Whether further anabolic effects are attainable on combining alkali with enhanced exercise (e.g. resistance exercise) merits further investigation.

  16. Peroxisomal Ubiquitin-Protein Ligases Peroxin2 and Peroxin10 Have Distinct But Synergistic Roles in Matrix Protein Import and Peroxin5 Retrotranslocation in Arabidopsis1[W][OPEN

    PubMed Central

    Burkhart, Sarah E.; Kao, Yun-Ting; Bartel, Bonnie

    2014-01-01

    Peroxisomal matrix proteins carry peroxisomal targeting signals (PTSs), PTS1 or PTS2, and are imported into the organelle with the assistance of peroxin (PEX) proteins. From a microscopy-based screen to identify Arabidopsis (Arabidopsis thaliana) mutants defective in matrix protein degradation, we isolated unique mutations in PEX2 and PEX10, which encode ubiquitin-protein ligases anchored in the peroxisomal membrane. In yeast (Saccharomyces cerevisiae), PEX2, PEX10, and a third ligase, PEX12, ubiquitinate a peroxisome matrix protein receptor, PEX5, allowing the PEX1 and PEX6 ATP-hydrolyzing enzymes to retrotranslocate PEX5 out of the membrane after cargo delivery. We found that the pex2-1 and pex10-2 Arabidopsis mutants exhibited defects in peroxisomal physiology and matrix protein import. Moreover, the pex2-1 pex10-2 double mutant exhibited severely impaired growth and synergistic physiological defects, suggesting that PEX2 and PEX10 function cooperatively in the wild type. The pex2-1 lesion restored the unusually low PEX5 levels in the pex6-1 mutant, implicating PEX2 in PEX5 degradation when retrotranslocation is impaired. PEX5 overexpression altered pex10-2 but not pex2-1 defects, suggesting that PEX10 facilitates PEX5 retrotranslocation from the peroxisomal membrane. Although the pex2-1 pex10-2 double mutant displayed severe import defects of both PTS1 and PTS2 proteins into peroxisomes, both pex2-1 and pex10-2 single mutants exhibited clear import defects of PTS1 proteins but apparently normal PTS2 import. A similar PTS1-specific pattern was observed in the pex4-1 ubiquitin-conjugating enzyme mutant. Our results indicate that Arabidopsis PEX2 and PEX10 cooperate to support import of matrix proteins into plant peroxisomes and suggest that some PTS2 import can still occur when PEX5 retrotranslocation is slowed. PMID:25214533

  17. DNA Ligase IV regulates XRCC4 nuclear localization.

    PubMed

    Francis, Dailia B; Kozlov, Mikhail; Chavez, Jose; Chu, Jennifer; Malu, Shruti; Hanna, Mary; Cortes, Patricia

    2014-09-01

    DNA Ligase IV, along with its interacting partner XRCC4, are essential for repairing DNA double strand breaks by non-homologous end joining (NHEJ). Together, they complete the final ligation step resolving the DNA break. Ligase IV is regulated by XRCC4 and XLF. However, the mechanism(s) by which Ligase IV control the NHEJ reaction and other NHEJ factor(s) remains poorly characterized. Here, we show that a C-terminal region of Ligase IV (aa 620-800), which encompasses a NLS, the BRCT I, and the XRCC4 interacting region (XIR), is essential for nuclear localization of its co-factor XRCC4. In Ligase IV deficient cells, XRCC4 showed deregulated localization remaining in the cytosol even after induction of DNA double strand breaks. DNA Ligase IV was also required for efficient localization of XLF into the nucleus. Additionally, human fibroblasts that harbor hypomorphic mutations within the Ligase IV gene displayed decreased levels of XRCC4 protein, implicating that DNA Ligase IV is also regulating XRCC4 stability. Our results provide evidence for a role of DNA Ligase IV in controlling the cellular localization and protein levels of XRCC4.

  18. DNA Ligase IV regulates XRCC4 nuclear localization

    PubMed Central

    Francis, Dailia B.; Kozlov, Mikhail; Chavez, Jose; Chu, Jennifer; Malu, Shruti; Hanna, Mary; Cortes, Patricia

    2014-01-01

    DNA Ligase IV, along with its interacting partner XRCC4, are essential for repairing DNA double strand breaks by non-homologous end joining (NHEJ). Together, they complete the final ligation step resolving the DNA break. Ligase IV is regulated by XRCC4 and XLF. However, the mechanism(s) by which Ligase IV control the NHEJ reaction and other NHEJ factor(s) remains poorly characterized. Here, we show that a C-terminal region of Ligase IV (aa 620 to 800), which encompasses a NLS, the BRCT I, and the XRCC4 interacting region (XIR), is essential for nuclear localization of its co-factor XRCC4. In Ligase IV deficient cells, XRCC4 showed deregulated localization remaining in the cytosol even after induction of DNA double strand breaks. DNA Ligase IV was also required for efficient localization of XLF into the nucleus. Additionally, human fibroblasts that harbor hypomorphic mutations within the Ligase IV gene displayed decreased levels of XRCC4 protein, implicating that DNA Ligase IV is also regulating XRCC4 stability. Our results provide evidence for a role of DNA Ligase IV in controlling the cellular localization and protein levels of XRCC4. PMID:24984242

  19. E3 Ligase SCFβTrCP-induced DYRK1A Protein Degradation Is Essential for Cell Cycle Progression in HEK293 Cells*

    PubMed Central

    Liu, Qiang; Tang, Yu; Chen, Long; Liu, Na; Lang, Fangfang; Liu, Heng; Wang, Pin; Sun, Xiulian

    2016-01-01

    DYRK1A, located on the Down syndrome (DS) critical region of chromosome 21, was found to be overexpressed in brains of DS and Alzheimer's disease individuals. DYRK1A was considered to play important roles in the pathogenesis of DS and Alzheimer's disease; however, the degradation mechanism of DYRK1A was still unclear. In this study, we found that DYRK1A was degraded through the ubiquitin-proteasome pathway in HEK293 cells. The N terminus of DYRK1A that was highly unstable in HEK293 cells contributed to proteolysis of DYRK1A. E3 ligase SCFβTrCP mediated ubiquitination and promoted degradation of DYRK1A through an unconserved binding motif (49SDQQVSALS57) lying in the N terminus. Any Ser-Ala substitution in this motif could decrease the binding between DYRK1A and β-transducin repeat containing protein (βTrCP), resulting in stabilization of DYRK1A. We also found DYRK1A protein was elevated in the G0/G1 phase and decreased in the S and G2/M phase, which was negatively correlated to βTrCP levels in the HEK293 cell cycle. Knockdown of βTrCP caused arrest of the G0/G1 phase, which could be partly rescued by down-regulation of DYRK1A. Our study uncovered a new regulatory mechanism of DYRK1A degradation by SCFβTrCP in HEK293 cell cycle progression. PMID:27807027

  20. Apoptosis by aloe-emodin is mediated through down-regulation of calpain-2 and ubiquitin-protein ligase E3A in human hepatoma Huh-7 cells.

    PubMed

    Jeon, Won; Jeon, Young Keul; Nam, Myeong Jin

    2012-02-01

    Natural flavonoids are associated with anti-proliferation of cancer growth. However, the antioxidant and anti-proliferation effects of AE (aloe-emodin) have not been well studied. We have investigated how AE affects the proliferation of hepatic hepatocellular carcinoma cells and exerts an anti-cancer effect. The cytotoxic effect of AE was demonstrated using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and Huh-7 cells were inhibited by AE treatment in both dose- and time-dependent manners. The IC(50) level of AE was ∼75 μM. AE also has anti-proliferative effects via induction of DNA damage and apoptosis. 2-DE (two-dimensional electrophoresis) revealed that several proteins were related to the anti-cancer effects of AE. CAPN2 (calpain-2) and UBE3A (ubiquitin-protein ligase E3A), which are associated with the apoptosis signalling pathway, were verified by Western blotting. AE exhibited potent anti-proliferative effects on Huh-7 cells via down-regulation of CAPN2 and UBE3A. The findings support the possibility of AE being a chemopreventative agent.

  1. Cancer stem-like cell related protein CD166 degrades through E3 ubiquitin ligase CHIP in head and neck cancer.

    PubMed

    Xiao, Meng; Yan, Ming; Zhang, Jianjun; Xu, Qin; Qi, Shengcai; Wang, Xu; Chen, Wantao

    2017-04-01

    Our previous studies have identified that CD166 works as a cancer stem-like cell (CSC) marker in epithelial cancers with a large repertoire of cellular functions. However, the post-translational regulatory mechanisms underlying CD166 turnover remain elusive. Several independent studies have reported that E3 ubiquitin ligase CHIP revealed significant biological effects through ubiquitin proteasome pathway on some kinds of malignant tumors. With analyzing the effects of CHIP expressions on stem-like cell populations, we found that CHIP represses CSC characteristics mainly targeting the CSC related protein CD166 in head and neck cancer (HNC). To investigate the role and relationship between CD166 and CHIP, HNC tissues and cell lines were used in this study. A significant negative correlation was observed between the expression levels of CHIP and CD166 in HNC patient samples. We also found that CHIP directly regulates the stability of CD166 protein through the ubiquitin proteasome system, which was also identified participating in the regulation of CSC behaviors in HNCs. Our findings demonstrate that CHIP-CD166-proteasome axis participates in regulating CSC properties in HNCs, suggesting that the regulation of CD166 by CHIP could provide new options for diagnosing and treating in the patients with HNCs.

  2. Integrated control of transporter endocytosis and recycling by the arrestin-related protein Rod1 and the ubiquitin ligase Rsp5

    PubMed Central

    Becuwe, Michel; Léon, Sébastien

    2014-01-01

    After endocytosis, membrane proteins can recycle to the cell membrane or be degraded in lysosomes. Cargo ubiquitylation favors their lysosomal targeting and can be regulated by external signals, but the mechanism is ill-defined. Here, we studied the post-endocytic trafficking of Jen1, a yeast monocarboxylate transporter, using microfluidics-assisted live-cell imaging. We show that the ubiquitin ligase Rsp5 and the glucose-regulated arrestin-related trafficking adaptors (ART) protein Rod1, involved in the glucose-induced internalization of Jen1, are also required for the post-endocytic sorting of Jen1 to the yeast lysosome. This new step takes place at the trans-Golgi network (TGN), where Rod1 localizes dynamically upon triggering endocytosis. Indeed, transporter trafficking to the TGN after internalization is required for their degradation. Glucose removal promotes Rod1 relocalization to the cytosol and Jen1 deubiquitylation, allowing transporter recycling when the signal is only transient. Therefore, nutrient availability regulates transporter fate through the localization of the ART/Rsp5 ubiquitylation complex at the TGN. DOI: http://dx.doi.org/10.7554/eLife.03307.001 PMID:25380227

  3. EXTRA-LARGE G PROTEINs Interact with E3 Ligases PUB4 and PUB2 and Function in Cytokinin and Developmental Processes1[OPEN

    PubMed Central

    Wang, Yiping; Wu, Yingying; Yu, Boying; Yin, Zhao

    2017-01-01

    Heterotrimeric GTP-binding proteins (G proteins) composed of Gα, Gβ, and Gγ subunits are conserved signal transduction molecules in animals and plants. In Arabidopsis (Arabidopsis thaliana), there are three Gα-like proteins named EXTRA-LARGE G PROTEINs (XLGs) in addition to the canonical Gα protein GPA1. XLGs have been reported to be implicated in multiple pathways, although the underlying mechanisms of their action remain elusive. Here, we report that all three XLGs interact with two closely related plant U-box (PUB) E3 ligases, PUB2 and PUB4. Three XLGs are predominantly localized at the plasma membrane, whereas XLG2 and XLG3 also show nuclear localization. The interactions between PUB2/4 and XLGs suggest that they might function in the same pathways. Indeed, we found that a newly obtained xlg1/2/3 triple knockout mutant, the pub4 mutant, and the pub2/4 double mutant all exhibited defects in cytokinin responses, stamen development, tapetum development, and male fertility. However, the xlg single mutants and the pub2 mutant did not exhibit an obvious defect in these processes, which suggests functional redundancy among the three XLGs and between PUB2 and PUB4. Overexpressing ARR10 to enhance the cytokinin response in pub4 or in the xlg1/2/3 triple mutant partially restored several phenotypes caused by the pub4 and xlg1/2/3 mutations. Our findings reveal that the XLGs and PUB2/4 are components in the complex cytokinin signaling networks regulating many developmental and physiological processes. PMID:27986866

  4. Reinvestigation of DNA ligase I in axolotl and Pleurodeles development.

    PubMed Central

    Aoufouchi, S; Hardy, S; Prigent, C; Philippe, M; Thiebaud, P

    1991-01-01

    We have recently shown that the exclusion process causing the replacement of DNA ligases II by DNA ligase I in amphibian eggs after fertilization does not occur in the case of Xenopus laevis [Hardy, S., Aoufouchi, S., Thiebaud, P., and Prigent, C., (1991) Nucleic Acids Res. 19, 701-705]. Since this result is in contradiction with the situation reported in axolotl and Pleurodeles we decided to reinvestigate such results in both species. Three different approaches have been used: (1) the substrate specificity of DNA ligase I; (2) the DNA ligase-AMP adduct reaction and (3) the immunological detection using antibodies raised against the X.laevis DNA ligase I. Our results clearly demonstrate that DNA ligase I activity is associated with a single polypeptide which is present in oocyte, unfertilized egg and embryo of both amphibians. Therefore, the hypothesis of a change in DNA ligase forms, resulting from an expression of the DNA ligase I gene in axolotl and Pleurodeles early development must be rejected. We also show that, in contradiction with published data, the unfertilized sea urchin egg contains a DNA ligase activity able to join blunt ended DNA molecules. Images PMID:1886765

  5. The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts.

    PubMed

    Wirbelauer, C; Sutterlüty, H; Blondel, M; Gstaiger, M; Peter, M; Reymond, F; Krek, W

    2000-10-16

    The ubiquitin protein ligase SCF(Skp2) is composed of Skp1, Cul1, Roc1/Rbx1 and the F-box protein Skp2, the substrate-recognition subunit. Levels of Skp2 decrease as cells exit the cell cycle and increase as cells re-enter the cycle. Ectopic expression of Skp2 in quiescent fibroblasts causes mitogen-independent S-phase entry. Hence, mechanisms must exist for limiting Skp2 protein expression during the G(0)/G(1) phases. Here we show that Skp2 is degraded by the proteasome in G(0)/G(1) and is stabilized when cells re-enter the cell cycle. Rapid degradation of Skp2 in quiescent cells depends on Skp2 sequences that contribute to Cul1 binding and interference with endogenous Cul1 function in serum-deprived cells induces Skp2 expression. Furthermore, recombinant Cul1-Roc1/Rbx1-Skp1 complexes can catalyse Skp2 ubiquitylation in vitro. These results suggest that degradation of Skp2 in G(0)/G(1) is mediated, at least in part, by an autocatalytic mechanism involving a Skp2-bound Cul1-based core ubiquitin ligase and imply a role for this mechanism in the suppression of SCF(Skp2) ubiquitin protein ligase function during the G(0)/G(1) phases of the cell cycle.

  6. Distinguishing Proteins From Arbitrary Amino Acid Sequences

    PubMed Central

    Yau, Stephen S.-T.; Mao, Wei-Guang; Benson, Max; He, Rong Lucy

    2015-01-01

    What kinds of amino acid sequences could possibly be protein sequences? From all existing databases that we can find, known proteins are only a small fraction of all possible combinations of amino acids. Beginning with Sanger's first detailed determination of a protein sequence in 1952, previous studies have focused on describing the structure of existing protein sequences in order to construct the protein universe. No one, however, has developed a criteria for determining whether an arbitrary amino acid sequence can be a protein. Here we show that when the collection of arbitrary amino acid sequences is viewed in an appropriate geometric context, the protein sequences cluster together. This leads to a new computational test, described here, that has proved to be remarkably accurate at determining whether an arbitrary amino acid sequence can be a protein. Even more, if the results of this test indicate that the sequence can be a protein, and it is indeed a protein sequence, then its identity as a protein sequence is uniquely defined. We anticipate our computational test will be useful for those who are attempting to complete the job of discovering all proteins, or constructing the protein universe. PMID:25609314

  7. The Matrix Protein of Nipah Virus Targets the E3-Ubiquitin Ligase TRIM6 to Inhibit the IKKε Kinase-Mediated Type-I IFN Antiviral Response

    PubMed Central

    Dawes, Brian E.; Yun, Tatyana E.; Park, Arnold; Yen, Benjamin; Basler, Christopher F.; Freiberg, Alexander N.; Lee, Benhur; Rajsbaum, Ricardo

    2016-01-01

    For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I) system. Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus), is known to encode for four P gene-derived viral proteins (P/C/W/V) with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M), which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε). We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258) in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNβ induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new potential target for

  8. Novel deletion of the E3A ubiquitin protein ligase gene detected by multiplex ligation-dependent probe amplification in a patient with Angelman syndrome

    PubMed Central

    Calì, Francesco; Ragalmuto, Alda; Chiavetta, Valeria; Calabrese, Giuseppe; Fichera, Marco; Vinci, Mirella; Ruggeri, Giuseppa; Schinocca, Pietro; Sturnio, Maurizio; Romano, Salvatore; Elia, Maurizio

    2010-01-01

    Angelman syndrome (AS) is a severe neurobehavioural disorder caused by failure of expression of the maternal copy of the imprinted domain located on 15q11-q13. There are different mechanisms leading to AS: maternal microdeletion, uniparental disomy, defects in a putative imprinting centre, mutations of the E3 ubiquitin protein ligase (UBE3A) gene. However, some of suspected cases of AS are still scored negative to all the latter mutations. Recently, it has been shown that a proportion of negative cases bear large deletions overlapping one or more exons of the UBE3A gene. These deletions are difficult to detect by conventional gene-scanning methods due to the masking effect by the non-deleted allele. In this study, we have used for the first time multiplex ligation-dependent probe amplification (MLPA) and comparative multiplex dosage analysis (CMDA) to search for large deletions affecting the UBE3A gene. Using this approach, we identified a novel causative deletion involving exon 8 in an affected sibling. Based on our results, we propose the use of MLPA as a fast, accurate and inexpensive test to detect large deletions in the UBE3A gene in a small but significant percentage of AS patients. PMID:21072004

  9. Protein Kinase C (PKC)-promoted Endocytosis of Glutamate Transporter GLT-1 Requires Ubiquitin Ligase Nedd4-2-dependent Ubiquitination but Not Phosphorylation*

    PubMed Central

    García-Tardón, Noemí; González-González, Inmaculada M.; Martínez-Villarreal, Jaime; Fernández-Sánchez, Enrique; Giménez, Cecilio; Zafra, Francisco

    2012-01-01

    Glutamate transporter-1 (GLT-1) is the main glutamate transporter in the central nervous system, and its concentration severely decreases in neurodegenerative diseases. The number of transporters in the plasma membrane reflects the balance between their insertion and removal, and it has been reported that the regulated endocytosis of GLT-1 depends on its ubiquitination triggered by protein kinase C (PKC) activation. Here, we identified serine 520 of GLT-1 as the primary target for PKC-dependent phosphorylation, although elimination of this serine did not impair either GLT-1 ubiquitination or endocytosis in response to phorbol esters. In fact, we present evidence indicating that the ubiquitin ligase Nedd4-2 mediates the PKC-dependent ubiquitination and down-regulation of GLT-1. Overexpression of Nedd4-2 increased the ubiquitination of the transporter and promoted its degradation. Moreover, phorbol myristate acetate enhanced Nedd4-2 phosphorylation and the formation of GLT-1·Nedd4-2 complexes, whereas siRNA knockdown of Nedd4-2 prevented ubiquitination, endocytosis, and the concomitant decrease in GLT-1 activity triggered by PKC activation. These results indicate that GLT-1 endocytosis is independent of its phosphorylation and that Nedd4-2 mediates PKC-dependent down-regulation of the transporter. PMID:22505712

  10. Neuregulin-induced ErbB3 downregulation is mediated by a protein stability cascade involving the E3 ubiquitin ligase Nrdp1.

    PubMed

    Cao, Zhongwei; Wu, Xiuli; Yen, Lily; Sweeney, Colleen; Carraway, Kermit L

    2007-03-01

    The molecular mechanisms underlying epidermal growth factor (EGF) receptor tyrosine kinase down-regulation in response to growth factor binding are coming into focus and involve cbl-mediated receptor ubiquitination followed by lysosomal degradation. However, mechanisms underlying the ligand-stimulated degradation of the related receptor tyrosine kinases of the ErbB family do not involve cbl and remain unexplored. Previous studies have demonstrated that the E3 ubiquitin ligase Nrdp1 contributes to the maintenance of steady-state ErbB3 levels by mediating its growth factor-independent degradation. Here we demonstrate that treatment of cells with the ErbB3 ligand neuregulin-1 (NRG1) stabilizes the deubiquitinating enzyme USP8, which in turn stabilizes Nrdp1. The catalytic activity of USP8 is required for NRG1-induced Nrdp1 stabilization. We provide evidence that Akt-mediated phosphorylation of USP8 threonine residue T907 contributes to USP8 stability. Finally, we demonstrate that Nrdp1 or USP8 knockdown suppresses NRG1-induced ErbB3 ubiquitination and degradation in MCF7 breast cancer cells. We conclude that an NRG1-induced protein stability cascade involving USP8 and Nrdp1 mediates the down-regulation of ErbB3. Our observations raise the possibility that the ligand-induced augmentation of pathways involved in the maintenance of basal levels of receptor tyrosine kinases can contribute to ligand-stimulated down-regulation.

  11. Neuregulin-Induced ErbB3 Downregulation Is Mediated by a Protein Stability Cascade Involving the E3 Ubiquitin Ligase Nrdp1▿

    PubMed Central

    Cao, Zhongwei; Wu, Xiuli; Yen, Lily; Sweeney, Colleen; Carraway, Kermit L.

    2007-01-01

    The molecular mechanisms underlying epidermal growth factor (EGF) receptor tyrosine kinase down-regulation in response to growth factor binding are coming into focus and involve cbl-mediated receptor ubiquitination followed by lysosomal degradation. However, mechanisms underlying the ligand-stimulated degradation of the related receptor tyrosine kinases of the ErbB family do not involve cbl and remain unexplored. Previous studies have demonstrated that the E3 ubiquitin ligase Nrdp1 contributes to the maintenance of steady-state ErbB3 levels by mediating its growth factor-independent degradation. Here we demonstrate that treatment of cells with the ErbB3 ligand neuregulin-1 (NRG1) stabilizes the deubiquitinating enzyme USP8, which in turn stabilizes Nrdp1. The catalytic activity of USP8 is required for NRG1-induced Nrdp1 stabilization. We provide evidence that Akt-mediated phosphorylation of USP8 threonine residue T907 contributes to USP8 stability. Finally, we demonstrate that Nrdp1 or USP8 knockdown suppresses NRG1-induced ErbB3 ubiquitination and degradation in MCF7 breast cancer cells. We conclude that an NRG1-induced protein stability cascade involving USP8 and Nrdp1 mediates the down-regulation of ErbB3. Our observations raise the possibility that the ligand-induced augmentation of pathways involved in the maintenance of basal levels of receptor tyrosine kinases can contribute to ligand-stimulated down-regulation. PMID:17210635

  12. Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein

    PubMed Central

    Xu, Shan; Cherok, Edward; Das, Shweta; Li, Sunan; Roelofs, Brian A.; Ge, Shealinna X.; Polster, Brian M.; Boyman, Liron; Lederer, W. Jonathan; Wang, Chunxin; Karbowski, Mariusz

    2016-01-01

    Ubiquitin- and proteasome-dependent outer mitochondrial membrane (OMM)-associated degradation (OMMAD) is critical for mitochondrial and cellular homeostasis. However, the scope and molecular mechanisms of the OMMAD pathways are still not well understood. We report that the OMM-associated E3 ubiquitin ligase MARCH5 controls dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and cell sensitivity to stress-induced apoptosis. MARCH5 knockout selectively inhibited ubiquitination and proteasomal degradation of MiD49, a mitochondrial receptor of Drp1, and consequently led to mitochondrial fragmentation. Mitochondrial fragmentation in MARCH5−/− cells was not associated with inhibition of mitochondrial fusion or bioenergetic defects, supporting the possibility that MARCH5 is a negative regulator of mitochondrial fission. Both MARCH5 re-expression and MiD49 knockout in MARCH5−/− cells reversed mitochondrial fragmentation and reduced sensitivity to stress-induced apoptosis. These findings and data showing MARCH5-dependent degradation of MiD49 upon stress support the possibility that MARCH5 regulation of MiD49 is a novel mechanism controlling mitochondrial fission and, consequently, the cellular response to stress. PMID:26564796

  13. CRYPTOCHROME 2 and PHOTOTROPIN 2 regulate resistance protein mediated viral 2 defense by negatively regulating a E3 ubiquitin ligase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Light harvested by plants is essential for the survival of most life forms. This light perception ability requires the activities of proteins termed photoreceptors. We report a function for photoreceptors in mediating resistance (R) protein-derived plant defense. The blue-light photoreceptors, crypt...

  14. Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs

    PubMed Central

    Beyer, Andrea R.; VieBrock, Lauren; Rodino, Kyle G.; Miller, Daniel P.; Tegels, Brittney K.; Marconi, Richard T.

    2015-01-01

    ABSTRACT A rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions. Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here, in silico and manual sequence analyses revealed that a large proportion of O. tsutsugamushi strain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAG-Ank4_01 or FLAG-Ank9, a glutathione S-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitated O. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. Aligning O. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate that O. tsutsugamushi strain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that an O. tsutsugamushi Ank does so during infection, and advance overall understanding of microbial F-box proteins. IMPORTANCE Ankyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with

  15. Effect of acetate formation pathway and long chain fatty acid CoA-ligase on the free fatty acid production in E. coli expressing acy-ACP thioesterase from Ricinus communis.

    PubMed

    Li, Mai; Zhang, Xiujun; Agrawal, Arpita; San, Ka-Yiu

    2012-07-01

    Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115

  16. Structure and function of the DNA ligases encoded by the mammalian LIG3 gene.

    PubMed

    Tomkinson, Alan E; Sallmyr, Annahita

    2013-12-01

    Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target.

  17. A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum

    PubMed Central

    Mareš, Jan; Hájek, Jan; Urajová, Petra; Kopecký, Jiří; Hrouzek, Pavel

    2014-01-01

    A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides. PMID:25369527

  18. Primary structure and genetic organization of phage T4 DNA ligase.

    PubMed Central

    Armstrong, J; Brown, R S; Tsugita, A

    1983-01-01

    The primary structure of phage T4 DNA ligase has been determined by DNA sequencing of a cloned restriction fragment containing its gene, and partial amino acid sequence analysis of the protein. The molecule has a Mr of 55,230, and contains 487 amino acids. The DNA sequence may also encode all of one and parts of two other, hitherto unidentified, T4 proteins. The four genes are closely packed, with overlaps between terminator and initiator codons of adjacent genes. Potential terminator and promoter sites for transcription are located within the coding sequence of one of the genes. PMID:6314278

  19. SLC27 fatty acid transport proteins.

    PubMed

    Anderson, Courtney M; Stahl, Andreas

    2013-01-01

    The uptake and metabolism of long chain fatty acids (LCFA) are critical to many physiological and cellular processes. Aberrant accumulation or depletion of LCFA underlie the pathology of numerous metabolic diseases. Protein-mediated transport of LCFA has been proposed as the major mode of LCFA uptake and activation. Several proteins have been identified to be involved in LCFA uptake. This review focuses on the SLC27 family of fatty acid transport proteins, also known as FATPs, with an emphasis on the gain- and loss-of-function animal models that elucidate the functions of FATPs in vivo and how these transport proteins play a role in physiological and pathological situations.

  20. The E3 Ubiquitin Ligase- and Protein Phosphatase 2A (PP2A)-binding Domains of the Alpha4 Protein Are Both Required for Alpha4 to Inhibit PP2A Degradation

    SciTech Connect

    LeNoue-Newton, Michele; Watkins, Guy R.; Zou, Ping; Germane, Katherine L.; McCorvey, Lisa R.; Wadzinski, Brian E.; Spiller, Benjamin W.

    2012-04-30

    Protein phosphatase 2A (PP2A) is regulated through a variety of mechanisms, including post-translational modifications and association with regulatory proteins. Alpha4 is one such regulatory protein that binds the PP2A catalytic subunit (PP2Ac) and protects it from polyubiquitination and degradation. Alpha4 is a multidomain protein with a C-terminal domain that binds Mid1, a putative E3 ubiquitin ligase, and an N-terminal domain containing the PP2Ac-binding site. In this work, we present the structure of the N-terminal domain of mammalian Alpha4 determined by x-ray crystallography and use double electron-electron resonance spectroscopy to show that it is a flexible tetratricopeptide repeat-like protein. Structurally, Alpha4 differs from its yeast homolog, Tap42, in two important ways: (1) the position of the helix containing the PP2Ac-binding residues is in a more open conformation, showing flexibility in this region; and (2) Alpha4 contains a ubiquitin-interacting motif. The effects of wild-type and mutant Alpha4 on PP2Ac ubiquitination and stability were examined in mammalian cells by performing tandem ubiquitin-binding entity precipitations and cycloheximide chase experiments. Our results reveal that both the C-terminal Mid1-binding domain and the PP2Ac-binding determinants are required for Alpha4-mediated protection of PP2Ac from polyubiquitination and degradation.

  1. Use of a Conditional Ubr5 Mutant Allele to Investigate the Role of an N-End Rule Ubiquitin-Protein Ligase in Hedgehog Signalling and Embryonic Limb Development

    PubMed Central

    Kinsella, Elaine; Dora, Natalie; Mellis, David; Lettice, Laura; Deveney, Paul

    2016-01-01

    Hedgehog (Hh) signalling is a potent regulator of cell fate and function. While much is known about the events within a Hh-stimulated cell, far less is known about the regulation of Hh-ligand production. Drosophila Hyperplastic Discs (Hyd), a ubiquitin-protein ligase, represents one of the few non-transcription factors that independently regulates both hh mRNA expression and pathway activity. Using a murine embryonic stem cell system, we revealed that shRNAi of the mammalian homologue of hyd, Ubr5, effectively prevented retinoic-acid-induced Sonic hedgehog (Shh) expression. We next investigated the UBR5:Hh signalling relationship in vivo by generating and validating a mouse bearing a conditional Ubr5 loss-of-function allele. Conditionally deleting Ubr5 in the early embryonic limb-bud mesenchyme resulted in a transient decrease in Indian hedgehog ligand expression and decreased Hh pathway activity, around E13.5. Although Ubr5-deficient limbs and digits were, on average, shorter than control limbs, the effects were not statistically significant. Hence, while loss of UBR5 perturbed Hedgehog signalling in the developing limb, there were no obvious morphological defects. In summary, we report the first conditional Ubr5 mutant mouse and provide evidence for a role for UBR5 in influencing Hh signalling, but are uncertain to whether the effects on Hedgehog signaling were direct (cell autonomous) or indirect (non-cell-autonomous). Elaboration of the cellular/molecular mechanism(s) involved may help our understanding on diseases and developmental disorders associated with aberrant Hh signalling. PMID:27299863

  2. HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation.

    PubMed

    Ho, Yik-Khuan; Zhi, Huijun; Bowlin, Tara; Dorjbal, Batsukh; Philip, Subha; Zahoor, Muhammad Atif; Shih, Hsiu-Ming; Semmes, Oliver John; Schaefer, Brian; Glover, J N Mark; Giam, Chou-Zen

    2015-08-01

    Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells.

  3. HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation

    PubMed Central

    Ho, Yik-Khuan; Zhi, Huijun; Bowlin, Tara; Dorjbal, Batsukh; Philip, Subha; Zahoor, Muhammad Atif; Shih, Hsiu-Ming; Semmes, Oliver John; Schaefer, Brian; Glover, J. N. Mark; Giam, Chou-Zen

    2015-01-01

    Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells. PMID:26285145

  4. An ankyrin repeat-containing protein, characterized as a ubiquitin ligase, is closely associated with membrane-enclosed organelles and required for pollen germination and pollen tube growth in lily.

    PubMed

    Huang, Jian; Chen, Feng; Del Casino, Cecilia; Autino, Antonella; Shen, Mouhua; Yuan, Shuai; Peng, Jia; Shi, Hexin; Wang, Chen; Cresti, Mauro; Li, Yiqin

    2006-04-01

    Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth.

  5. Prokaryotic BirA ligase biotinylates K4, K9, K18 and K23 in eukaryotic histone H3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BirA ligase, a prokaryotic ortholog of human holocarboxylase synthetase (HCS), is known to biotinylate proteins. Here, we tested the hypothesis that BirA ligase may also catalyze biotinylation of eukaryotic histones. If so, this would render recombinant BirA ligase a useful surrogate for HCS in stud...

  6. Detection of non-protein amino acids in the presence of protein amino acids. II.

    NASA Technical Reports Server (NTRS)

    Shapshak, P.; Okaji, M.

    1972-01-01

    Studies conducted with the JEOL 5AH amino acid analyzer are described. This instrument makes possible the programming of the chromatographic process. Data are presented showing the separations of seventeen non-protein amino acids in the presence of eighteen protein amino acids. It is pointed out that distinct separations could be obtained in the case of a number of chemically similar compounds, such as ornithine and lysine, N-amidino alanine and arginine, and iminodiacetic acid and S-carboxymethyl cysteine and aspartic acid.

  7. Nucleic acids, proteins, and chirality

    NASA Technical Reports Server (NTRS)

    Usher, D. A.; Profy, A. T.; Walstrum, S. A.; Needels, M. C.; Bulack, S. C.; Lo, K. M.

    1984-01-01

    The present investigation is concerned with experimental results related, in one case, to the chirality of nucleotides, and, in another case, to the possibility of a link between the chirality of nucleic acids, and that of peptides. It has been found that aminoacylation of the 'internal' hydroxyl group of a dinucleoside monophosphate can occur stereoselectively. However, this reaction has not yet been made a part of a working peptide synthesis scheme. The formation and cleavage of oligonucleotides is considered. In the event of the formation of a helical complex between the oligonucleotide and the polymer, 1-prime,5-prime-bonds in the oligomer are found to become more resistant towards cleavage. The conditions required for peptide bond formation are examined, taking into account the known structures of RNA and possible mechanisms for prebiotic peptide bond formation. The possibility is considered that the 2-prime,5-prime-internucleotide linkage could have played an important part in the early days of biological peptide synthesis.

  8. The Arabidopsis EDR1 Protein Kinase Negatively Regulates the ATL1 E3 Ubiquitin Ligase to Suppress Cell Death[W

    PubMed Central

    Serrano, Irene; Gu, Yangnan; Qi, Dong; Dubiella, Ullrich

    2014-01-01

    Loss-of-function mutations in the Arabidopsis thaliana ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced programmed cell death under a variety of abiotic and biotic stress conditions. All edr1 mutant phenotypes can be suppressed by missense mutations in the KEEP ON GOING gene, which encodes a trans-Golgi network/early endosome (TGN/EE)-localized E3 ubiquitin ligase. Here, we report that EDR1 interacts with a second E3 ubiquitin ligase, ARABIDOPSIS TOXICOS EN LEVADURA1 (ATL1), and negatively regulates its activity. Overexpression of ATL1 in transgenic Arabidopsis induced severe growth inhibition and patches of cell death, while transient overexpression in Nicotiana benthamiana leaves induced cell death and tissue collapse. The E3 ligase activity of ATL1 was required for both of these processes. Importantly, we found that ATL1 interacts with EDR1 on TGN/EE vesicles and that EDR1 suppresses ATL1-mediated cell death in N. benthamiana and Arabidopsis. Lastly, knockdown of ATL1 expression suppressed cell death phenotypes associated with the edr1 mutant and made Arabidopsis hypersusceptible to powdery mildew infection. Taken together, our data indicate that ATL1 is a positive regulator of programmed cell death and EDR1 negatively regulates ATL1 activity at the TGN/EE and thus controls stress responses initiated by ATL1-mediated ubiquitination events. PMID:25398498

  9. Direct role for proliferating cell nuclear antigen in substrate recognition by the E3 ubiquitin ligase CRL4Cdt2.

    PubMed

    Havens, Courtney G; Shobnam, Nadia; Guarino, Estrella; Centore, Richard C; Zou, Lee; Kearsey, Stephen E; Walter, Johannes C

    2012-03-30

    The E3 ubiquitin ligase Cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) is emerging as an important cell cycle regulator that targets numerous proteins for destruction in S phase and after DNA damage, including Cdt1, p21, and Set8. CRL4(Cdt2) substrates contain a "PIP degron," which consists of a canonical proliferating cell nuclear antigen (PCNA) interaction motif (PIP box) and an adjacent basic amino acid. Substrates use their PIP box to form a binary complex with PCNA on chromatin and the basic residue to recruit CRL4(Cdt2) for substrate ubiquitylation. Using Xenopus egg extracts, we identify an acidic residue in PCNA that is essential to support destruction of all CRL4(Cdt2) substrates. This PCNA residue, which adjoins the basic amino acid of the bound PIP degron, is dispensable for substrate binding to PCNA but essential for CRL4(Cdt2) recruitment to chromatin. Our data show that the interaction of CRL4(Cdt2) with substrates requires molecular determinants not only in the substrate degron but also on PCNA. The results illustrate a potentially general mechanism by which E3 ligases can couple ubiquitylation to the formation of protein-protein interactions.

  10. Alimentary proteins, amino acids and cholesterolemia.

    PubMed

    Blachier, François; Lancha, Antonio H; Boutry, Claire; Tomé, Daniel

    2010-01-01

    Numerous data from both epidemiological and experimental origins indicate that some alimentary proteins and amino acids in supplements can modify the blood LDL cholesterol, HDL cholesterol and total cholesterol. After an initial approval of the health claim for soy protein consumption for the prevention of coronary heart disease, more recently it has been concluded from an overall analysis of literature that isolated soy protein with isoflavones only slightly decrease LDL and total cholesterol. Other plant extracts and also some proteins from animal origin have been reported to exert a lowering effect on blood cholesterol when compared with a reference protein (often casein). The underlying mechanisms are still little understood. Individual amino acids and mixture of amino acids have also been tested (mostly in animal studies) for their effects on cholesterol parameters and on cholesterol metabolism. Methionine, lysine, cystine, leucine, aspartate and glutamate have been tested individually and in combination in different models of either normo or hypercholesterolemic animals and found to be able to modify blood cholesterol and/or LDL cholesterol and/or HDL cholesterol. It is however not known if these results are relevant to human nutrition.

  11. Electrocatalysis in proteins, nucleic acids and carbohydrates.

    PubMed

    Paleček, Emil; Bartošík, Martin; Ostatná, Veronika; Trefulka, Mojmír

    2012-02-01

    The ability of proteins to catalyze hydrogen evolution has been known for more than 80 years, but the poorly developed d.c. polarographic "pre-sodium wave" was of little analytical use. Recently, we have shown that by using constant current chronopotentiometric stripping analysis, proteins produce a well-developed peak H at hanging mercury drop and solid amalgam electrodes. Peak H sensitively reflects changes in protein structures due to protein denaturation, single amino acid exchange, etc. at the picomole level. Unmodified DNA and RNA do not yield such a peak, but they produce electrocatalytic voltammetric signals after modification with osmium tetroxide complexes with nitrogen ligands [Os(VIII)L], binding covalently to pyrimidine bases in nucleic acids. Recently, it has been shown that six-valent [Os(VI)L] complexes bind to 1,2-diols in polysaccharides and oligosaccharides, producing voltammetric responses similar to those of DNA-Os(VIII)L adducts. Electrocatalytic peaks produced by Os-modified nucleic acids, proteins (reaction with tryptophan residues) and carbohydrates are due to the catalytic hydrogen evolution, allowing determination of oligomers at the picomolar level.

  12. Defining interactions between DNA-PK and ligase IV/XRCC4

    SciTech Connect

    Hsu, Hsin-Ling; Yannone, Steven M.; Chen, David J.

    2001-04-10

    Non-homologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks in mammalian cells. DNA-dependent protein kinase (DNA-PK), ligase IV, and XRCC4 are all critical components of the NHEJ repair pathway. DNA-PK is composed of a heterodimeric DNA-binding component, Ku, and a large catalytic subunit, DNA-PKcs. Ligase IV and XRCC4 associate to form a multimeric complex that is also essential for NHEJ. DNA-PK and ligase IV/XRCC4 interact at DNA termini which results in stimulated ligase activity. Here we define interactions between the components of these two essential complexes, DNA-PK and ligase IV/XRCC4. We find that ligase IV/XRCC4 associates with DNA-PK in a DNA-independent manner. The specific protein-protein interactions that mediate the interaction between these two complexes are further identified. Direct physical interactions between ligase IV and Ku as well as between XRCC4 and DNA-PKcs are shown. No direct interactions are observed between ligase IV and DNA-PKcs or between XRCC4 and Ku. Our data defines the specific protein pairs involved in the association of DNA-PK and ligase IV/XRCC4, and suggests a molecular mechanism for coordinating the assembly of the DNA repair complex at DNA breaks.

  13. Conformations of amino acids in proteins.

    PubMed

    Hovmöller, Sven; Zhou, Tuping; Ohlson, Tomas

    2002-05-01

    The main-chain conformations of 237 384 amino acids in 1042 protein subunits from the PDB were analyzed with Ramachandran plots. The populated areas of the empirical Ramachandran plot differed markedly from the classical plot in all regions. All amino acids in alpha-helices are found within a very narrow range of phi, psi angles. As many as 40% of all amino acids are found in this most populated region, covering only 2% of the Ramachandran plot. The beta-sheet region is clearly subdivided into two distinct regions. These do not arise from the parallel and antiparallel beta-strands, which have quite similar conformations. One beta region is mainly from amino acids in random coil. The third and smallest populated area of the Ramachandran plot, often denoted left-handed alpha-helix, has a different position than that originally suggested by Ramachandran. Each of the 20 amino acids has its own very characteristic Ramachandran plot. Most of the glycines have conformations that were considered to be less favoured. These results may be useful for checking secondary-structure assignments in the PDB and for predicting protein folding.

  14. Measurement of protein using bicinchoninic acid.

    PubMed

    Smith, P K; Krohn, R I; Hermanson, G T; Mallia, A K; Gartner, F H; Provenzano, M D; Fujimoto, E K; Goeke, N M; Olson, B J; Klenk, D C

    1985-10-01

    Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.

  15. Origin and diversification of TRIM ubiquitin ligases.

    PubMed

    Marín, Ignacio

    2012-01-01

    Most proteins of the TRIM family (also known as RBCC family) are ubiquitin ligases that share a peculiar protein structure, characterized by including an N-terminal RING finger domain closely followed by one or two B-boxes. Additional protein domains found at their C termini have been used to classify TRIM proteins into classes. TRIMs are involved in multiple cellular processes and many of them are essential components of the innate immunity system of animal species. In humans, it has been shown that mutations in several TRIM-encoding genes lead to diverse genetic diseases and contribute to several types of cancer. They had been hitherto detected only in animals. In this work, by comprehensively analyzing the available diversity of TRIM and TRIM-like protein sequences and evaluating their evolutionary patterns, an improved classification of the TRIM family is obtained. Members of one of the TRIM subfamilies defined, called Subfamily A, turn to be present not only in animals, but also in many other eukaryotes, such as fungi, apusozoans, alveolates, excavates and plants. The rest of subfamilies are animal-specific and several of them originated only recently. Subfamily A proteins are characterized by containing a MATH domain, suggesting a potential evolutionary connection between TRIM proteins and a different type of ubiquitin ligases, known as TRAFs, which contain quite similar MATH domains. These results indicate that the TRIM family emerged much earlier than so far thought and contribute to our understanding of its origin and diversification. The structural and evolutionary links with the TRAF family of ubiquitin ligases can be experimentally explored to determine whether functional connections also exist.

  16. Regulation of Parkin E3 ubiquitin ligase activity.

    PubMed

    Walden, Helen; Martinez-Torres, R Julio

    2012-09-01

    Parkin is an E3 ubiquitin ligase mutated in autosomal recessive juvenile Parkinson's disease. In addition, it is a putative tumour suppressor, and has roles outside its enzymatic activity. It is critical for mitochondrial clearance through mitophagy, and is an essential protein in most eukaryotes. As such, it is a tightly controlled protein, regulated through an array of external interactions with multiple proteins, posttranslational modifications including phosphorylation and S-nitrosylation, and self-regulation through internal associations. In this review, we highlight some of the recent studies into Parkin regulation and discuss future challenges for gaining a full molecular understanding of the regulation of Parkin E3 ligase activity.

  17. Structure-based design of a new series of D-glutamic acid based inhibitors of bacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD).

    PubMed

    Tomasić, Tihomir; Zidar, Nace; Sink, Roman; Kovac, Andreja; Blanot, Didier; Contreras-Martel, Carlos; Dessen, Andréa; Müller-Premru, Manica; Zega, Anamarija; Gobec, Stanislav; Kikelj, Danijel; Masic, Lucija Peterlin

    2011-07-14

    MurD ligase is one of the key enzymes participating in the intracellular steps of peptidoglycan biosynthesis and constitutes a viable target in the search for novel antibacterial drugs to combat bacterial drug-resistance. We have designed, synthesized, and evaluated a new series of D-glutamic acid-based Escherichia coli MurD inhibitors incorporating the 5-benzylidenethiazolidin-4-one scaffold. The crystal structure of 16 in the MurD active site has provided a good starting point for the design of structurally optimized inhibitors 73-75 endowed with improved MurD inhibitory potency (IC(50) between 3 and 7 μM). Inhibitors 74 and 75 showed weak activity against Gram-positive Staphylococcus aureus and Enterococcus faecalis. Compounds 73-75, with IC(50) values in the low micromolar range, represent the most potent D-Glu-based MurD inhibitors reported to date.

  18. Overview of the membrane-associated RING-CH (MARCH) E3 ligase family.

    PubMed

    Bauer, Johannes; Bakke, Oddmund; Morth, J Preben

    2016-12-14

    E3 ligases are critical checkpoints for protein ubiquitination, a signal that often results in protein sorting and degradation but has also been linked to regulation of transcription and DNA repair. In line with their key role in cellular trafficking and cell-cycle control, malfunction of E3 ligases is often linked to human disease. Thus, they have emerged as prime drug targets. However, the molecular basis of action of membrane-bound E3 ligases is still unknown. Here, we review the current knowledge on the membrane-embedded MARCH E3 ligases (MARCH-1-6,7,8,11) with a focus on how the transmembrane regions can contribute via GxxxG-motifs to the selection and recognition of other membrane proteins as substrates for ubiquitination. Further understanding of the molecular parameters that govern target protein recognition of MARCH E3 ligases will contribute to development of strategies for therapeutic regulation of MARCH-induced ubiquitination.

  19. Inadequacy of prebiotic synthesis as origin of proteinous amino acids.

    PubMed

    Wong, J T; Bronskill, P M

    1979-07-18

    The production of some nonproteinous, and lack of production of other proteinous, amino acids in model prebiotic synthesis, along with the instability of glutamine and asparagine, suggest that not all of the 20 present day proteinous amino acids gained entry into proteins directly from the primordial soup. Instead, a process of active co-evolution of the genetic code and its constituent amino acids would have to precede the final selection of these proteinous amono acids.

  20. Biochemical characterization of the DNA ligase I from Entamoeba histolytica.

    PubMed

    Cardona-Felix, Cesar S; Pastor-Palacios, Guillermo; Cardenas, Helios; Azuara-Liceaga, Elisa; Brieba, Luis G

    2010-11-01

    DNA ligases play an essential role in DNA replication and repair. Herein, we report the cloning and biochemical characterization of DNA ligase I from the protozoan parasite Entamoeba histolytica (EhDNAligI). EhDNAligI is an ATP-dependent DNA ligase of 685 amino acids with 35% identity to human DNA ligase I. This report shows that heterologous expressed EhDNAligI is able to perform the three conserved steps of a DNA ligation reaction: adenylation, binding to a 5'-phosphorylated nicked DNA substrate and sealing of the nick. EhDNAligI is strongly inhibited by NaCl and displays optimal activity at pH 7.5. EhDNAligI uses Mn2+ or Mg2+ as metal cofactors and ATP as nucleotide cofactor. EhDNAligI has a nicked DNA binding constant of 6.6microM and follows Michaelis-Menten steady-state kinetics with a K(m) ATP of 64nM and a k(cat) of 2.4min(-1). Accordingly to its properties as a family I DNA ligase, EhDNAligI is able to ligate a RNA strand upstream of a nucleic acid nick, but not in the downstream or the template position. We propose that EhDNAligI is involved in sealing DNA nicks during lagging strand synthesis and may have a role in base excision repair in E. histolytica.

  1. Protein and Amino Acid Profiles of Different Whey Protein Supplements.

    PubMed

    Almeida, Cristine C; Alvares, Thiago S; Costa, Marion P; Conte-Junior, Carlos A

    2016-01-01

    Whey protein (WP) supplements have received increasing attention by consumers due to the high nutritional value of the proteins and amino acids they provide. However, some WP supplements may not contain the disclosed amounts of the ingredients listed on the label, compromising the nutritional quality and the effectiveness of these supplements. The aim of this study was to evaluate and compare the contents of total protein (TP), α-lactalbumin (α-LA), β-lactoglobulin (β-LG), free essential amino acids (free EAA), and free branched-chain amino acids (free BCAA), amongst different WP supplements produced by U.S. and Brazilian companies. Twenty commercial brands of WP supplements were selected, ten manufactured in U.S. (WP-USA) and ten in Brazil (WP-BRA). The TP was analyzed using the Kjeldahl method, while α-LA, β-LG, free EAA, and free BCAA were analyzed using HPLC system. There were higher (p < 0.05) concentrations of TP, α-LA, β-LG, and free BCAA in WP-USA supplements, as compared to the WP-BRA supplements; however, there was no difference (p > 0.05) in the content of free EAA between WP-USA and WP-BRA. Amongst the 20 brands evaluated, four WP-USA and seven WP-BRA had lower (p < 0.05) values of TP than those specified on the label. In conclusion, the WP-USA supplements exhibited better nutritional quality, evaluated by TP, α-LA, β-LG, and free BCAA when compared to WP-BRA.

  2. SVIP regulates Z variant alpha-1 antitrypsin retro-translocation by inhibiting ubiquitin ligase gp78

    PubMed Central

    Khodayari, Nazli; Wang, Rejean liqun; Marek, George; Krotova, Karina; Kirst, Mariana; Liu, Chen; Rouhani, Farshid; Brantly, Mark

    2017-01-01

    Alpha-1 antitrypsin deficiency (AATD) is an inherited disorder characterized by early-onset emphysema and liver disease. The most common disease-causing mutation is a single amino acid substitution (Glu/Lys) at amino acid 342 of the mature protein, resulting in disruption of the 290–342 salt bridge (an electrophoretic abnormality defining the mutation [Z allele, or ZAAT]), protein misfolding, polymerization, and accumulation in the endoplasmic reticulum of hepatocytes and monocytes. The Z allele causes a toxic gain of function, and the E3 ubiquitin ligase gp78 promotes degradation and increased solubility of endogenous ZAAT. We hypothesized that the accumulation of ZAAT is influenced by modulation of gp78 E3 ligase and SVIP (small VCP-interacting protein) interaction with p97/VCP in ZAAT-expressing hepatocytes. We showed that the SVIP inhibitory effect on ERAD due to overexpression causes the accumulation of ZAAT in a human Z hepatocyte–like cell line (AT01). Overexpression of gp78, as well as SVIP suppression, induces gp78-VCP/p97 interaction in AT01 cells. This interaction leads to retro-translocation of ZAAT and reduction of the SVIP inhibitory role in ERAD. In this context, overexpression of gp78 or SVIP suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD. PMID:28301499

  3. Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex.

    PubMed

    Peng, Zhimin; Liao, Zhongping; Matsumoto, Yoshihiro; Yang, Austin; Tomkinson, Alan E

    2016-10-14

    The synthesis, processing, and joining of Okazaki fragments during DNA replication is complex, requiring the sequential action of a large number of proteins. Proliferating cell nuclear antigen, a DNA sliding clamp, interacts with and coordinates the activity of several DNA replication proteins, including the enzymes flap endonuclease 1 (FEN-1) and DNA ligase I that complete the processing and joining of Okazaki fragments, respectively. Although it is evident that maintaining the appropriate relative stoichiometry of FEN-1 and DNA ligase I, which compete for binding to proliferating cell nuclear antigen, is critical to prevent genomic instability, little is known about how the steady state levels of DNA replication proteins are regulated, in particular the proteolytic mechanisms involved in their turnover. Because DNA ligase I has been reported to be ubiquitylated, we used a proteomic approach to map ubiquitylation sites and screen for DNA ligase I-associated E3 ubiquitin ligases. We identified three ubiquitylated lysine residues and showed that DNA ligase I interacts with and is targeted for ubiquitylation by DCAF7, a specificity factor for the Cul4-DDB1 complex. Notably, knockdown of DCAF7 reduced the degradation of DNA ligase I in response to inhibition of proliferation and replacement of ubiquitylated lysine residues reduced the in vitro ubiquitylation of DNA ligase I by Cul4-DDB1 and DCAF7. In contrast, a different E3 ubiquitin ligase regulates FEN-1 turnover. Thus, although the expression of many of the genes encoding DNA replication proteins is coordinately regulated, our studies reveal that different mechanisms are involved in the turnover of these proteins.

  4. Hypochlorous and peracetic acid induced oxidation of dairy proteins.

    PubMed

    Kerkaert, Barbara; Mestdagh, Frédéric; Cucu, Tatiana; Aedo, Philip Roger; Ling, Shen Yan; De Meulenaer, Bruno

    2011-02-09

    Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.

  5. A Point Mutation in the Ubiquitin Ligase RNF170 That Causes Autosomal Dominant Sensory Ataxia Destabilizes the Protein and Impairs Inositol 1,4,5-Trisphosphate Receptor-mediated Ca2+ Signaling*

    PubMed Central

    Wright, Forrest A.; Lu, Justine P.; Sliter, Danielle A.; Dupré, Nicolas; Rouleau, Guy A.; Wojcikiewicz, Richard J. H.

    2015-01-01

    RNF170 is an endoplasmic reticulum membrane ubiquitin ligase that contributes to the ubiquitination of activated inositol 1,4,5-trisphosphate (IP3) receptors, and also, when point mutated (arginine to cysteine at position 199), causes autosomal dominant sensory ataxia (ADSA), a disease characterized by neurodegeneration in the posterior columns of the spinal cord. Here we demonstrate that this point mutation inhibits RNF170 expression and signaling via IP3 receptors. Inhibited expression of mutant RNF170 was seen in cells expressing exogenous RNF170 constructs and in ADSA lymphoblasts, and appears to result from enhanced RNF170 autoubiquitination and proteasomal degradation. The basis for these effects was probed via additional point mutations, revealing that ionic interactions between charged residues in the transmembrane domains of RNF170 are required for protein stability. In ADSA lymphoblasts, platelet-activating factor-induced Ca2+ mobilization was significantly impaired, whereas neither Ca2+ store content, IP3 receptor levels, nor IP3 production were altered, indicative of a functional defect at the IP3 receptor locus, which may be the cause of neurodegeneration. CRISPR/Cas9-mediated genetic deletion of RNF170 showed that RNF170 mediates the addition of all of the ubiquitin conjugates known to become attached to activated IP3 receptors (monoubiquitin and Lys48- and Lys63-linked ubiquitin chains), and that wild-type and mutant RNF170 have apparently identical ubiquitin ligase activities toward IP3 receptors. Thus, the Ca2+ mobilization defect seen in ADSA lymphoblasts is apparently not due to aberrant IP3 receptor ubiquitination. Rather, the defect likely reflects abnormal ubiquitination of other substrates, or adaptation to the chronic reduction in RNF170 levels. PMID:25882839

  6. A Point Mutation in the Ubiquitin Ligase RNF170 That Causes Autosomal Dominant Sensory Ataxia Destabilizes the Protein and Impairs Inositol 1,4,5-Trisphosphate Receptor-mediated Ca2+ Signaling.

    PubMed

    Wright, Forrest A; Lu, Justine P; Sliter, Danielle A; Dupré, Nicolas; Rouleau, Guy A; Wojcikiewicz, Richard J H

    2015-05-29

    RNF170 is an endoplasmic reticulum membrane ubiquitin ligase that contributes to the ubiquitination of activated inositol 1,4,5-trisphosphate (IP3) receptors, and also, when point mutated (arginine to cysteine at position 199), causes autosomal dominant sensory ataxia (ADSA), a disease characterized by neurodegeneration in the posterior columns of the spinal cord. Here we demonstrate that this point mutation inhibits RNF170 expression and signaling via IP3 receptors. Inhibited expression of mutant RNF170 was seen in cells expressing exogenous RNF170 constructs and in ADSA lymphoblasts, and appears to result from enhanced RNF170 autoubiquitination and proteasomal degradation. The basis for these effects was probed via additional point mutations, revealing that ionic interactions between charged residues in the transmembrane domains of RNF170 are required for protein stability. In ADSA lymphoblasts, platelet-activating factor-induced Ca(2+) mobilization was significantly impaired, whereas neither Ca(2+) store content, IP3 receptor levels, nor IP3 production were altered, indicative of a functional defect at the IP3 receptor locus, which may be the cause of neurodegeneration. CRISPR/Cas9-mediated genetic deletion of RNF170 showed that RNF170 mediates the addition of all of the ubiquitin conjugates known to become attached to activated IP3 receptors (monoubiquitin and Lys(48)- and Lys(63)-linked ubiquitin chains), and that wild-type and mutant RNF170 have apparently identical ubiquitin ligase activities toward IP3 receptors. Thus, the Ca(2+) mobilization defect seen in ADSA lymphoblasts is apparently not due to aberrant IP3 receptor ubiquitination. Rather, the defect likely reflects abnormal ubiquitination of other substrates, or adaptation to the chronic reduction in RNF170 levels.

  7. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  8. Kinetic Characterization of Lipid II-Ala:Alanyl-tRNA Ligase (MurN) from Streptococcus pneumoniae using Semisynthetic Aminoacyl-lipid II Substrates*S⃞

    PubMed Central

    De Pascale, Gianfranco; Lloyd, Adrian J.; Schouten, James A.; Gilbey, Andrea M.; Roper, David I.; Dowson, Christopher G.; Bugg, Timothy D. H.

    2008-01-01

    MurM and MurN are tRNA-dependent ligases that catalyze the addition of the first (l-Ala/l-Ser) and second (l-Ala) amino acid onto lipid II substrates in the biosynthesis of the peptidoglycan layer of Streptococcus pneumoniae. We have previously characterized the first ligase, MurM (Lloyd, A. J., Gilbey, A. M., Blewett, A. M., De Pascale, G., El Zoeiby, A., Levesque, R. C., Catherwood, A. C., Tomasz, A., Bugg, T. D., Roper, D. I., and Dowson, C. G. (2008) J. Biol. Chem. 283, 6402–6417). In order to characterize the second ligase MurN, we have developed a chemoenzymatic route to prepare the lipid II-Ala and lipid II-Ser substrates. Recombinant MurN enzymes from penicillin-resistant (159) and -sensitive (Pn16) S. pneumoniae were expressed and purified as MBP fusion proteins and reconstituted using a radiochemical assay. MurN ligases from strains 159 and Pn16 both showed a 20-fold higher catalytic efficiency for lipid II-l-Ala over lipid II-l-Ser, with no activity against unmodified lipid II, and similar kinetic parameters were measured for MurN from penicillin-resistant and penicillin-sensitive strains. These results concur with the peptidoglycan analysis of S. pneumoniae, in which the major cross-link observed is l-Ala-l-Ala. The combined action of ligases MurM and MurN is therefore required in order to rationalize the high level of dipeptide cross-links in penicillin-resistant S. pneumoniae, with ligase MurM showing the major difference between penicillin-resistant and penicillin-sensitive strains. PMID:18842590

  9. Kinetic characterization of lipid II-Ala:alanyl-tRNA ligase (MurN) from Streptococcus pneumoniae using semisynthetic aminoacyl-lipid II substrates.

    PubMed

    De Pascale, Gianfranco; Lloyd, Adrian J; Schouten, James A; Gilbey, Andrea M; Roper, David I; Dowson, Christopher G; Bugg, Timothy D H

    2008-12-12

    MurM and MurN are tRNA-dependent ligases that catalyze the addition of the first (L-Ala/L-Ser) and second (L-Ala) amino acid onto lipid II substrates in the biosynthesis of the peptidoglycan layer of Streptococcus pneumoniae. We have previously characterized the first ligase, MurM (Lloyd, A. J., Gilbey, A. M., Blewett, A. M., De Pascale, G., El Zoeiby, A., Levesque, R. C., Catherwood, A. C., Tomasz, A., Bugg, T. D., Roper, D. I., and Dowson, C. G. (2008) J. Biol. Chem. 283, 6402-6417). In order to characterize the second ligase MurN, we have developed a chemoenzymatic route to prepare the lipid II-Ala and lipid II-Ser substrates. Recombinant MurN enzymes from penicillin-resistant (159) and -sensitive (Pn16) S. pneumoniae were expressed and purified as MBP fusion proteins and reconstituted using a radiochemical assay. MurN ligases from strains 159 and Pn16 both showed a 20-fold higher catalytic efficiency for lipid II-L-Ala over lipid II-l-Ser, with no activity against unmodified lipid II, and similar kinetic parameters were measured for MurN from penicillin-resistant and penicillin-sensitive strains. These results concur with the peptidoglycan analysis of S. pneumoniae, in which the major cross-link observed is L-Ala-L-Ala. The combined action of ligases MurM and MurN is therefore required in order to rationalize the high level of dipeptide cross-links in penicillin-resistant S. pneumoniae, with ligase MurM showing the major difference between penicillin-resistant and penicillin-sensitive strains.

  10. Manipulation of cellular GSH biosynthetic capacity via TAT-mediated protein transduction of wild-type or a dominant-negative mutant of glutamate cysteine ligase alters cell sensitivity to oxidant-induced cytotoxicity

    SciTech Connect

    Backos, Donald S.; Brocker, Chad N.; Franklin, Christopher C.

    2010-02-15

    The glutathione (GSH) antioxidant defense system plays a central role in protecting mammalian cells against oxidative injury. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme in GSH biosynthesis and is a heterodimeric holoenzyme composed of catalytic (GCLC) and modifier (GCLM) subunits. As a means of assessing the cytoprotective effects of enhanced GSH biosynthetic capacity, we have developed a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain. Bacterial expression vectors encoding TAT fusion proteins of both GCL subunits were generated and recombinant fusion proteins were synthesized and purified to near homogeneity. The TAT-GCL fusion proteins were capable of heterodimerization and formation of functional GCL holoenzyme in vitro. Exposure of Hepa-1c1c7 cells to the TAT-GCL fusion proteins resulted in the time- and dose-dependent transduction of both GCL subunits and increased cellular GCL activity and GSH levels. A heterodimerization-competent, enzymatically deficient GCLC-TAT mutant was also generated in an attempt to create a dominant-negative suppressor of GCL. Transduction of cells with a catalytically inactive GCLC(E103A)-TAT mutant decreased cellular GCL activity in a dose-dependent manner. TAT-mediated manipulation of cellular GCL activity was also functionally relevant as transduction with wild-type GCLC(WT)-TAT or mutant GCLC(E103A)-TAT conferred protection or enhanced sensitivity to H{sub 2}O{sub 2}-induced cell death, respectively. These findings demonstrate that TAT-mediated transduction of wild-type or dominant-inhibitory mutants of the GCL subunits is a viable means of manipulating cellular GCL activity to assess the effects of altered GSH biosynthetic capacity.

  11. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    2011-12-06

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  12. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  13. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    2011-03-22

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  14. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2008-10-07

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  15. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2009-04-28

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  16. An E3 Ubiquitin Ligase-BAG Protein Module Controls Plant Innate Immunity and Broad-Spectrum Disease Resistance.

    PubMed

    You, Quanyuan; Zhai, Keran; Yang, Donglei; Yang, Weibing; Wu, Jingni; Liu, Junzhong; Pan, Wenbo; Wang, Jianjun; Zhu, Xudong; Jian, Yikun; Liu, Jiyun; Zhang, Yingying; Deng, Yiwen; Li, Qun; Lou, Yonggen; Xie, Qi; He, Zuhua

    2016-12-14

    Programmed cell death (PCD) and immunity in plants are tightly controlled to promote antimicrobial defense while preventing autoimmunity. However, the mechanisms contributing to this immune homeostasis are poorly understood. Here, we isolated a rice mutant ebr1 (enhanced blight and blast resistance 1) that shows enhanced broad-spectrum bacterial and fungal disease resistance, but displays spontaneous PCD, autoimmunity, and stunted growth. EBR1 encodes an E3 ubiquitin ligase that interacts with OsBAG4, which belongs to the BAG (Bcl-2-associated athanogene) family that functions in cell death, growth arrest, and immune responses in mammals. EBR1 directly targets OsBAG4 for ubiquitination-mediated degradation. Elevated levels of OsBAG4 in rice are necessary and sufficient to trigger PCD and enhanced disease resistance to pathogenic infection, most likely by activating pathogen-associated molecular patterns-triggered immunity (PTI). Together, our study suggests that an E3-BAG module orchestrates innate immune homeostasis and coordinates the trade-off between defense and growth in plants.

  17. Forkhead-Associated Domain of Yeast Xrs2, a Homolog of Human Nbs1, Promotes Nonhomologous End Joining Through Interaction With a Ligase IV Partner Protein, Lif1

    PubMed Central

    Matsuzaki, Kenichiro; Shinohara, Akira; Shinohara, Miki

    2008-01-01

    DNA double-strand breaks (DSB) are repaired through two different pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). Yeast Xrs2, a homolog of human Nbs1, is a component of the Mre11-Rad50-Xrs2 (MRX) complex required for both HR and NHEJ. Previous studies showed that the N-terminal forkhead-associated (FHA) domain of Xrs2/Nbs1 in yeast is not involved in HR, but is likely to be in NHEJ. In this study, we showed that the FHA domain of Xrs2 plays a critical role in efficient DSB repair by NHEJ. The FHA domain of Xrs2 specifically interacts with Lif1, a component of the ligase IV complex, Dnl4-Nej1-Lif1 (DNL). Lif1, which is phosphorylated in vivo, contains two Xrs2-binding regions. Serine 383 of Lif1 plays an important role in the interaction with Xrs2 as well as in NHEJ. Interestingly, the phospho-mimetic substitutions of serine 383 enhance the NHEJ activity of Lif1. Our results suggest that the phosphorylation of Lif1 at serine 383 is recognized by the Xrs2 FHA domain, which in turn may promote recruitment of the DNL complex to DSB for NHEJ. The interaction between Xrs2 and Lif1 through the FHA domain is conserved in humans; the FHA domain Nbs1 interacts with Xrcc4, a Lif1 homolog of human. PMID:18458108

  18. Unique ligation properties of eukaryotic NAD+-dependent DNA ligase from Melanoplus sanguinipes entomopoxvirus.

    PubMed

    Lu, Jing; Tong, Jie; Feng, Hong; Huang, Jianmin; Afonso, Claudio L; Rock, Dan L; Barany, Francis; Cao, Weiguo

    2004-09-01

    The eukaryotic Melanoplus sanguinipes entomopoxvirus (MsEPV) genome reveals a homologous sequence to eubacterial nicotinamide adenine dinucleotide (NAD(+))-dependent DNA ligases [J. Virol. 73 (1999) 533]. This 522-amino acid open reading frame (ORF) contains all conserved nucleotidyl transferase motifs but lacks the zinc finger motif and BRCT domain found in conventional eubacterial NAD(+) ligases. Nevertheless, cloned MsEPV ligase seals DNA nicks in a NAD(+)-dependent fashion, while adenosine 5'-monophosphate (ATP) cannot serve as an adenylation cofactor. The ligation activity of MsEPV ligase requires Mg(2+) or Mn(2+). MsEPV ligase seals sticky ends efficiently, but has little activity on 1-nucleotide gap or blunt-ended DNA substrates even in the presence of polyethylene glycol. In comparison, bacterial NAD(+)-dependent ligases seal blunt-ended DNA substrates in the presence of polyethylene glycol. MsEPV DNA ligase readily joins DNA nicks with mismatches at either side of the nick junction, except for mismatches at the nick junction containing an A base in the template strand (A/A, G/A, and C/A). MsEPV NAD(+)-dependent DNA ligase can join DNA probes on RNA templates, a unique property that distinguishes this enzyme from other conventional bacterial NAD(+) DNA ligases. T4 ATP-dependent DNA ligase shows no detectable mismatch ligation at the 3' side of the nick but substantial 5' T/G mismatch ligation on an RNA template. In contrast, MsEPV ligase joins mismatches at the 3' side of the nick more frequently than at the 5' side of the nick on an RNA template. The complementary specificities of these two enzymes suggest alternative primer design for genomic profiling approaches that use allele-specific detection directly from RNA transcripts.

  19. Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity.

    PubMed

    Raychaudhuri, Sumana; Espenshade, Peter J

    2015-06-05

    Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1-Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking.

  20. Rational design of human DNA ligase inhibitors that target cellular DNA replication and repair.

    PubMed

    Chen, Xi; Zhong, Shijun; Zhu, Xiao; Dziegielewska, Barbara; Ellenberger, Tom; Wilson, Gerald M; MacKerell, Alexander D; Tomkinson, Alan E

    2008-05-01

    Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA ligases. Three compounds that differed in their specificity for the three human DNA ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA ligases I and III, and L189 inhibited DNA ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents.

  1. Substrates of IAP ubiquitin ligases identified with a designed orthogonal E3 ligase, the NEDDylator

    PubMed Central

    Zhuang, Min; Guan, Shenheng; Wang, Haopeng; Burlingame, Alma L.; Wells, James A.

    2012-01-01

    SUMMARY Inhibitors of Apoptosis Proteins (IAPs) are guardian ubiquitin ligases that keep classic pro-apoptotic proteins in check. Systematic identification of additional IAP substrates is challenged by the heterogeneity and sheer number of ubiquitinated proteins (>5000). Here we report a powerful catalytic tagging tool, the NEDDylator, which fuses a NEDD8 E2 conjugating enzyme, Ubc12, to the ubiquitin ligase, XIAP or cIAP1. This permits transfer of the rare ubiquitin homolog NEDD8 to the ubiquitin E3 substrates allowing them to be efficiently purified for LC/MS/MS identification. We have identified >50 potential IAP substrates of both cytosolic and mitochondrial origin that bear hallmark N-terminal IAP binding motifs. These substrates include the recently discovered protein phosphatase, PGAM5, which we show is proteolytically processed, accumulates in cytosol during apoptosis, and sensitizes cells to death. These studies reveal mechanisms and antagonistic partners for specific IAPs, and provide a powerful technology for labeling binding partners in transient protein-protein complexes. PMID:23201124

  2. Cellular Retinoic Acid Binding Protein and Breast Cancer

    DTIC Science & Technology

    2006-05-01

    fatty acid probe anilinonaphtalene-8- sulphonic acid (ANS) was measured. ANS readily associates with various FABPs and its fluorescence is highly...DAMD17-03-1-0249 TITLE: Cellular Retinoic Acid Binding Protein and Breast Cancer PRINCIPAL INVESTIGATOR: Leslie J. (Willmert) Donato...DATES COVERED (From - To) 14 Apr 03 – 13 Apr 06 5a. CONTRACT NUMBER Cellular Retinoic Acid Binding Protein and Breast Cancer 5b. GRANT NUMBER

  3. Signal-induced disassembly of the SCF ubiquitin ligase complex by Cdc48/p97

    PubMed Central

    Yen, James L.; Flick, Karin; Papagiannis, Christie V.; Mathur, Radhika; Tyrrell, An; Ouni, Ikram; Kaake, Robyn M.; Huang, Lan; Kaiser, Peter

    2012-01-01

    Summary A large group of E3 ubiquitin ligases is formed by the multisubunit SCF complex, whose core complex (Rbx1/Cul1-Cdc53/Skp1) binds one of many substrate recruiting F-box proteins to form an array of SCF ligases with diverse substrate specificities. It has long been thought that ubiquitylation by SCF ligases is regulated at the level of substrate binding. Here we describe an alternative mechanism of SCF regulation by active dissociation of the F-box subunit. We show that cadmium stress induces selective recruitment of the AAA+ ATPase Cdc48/p97 to catalyze dissociation of the F-box subunit from the yeast SCFMet30 ligase to block substrate ubiquitylation and trigger downstream events. Our results not only provide an additional layer of ubiquitin ligase regulation but also suggest that targeted, signal-dependent dissociation of multisubunit enzyme complexes is an important mechanism in control of enzyme function. PMID:23000173

  4. Selective multifaceted E3 ubiquitin ligases barricade extreme defense: Potential therapeutic targets for neurodegeneration and ageing.

    PubMed

    Upadhyay, Arun; Amanullah, Ayeman; Chhangani, Deepak; Mishra, Ribhav; Mishra, Amit

    2015-11-01

    Efficient and regular performance of Ubiquitin Proteasome System and Autophagy continuously eliminate deleterious accumulation of nonnative protiens. In cellular quality control system, E3 ubiquitin ligases are significant employees for defense mechanism against abnormal toxic proteins. Few findings indicate that lack of functions of E3 ubiquitin ligases can be a causative factor of neurodevelopmental disorders, neurodegeneration, cancer and ageing. However, the detailed molecular pathomechanism implying E3 ubiquitin ligases in cellular functions in multifactorial disease conditions are not well understood. This article systematically represents the unique characteristics, molecular nature, and recent developments in the knowledge of neurobiological functions of few crucial E3 ubiquitin ligases. Here, we review recent literature on the roles of E6-AP, HRD1 and ITCH E3 ubiquitin ligases in the neuro-pathobiological mechanisms, with precise focus on the processes of neurodegeneration, and thereby propose new lines of potential targets for therapeutic interventions.

  5. Functional conservation between mammalian MGRN1 and plant LOG2 ubiquitin ligases.

    PubMed

    Guerra, Damian D; Pratelli, Réjane; Kraft, Edward; Callis, Judy; Pilot, Guillaume

    2013-11-01

    Plant LOSS OF GDU 2 (LOG2) and Mammalian Mahogunin Ring Finger 1 (MGRN1) proteins are RING-type E3 ligases sharing similarity N-terminal to the RING domain. Deletion of this region disrupts the interaction of LOG2 with the plant membrane protein GLUTAMINE DUMPER1 (GDU1). Phylogenetic analysis identified two clades of LOG2/MGRN1-like proteins in vertebrates and plants. The ability of MGRN1 to functionally replace LOG2 was tested. MGRN1 ubiquitylates GDU1 in vitro and can partially substitute for LOG2 in the plant, partially restoring amino acid resistance to a GDU1-myc over-expression, log2-2 background. Altogether, these results suggest a conserved function for the N-terminal domain in evolution.

  6. Essential Roles of E3 Ubiquitin Ligases in p53 Regulation

    PubMed Central

    Sane, Sanam; Rezvani, Khosrow

    2017-01-01

    The ubiquitination pathway and proteasomal degradation machinery dominantly regulate p53 tumor suppressor protein stability, localization, and functions in both normal and cancerous cells. Selective E3 ubiquitin ligases dominantly regulate protein levels and activities of p53 in a large range of physiological conditions and in response to cellular changes induced by exogenous and endogenous stresses. The regulation of p53’s functions by E3 ubiquitin ligases is a complex process that can lead to positive or negative regulation of p53 protein in a context- and cell type-dependent manner. Accessory proteins bind and modulate E3 ubiquitin ligases, adding yet another layer of regulatory control for p53 and its downstream functions. This review provides a comprehensive understanding of p53 regulation by selective E3 ubiquitin ligases and their potential to be considered as a new class of biomarkers and therapeutic targets in diverse types of cancers. PMID:28218667

  7. Two divergent members of a tobacco 4-coumarate:coenzyme A ligase (4CL) gene family. cDNA structure, gene inheritance and expression, and properties of recombinant proteins.

    PubMed Central

    Lee, D; Douglas, C J

    1996-01-01

    Several cDNA clones encoding 4-coumarate:coenzyme A ligase (4CL) were isolated from a tobacco (Nicotiana tabacum) cDNA library and grouped into two classes. Sequencing of one cDNA from each class showed that the clones were similar to other 4CL genes and about 80% identical with each other. Genomic Southern blots using DNA from Nicotiana sylvestris, Nicotiana tomentosiformis, and N. tabacum demonstrated the presence of both classes of 4CL sequences (4CL1 and 4CL2) in the progenitor species and in tobacco. Northern blots indicated that 4CL mRNA transcripts are highest in old stems and higher in the unpigmented corolla tubes than in the pigmented limbs of tobacco flowers. The 4CL genes are developmentally regulated and are wound and methyl jasmonate inducible. The relative abilities of recombinant 4CL1 and 4CL2 proteins to utilize 4-coumarate, ferulate, and caffeate as substrates were similar and comparable with that of 4CL in tobacco stem extracts. Surprisingly, both recombinant 4CL proteins utilized cinnamate as a substrate, an activity not observed in stem extracts. This activity was inhibited by a heat-labile, high-molecular-weight factor found in tobacco stem extracts, suggesting that the substrate specificity of 4CL is, in part, determined by the activity of proteinaceous cellular components. PMID:8819324

  8. A third MRX family (MRX68) is the result of mutation in the long chain fatty acid-CoA ligase 4 (FACL4) gene: proposal of a rapid enzymatic assay for screening mentally retarded patients

    PubMed Central

    Longo, I; Frints, S; Fryns, J; Meloni, I; Pescucci, C; Ariani, F; Borghgraef, M; Raynaud, M; Marynen, P; Schwartz, C; Renieri, A; Froyen, G

    2003-01-01

    Background: The gene encoding fatty acid CoA ligase 4 (FACL4) is mutated in families with non-specific X linked mental retardation (MRX) and is responsible for cognitive impairment in the contiguous gene syndrome ATS-MR (Alport syndrome and mental retardation), mapped to Xq22.3. This finding makes this gene a good candidate for other mental retardation disorders mapping in this region. Methods: We have screened the FACL4 gene in eight families, two MRX and six syndromic X linked mental retardation (MRXS), mapping in a large interval encompassing Xq22.3. Results: We have found a missense mutation in MRX68. The mutation (c.1001C>T in the brain isoform) cosegregates with the disease and changes a highly conserved proline into a leucine (p.P375L) in the first luciferase domain, which markedly reduces the enzymatic activity. Furthermore, all heterozygous females showed completely skewed X inactivation in blood leucocytes, as happens in all reported females with other FACL4 point mutations or deletions. Conclusions: Since the FACL4 gene is highly expressed in brain, where it encodes a brain specific isoform, and is located in hippocampal and cerebellar neurones, a role for this gene in cognitive processes can be expected. Here we report the third MRX family with a FACL4 mutation and describe the development of a rapid enzymatic assay on peripheral blood that we propose as a sensitive, robust, and efficient diagnostic tool in mentally retarded males. PMID:12525535

  9. Phosphorylation of a conserved Thr357 in yeast Nedd4-like ubiquitin ligase Rsp5 is involved in down-regulation of the general amino acid permease Gap1.

    PubMed

    Sasaki, Toshiya; Takagi, Hiroshi

    2013-06-01

    Rsp5, an essential HECT-type ubiquitin ligase, is the only yeast Saccharomyces cerevisiae member of the Nedd4 family. Rsp5 triggers the ubiquitination-dependent endocytosis of the general amino acid permease Gap1 in response to a good nitrogen source. Previously, we showed that the Thr357Ala/Lys764Glu variant Rsp5 induces the constitutive inactivation of Gap1, which is mainly involved in uptake of the toxic proline analogue, l-azetidine-2-carboxylate (AZC). Here, our experimental results indicated that the Thr357Ala substitution in the substrate-recognizing WW2 domain of Rsp5 constitutively causes the down-regulation of four proline permeases (Gap1, Put4, Agp1 and Gnp1), leading to AZC tolerance to yeast cells. In RSP5(T357A) cells, Gap1 was highly ubiquitinated and constantly delivered to the vacuole from the Golgi without sorting to the plasma membrane. Analyses of RSP5 mutants using antiphosphopeptide antibody suggest that Thr phosphorylation occurred in all three WW domains and, interestingly, that Thr357 in the WW2 domain was phosphorylated, in agreement with the in vitro result for the mouse Rsp5 orthologue. Furthermore, the phosphorylation-mimic mutant (Thr357Asp) showed strong sensitivity to AZC. From these results, we propose a possible mechanism involved in the regulation of Rsp5 activity for Gap1 down-regulation via the phosphorylation of a conserved Thr357 in the Nedd4 family.

  10. A conserved physical and functional interaction between the cell cycle checkpoint clamp loader and DNA ligase I of eukaryotes.

    PubMed

    Song, Wei; Levin, David S; Varkey, Johnson; Post, Sean; Bermudez, Vladimir P; Hurwitz, Jerard; Tomkinson, Alan E

    2007-08-03

    DNA ligase I joins Okazaki fragments during DNA replication and completes certain excision repair pathways. The participation of DNA ligase I in these transactions is directed by physical and functional interactions with proliferating cell nuclear antigen, a DNA sliding clamp, and, replication factor C (RFC), the clamp loader. Here we show that DNA ligase I also interacts with the hRad17 subunit of the hRad17-RFC cell cycle checkpoint clamp loader, and with each of the subunits of its DNA sliding clamp, the heterotrimeric hRad9-hRad1-hHus1 complex. In contrast to the inhibitory effect of RFC, hRad17-RFC stimulates joining by DNA ligase I. Similar results were obtained with the homologous Saccharomyces cerevisiae proteins indicating that the interaction between the replicative DNA ligase and checkpoint clamp is conserved in eukaryotes. Notably, we show that hRad17 preferentially interacts with and specifically stimulates dephosphorylated DNA ligase I. Moreover, there is an increased association between DNA ligase I and hRad17 in S phase following DNA damage and replication blockage that occurs concomitantly with DNA damage-induced dephosphorylation of chromatin-associated DNA ligase I. Thus, our results suggest that the in vivo interaction between DNA ligase I and the checkpoint clamp loader is regulated by post-translational modification of DNA ligase I.

  11. Prokaryotic BirA ligase biotinylates K4, K9, K18 and K23 in histone H3.

    PubMed

    Kobza, Keyna; Sarath, Gautam; Zempleni, Janos

    2008-04-30

    BirA ligase is a prokaryotic ortholog of holocarboxylase synthetase (HCS) that can biotinylate proteins. This study tested the hypothesis that BirA ligase catalyzes the biotinylation of eukaryotic histones. If so, this would mean that recombinant BirA ligase is a useful surrogate for HCS in studies of histone biotinylation. The biological activity of recombinant BirA ligase was confirmed by enzymatic biotinylation of p67. In particular, it was found that BirA ligase biotinylated both calf thymus histone H1 and human bulk histone extracts. Incubation of recombinant BirA ligase with H3-based synthetic peptides showed that lysines 4, 9, 18, and 23 in histone H3 are the targets for the biotinylation by BirA ligase. Modification of the peptides (e.g., serine phosphorylation) affected the subsequent biotinylation by BirA ligase, suggesting crosstalk between modifications. In conclusion, this study suggests that prokaryotic BirA ligase is a promiscuous enzyme and biotinylates eukaryotic histones. Moreover the biotinylation of histones by BirA ligase is consistent with the proposed role of human HCS in chromatin.

  12. The transport of phenylacetic acid across the peroxisomal membrane is mediated by the PaaT protein in Penicillium chrysogenum.

    PubMed

    Fernández-Aguado, Marta; Ullán, Ricardo V; Teijeira, Fernando; Rodríguez-Castro, Raquel; Martín, Juan F

    2013-04-01

    Penicillium chrysogenum, an industrial microorganism used worldwide for penicillin production, is an excellent model to study the biochemistry and the cell biology of enzymes involved in the synthesis of secondary metabolites. The well-known peroxisomal location of the last two steps of penicillin biosynthesis (phenylacetyl-CoA ligase and isopenicillin N acyltransferase) requires the import into the peroxisomes of the intermediate isopenicillin N and the precursors phenylacetic acid and coenzyme A. The mechanisms for the molecular transport of these precursors are still poorly understood. In this work, a search was made, in the genome of P. chrysogenum, in order to find a Major Facilitator Superfamily (MFS) membrane protein homologous to CefT of Acremonium chrysogenum, which is known to confer resistance to phenylacetic acid. The paaT gene was found to encode a MFS membrane protein containing 12 transmembrane spanners and one Pex19p-binding domain for Pex19-mediated targeting to peroxisomal membranes. RNA interference-mediated silencing of the paaT gene caused a clear reduction of benzylpenicillin secretion and increased the sensitivity of P. chrysogenum to the penicillin precursor phenylacetic acid. The opposite behavior was found when paaT was overexpressed from the glutamate dehydrogenase promoter that increases phenylacetic acid resistance and penicillin production. Localization studies by fluorescent laser scanning microscopy using PaaT-DsRed and EGFP-SKL fluorescent fusion proteins clearly showed that the protein was located in the peroxisomal membrane. The results suggested that PaaT is involved in penicillin production, most likely through the translocation of side-chain precursors (phenylacetic acid and phenoxyacetic acid) from the cytosol to the peroxisomal lumen across the peroxisomal membrane of P. chrysogenum.

  13. Lack of SMALL ACIDIC PROTEIN 1 (SMAP1) causes increased sensitivity to an inhibitor of RUB/NEDD8-activating enzyme in Arabidopsis seedlings.

    PubMed

    Oono, Yutaka; Nakasone, Akari

    2013-10-01

    SMALL ACIDIC PROTEIN 1 (SMAP1) functions upstream of the degradation of AUX/IAA-proteins in the response to 2,4-dichlorophenoxyacetic acid and physically interacts with the COP9 SIGNALOSOME (CSN). Also, its function is linked to RELATED TO UBIQUITIN (RUB) modification. To further investigate the relationship between SMAP1 and the RUB modification system, we examined the effect of MLN4924, an inhibitor of RUB/NEDD8-activating E1 enzyme, on the growth of Arabidopsis thaliana. We found that the anti-auxin resistant 1 mutants, which lack SMAP1, are more sensitive to MLN4924 than wild type and that SMAP1 is responsible for this hypersensitivity. This new evidence supports our previous speculation that SMAP1 acts in Cullin-RING ubiquitin E3 ligase regulated signaling processes via its interaction with components associated with the RUB modification system.

  14. Heterologous expression of the gourd E3 ubiquitin ligase gene LsRZF1 compromises the drought stress tolerance in Arabidopsis thaliana.

    PubMed

    Min, Ji-Hee; Ju, Hyun-Woo; Yang, Kwang-Yeol; Chung, Jung-Sung; Cho, Baik-Ho; Kim, Cheol Soo

    2014-04-01

    Protein ubiquitination is one of the major regulatory processes used by eukaryotic cells. The ubiquitin E3 ligase acts as a main determinant of substrate specificity. However, the precise roles of E3 ligase in plants to drought stress are poorly understood. In this study, a gourd family (Lagenaria siceraria) ortholog of Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1) gene, designated LsRZF1, was identified and characterized. LsRZF1 was reduced by abscisic acid (ABA), osmotic stress, and drought conditions. Compared to wild type, transgenic Arabidopsis plants ectopic expressing LsRZF1 were hypersensitive to ABA and osmotic stress during early seedling development, indicating that LsRZF1 negatively regulates drought-mediated control of early seedling development. Moreover, the ectopic expression of the LsRZF1 gene was very influential in drought sensitive parameters including proline content, water loss, and the expression of dehydration stress-related genes. Furthermore, ubiquitin E3 ligase activity and genetic data indicate that AtRZF1 and LsRZF1 function in similar pathway to control proline metabolism in Arabidopsis under drought condition. Together, these results suggest that the E3 ligase LsRZF1 is an important regulator of water deficit stress during early seedling development.

  15. Systematic approaches to identify E3 ligase substrates

    PubMed Central

    Iconomou, Mary; Saunders, Darren N.

    2016-01-01

    Protein ubiquitylation is a widespread post-translational modification, regulating cellular signalling with many outcomes, such as protein degradation, endocytosis, cell cycle progression, DNA repair and transcription. E3 ligases are a critical component of the ubiquitin proteasome system (UPS), determining the substrate specificity of the cascade by the covalent attachment of ubiquitin to substrate proteins. Currently, there are over 600 putative E3 ligases, but many are poorly characterized, particularly with respect to individual protein substrates. Here, we highlight systematic approaches to identify and validate UPS targets and discuss how they are underpinning rapid advances in our understanding of the biochemistry and biology of the UPS. The integration of novel tools, model systems and methods for target identification is driving significant interest in drug development, targeting various aspects of UPS function and advancing the understanding of a diverse range of disease processes. PMID:27834739

  16. Ubiquitylation-dependent oligomerization regulates activity of Nedd4 ligases.

    PubMed

    Attali, Ilan; Tobelaim, William Sam; Persaud, Avinash; Motamedchaboki, Khatereh; Simpson-Lavy, Kobi J; Mashahreh, Bayan; Levin-Kravets, Olga; Keren-Kaplan, Tal; Pilzer, Inbar; Kupiec, Martin; Wiener, Reuven; Wolf, Dieter A; Rotin, Daniela; Prag, Gali

    2017-02-15

    Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.

  17. PEX2 is the E3 ubiquitin ligase required for pexophagy during starvation

    PubMed Central

    Sargent, Graeme; van Zutphen, Tim; Shatseva, Tatiana; Zhang, Ling; Di Giovanni, Valeria

    2016-01-01

    Peroxisomes are metabolic organelles necessary for anabolic and catabolic lipid reactions whose numbers are highly dynamic based on the metabolic need of the cells. One mechanism to regulate peroxisome numbers is through an autophagic process called pexophagy. In mammalian cells, ubiquitination of peroxisomal membrane proteins signals pexophagy; however, the E3 ligase responsible for mediating ubiquitination is not known. Here, we report that the peroxisomal E3 ubiquitin ligase peroxin 2 (PEX2) is the causative agent for mammalian pexophagy. Expression of PEX2 leads to gross ubiquitination of peroxisomes and degradation of peroxisomes in an NBR1-dependent autophagic process. We identify PEX5 and PMP70 as substrates of PEX2 that are ubiquitinated during amino acid starvation. We also find that PEX2 expression is up-regulated during both amino acid starvation and rapamycin treatment, suggesting that the mTORC1 pathway regulates pexophagy by regulating PEX2 expression levels. Finally, we validate our findings in vivo using an animal model. PMID:27597759

  18. Enzyme-regulated activation of DNAzyme: a novel strategy for a label-free colorimetric DNA ligase assay and ligase-based biosensing.

    PubMed

    He, Kaiyu; Li, Wang; Nie, Zhou; Huang, Yan; Liu, Zhuoliang; Nie, Lihua; Yao, Shouzhuo

    2012-03-26

    The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label-free and DNAzyme-based strategy to detect DNA ligase activity. This novel strategy relies on the ligation-trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin-DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40 U mL(-1) and a detection limit of 0.2 U mL(-1). Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a "split DNA machine" to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01 U mL(-1).

  19. Oxaloacetate synthesis in the methanarchaeon Methanosarcina barkeri: pyruvate carboxylase genes and a putative Escherichia coli-type bifunctional biotin protein ligase gene (bpl/birA) exhibit a unique organization.

    PubMed

    Mukhopadhyay, B; Purwantini, E; Kreder, C L; Wolfe, R S

    2001-06-01

    Evidence is presented that, in Methanosarcina barkeri oxaloacetate synthesis, an essential and major CO(2) fixation reaction is catalyzed by an apparent alpha(4)beta(4)-type acetyl coenzyme A-independent pyruvate carboxylase (PYC), composed of 64.2-kDa biotinylated and 52.9-kDa ATP-binding subunits. The purified enzyme was most active at 70 degrees C, insensitive to aspartate and glutamate, mildly inhibited by alpha-ketoglutarate, and severely inhibited by ATP, ADP, and excess Mg(2+). It showed negative cooperativity towards bicarbonate at 70 degrees C but not at 37 degrees C. The organism expressed holo-PYC without an external supply of biotin and, thus, synthesized biotin. pycA, pycB, and a putative bpl gene formed a novel operon-like arrangement. Unlike other archaeal homologs, the putative biotin protein ligases (BPLs) of M. barkeri and the closely related euryarchaeon Archaeoglobus fulgidus appeared to be of the Escherichia coli-type (bifunctional, with two activities: BirA or a repressor of the biotin operon and BPL). We found the element Tyr(Phe)ProX(5)Phe(Tyr) to be fully conserved in biotin-dependent enzymes; it might function as the hinge for their "swinging arms."

  20. Protein and sulfur amino acid requirements of broiler breeder hens.

    PubMed

    Harms, R H; Wilson, H R

    1980-02-01

    Two experiments were conducted with Cobb color-sexed broiler breeder hens to determine their protein and sulfur amino acid requirement. A daily intake between 400 and 478 mg of methionine and between 722 and 839 mg of total sulfur amino acids was necessary for maximum egg production, the latter in a diet of 13.07% protein. Slightly lower levels supported maximum body weights. Hens laying at the highest rate consumed 23.4 g of protein per day.

  1. Los Alamos sequence analysis package for nucleic acids and proteins.

    PubMed Central

    Kanehisa, M I

    1982-01-01

    An interactive system for computer analysis of nucleic acid and protein sequences has been developed for the Los Alamos DNA Sequence Database. It provides a convenient way to search or verify various sequence features, e.g., restriction enzyme sites, protein coding frames, and properties of coded proteins. Further, the comprehensive analysis package on a large-scale database can be used for comparative studies on sequence and structural homologies in order to find unnoted information stored in nucleic acid sequences. PMID:6174934

  2. TRIM E3 ligases in HIV infection: can these intrinsic immunity factors be harnessed for novel vaccines or therapies?

    PubMed

    Ndung'u, Thumbi

    2011-01-01

    Tripartite motif-containing (TRIM) E3 ligases are a recently identified family of proteins with potent antiviral activity in mammalian cells. The prototype TRIM E3 ligase, TRIM5α was initially identified as a species-specific antiviral restriction factor but subsequent studies suggest some antiviral activity by several TRIM E3 ligases in human cells. However, the mechanisms of antiviral activity by these proteins and their transcriptional, translational and post-translational regulation are poorly understood. Furthermore, the contribution of TRIM E3 ligases to relative resistance or viral control in vivo is largely unknown. Emerging data from our laboratory and other groups suggests that these proteins may have antiviral activity in vivo and contribute to HIV pathogenesis. Considering the significant difficulties so far encountered in developing an effective HIV vaccine and with the use of antiretroviral therapies, it will be important to further investigate the potential of TRIM E3 ligases as novel prophylactics or therapies.

  3. Amino acid sequences of proteins from Leptospira serovar pomona.

    PubMed

    Alves, S F; Lefebvre, R B; Probert, W

    2000-01-01

    This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.

  4. NRIP/DCAF6 stabilizes the androgen receptor protein by displacing DDB2 from the CUL4A-DDB1 E3 ligase complex in prostate cancer.

    PubMed

    Chen, Hsin-Hsiung; Fan, Ping; Chang, Szu-Wei; Tsao, Yeou-Ping; Huang, Hsiang-Po; Chen, Show-Li

    2017-02-14

    Both nuclear receptor interaction protein (NRIP) and DNA damage binding protein 2 (DDB2) belong to the Cullin 4 (CUL4)-DDB1 binding protein family and are androgen receptor (AR)-interacting proteins. Here, we investigated the expression patterns of the NRIP, DDB2 and AR proteins in human prostate cancer tissues and found that the expression levels of NRIP and AR were higher, but the DDB2 level was lower, in prostate cancer tissues than in non-neoplastic controls, suggesting NRIP as a candidate tumor promoter and DDB2 as a tumor suppressor in prostate cancer. Furthermore, both NRIP and DDB2 shared the same AR binding domain; they were competitors for the AR, but not for DDB1 binding, in the AR-DDB2-DDB1-CUL4A complex. Conclusively, NRIP stabilizes the AR protein by displacing DDB2 from the AR-DDB2 complex. Consistent with our hypothesis, a specific expression pattern with high levels of NRIP and AR, together with a low level of DDB2, was found more frequently in the human prostate cancer tissues with a cribriform pattern than in non-cribriform tumors, suggesting that disruption of the balance between NRIP and DDB2 may change AR protein homeostasis and contribute to pathogenesis in certain aggressive types of prostate cancer.

  5. Cloning and characterization of a novel CoA-ligase gene from Penicillium chrysogenum.

    PubMed

    Yu, Zhou-Liang; Liu, Jing; Wang, Fu-Qiang; Dai, Meng; Zhao, Bao-Hua; He, Jian-Gong; Zhang, Hua

    2011-05-01

    A novel phenylacetic acid (PAA)-induced CoA-ligase-encoding gene, designated as phlC, has been cloned from penicillin-producing fungus Penicillium chrysogenum. The open reading frame of phlC cDNA was 1671 bp and encoded a 556 amino acid residues protein with the consensus AMP binding site and a peroxisomal targeting signal 1 on its C terminus. The deduced amino acid sequence showed 37% and 38% identity with characterized P. chrysogenum Phl and PhlB protein, respectively. Functional recombinant PhlC protein was overexpressed in Escherichia coli. The purified recombinant enzyme was capable to convert PAA into its corresponding CoA ester with a specific activity of 129.5 ± 3.026 pmol/min per mg protein. Similar to Phl and PhlB, PhlC displayed broad substrate spectrum and showed higher activities to medium- and long-chain fatty acids. The catalytic properties of PhlC have been determined and compared to those of Phl and PhlB.

  6. Kainic acid inhibits protein amino acid incorporation in select rat brain regions.

    PubMed

    Planas, A M; Soriano, M A; Ferrer, I; Rodríguez-Farré, E

    1994-11-21

    Regional incorporation of labelled methionine into proteins was studied with quantitative autoradiography in different regions of the rat brain 2.5 h following systemic kainic acid administration. Labelled protein concentration was found reduced to approximately 40% of control values in the pyramidal cell layer of hippocampus, piriform, entorhinal and perirhinal cortices, ventral lateral septum and mediodorsal thalamic nucleus. These regions showed increased levels of label not incorporated into proteins, indicating that free labelled methionine was available for protein synthesis. Reduction of protein amino acid incorporation in those brain regions selectively affected by kainic acid may be involved in subsequent tissue damage.

  7. Amino acid metabolism and protein synthesis in malarial parasites*

    PubMed Central

    Sherman, I. W.

    1977-01-01

    Malaria-infected red cells and free parasites have limited capabilities for the biosynthesis of amino acids. Therefore, the principal amino acid sources for parasite protein synthesis are the plasma free amino acids and host cell haemoglobin. Infected cells and plasmodia incorporate exogenously supplied amino acids into protein. However, the hypothesis that amino acid utilization (from an external source) is related to availability of that amino acid in haemoglobin is without universal support: it is true for isoleucine and for Plasmodium knowlesi and P. falciparum, but not for methionine, cysteine, and other amino acids, and it does not apply to P. lophurae. More by default than by direct evidence, haemoglobin is believed to be the main amino acid reservoir available to the intraerythrocytic plasmodium. Haemoglobin, ingested via the cytostome, is held in food vacuoles where auto-oxidation takes place. As a consequence, haem is released and accumulates in the vacuole as particulate haemozoin (= malaria pigment). Current evidence favours the view that haemozoin is mainly haematin. Acid and alkaline proteases (identified in crude extracts from mammalian and avian malarias) are presumably secreted directly into the food vacuole. They then digest the denatured globin and the resulting amino acids are incorporated into parasite protein. Cell-free protein synthesizing systems have been developed using P. knowlesi and P. lophurae ribosomes. In the main these systems are typically eukaryotic. Studies of amino acid metabolism are exceedingly limited. Arginine, lysine, methionine, and proline are incorporated into protein, whereas glutamic acid is metabolized via an NADP-specific glutamic dehydrogenase. Glutamate oxidation generates NADPH and auxiliary energy (in the form of α-ketoglutarate). The role of red cell glutathione in the economy of the parasite remains obscure. Important goals for future research should be: quantitative assessment of the relative importance of

  8. Purification and characterization of benzoate-coenzyme A ligase and 2-aminobenzoate-coenzyme A ligases from a denitrifying Pseudomonas sp.

    PubMed Central

    Altenschmidt, U; Oswald, B; Fuchs, G

    1991-01-01

    The enzymes catalyzing the formation of coenzyme A (CoA) thioesters of benzoate and 2-aminobenzoate were studied in a denitrifying Pseudomonas sp. anaerobically grown with these aromatic acids and nitrate as sole carbon and energy sources. Three different rather specific aromatic acyl-CoA ligases, E1, E2, and E3, were found which catalyze the formation of CoA thioesters of benzoate, fluorobenzoates, and 2-aminobenzoate. ATP is cleaved into AMP and pyrophosphate. The enzymes were purified, their N-terminal amino acid sequences were determined, and their catalytic and molecular properties were studied. Cells anaerobically grown on benzoate and nitrate contain one CoA ligase (AMP forming) for benzoic acid (E1). It is a homodimer of Mr 120,000 which prefers benzoate as a substrate but shows some activity also with 2-aminobenzoate and fluorobenzoates, although with lower Km. Cells anaerobically grown on 2-aminobenzoate and nitrate contain three different CoA ligases for aromatic acids. The first one is identical with benzoate-CoA ligase (E1). The second enzyme is a 2-aminobenzoate-CoA ligase (E2). It is a monomer of Mr 60,000 which prefers 2-aminobenzoate but also activates benzoate, fluorobenzoates and, less effectively, 2-methylbenzoate, with lower affinities to the latter substrates. The enzymes E1 and E2 have similar activity levels; a third minor CoA ligase activity is due to a different 2-aminobenzoate-CoA ligase. The enzyme (E3) is a monomer of Mr, 65,000 which 2-aminobenzoate pathway (U. Altenschmidt, C. Eckerskorn, and G. Fuchs, Eur. J. Biochem. 194:647-653, 1990); apparently, it is not completely repressed under anaerobic conditions and therefore also is induced to a small extent by 2-aminobenzoate under anaerobic growth conditions. Images PMID:1885526

  9. Pseudomonas aeruginosa PqsA is an anthranilate-coenzyme A ligase.

    PubMed

    Coleman, James P; Hudson, L Lynn; McKnight, Susan L; Farrow, John M; Calfee, M Worth; Lindsey, Claire A; Pesci, Everett C

    2008-02-01

    Pseudomonas aeruginosa is an opportunistic human pathogen which relies on several intercellular signaling systems for optimum population density-dependent regulation of virulence genes. The Pseudomonas quinolone signal (PQS) is a 3-hydroxy-4-quinolone with a 2-alkyl substitution which is synthesized by the condensation of anthranilic acid with a 3-keto-fatty acid. The pqsABCDE operon has been identified as being necessary for PQS production, and the pqsA gene encodes a predicted protein with homology to acyl coenzyme A (acyl-CoA) ligases. In order to elucidate the first step of the 4-quinolone synthesis pathway in P. aeruginosa, we have characterized the function of the pqsA gene product. Extracts prepared from Escherichia coli expressing PqsA were shown to catalyze the formation of anthraniloyl-CoA from anthranilate, ATP, and CoA. The PqsA protein was purified as a recombinant His-tagged polypeptide, and this protein was shown to have anthranilate-CoA ligase activity. The enzyme was active on a variety of aromatic substrates, including benzoate and chloro and fluoro derivatives of anthranilate. Inhibition of PQS formation in vivo was observed for the chloro- and fluoroanthranilate derivatives, as well as for several analogs which were not PqsA enzymatic substrates. These results indicate that the PqsA protein is responsible for priming anthranilate for entry into the PQS biosynthetic pathway and that this enzyme may serve as a useful in vitro indicator for potential agents to disrupt quinolone signaling in P. aeruginosa.

  10. KINETICS OF AMINO ACID INCORPORATION INTO SERUM PROTEINS

    PubMed Central

    Green, H.; Anker, H. S.

    1955-01-01

    1. The effect of varying body temperature on the rate of amino acid incorporation into serum protein does not give support to the idea that the rate of this process is adjusted in vivo to restore those protein molecules destroyed by thermal denaturation. The experimentally observed Q10 was about 3.9. 2. When amino acids are injected into the blood of animals in a steady state of serum protein turnover, a period of time elapses before these amino acids can be found in the serum proteins. This has been called transit time. At a given temperature (31°) it is the same in rabbits, turtles, and Limulus (1 hour). In rabbits and turtles it has a Q10 of 3.2. It appears to be specifically related to the process of synthesis (or release) of serum proteins. 3. It was not possible to affect the transit time or the incorporation rate by the administration of amino acid analogues. PMID:13221773

  11. Interaction of milk whey protein with common phenolic acids

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Yu, Dandan; Sun, Jing; Guo, Huiyuan; Ding, Qingbo; Liu, Ruihai; Ren, Fazheng

    2014-01-01

    Phenolics-rich foods such as fruit juices and coffee are often consumed with milk. In this study, the interactions of α-lactalbumin and β-lactoglobulin with the phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, and coumalic acid) were examined. Fluorescence, CD, and FTIR spectroscopies were used to analyze the binding modes, binding constants, and the effects of complexation on the conformation of whey protein. The results showed that binding constants of each whey protein-phenolic acid interaction ranged from 4 × 105 to 7 × 106 M-n and the number of binding sites n ranged from 1.28 ± 0.13 to 1.54 ± 0.34. Because of these interactions, the conformation of whey protein was altered, with a significant reduction in the amount of α-helix and an increase in the amounts of β-sheet and turn structures.

  12. Recombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicida.

    PubMed

    Williamson, Adele; Pedersen, Hege

    2014-05-01

    The genome of the psychrophilic fish-pathogen Aliivibrio salmonicida encodes a putative ATP-dependent DNA ligase in addition to a housekeeping NAD-dependent enzyme. In order to study the structure and activity of the ATP dependent ligase in vitro we have undertaken its recombinant production and purification from an Escherichia coli based expression system. Expression and purification of this protein presented two significant challenges. First, the gene product was moderately toxic to E. coli cells, second it was necessary to remove the large amounts of E. coli DNA present in bacterial lysates without contamination of the protein preparation by nucleases which might interfere with future assaying. The toxicity problem was overcome by fusion of the putative ligase to large solubility tags such as maltose-binding protein (MBP) or Glutathione-S-transferase (GST), and DNA was removed by treatment with a nuclease which could be inhibited by reducing agents. As the A. salmonicida ATP-dependent DNA ligase gene encodes a predicted leader peptide, both the full-length and mature forms of the protein were produced. Both possessed ATP-dependent DNA ligase activity, but the truncated form was significantly more active. Here we detail the first reported production, purification and preliminary characterization of active A. salmonicida ATP-dependent DNA ligase.

  13. Pressure-induced endocytic degradation of the Saccharomyces cerevisiae low-affinity tryptophan permease Tat1 is mediated by Rsp5 ubiquitin ligase and functionally redundant PPxY motif proteins.

    PubMed

    Suzuki, Asaha; Mochizuki, Takahiro; Uemura, Satoshi; Hiraki, Toshiki; Abe, Fumiyoshi

    2013-07-01

    Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1(K29R-K31R)-GFP remained. The HPG1-1 (Rsp5(P514T)) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.

  14. Suramin inhibits cullin-RING E3 ubiquitin ligases

    PubMed Central

    Wu, Kenneth; Chong, Robert A.; Yu, Qing; Bai, Jin; Spratt, Donald E.; Ching, Kevin; Lee, Chan; Miao, Haibin; Tappin, Inger; Hurwitz, Jerard; Zheng, Ning; Shaw, Gary S.; Sun, Yi; Felsenfeld, Dan P.; Sanchez, Roberto; Zheng, Jun-nian; Pan, Zhen-Qiang

    2016-01-01

    Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3′s cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1’s conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2–E3 interface through small-molecule modulators. PMID:27001857

  15. Amino acid composition of proteins reduces deleterious impact of mutations

    PubMed Central

    Hormoz, Sahand

    2013-01-01

    The evolutionary origin of amino acid occurrence frequencies in proteins (composition) is not yet fully understood. We suggest that protein composition works alongside the genetic code to minimize impact of mutations on protein structure. First, we propose a novel method for estimating thermodynamic stability of proteins whose sequence is constrained to a fixed composition. Second, we quantify the average deleterious impact of substituting one amino acid with another. Natural proteome compositions are special in at least two ways: 1) Natural compositions do not generate more stable proteins than the average random composition, however, they result in proteins that are less susceptible to damage from mutations. 2) Natural proteome compositions that result in more stable proteins (i.e. those of thermophiles) are also tuned to have a higher tolerance for mutations. This is consistent with the observation that environmental factors selecting for more stable proteins also enhance the deleterious impact of mutations. PMID:24108121

  16. The protein digestibility-corrected amino acid score.

    PubMed

    Schaafsma, G

    2000-07-01

    The protein digestibility-corrected amino acid score (PDCAAS) has been adopted by FAO/WHO as the preferred method for the measurement of the protein value in human nutrition. The method is based on comparison of the concentration of the first limiting essential amino acid in the test protein with the concentration of that amino acid in a reference (scoring) pattern. This scoring pattern is derived from the essential amino acid requirements of the preschool-age child. The chemical score obtained in this way is corrected for true fecal digestibility of the test protein. PDCAAS values higher than 100% are not accepted as such but are truncated to 100%. Although the principle of the PDCAAS method has been widely accepted, critical questions have been raised in the scientific community about a number of issues. These questions relate to 1) the validity of the preschool-age child amino acid requirement values, 2) the validity of correction for fecal instead of ileal digestibility and 3) the truncation of PDCAAS values to 100%. At the time of the adoption of the PDCAAS method, only a few studies had been performed on the amino acid requirements of the preschool-age child, and there is still a need for validation of the scoring pattern. Also, the scoring pattern does not include conditionally indispensable amino acids. These amino acids also contribute to the nutrition value of a protein. There is strong evidence that ileal, and not fecal, digestibility is the right parameter for correction of the amino acid score. The use of fecal digestibility overestimates the nutritional value of a protein, because amino acid nitrogen entering the colon is lost for protein synthesis in the body and is, at least in part, excreted in urine as ammonia. The truncation of PDCAAS values to 100% can be defended only for the limited number of situations in which the protein is to be used as the sole source of protein in the diet. For evaluation of the nutritional significance of proteins as

  17. ATM mediates oxidative stress-induced dephosphorylation of DNA ligase IIIalpha.

    PubMed

    Dong, Zhiwan; Tomkinson, Alan E

    2006-01-01

    Among the three mammalian genes encoding DNA ligases, only the LIG3 gene does not have a homolog in lower eukaryotes. In somatic mammalian cells, the nuclear form of DNA ligase IIIalpha forms a stable complex with the DNA repair protein XRCC1 that is also found only in higher eukaryotes. Recent studies have shown that XRCC1 participates in S phase-specific DNA repair pathways independently of DNA ligase IIIalpha and is constitutively phosphorylated by casein kinase II. In this study we demonstrate that DNA ligase IIIalpha, unlike XRCC1, is phosphorylated in a cell cycle-dependent manner. Specifically, DNA ligase IIIalpha is phosphorylated on Ser123 by the cell division cycle kinase Cdk2 beginning early in S phase and continuing into M phase. Interestingly, treatment of S phase cells with agents that cause oxygen free radicals induces the dephosphorylation of DNA ligase IIIalpha. This oxidative stress-induced dephosphorylation of DNA ligase IIIalpha is dependent upon the ATM (ataxia-telangiectasia mutated) kinase and appears to involve inhibition of Cdk2 and probably activation of a phosphatase.

  18. ATM mediates oxidative stress-induced dephosphorylation of DNA ligase IIIα

    PubMed Central

    Dong, Zhiwan; Tomkinson, Alan E.

    2006-01-01

    Among the three mammalian genes encoding DNA ligases, only the LIG3 gene does not have a homolog in lower eukaryotes. In somatic mammalian cells, the nuclear form of DNA ligase IIIα forms a stable complex with the DNA repair protein XRCC1 that is also found only in higher eukaryotes. Recent studies have shown that XRCC1 participates in S phase-specific DNA repair pathways independently of DNA ligase IIIα and is constitutively phosphorylated by casein kinase II. In this study we demonstrate that DNA ligase IIIα, unlike XRCC1, is phosphorylated in a cell cycle-dependent manner. Specifically, DNA ligase IIIα is phosphorylated on Ser123 by the cell division cycle kinase Cdk2 beginning early in S phase and continuing into M phase. Interestingly, treatment of S phase cells with agents that cause oxygen free radicals induces the dephosphorylation of DNA ligase IIIα. This oxidative stress-induced dephosphorylation of DNA ligase IIIα is dependent upon the ATM (ataxia-telangiectasia mutated) kinase and appears to involve inhibition of Cdk2 and probably activation of a phosphatase. PMID:17040896

  19. The RING E3 Ligase KEEP ON GOING Modulates JASMONATE ZIM-DOMAIN12 Stability1[OPEN

    PubMed Central

    Pauwels, Laurens; Ritter, Andrés; Goossens, Jonas; Durand, Astrid Nagels; Liu, Hongxia; Gu, Yangnan; Geerinck, Jan; Boter, Marta; Vanden Bossche, Robin; De Clercq, Rebecca; Van Leene, Jelle; Gevaert, Kris; De Jaeger, Geert; Solano, Roberto; Stone, Sophia; Innes, Roger W.; Callis, Judy; Goossens, Alain

    2015-01-01

    Jasmonate (JA) signaling in plants is mediated by the JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of several transcription factors regulating JA-inducible gene expression. The hormone JA-isoleucine triggers the interaction of JAZ repressor proteins with the F-box protein CORONATINE INSENSITIVE1 (COI1), part of an S-phase kinase-associated protein1/Cullin1/F-box protein COI1 (SCFCOI1) E3 ubiquitin ligase complex, and their degradation by the 26S proteasome. In Arabidopsis (Arabidopsis thaliana), the JAZ family consists of 13 members. The level of redundancy or specificity among these members is currently not well understood. Here, we characterized JAZ12, encoded by a highly expressed JAZ gene. JAZ12 interacted with the transcription factors MYC2, MYC3, and MYC4 in vivo and repressed MYC2 activity. Using tandem affinity purification, we found JAZ12 to interact with SCFCOI1 components, matching with observed in vivo ubiquitination and with rapid degradation after treatment with JA. In contrast to the other JAZ proteins, JAZ12 also interacted directly with the E3 RING ligase KEEP ON GOING (KEG), a known repressor of the ABSCISIC ACID INSENSITIVE5 transcription factor in abscisic acid signaling. To study the functional role of this interaction, we circumvented the lethality of keg loss-of-function mutants by silencing KEG using an artificial microRNA approach. Abscisic acid treatment promoted JAZ12 degradation, and KEG knockdown led to a decrease in JAZ12 protein levels. Correspondingly, KEG overexpression was capable of partially inhibiting COI1-mediated JAZ12 degradation. Our results provide additional evidence for KEG as an important factor in plant hormone signaling and a positive regulator of JAZ12 stability. PMID:26320228

  20. Protein packing: dependence on protein size, secondary structure and amino acid composition.

    PubMed

    Fleming, P J; Richards, F M

    2000-06-02

    We have used the occluded surface algorithm to estimate the packing of both buried and exposed amino acid residues in protein structures. This method works equally well for buried residues and solvent-exposed residues in contrast to the commonly used Voronoi method that works directly only on buried residues. The atomic packing of individual globular proteins may vary significantly from the average packing of a large data set of globular proteins. Here, we demonstrate that these variations in protein packing are due to a complex combination of protein size, secondary structure composition and amino acid composition. Differences in protein packing are conserved in protein families of similar structure despite significant sequence differences. This conclusion indicates that quality assessments of packing in protein structures should include a consideration of various parameters including the packing of known homologous proteins. Also, modeling of protein structures based on homologous templates should take into account the packing of the template protein structure.

  1. Re-evaluation of turbidimetry of proteins by use of aromatic sulfonic acids and chloroacetic acids.

    PubMed

    Ebina, S; Nagai, Y

    1979-02-01

    From studies on 11 different proteins (including native albumin and albumin with reduced disulfide-bridges) treated with sulfosalicylic, 2-naphthalenesulfonic, toluenesulfonic, dichloroacetic, or trichloroacetic acids, we elucidate the interactions determining the resulting turbidities and other factors affecting turbidities, and we discuss the clinical utility of such turbidimetry. At least three interactions are important in determining turbidity: reduction of positive charges on the protein, hydrogen bonding of the non-ionized chloroacetic acids with the protein, and hydrophobic interaction of the aromatic sulfonic acids with albumin. Turbidity varies appreciably with the species of acid and protein, concentrations of acid, temperature, and standing time after acid is added. We conclude that this technique should be restricted to confirming proteinuria.

  2. Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase.

    PubMed Central

    Lahav-Baratz, S; Sudakin, V; Ruderman, J V; Hershko, A

    1995-01-01

    Cyclin B/cdc2 is responsible both for driving cells into mitosis and for activating the ubiquitin-dependent degradation of mitotic cyclins near the end of mitosis, an event required for the completion of mitosis and entry into interphase of the next cell cycle. Previous work with cell-free extracts of rapidly dividing clam embryos has identified two specific components required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase that purifies as part of a approximately 1500-kDa complex, termed the cyclosome, and which is active only near the end of mitosis. Here, we have separated the cyclosome from its ultimate upstream activator, cdc2. The mitotic, active form of the cyclosome can be inactivated by incubation with a partially purified, endogenous okadaic acid-sensitive phosphatase; addition of cdc2 restores activity to the cyclosome after a lag that reproduces that seen previously in intact cells and in crude extracts. These results demonstrate that activity of cyclin-ubiquitin ligase is controlled by reversible phosphorylation of the cyclosome complex. Images Fig. 3 PMID:7568122

  3. Overexpression of Sna3 stabilizes tryptophan permease Tat2, potentially competing for the WW domain of Rsp5 ubiquitin ligase with its binding protein Bul1.

    PubMed

    Hiraki, Toshiki; Abe, Fumiyoshi

    2010-01-04

    Tryptophan permease Tat2 in Saccharomyces cerevisiae undergoes Rsp5-dependent degradation upon exposure to high hydrostatic pressure and it limits the growth of tryptophan auxotrophs. Overexpression of SNA3 encoding an endosomal/vacuolar protein possessing the PPAY motif allowed growth at 25 MPa, which was potentiated by marked stabilization of Tat2. This appeared to depend on the PPAY motif, which interacted with the WW domain of Rsp5. Subcellular localization of Rsp5 was unchanged by overexpression of either SNA3 or SNA3-AAAY. While the loss of Bul1, a binding protein of Rsp5, or the rsp5-ww3 mutation allowed high-pressure growth, overexpression of BUL1 abolished the Sna3-mediated growth at 25 MPa. These results suggest that Sna3 and Bul1 compete for the WW domain of Rsp5 upon Tat2 ubiquitination.

  4. SUMO E3 Ligases GmSIZ1a and GmSIZ1b regulate vegetative growth in soybean† 

    PubMed Central

    Cai, Bin; Kong, Xiangxiong; Zhong, Chao; Sun, Suli; Zhou, Xiao Feng; Jin, Yin Hua; Wang, Youning; Li, Xia; Zhu, Zhendong

    2017-01-01

    Abstract SIZ1 is a small ubiquitin‐related modifier (SUMO) E3 ligase that mediates post‐translational SUMO modification of target proteins and thereby regulates developmental processes and hormonal and environmental stress responses in Arabidopsis. However, the role of SUMO E3 ligases in crop plants is largely unknown. Here, we identified and characterized two Glycine max (soybean) SUMO E3 ligases, GmSIZ1a and GmSIZ1b. Expression of GmSIZ1a and GmSIZ1b was induced in response to salicylic acid (SA), heat, and dehydration treatment, but not in response to cold, abscisic acid (ABA), and NaCl treatment. Although GmSIZ1a was expressed at higher levels than GmSIZ1b, both genes encoded proteins with SUMO E3 ligase activity in vivo. Heterologous expression of GmSIZ1a or GmSIZ1b rescued the mutant phenotype of Arabidopsis siz1‐2, including dwarfism, constitutively activated expression of pathogen‐related genes, and ABA‐sensitive seed germination. Simultaneous downregulation of GmSIZ1a and GmSIZ1b (GmSIZ1a/b) using RNA interference (RNAi)‐mediated gene silencing decreased heat shock‐induced SUMO conjugation in soybean. Moreover, GmSIZ1RNAi plants exhibited reduced plant height and leaf size. However, unlike Arabidopsis siz1‐2 mutant plants, flowering time and SA levels were not significantly altered in GmSIZ1RNAi plants. Taken together, our results indicate that GmSIZ1a and GmSIZ1b mediate SUMO modification and positively regulate vegetative growth in soybean. PMID:27762067

  5. Identification of the endocytic sorting signal recognized by the Art1-Rsp5 ubiquitin ligase complex

    PubMed Central

    Guiney, Evan L.; Klecker, Till; Emr, Scott D.

    2016-01-01

    Targeted endocytosis of plasma membrane (PM) proteins allows cells to adjust their complement of membrane proteins to changing extracellular conditions. For a wide variety of PM proteins, initiation of endocytosis is triggered by ubiquitination. In yeast, arrestin-related trafficking adaptors (ARTs) enable a single ubiquitin ligase, Rsp5, to specifically and selectively target a wide range of PM proteins for ubiquitination and endocytosis. However, the mechanisms that allow ARTs to specifically recognize their appropriate substrates are unknown. We present the molecular features in the methionine permease Mup1 that are required for Art1-Rsp5–mediated ubiquitination and endocytosis. A combination of genetics, fluorescence microscopy, and biochemistry reveals three critical features that comprise an ART sorting signal in the Mup1 N-terminal cytosolic tail: 1) an extended acidic patch, 2) in close proximity to the first Mup1 transmembrane domain, and 3) close to the ubiquitinated lysines. We show that a functionally similar ART sorting signal is also required for the endocytosis of a second Art1-dependent cargo, Can1, suggesting a common mechanism for recognition of Art1 substrates. We isolate two separate suppressor mutations in the Art1 C-terminal domain that allele-specifically restore endocytosis of two Mup1 acidic patch mutants, consistent with an interaction between the Art1 C-terminus and the Mup1 acidic patch. We propose that this interaction is required for recruitment of the Art1-Rsp5 ubiquitination complex. PMID:27798240

  6. BIOACTIVE PROTEINS, PEPTIDES, AND AMINO ACIDS FROM MACROALGAE(1).

    PubMed

    Harnedy, Pádraigín A; FitzGerald, Richard J

    2011-04-01

    Macroalgae are a diverse group of marine organisms that have developed complex and unique metabolic pathways to ensure survival in highly competitive marine environments. As a result, these organisms have been targeted for mining of natural biologically active components. The exploration of marine organisms has revealed numerous bioactive compounds that are proteinaceous in nature. These include proteins, linear peptides, cyclic peptides and depsipeptides, peptide derivatives, amino acids, and amino acid-like components. Furthermore, some species of macroalgae have been shown to contain significant levels of protein. While some protein-derived bioactive peptides have been characterized from macroalgae, macroalgal proteins currently still represent good candidate raw materials for biofunctional peptide mining. This review will provide an overview of the important bioactive amino-acid-containing compounds that have been identified in macroalgae. Moreover, the potential of macroalgal proteins as substrates for the generation of biofunctional peptides for utilization as functional foods to provide specific health benefits will be discussed.

  7. Pressure-induced differential regulation of the two tryptophan permeases Tat1 and Tat2 by ubiquitin ligase Rsp5 and its binding proteins, Bul1 and Bul2.

    PubMed

    Abe, Fumiyoshi; Iida, Hidetoshi

    2003-11-01

    Tryptophan uptake appears to be the Achilles' heel in yeast physiology, since under a variety of seemingly diverse toxic conditions, it becomes the limiting factor for cell growth. When growing cells of Saccharomyces cerevisiae are subjected to high hydrostatic pressure, tryptophan uptake is down-regulated, leading to cell cycle arrest in the G(1) phase. Here we present evidence that the two tryptophan permeases Tat1 and Tat2 are differentially regulated by Rsp5 ubiquitin ligase in response to high hydrostatic pressure. Analysis of high-pressure growth mutants revealed that the HPG1 gene was allelic to RSP5. The HPG1 mutation or the bul1Delta bul2Delta double mutation caused a marked increase in the steady-state level of Tat2 but not of Tat1, although both permeases were degraded at high pressure in an Rsp5-dependent manner. There were marked differences in subcellular localization. Tat1 localized predominantly in the plasma membrane, whereas Tat2 was abundant in the internal membranes. Moreover, Tat1 was associated with lipid rafts, whereas Tat2 localized in bulk lipids. Surprisingly, Tat2 became associated with lipid rafts upon the occurrence of a ubiquitination defect. These results suggest that ubiquitination is an important determinant of the localization and regulation of these tryptophan permeases. Determination of the activation volume (DeltaV( not equal )) for Tat1- and Tat2-mediated tryptophan uptake (89.3 and 50.8 ml/mol, respectively) revealed that both permeases are highly sensitive to membrane perturbation and that Tat1 rather than Tat2 is likely to undergo a dramatic conformational change during tryptophan import. We suggest that hydrostatic pressure is a unique tool for elucidating the dynamics of integral membrane protein functions as well as for probing lipid microenvironments where they localize.

  8. A novel interaction between DNA ligase III and DNA polymerase gamma plays an essential role in mitochondrial DNA stability.

    PubMed

    De, Ananya; Campbell, Colin

    2007-02-15

    The data in the present study show that DNA polymerase gamma and DNA ligase III interact in mitochondrial protein extracts from cultured HT1080 cells. An interaction was also observed between the two recombinant proteins in vitro. Expression of catalytically inert versions of DNA ligase III that bind DNA polymerase gamma was associated with reduced mitochondrial DNA copy number and integrity. In contrast, overexpression of wild-type DNA ligase III had no effect on mitochondrial DNA copy number or integrity. Experiments revealed that wild-type DNA ligase III facilitates the interaction of DNA polymerase gamma with a nicked DNA substrate in vitro, and that the zinc finger domain of DNA ligase III is required for this activity. Mitochondrial protein extracts prepared from cells overexpressing a DNA ligase III protein that lacked the zinc finger domain had reduced base excision repair activity compared with extracts from cells overexpressing the wild-type protein. These data support the interpretation that the interaction of DNA ligase III and DNA polymerase gamma is required for proper maintenance of the mammalian mitochondrial genome.

  9. E3 ubiquitin ligase gene CMPG1-V from Haynaldia villosa L. contributes to powdery mildew resistance in common wheat (Triticum aestivum L.).

    PubMed

    Zhu, Yanfei; Li, Yingbo; Fei, Fei; Wang, Zongkuan; Wang, Wei; Cao, Aizhong; Liu, Yuan; Han, Shuang; Xing, Liping; Wang, Haiyan; Chen, Wei; Tang, Sanyuan; Huang, Xiahe; Shen, Qianhua; Xie, Qi; Wang, Xiue

    2015-10-01

    Powdery mildew is one of the most devastating wheat fungal diseases. A diploid wheat relative, Haynaldia villosa L., is highly resistant to powdery mildew, and its genetic resource of resistances, such as the Pm21 locus, is now widely used in wheat breeding. Here we report the cloning of a resistance gene from H. villosa, designated CMPG1-V, that encodes a U-box E3 ubiquitin ligase. Expression of the CMPG1-V gene was induced in the leaf and stem of H. villosa upon inoculation with Blumeria graminis f. sp. tritici (Bgt) fungus, and the presence of Pm21 is essential for its rapid induction of expression. CMPG1-V has conserved key residues for E3 ligase, and possesses E3 ligase activity in vitro and in vivo. CMPG1-V is localized in the nucleus, endoplasmic reticulum, plasma membrane and partially in trans-Golgi network/early endosome vesicles. Transgenic wheat over-expressing CMPG1-V showed improved broad-spectrum powdery mildew resistance at seedling and adult stages, associated with an increase in expression of salicylic acid-responsive genes, H2 O2 accumulation, and cell-wall protein cross-linking at the Bgt infection sites, and the expression of CMPG1-V in H. villosa was increased when treated with salicylic acid, abscisic acid and H2 O2 . These results indicate the involvement of E3 ligase in defense responses to Bgt fungus in wheat, particularly in broad-spectrum disease resistance, and suggest association of reactive oxidative species and the phytohormone pathway with CMPG1-V-mediated powdery mildew resistance.

  10. Cloning, molecular characterization and expression of a DNA-ligase from a new bacteriophage: Phax1.

    PubMed

    Setayesh, Neda; Sabouri-Shahrbabak, Saleheh; Bakherad, Hamid; Sepehrizadeh, Zargham

    2013-12-01

    DNA ligases join 3' hydroxyl and 5' phosphate ends in double stranded DNA and are necessary for maintaining the integrity of genome. The gene encoding a new Escherichia phage (Phax1) DNA ligase was cloned and sequenced. The gene contains an open reading frame with 1,428 base pairs, encoding 475 amino acid residues. Alignment of the entire amino acid sequence showed that Phax1 DNA ligase has a high degree of sequence homology with ligases from Escherichia (vB_EcoM_CBA120), Salmonella (PhiSH19 and SFP10), Shigella (phiSboM-AG3), and Deftia (phiW-14) phages. The Phax1 DNA ligase gene was expressed under the control of the T7lac promoter on the pET-16b (+) in Escherichia coli Rossetta gami. The enzyme was then homogeneously purified by a metal affinity column. Enzymatic activity of the recombinant DNA ligase was assayed by an in-house PCR-based method.

  11. Conformational Entropy of Intrinsically Disordered Proteins from Amino Acid Triads

    PubMed Central

    Baruah, Anupaul; Rani, Pooja; Biswas, Parbati

    2015-01-01

    This work quantitatively characterizes intrinsic disorder in proteins in terms of sequence composition and backbone conformational entropy. Analysis of the normalized relative composition of the amino acid triads highlights a distinct boundary between globular and disordered proteins. The conformational entropy is calculated from the dihedral angles of the middle amino acid in the amino acid triad for the conformational ensemble of the globular, partially and completely disordered proteins relative to the non-redundant database. Both Monte Carlo (MC) and Molecular Dynamics (MD) simulations are used to characterize the conformational ensemble of the representative proteins of each group. The results show that the globular proteins span approximately half of the allowed conformational states in the Ramachandran space, while the amino acid triads in disordered proteins sample the entire range of the allowed dihedral angle space following Flory’s isolated-pair hypothesis. Therefore, only the sequence information in terms of the relative amino acid triad composition may be sufficient to predict protein disorder and the backbone conformational entropy, even in the absence of well-defined structure. The predicted entropies are found to agree with those calculated using mutual information expansion and the histogram method. PMID:26138206

  12. Molecular cloning and functional identification of a novel phenylacetyl-CoA ligase gene from Penicillium chrysogenum.

    PubMed

    Wang, Fu-Qiang; Liu, Jing; Dai, Meng; Ren, Zhi-Hong; Su, Cai-Yun; He, Jian-Gong

    2007-08-24

    A novel phenylacetyl-CoA ligase gene, designated phlB, was cloned and identified from the penicillin producing strain Penicillium chrysogenum based on subtractive suppression hybridization approach. The phlB gene contains a 1686-bp open-reading frame and encodes a protein of approximately 62.6 kDa. The deduced amino acid sequence shows about 35% identity to the characterized P. chrysogenum phenylacetyl-CoA ligase Phl and has a peroxisomal targeting signal on its C-terminal. Recombinant PhlB protein was overexpressed in Escherichia coli and purified by nickel affinity chromatography. Enzymatic assay confirmed that recombinant PhlB can catalyze the reaction of phenylacetic acid (PAA) with CoA to yield phenylacetyl-CoA. The expression level of phlB in the penicillin producing medium supplemented with PAA, the side chain precursor of penicillin G, was about 2.5-fold higher than that in medium without PAA. The study suggested that PhlB might participate in the activation of PAA during penicillin biosynthesis in P. chrysogenum.

  13. Protein turnover, amino acid profile and amino acid flux in juvenile shrimp Litopenaeus vannamei: effects of dietary protein source.

    PubMed

    Mente, Eleni; Coutteau, Peter; Houlihan, Dominic; Davidson, Ian; Sorgeloos, Patrick

    2002-10-01

    The effect of dietary protein on protein synthesis and growth of juvenile shrimps Litopenaeus vannamei was investigated using three different diets with equivalent protein content. Protein synthesis was investigated by a flooding dose of tritiated phenylalanine. Survival, specific growth and protein synthesis rates were higher, and protein degradation was lower, in shrimps fed a fish/squid/shrimp meal diet, or a 50% laboratory diet/50% soybean meal variant diet, than in those fed a casein-based diet. The efficiency of retention of synthesized protein as growth was 94% for shrimps fed the fish meal diet, suggesting a very low protein turnover rate; by contrast, the retention of synthesized protein was only 80% for shrimps fed the casein diet. The amino acid profile of the casein diet was poorly correlated with that of the shrimps. 4 h after a single meal the protein synthesis rates increased following an increase in RNA activity. A model was developed for amino acid flux, suggesting that high growth rates involve a reduction in the turnover of proteins, while amino acid loss appears to be high.

  14. Mechanism of replication machinery assembly as revealed by the DNA ligase-PCNA-DNA complex architecture.

    PubMed

    Mayanagi, Kouta; Kiyonari, Shinichi; Saito, Mihoko; Shirai, Tsuyoshi; Ishino, Yoshizumi; Morikawa, Kosuke

    2009-03-24

    The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.

  15. Cullin RING Ligases: Glommed by Glomulin

    PubMed Central

    Hristova, Ventzislava A.; Stringer, Daniel K.; Weissman, Allan M.

    2012-01-01

    Cullin ring ligases (CRLs) constitute the largest group of RING finger ubiquitin ligases. Two recent studies in Molecular Cell describe glomulin as a CRL1 inhibitor that blocks interactions with its ubiquitin-conjugating enzyme (E2) (Duda et al., 2012; Tron et al., 2012). These findings and their significance are discussed. PMID:22883621

  16. A High-Fidelity Codon Set for the T4 DNA Ligase-Catalyzed Polymerization of Modified Oligonucleotides.

    PubMed

    Lei, Yi; Kong, Dehui; Hili, Ryan

    2015-12-14

    In vitro selection of nucleic acid polymers can readily deliver highly specific receptors and catalysts for a variety of applications; however, it is suspected that the functional group deficit of nucleic acids has limited their potential with respect to proteinogenic polymers. This has stimulated research toward expanding their chemical diversity to bridge the functional gap between nucleic acids and proteins to develop a superior biopolymer. In this study, we investigate the effect of codon library size and composition on the sequence specificity of T4 DNA ligase in the DNA-templated polymerization of both unmodified and modified oligonucleotides. Using high-throughput DNA sequencing of duplex pairs, we have uncovered a 256-membered codon set that yields sequence-defined modified ssDNA polymers in high yield and with high fidelity.

  17. Mitotic apparatus: the selective extraction of protein with mild acid.

    PubMed

    Bibring, T; Baxandall, J

    1968-07-26

    The treatment of isolated mitotic apparatus with mild (pH 3) hydrochloric acid results in the extraction of less than 10 percent of its protein, accompanied by the selective morphological disappearance of the microtubules. The same extraction can be shown to dissolve outer doublet microtubules from sperm flagella. A protein with points of similarity to the flagellar microtubule protein is the major component of the extract from mitotic apparatus.

  18. Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus.

    PubMed

    Arai, Toshinobu; Noguchi, Atsushi; Takano, Eriko; Kino, Kuniki

    2013-04-01

    Dipeptides exhibit unique physiological functions and physical properties, e.g., l-aspartyl-l-phenylalanine-methyl ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an l-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, l-asparagine; Gln, l-glutamine; and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, l-aspartic acid; and Glu, l-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated l-asparaginyl-l-valine (Asn-Val) and l-glutaminyl-glycine (Gln-Gly), but did not deamidate l-valyl-l-asparagine and l-alanyl-l-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 ± 30 nmol min(-1) mg(-1)·protein and 136 ± 6 nmol min(-1) mg(-1)·protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa.

  19. Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

    PubMed Central

    Blatti, Jillian L.; Beld, Joris; Behnke, Craig A.; Mendez, Michael; Mayfield, Stephen P.; Burkart, Michael D.

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes. PMID:23028438

  20. Manipulating fatty acid biosynthesis in microalgae for biofuel through protein-protein interactions.

    PubMed

    Blatti, Jillian L; Beld, Joris; Behnke, Craig A; Mendez, Michael; Mayfield, Stephen P; Burkart, Michael D

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.

  1. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    NASA Astrophysics Data System (ADS)

    Refsgaard, Hanne H. F.; Tsai, Lin; Stadtman, Earl R.

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O2] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts.

  2. DNA looping by a ligase under nanoconfinement

    NASA Astrophysics Data System (ADS)

    Heidarpour-Roushan, Maedeh; Riehn, Robert

    2013-03-01

    DNA looping is essential for the function and maintenance of genetic information. We have investigated the kinetic evolution of DNA loops (48500 bp) induced by T4 ligase inside a nanofabricated channel system with a channel cross-section of 100x100 nm2, and a few hundred microns channel length. We found that addition of the ligase profoundly alters the behavior of DNA. In particular, ligase acts to stabilize hairpin geometries in which the extended forward and backward arms of the hairpin scan past each other. From the linear density of DNA inside the channel, we deduce that the effective excluded volume vanishes upon addition of T4 ligase and ATP. We conclude that the two strands are effectively stapled together through a large number of weak bonds involving T4 ligase.

  3. IR-UV photochemistry of protein-nucleic acid systems

    SciTech Connect

    Kozub, J.; Edwards, G.

    1995-12-31

    UV light has often been used to induce the formation of covalent bonds between DNA (or RNA) and tightly-bound protein molecules. However, the internal photoreactions of nucleic acids and proteins limit the yield and complicate the analysis of intermolecular crosslinks. In an ongoing search for improved reaction specificity or new photoreactions in these systems, we have employed UV photons from a Nd:YAG-pumped dye laser and mid-IR photons from the Vanderbilt FEL. Having crosslinked several protein-nucleic acid systems with nanosecond UV laser pulses, we are currently studying the effect of various IR wavelengths on a model system (gene 32 protein and poly[dT]). We have found that irradiation with sufficiently intense FEL macropulses creates an altered form of gene 32 protein which was not observed with UV-only irradiation. The electrophoretic nobility of the product is consistent with the formation of a specific protein-protein crosslink. No evidence of the non-specific protein damage typically induced by UV light is found. The yield of the new photoproduct is apparently enhanced by exposure to FEL macropulses which are synchronized with UV laser pulses. With ideal exposure parameters, the two-color reaction effectively competes with UV-only reactions. Experiments designed to determine the reaction mechanism and to demonstrate FEL-induced reactions in other protein-nucleic acid systems are currently underway.

  4. Ubiquitin Protein Ligase Ring2 Is Involved in S-phase Checkpoint and DNA Damage in Cells Exposed to Benzo[a]pyrene.

    PubMed

    Yang, Jin; Chen, Wentao; Fan, Yanfeng; Zhang, Huitao; Wang, Wubin; Zhang, Hongjie

    2016-10-01

    Previous studies in our laboratory demonstrated that Ring2 may affect DNA damage and repair through pathways other than through regulating the expression of the nucleotide excision repair protein. In a series of experiments using wild-type cell (16HBE and WI38) and small interfering RNA (siRNA) Ring2 cells exposed to benzo[a]pyrene (BaP), we evaluated the cell cycle and DNA damage. The benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE-DNA) adduct assay demonstrated that in vitro exposure to BaP increased DNA damage in a time- and dose-dependent manner in wild-type and siRNA Ring2 cells. Analysis of covariance showed that a decrease of Ring2 caused DNA hypersensitivity to BaP. Flow cytometry results and proliferating cell nuclear antigen levels indicated that inhibition of Ring2 attenuated the effect of BaP on S-phase arrest. Taken together, these data implied that the lower proportion of cells in the S phase induced by inhibition of Ring2 may play an important role in DNA hypersensitivity to BaP.

  5. 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.

  6. Nucleic acid compositions and the encoding proteins

    DOEpatents

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  7. Quality control of a cytoplasmic protein complex: chaperone motors and the ubiquitin-proteasome system govern the fate of orphan fatty acid synthase subunit Fas2 of yeast.

    PubMed

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H

    2015-02-20

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2.

  8. SMALL ACIDIC PROTEIN1 acts with RUB modification components, the COP9 signalosome, and AXR1 to regulate growth and development of Arabidopsis.

    PubMed

    Nakasone, Akari; Fujiwara, Masayuki; Fukao, Yoichiro; Biswas, Kamal Kanti; Rahman, Abidur; Kawai-Yamada, Maki; Narumi, Issay; Uchimiya, Hirofumi; Oono, Yutaka

    2012-09-01

    Previously, a dysfunction of the SMALL ACIDIC PROTEIN1 (SMAP1) gene was identified as the cause of the anti-auxin resistant1 (aar1) mutant of Arabidopsis (Arabidopsis thaliana). SMAP1 is involved in the response pathway of synthetic auxin, 2,4-dichlorophenoxyacetic acid, and functions upstream of the auxin/indole-3-acetic acid protein degradation step in auxin signaling. However, the exact mechanism by which SMAP1 functions in auxin signaling remains unknown. Here, we demonstrate that SMAP1 is required for normal plant growth and development and the root response to indole-3-acetic acid or methyl jasmonate in the auxin resistant1 (axr1) mutation background. Deletion analysis and green fluorescent protein/glutathione S-transferase pull-down assays showed that SMAP1 physically interacts with the CONSTITUTIVE PHOTOMORPHOGENIC9 SIGNALOSOME (CSN) via the SMAP1 F/D region. The extremely dwarf phenotype of the aar1-1 csn5a-1 double mutant confirms the functional role of SMAP1 in plant growth and development under limiting CSN functionality. Our findings suggest that SMAP1 is involved in the auxin response and possibly in other cullin-RING ubiquitin ligase-regulated signaling processes via its interaction with components associated with RELATED TO UBIQUITIN modification.

  9. Real-time measurements of amino acid and protein hydroperoxides using coumarin boronic acid.

    PubMed

    Michalski, Radoslaw; Zielonka, Jacek; Gapys, Ewa; Marcinek, Andrzej; Joseph, Joy; Kalyanaraman, Balaraman

    2014-08-08

    Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7-23 M(-1) s(-1)) were significantly higher than that measured for H2O2 (1.5 M(-1) s(-1)). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1-1.5 × 10(3) M(-1) s(-1). Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems.

  10. Cullin E3 Ligases and Their Rewiring by Viral Factors

    PubMed Central

    Mahon, Cathal; Krogan, Nevan J.; Craik, Charles S.; Pick, Elah

    2014-01-01

    The ability of viruses to subvert host pathways is central in disease pathogenesis. Over the past decade, a critical role for the Ubiquitin Proteasome System (UPS) in counteracting host immune factors during viral infection has emerged. This counteraction is commonly achieved by the expression of viral proteins capable of sequestering host ubiquitin E3 ligases and their regulators. In particular, many viruses hijack members of the Cullin-RING E3 Ligase (CRL) family. Viruses interact in many ways with CRLs in order to impact their ligase activity; one key recurring interaction involves re-directing CRL complexes to degrade host targets that are otherwise not degraded within host cells. Removal of host immune factors by this mechanism creates a more amenable cellular environment for viral propagation. To date, a small number of target host factors have been identified, many of which are degraded via a CRL-proteasome pathway. Substantial effort within the field is ongoing to uncover the identities of further host proteins targeted in this fashion and the underlying mechanisms driving their turnover by the UPS. Elucidation of these targets and mechanisms will provide appealing anti-viral therapeutic opportunities. This review is focused on the many methods used by viruses to perturb host CRLs, focusing on substrate sequestration and viral regulation of E3 activity. PMID:25314029

  11. Splint ligation of RNA with T4 DNA ligase

    PubMed Central

    Kershaw, Christopher J.; O’Keefe, Raymond T.

    2014-01-01

    Splint ligation of RNA, whereby specific RNA molecules are ligated together, can be carried out using T4 DNA ligase and a bridging DNA oligonucleotide complementary to the RNAs. This method takes advantage of the property of T4 DNA ligase to join RNA molecules when they are in an RNA:DNA hybrid. Splint ligation is a useful tool for the introduction of modified nucleotides into RNA molecules, insertion of a radiolabel into a specific position within an RNA and for the assembly of smaller synthetic RNAs into longer RNA molecules. Such modifications enable a wide range of experiments to be carried out with the modified RNA including structural studies, co-immunoprecipitations, and the ability to map sites of RNA:RNA and RNA:protein interactions. PMID:23065567

  12. Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage

    PubMed Central

    Kang, Ho Chul; Lee, Yun-Il; Shin, Joo-Ho; Andrabi, Shaida A.; Chi, Zhikai; Gagné, Jean-Philippe; Lee, Yunjong; Ko, Han Seok; Lee, Byoung Dae; Poirier, Guy G.; Dawson, Valina L.; Dawson, Ted M.

    2011-01-01

    Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3 ligase. Iduna’s E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate Iduna’s PAR binding and Iduna’s E3 ligase activity. Iduna’s E3 ligase activity also requires an intact really interesting new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86, XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna’s PAR-dependent ubiquitination of PARP1 targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA repair. PMID:21825151

  13. Cloning and functional characterization of a 4-coumarate CoA ligase from liverwort Plagiochasma appendiculatum.

    PubMed

    Gao, Shuai; Yu, Hai-Na; Xu, Rui-Xue; Cheng, Ai-Xia; Lou, Hong-Xiang

    2015-03-01

    Plant phenylpropanoids represent a large group of secondary metabolites which have played an important role in terrestrial plant life, beginning with the evolution of land plants from primitive green algae. 4-Coumarate: coenzyme A ligase (4CL) is a provider of activated thioester substrates within the phenylpropanoid synthesis pathway. Although 4CLs have been extensively characterized in angiosperm, gymnosperm and moss species, little is known of their functions in liverworts. Here, a 4CL homolog (designated as Pa4CL1) was isolated from the liverwort species Plagiochasma appendiculatum. The full-length cDNA sequence of Pa4CL1 contains 1644bp and is predicted to encode a protein with 547amino acids. The gene products were 40-50% identical with 4CL sequences reported in public databases. The recombinant protein was heterologously expressed in Escherichia coli and exhibited a high level of 4CL activity, catalyzing formation of hydroxycinnamate-CoA thioesters by a two-step reaction mechanism from corresponding hydroxycinnamic acids. Kinetic analysis indicated that the most favorable substrate for Pa4CL1 is p-coumaric acid. The transcription of Pa4CL1 was induced when P. appendiculatum thallus was treated with either salicylic acid or methyl jasmonate.

  14. Determinants of Small Ubiquitin-like Modifier 1 (SUMO1) Protein Specificity, E3 Ligase, and SUMO-RanGAP1 Binding Activities of Nucleoporin RanBP2

    SciTech Connect

    Gareau, Jaclyn R.; Reverter, David; Lima, Christopher D.

    2012-02-16

    The RanBP2 nucleoporin contains an internal repeat domain (IR1-M-IR2) that catalyzes E3 ligase activity and forms a stable complex with SUMO-modified RanGAP1 and UBC9 at the nuclear pore complex. RanBP2 exhibits specificity for SUMO1 as RanGAP1-SUMO1/UBC9 forms a more stable complex with RanBP2 compared with RanGAP1-SUMO2 that results in greater protection of RanGAP-SUMO1 from proteases. The IR1-M-IR2 SUMO E3 ligase activity also shows a similar preference for SUMO1. We utilized deletions and domain swap constructs in protease protection assays and automodification assays to define RanBP2 domains responsible for RanGAP1-SUMO1 protection and SUMO1-specific E3 ligase activity. Our data suggest that elements in both IR1 and IR2 exhibit specificity for SUMO1. IR1 protects RanGAP1-SUMO1/UBC9 and functions as the primary E3 ligase of RanBP2, whereas IR2 retains the ability to interact with SUMO1 to promote SUMO1-specific E3 ligase activity. To determine the structural basis for SUMO1 specificity, a hybrid IR1 construct and IR1 were used to determine three new structures for complexes containing UBC9 with RanGAP1-SUMO1/2. These structures show more extensive contacts among SUMO, UBC9, and RanBP2 in complexes containing SUMO1 compared with SUMO2 and suggest that differences in SUMO specificity may be achieved through these subtle conformational differences.

  15. Determinants of Small Ubiquitin-like Modifier 1 (SUMO1) Protein Specificity, E3 Ligase, and SUMO-RanGAP1 Binding Activities of Nucleoporin RanBP2*

    PubMed Central

    Gareau, Jaclyn R.; Reverter, David; Lima, Christopher D.

    2012-01-01

    The RanBP2 nucleoporin contains an internal repeat domain (IR1-M-IR2) that catalyzes E3 ligase activity and forms a stable complex with SUMO-modified RanGAP1 and UBC9 at the nuclear pore complex. RanBP2 exhibits specificity for SUMO1 as RanGAP1-SUMO1/UBC9 forms a more stable complex with RanBP2 compared with RanGAP1-SUMO2 that results in greater protection of RanGAP-SUMO1 from proteases. The IR1-M-IR2 SUMO E3 ligase activity also shows a similar preference for SUMO1. We utilized deletions and domain swap constructs in protease protection assays and automodification assays to define RanBP2 domains responsible for RanGAP1-SUMO1 protection and SUMO1-specific E3 ligase activity. Our data suggest that elements in both IR1 and IR2 exhibit specificity for SUMO1. IR1 protects RanGAP1-SUMO1/UBC9 and functions as the primary E3 ligase of RanBP2, whereas IR2 retains the ability to interact with SUMO1 to promote SUMO1-specific E3 ligase activity. To determine the structural basis for SUMO1 specificity, a hybrid IR1 construct and IR1 were used to determine three new structures for complexes containing UBC9 with RanGAP1-SUMO1/2. These structures show more extensive contacts among SUMO, UBC9, and RanBP2 in complexes containing SUMO1 compared with SUMO2 and suggest that differences in SUMO specificity may be achieved through these subtle conformational differences. PMID:22194619

  16. Functional domains of the fatty acid transport proteins: studies using protein chimeras.

    PubMed

    DiRusso, Concetta C; Darwis, Dina; Obermeyer, Thomas; Black, Paul N

    2008-03-01

    Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of the FATP family. This latter motif has been implicated in fatty acid transport in the yeast FATP orthologue Fat1p. In the present studies using a yeast strain containing deletions in FAT1 (encoding Fat1p) and FAA1 (encoding the major acyl CoA synthetase (Acsl) Faa1p) as an experimental platform, the phenotypic and functional properties of specific murine FATP1-FATP4 and FATP6-FATP4 protein chimeras were evaluated in order to define elements within these proteins that further distinguish the fatty acid transport and activation functions. As expected from previous work FATP1 and FATP4 were functional in the fatty acid transport pathway, while and FATP6 was not. All three isoforms were able to activate the very long chain fatty acids arachidonate (C(20:4)) and lignocerate (C(24:0)), but with distinguishing activities between saturated and highly unsaturated ligands. A 73 amino acid segment common to FATP1 and FATP4 and between the ATP/AMP and FATP/VLACS motifs was identified by studying the chimeras, which is hypothesized to contribute to the transport function.

  17. Plasma amino acid response to graded levels of escape protein.

    PubMed

    Gibb, D J; Klopfenstein, T J; Britton, R A; Lewis, A J

    1992-09-01

    A trial was conducted to examine the potential of using plasma amino acid responses to graded levels of escape protein to determine limiting amino acids in cattle. Growing calves (n = 120; mean BW = 220 +/- 21 kg) were fed a basal diet of corncob:sorghum silage (61:39) and were individually supplemented with distillers' dried grains (DDG), heat-damaged DDG (H-DDG), feather meal (FTH), or urea. The urea supplement was mixed with DDG and H-DDG to allow 0, 20, 35, 50, 65, or 80% of the supplemental CP to come from distillers' protein and maintain an 11.5% CP diet. Urea supplement was mixed with FTH to allow 0, 22, 39, 56, 73, or 90% of the supplemental CP to come from FTH. Dietary CP ranged from 11.5% at the 0% level to 17.3% at the 90% level. Plasma concentration of most essential plasma amino acids responded (P less than .10) linearly and(or) quadratically to increased escape protein. The broken-line response of plasma methionine at low DDG intake suggested that methionine was limiting at low levels of escape protein. An initial decrease followed by a plateau fit by a broken line indicated that histidine became limiting in FTH diets, and lysine eventually became limiting for DDG, H-DDG, and FTH diets before maximum BW gain was reached. Results indicate that plasma amino acid responses may identify amino acids that become limiting with increasing escape protein.

  18. Suppressor mutations in the Glutamine Dumper1 protein dissociate disturbance in amino acid transport from other characteristics of the Gdu1D phenotype.

    PubMed

    Yu, Shi; Pratelli, Réjane; Denbow, Cynthia; Pilot, Guillaume

    2015-01-01

    Intracellular amino acid transport across plant membranes is critical for metabolic pathways which are often split between different organelles. In addition, transport of amino acids across the plasma membrane enables the distribution of organic nitrogen through the saps between leaves and developing organs. Amino acid importers have been studied for more than two decades, and their role in this process is well-documented. While equally important, amino acid exporters are not well-characterized. The over-expression of GDU1, encoding a small membrane protein with one transmembrane domain, leads to enhancement of amino acid export by Arabidopsis cells, glutamine secretion at the leaf margin, early senescence and size reduction of the plant, possibly caused by the stimulation of amino acid exporter(s). Previous work reported the identification of suppressor mutations of the GDU1 over-expression phenotype, which affected the GDU1 and LOG2 genes, the latter encoding a membrane-bound ubiquitin ligase interacting with GDU1. The present study focuses on the characterization of three additional suppressor mutations affecting GDU1. Size, phenotype, glutamine transport and amino acid tolerance were recorded for recapitulation plants and over-expressors of mutagenized GDU1 proteins. Unexpectedly, the over-expression of most mutated GDU1 led to plants with enhanced amino acid export, but failing to display secretion of glutamine and size reduction. The results show that the various effects triggered by GDU1 over-expression can be dissociated from one another by mutagenizing specific residues. The fact that these residues are not necessarily conserved suggests that the diverse biochemical properties of the GDU1 protein are not only born by the characterized transmembrane and VIMAG domains. These data provide a better understanding of the structure/function relationships of GDU1 and may enable modifying amino acid export in plants without detrimental effects on plant fitness.

  19. Stimulation of nonselective amino acid export by glutamine dumper proteins.

    PubMed

    Pratelli, Réjane; Voll, Lars M; Horst, Robin J; Frommer, Wolf B; Pilot, Guillaume

    2010-02-01

    Phloem and xylem transport of amino acids involves two steps: export from one cell type to the apoplasm, and subsequent import into adjacent cells. High-affinity import is mediated by proton/amino acid cotransporters, while the mechanism of export remains unclear. Enhanced expression of the plant-specific type I membrane protein Glutamine Dumper1 (GDU1) has previously been shown to induce the secretion of glutamine from hydathodes and increased amino acid content in leaf apoplasm and xylem sap. In this work, tolerance to low concentrations of amino acids and transport analyses using radiolabeled amino acids demonstrate that net amino acid uptake is reduced in the glutamine-secreting GDU1 overexpressor gdu1-1D. The net uptake rate of phenylalanine decreased over time, and amino acid net efflux was increased in gdu1-1D compared with the wild type, indicating increased amino acid export from cells. Independence of the export from proton gradients and ATP suggests that overexpression of GDU1 affects a passive export system. Each of the seven Arabidopsis (Arabidopsis thaliana) GDU genes led to similar phenotypes, including increased efflux of a wide spectrum of amino acids. Differences in expression profiles and functional properties suggested that the GDU genes fulfill different roles in roots, vasculature, and reproductive organs. Taken together, the GDUs appear to stimulate amino acid export by activating nonselective amino acid facilitators.

  20. The multiple roles of fatty acid handling proteins in brain

    PubMed Central

    Moullé, Valentine S. F.; Cansell, Céline; Luquet, Serge; Cruciani-Guglielmacci, Céline

    2012-01-01

    Lipids are essential components of a living organism as energy source but also as constituent of the membrane lipid bilayer. In addition fatty acid (FA) derivatives interact with many signaling pathways. FAs have amphipathic properties and therefore require being associated to protein for both transport and intracellular trafficking. Here we will focus on several FA handling proteins, among which the fatty acid translocase/CD36 (FAT/CD36), members of fatty acid transport proteins (FATPs), and lipid chaperones fatty acid-binding proteins (FABPs). A decade of extensive studies has helped decipher the mechanism of action of these proteins in peripheral tissue with high lipid metabolism. However, considerably less information is available regarding their role in the brain, despite the high lipid content of this tissue. This review will primarily focus on the recent studies that have highlighted the crucial role of lipid handling proteins in brain FA transport, neuronal differentiation and development, cognitive processes and brain diseases. Finally a special focus will be made on the recent studies that have revealed the role of FAT/CD36 in brain lipid sensing and nervous control of energy balance. PMID:23060810

  1. Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor.

    PubMed

    Dounay, A B; Forsyth, C J

    2002-11-01

    As the first recognized member of the "okadaic acid class" of phosphatase inhibitors, the marine natural product okadaic acid is perhaps the most well-known member of a diverse array of secondary metabolites that have emerged as valuable probes for studying the roles of various cellular protein serine/threonine phosphatases. This review provides a historical perspective on the role that okadaic acid has played in stimulating a broad spectrum of modern scientific research as a result of the natural product's ability to bind to and inhibit important classes of protein serine / threonine phosphatases. The relationships between the structure and biological activities of okadaic acid are briefly reviewed, as well as the structural information regarding the particular cellular receptors protein phosphatases 1 (PP1) and 2A. Laboratory syntheses of okadaic acid and its analogs are thoroughly reviewed. Finally, an interpretation of the critical contacts observed between okadaic acid and PP1 by X-ray crystallography is provided, and specific molecular recognition hypotheses that are testable via the synthesis and assay of non-natural analogs of okadaic acid are suggested.

  2. The E3 ubiquitin ligase neuregulin receptor degradation protein 1 (Nrdp1) promotes M2 macrophage polarization by ubiquitinating and activating transcription factor CCAAT/enhancer-binding Protein β (C/EBPβ).

    PubMed

    Ye, Shuo; Xu, Hongmei; Jin, Jing; Yang, Mingjin; Wang, Chunmei; Yu, Yizhi; Cao, Xuetao

    2012-08-03

    Macrophage activation, including classical (M1) activation and alternative (M2) activation, plays important roles in host immune response and pathogenesis of diseases. Ubiquitination has been shown to be involved in the differentiation of immune cells and in the regulation of immune responses. However, the role of ubiquitination during M1 versus M2 polarization is poorly explored. Here, we showed that arginase 1 (Arg1), a well recognized marker of M2 macrophages, is highly up-regulated in peritoneal macrophages derived from E3 ubiquitin ligase Nrdp1 transgenic (Nrdp1-TG) mice. Furthermore, other M2 feature markers such as MR, Ym1, and Fizz1, as well as Th2 cytokine IL-10, are also up-regulated in Nrdp1-TG macrophages after IL-4 stimulation. Knockdown of Nrdp1 expression effectively inhibits IL-4-induced expression of M2-related genes in macrophages. Moreover, Nrdp1 inhibits LPS-induced production of inducible NOS and pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in macrophages. Immunoprecipitation assays show that Nrdp1 interacts with and ubiquitinates transcriptional factor C/EBPβ via Lys-63-linked ubiquitination. Nrdp1 enhances C/EBPβ-triggered transcriptional activation of the Arg1 reporter gene in the presence of IL-4 stimulation. Thus, we demonstrate that Nrdp1-mediated ubiquitination and activation of C/EBPβ contributes to a ubiquitin-dependent nonproteolytic pathway that up-regulates Arg1 expression and promotes M2 macrophage polarization.

  3. The E3 Ubiquitin Ligase Neuregulin Receptor Degradation Protein 1 (Nrdp1) Promotes M2 Macrophage Polarization by Ubiquitinating and Activating Transcription Factor CCAAT/Enhancer-binding Protein β (C/EBPβ)*

    PubMed Central

    Ye, Shuo; Xu, Hongmei; Jin, Jing; Yang, Mingjin; Wang, Chunmei; Yu, Yizhi; Cao, Xuetao

    2012-01-01

    Macrophage activation, including classical (M1) activation and alternative (M2) activation, plays important roles in host immune response and pathogenesis of diseases. Ubiquitination has been shown to be involved in the differentiation of immune cells and in the regulation of immune responses. However, the role of ubiquitination during M1 versus M2 polarization is poorly explored. Here, we showed that arginase 1 (Arg1), a well recognized marker of M2 macrophages, is highly up-regulated in peritoneal macrophages derived from E3 ubiquitin ligase Nrdp1 transgenic (Nrdp1-TG) mice. Furthermore, other M2 feature markers such as MR, Ym1, and Fizz1, as well as Th2 cytokine IL-10, are also up-regulated in Nrdp1-TG macrophages after IL-4 stimulation. Knockdown of Nrdp1 expression effectively inhibits IL-4-induced expression of M2-related genes in macrophages. Moreover, Nrdp1 inhibits LPS-induced production of inducible NOS and pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in macrophages. Immunoprecipitation assays show that Nrdp1 interacts with and ubiquitinates transcriptional factor C/EBPβ via Lys-63-linked ubiquitination. Nrdp1 enhances C/EBPβ-triggered transcriptional activation of the Arg1 reporter gene in the presence of IL-4 stimulation. Thus, we demonstrate that Nrdp1-mediated ubiquitination and activation of C/EBPβ contributes to a ubiquitin-dependent nonproteolytic pathway that up-regulates Arg1 expression and promotes M2 macrophage polarization. PMID:22707723

  4. Molecular Characterization of NAD+-Dependent DNA Ligase from Wolbachia Endosymbiont of Lymphatic Filarial Parasite Brugia malayi

    PubMed Central

    Shrivastava, Nidhi; Nag, Jeetendra Kumar; Misra-Bhattacharya, Shailja

    2012-01-01

    The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD+-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD+-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA) for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD+-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH) and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid) was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies. PMID:22815933

  5. Dysregulation of ubiquitin ligases in cancer

    PubMed Central

    Ronai, Ze’ev A.

    2015-01-01

    Ubiquitin ligases are critical components of the ubiquitin proteasome system (UPS), which governs fundamental processes regulating normal cellular homeostasis, metabolism, and cell cycle in response to external stress signals and DNA damage. Among multiple steps of the UPS system required to regulate protein ubiquitination and stability, UBLs define specificity, as they recognize and interact with substrates in a temporally- and spatially-regulated manner. Such interactions are required for substrate modification by ubiquitin chains, which marks proteins for recognition and degradation by the proteasome, or alters their subcellular localization or assembly into functional complexes. UBLs are often deregulated in cancer, altering substrate availability or activity in a manner that can promote cellular transformation. Such deregulation can occur at the epigenetic, genomic, or post-translational levels. Alterations in UBL can be used to predict their contributions, affecting tumor suppressors or oncogenes in select tumors. Better understanding of mechanisms underlying UBL expression and activities is expected to drive the development of next generation modulators that can serve as novel therapeutic modalities. This review summarizes our current understanding of UBL deregulation in cancer and highlights novel opportunities for therapeutic interventions. PMID:26690337

  6. Roles of intrinsic disorder in protein-nucleic acid interactions.

    PubMed

    Dyson, H Jane

    2012-01-01

    Interactions between proteins and nucleic acids typify the role of disordered segments, linkers, tails and other entities in the function of complexes that must form with high affinity and specificity but which must be capable of dissociating when no longer needed. While much of the emphasis in the literature has been on the interactions of disordered proteins with other proteins, disorder is also frequently observed in nucleic acids (particularly RNA) and in the proteins that interact with them. The interactions of disordered proteins with DNA most often manifest as molding of the protein onto the B-form DNA structure, although some well-known instances involve remodeling of the DNA structure that seems to require that the interacting proteins be disordered to various extents in the free state. By contrast, induced fit in RNA-protein interactions has been recognized for many years-the existence and prevalence of this phenomenon provides the clearest possible evidence that RNA and its interactions with proteins must be considered as highly dynamic, and the dynamic nature of RNA and its multiplicity of folded and unfolded states is an integral part of its nature and function.

  7. Lithium promotes DNA stability and survival of ischemic retinal neurocytes by upregulating DNA ligase IV

    PubMed Central

    Yang, Ying; Wu, Nandan; Tian, Sijia; Li, Fan; Hu, Huan; Chen, Pei; Cai, Xiaoxiao; Xu, Lijun; Zhang, Jing; Chen, Zhao; Ge, Jian; Yu, Keming; Zhuang, Jing

    2016-01-01

    Neurons display genomic fragility and show fragmented DNA in pathological degeneration. A failure to repair DNA breaks may result in cell death or apoptosis. Lithium protects retinal neurocytes following nutrient deprivation or partial nerve crush, but the underlying mechanisms are not well defined. Here we demonstrate that pretreatment with lithium protects retinal neurocytes from ischemia-induced damage and enhances light response in rat retina following ischemia–reperfusion injury. Moreover, we found that DNA nonhomologous end-joining (NHEJ) repair is implicated in this process because in ischemic retinal neurocytes, lithium significantly reduces the number of γ-H2AX foci (well-characterized markers of DNA double-strand breaks in situ) and increases the DNA ligase IV expression level. Furthermore, we also demonstrate that nuclear respiratory factor 1 (Nrf-1) and phosphorylated cyclic AMP-response element binding protein-1 (P-CREB1) bind to ligase IV promoter to cause upregulation of ligase IV in neurocytes. The ischemic upregulation of Nrf-1 and lithium-induced increase of P-CREB1 cooperate to promote transcription of ligase IV. Short hairpin RNAs against Nrf-1 and CREB1 could significantly inhibit the increase in promoter activity and expression of ligase IV observed in the control oligos following lithium treatment in retinal neurocytes. More importantly, ischemic stimulation triggers the expression of ligase IV. Taken together, our results thus reveal a novel mechanism that lithium offers neuroprotection from ischemia-induced damage by enhancing DNA NHEJ repair. PMID:27853172

  8. THE ROLE OF E3 LIGASES IN THE UBIQUITIN-DEPENDENT REGULATION OF SPERMATOGENESIS*

    PubMed Central

    Richburg, John H.; Myers, Jessica L.; Bratton, Shawn B.

    2014-01-01

    The ubiquitination of proteins is a post-translational modification that was first described as a means to target misfolded or unwanted proteins for degradation by the proteasome. It is now appreciated that the ubiquitination of proteins also serves as a mechanism to modify protein function and cellular functions such as protein trafficking, cell signaling, DNA repair, chromatin modifications, cell-cycle progression and cell death. The ubiquitination of proteins occurs through the hierarchal transfer of ubiquitin from an E1 ubiquitin-activating enzyme to an E2 ubiquitin-conjugating enzyme and finally to an E3 ubiquitin ligase that transfers the ubiquitin to its target protein. It is the final E3 ubiquitin ligase that confers the substrate specificity for ubiquitination and is the focus of this review. Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells undergo mitotic proliferation and expansion of the diploid spermatogonial population, differentiate into spermatocytes and progress through two meiotic divisions to produce haploid spermatids that proceed through a final morphogenesis to generate mature spermatozoa. The ubiquitination of proteins in the cells of the testis occurs in many of the processes required for the progression of mature spermatozoa. Since it is the E3 ubiquitin ligase that recognizes the target protein and provides the specificity and selectivity for ubiquitination, this review highlights known examples of E3 ligases in the testis and the differing roles that they play in maintaining functional spermatogenesis. PMID:24632385

  9. 4-coumarate:coenzyme a ligase from loblolly pine xylem. Isolation, characterization, and complementary DNA cloning.

    PubMed Central

    Voo, K S; Whetten, R W; O'Malley, D M; Sederoff, R R

    1995-01-01

    4-Coumarate:CoA ligase (4CL, EC 6.2.1.12) was purified from differentiating xylem of loblolly pine (Pinus taeda L.). The pine enzyme had an apparent molecular mass of 64 kD and was similar in size and kinetic properties to 4CL isolated from Norway spruce. The pine enzyme used 4-coumaric acid, caffeic acid, ferulic acid, and cinnamic acid as substrates but had no detectable activity using sinapic acid. 4CL was inhibited by naringenin and coniferin, products of phenylpropanoid metabolism. Although the lignin composition in compression wood is higher in p-hydroxyphenyl units than lignin from normal wood, there was no evidence for a different form of 4CL enzyme in differentiating xylem that was forming compression wood. cDNA clones for 4CL were obtained from a xylem expression library. The cDNA sequences matched pine xylem 4CL protein sequences and showed 60 to 66% DNA sequence identity with 4CL sequences from herbaceous angiosperms. There were two classes of cDNA obtained from pine xylem, and the genetic analysis showed that they were products of a single gene. PMID:7784527

  10. A general method of protein purification for recombinant unstructured non-acidic proteins.

    PubMed

    Campos, Francisco; Guillén, Gabriel; Reyes, José L; Covarrubias, Alejandra A

    2011-11-01

    Typical late embryogenesis abundant (LEA) proteins accumulate in response to water deficit imposed by the environment or by plant developmental programs. Because of their physicochemical properties, they can be considered as hydrophilins and as a paradigm of intrinsically unstructured proteins (IUPs) in plants. To study their biophysical and biochemical characteristics large quantities of highly purified protein are required. In this work, we report a fast and simple purification method for non-acidic recombinant LEA proteins that does not need the addition of tags and that preserves their in vitro protective activity. The method is based on the enrichment of the protein of interest by boiling the bacterial protein extract, followed by a differential precipitation with trichloroacetic acid (TCA). Using this procedure we have obtained highly pure recombinant LEA proteins of groups 1, 3, and 4 and one recombinant bacterial hydrophilin. This protocol will facilitate the purification of this type of IUPs, and could be particularly useful in proteomic projects/analyses.

  11. RBR E3 ubiquitin ligases: new structures, new insights, new questions

    PubMed Central

    Spratt, Donald E.; Walden, Helen; Shaw, Gary S.

    2014-01-01

    The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology. PMID:24576094

  12. Substitution of aspartic acid with glutamic acid increases the unfolding transition temperature of a protein.

    PubMed

    Lee, Duck Yeon; Kim, Kyeong-Ae; Yu, Yeon Gyu; Kim, Key-Sun

    2004-07-30

    Proteins from thermophiles are more stable than those from mesophiles. Several factors have been suggested as causes for this greater stability, but no general rule has been found. The amino acid composition of thermophile proteins indicates that the content of polar amino acids such as Asn, Gln, Ser, and Thr is lower, and that of charged amino acids such as Arg, Glu, and Lys is higher than in mesophile proteins. Among charged amino acids, however, the content of Asp is even lower in thermophile proteins than in mesophile proteins. To investigate the reasons for the lower occurrence of Asp compared to Glu in thermophile proteins, Glu was substituted with Asp in a hyperthermophile protein, MjTRX, and Asp was substituted with Glu in a mesophile protein, ETRX. Each substitution of Glu with Asp decreased the Tm of MjTRX by about 2 degrees C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 degrees C. The change of Tm destabilizes the MjTRX by 0.55 kcal/mol and stabilizes the ETRX by 0.45 kcal/mol in free energy.

  13. Comparative proteomic analysis of differentially expressed proteins in β-aminobutyric acid enhanced Arabidopsis thaliana tolerance to simulated acid rain.

    PubMed

    Liu, Tingwu; Jiang, Xinwu; Shi, Wuliang; Chen, Juan; Pei, Zhenming; Zheng, Hailei

    2011-05-01

    Acid rain is a worldwide environmental issue that has seriously destroyed forest ecosystems. As a highly effective and broad-spectrum plant resistance-inducing agent, β-aminobutyric acid could elevate the tolerance of Arabidopsis when subjected to simulated acid rain. Using comparative proteomic strategies, we analyzed 203 significantly varied proteins of which 175 proteins were identified responding to β-aminobutyric acid in the absence and presence of simulated acid rain. They could be divided into ten groups according to their biological functions. Among them, the majority was cell rescue, development and defense-related proteins, followed by transcription, protein synthesis, folding, modification and destination-associated proteins. Our conclusion is β-aminobutyric acid can lead to a large-scale primary metabolism change and simultaneously activate antioxidant system and salicylic acid, jasmonic acid, abscisic acid signaling pathways. In addition, β-aminobutyric acid can reinforce physical barriers to defend simulated acid rain stress.

  14. Parkin is a component of an SCF-like ubiquitin ligase complex and protects postmitotic neurons from kainate excitotoxicity.

    PubMed

    Staropoli, John F; McDermott, Caroline; Martinat, Cécile; Schulman, Brenda; Demireva, Elena; Abeliovich, Asa

    2003-03-06

    Mutations in parkin, which encodes a RING domain protein associated with ubiquitin ligase activity, lead to autosomal recessive Parkinson's disease characterized by midbrain dopamine neuron loss. Here we show that parkin functions in a multiprotein ubiquitin ligase complex that includes the F-box/WD repeat protein hSel-10 and Cullin-1. HSel-10 serves to target the parkin ubiquitin ligase activity to cyclin E, an hSel-10-interacting protein previously implicated in the regulation of neuronal apoptosis. Consistent with the notion that cyclin E is a substrate of the parkin ubiquitin ligase complex, parkin deficiency potentiates the accumulation of cyclin E in cultured postmitotic neurons exposed to the glutamatergic excitotoxin kainate and promotes their apoptosis. Furthermore, parkin overexpression attenuates the accumulation of cyclin E in toxin-treated primary neurons, including midbrain dopamine neurons, and protects them from apoptosis.

  15. SAG/ROC-SCF beta-TrCP E3 ubiquitin ligase promotes pro-caspase-3 degradation as a mechanism of apoptosis protection.

    PubMed

    Tan, Mingjia; Gallegos, Jayme R; Gu, Qingyang; Huang, Yuanhui; Li, Jun; Jin, Yetao; Lu, Hua; Sun, Yi

    2006-12-01

    Skp1-cullin-F-box protein (SCF) is a multicomponent E3 ubiquitin (Ub) ligase that ubiquitinates a number of important biologic molecules such as p27, beta-catenin, and IkappaB for proteasomal degradation, thus regulating cell proliferation and survival. One SCF component, SAG/ROC2/Rbx2/Hrt2, a RING finger protein, was first identified as a redox-inducible protein, which, when overexpressed, inhibited apoptosis both in vitro and in vivo. We report here that sensitive to apoptosis gene (SAG), as well as its family member ROC1/Rbx1, bound to the proinactive form of caspase-3 (pro-caspase-3). Binding was likely mediated through F-box protein, beta-transducin repeat-containing protein (beta-TrCP), which binds to the first 38 amino acids of pro-caspase-3. Importantly, beta-TrCP1 expression significantly shortened the protein half-life of pro-caspase-3, whereas expression of a dominant-negative beta-TrCP1 mutant with the F-box domain deleted extended it. An in vitro ubiquitination assay showed that SAG/ROC-SCF(beta-TrCP) promoted ubiquitination of pro-caspase-3. Furthermore, endogenous levels of pro-caspase-3 were decreased by overexpression of SAG/ROC-SCF(beta-TrCP) E3 Ub ligases, but increased on siRNA silencing of SAG, regulator of cullin-1 (ROC1), or beta-TrCPs, leading to increased apoptosis by etoposide and TNF-related apoptosis-inducing ligand through increased activation of caspase-3. Thus, pro-caspase-3 appears to be a substrate of SAG/ROC-SCF(beta-TrCP) E3 Ub ligase, which protects cells from apoptosis through increased apoptosis threshold by reducing the basal level of pro-caspase-3.

  16. Predicting the Function of 4-Coumarate:CoA Ligase (LJ4CL1) in Lonicera japonica

    PubMed Central

    Yuan, Yuan; Yu, Shulin; Yu, Jun; Zhan, Zhilai; Li, Minhui; Liu, Guiming; Wang, Xumin; Huang, Luqi

    2014-01-01

    4-Coumarate:CoA ligases (4CLs) are a group of essential enzymes involved in the pathway of phenylpropanoid-derived compound metabolisms; however it is still difficult to identify orthologs and paralogs of these important enzymes just based on sequence similarity of the conserved domains. Using sequence data of 20 plant species from the public databases and sequences from Lonicera japonica, we define 1252 adenosine monophosphate (AMP)-dependent synthetase/ligase sequences and classify them into three phylogenetic clades. 4CLs are in one of the four subgroups, according to their partitioning, with known proteins characterized in A. thaliana and Oryza sativa. We also defined 184 non-redundant sequences that encode proteins containing the GEICIRG motif and the taxonomic distribution of these GEICIRG-containing proteins suggests unique catalytic activities in plants. We further analyzed their transcription levels in L. japonica and L. japonica. var. chinensis flowers and chose the highest expressed genes representing the subgroups for structure and binding site predictions. Coupled with liquid chromatography-mass spectrometry (LC-MS) analysis of the L. japonica flowers, the structural study on putative substrate binding amino acid residues, ferulate, and 4-coumaric acid of the conserved binding-site of LJ4CL1 leads to a conclusion that this highly expressed protein group in the flowers may process 4-coumarate that represents 90% of the known phenylpropanoid-derived compounds. The activity of purified crude LJ4CL1 protein was analyzed using 4-coumarate as template and high activity indicating that 4-coumarate is one of the substrates of LJ4CL1. PMID:24518682

  17. Predicting the function of 4-coumarate:CoA ligase (LJ4CL1) in Lonicera japonica.

    PubMed

    Yuan, Yuan; Yu, Shulin; Yu, Jun; Zhan, Zhilai; Li, Minhui; Liu, Guiming; Wang, Xumin; Huang, Luqi

    2014-02-10

    4-Coumarate:CoA ligases (4CLs) are a group of essential enzymes involved in the pathway of phenylpropanoid-derived compound metabolisms; however it is still difficult to identify orthologs and paralogs of these important enzymes just based on sequence similarity of the conserved domains. Using sequence data of 20 plant species from the public databases and sequences from Lonicera japonica, we define 1252 adenosine monophosphate (AMP)-dependent synthetase/ligase sequences and classify them into three phylogenetic clades. 4CLs are in one of the four subgroups, according to their partitioning, with known proteins characterized in A. thaliana and Oryza sativa. We also defined 184 non-redundant sequences that encode proteins containing the GEICIRG motif and the taxonomic distribution of these GEICIRG-containing proteins suggests unique catalytic activities in plants. We further analyzed their transcription levels in L. japonica and L. japonica. var. chinensis flowers and chose the highest expressed genes representing the subgroups for structure and binding site predictions. Coupled with liquid chromatography-mass spectrometry (LC-MS) analysis of the L. japonica flowers, the structural study on putative substrate binding amino acid residues, ferulate, and 4-coumaric acid of the conserved binding-site of LJ4CL1 leads to a conclusion that this highly expressed protein group in the flowers may process 4-coumarate that represents 90% of the known phenylpropanoid-derived compounds. The activity of purified crude LJ4CL1 protein was analyzed using 4-coumarate as template and high activity indicating that 4-coumarate is one of the substrates of LJ4CL1.

  18. Small Molecule Inhibitors of the Interaction Between the E3 Ligase VHL and HIF1α

    PubMed Central

    Buckley, Dennis L.; Gustafson, Jeffrey L.; Van Molle, Inge; Roth, Anke G.; Tae, Hyun Seop; Gareiss, Peter C.; Jorgensen, William L.; Ciulli, Alessio

    2012-01-01

    E3 ubiquitin ligases, such as the therapeutically relevant VHL, are challenging targets for traditional medicinal chemistry, as their modulation requires targeting protein-protein interactions. We report novel small-molecule inhibitors of the interaction between VHL and its molecular target HIF1α, a transcription factor involved in oxygen sensing. PMID:23065727

  19. Nucleic Acid Programmable Protein Array: A Just-In-Time Multiplexed Protein Expression and Purification Platform

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. PMID:21943897

  20. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    PubMed

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally.

  1. Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency

    NASA Technical Reports Server (NTRS)

    Tawa, N. E. Jr; Goldberg, A. L.

    1992-01-01

    To define the adaptations that conserve amino acids and muscle protein when dietary protein intake is inadequate, rats (60-70 g final wt) were fed a normal or protein-deficient (PD) diet (18 or 1% lactalbumin), and their muscles were studied in vitro. After 7 days on the PD diet, both protein degradation and synthesis fell 30-40% in skeletal muscles and atria. This fall in proteolysis did not result from reduced amino acid supply to the muscle and preceded any clear decrease in plasma amino acids. Oxidation of branched-chain amino acids, glutamine and alanine synthesis, and uptake of alpha-aminoisobutyrate also fell by 30-50% in muscles and adipose tissue of PD rats. After 1 day on the PD diet, muscle protein synthesis and amino acid uptake decreased by 25-40%, and after 3 days proteolysis and leucine oxidation fell 30-45%. Upon refeeding with the normal diet, protein synthesis also rose more rapidly (+30% by 1 day) than proteolysis, which increased significantly after 3 days (+60%). These different time courses suggest distinct endocrine signals for these responses. The high rate of protein synthesis and low rate of proteolysis during the first 3 days of refeeding a normal diet to PD rats contributes to the rapid weight gain ("catch-up growth") of such animals.

  2. Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli.

    PubMed

    Liu, Xinxin; Huang, Anliang; Luo, Dan; Liu, Haipeng; Han, Huzi; Xu, Yang; Liang, Peng

    2015-05-01

    The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in E. coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. E.coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in E. coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni-NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification.

  3. Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation.

    PubMed

    Riling, Christopher; Kamadurai, Hari; Kumar, Suresh; O'Leary, Claire E; Wu, Kuen-Phon; Manion, Erica E; Ying, Mingjie; Schulman, Brenda A; Oliver, Paula M

    2015-09-25

    Nedd4-family E3 ubiquitin ligases regulate an array of biologic processes. Autoinhibition maintains these catalytic ligases in an inactive state through several mechanisms. However, although some Nedd4 family members are activated by binding to Nedd4 family-interacting proteins (Ndfips), how binding activates E3 function remains unclear. Our data reveal how these two regulatory processes are linked functionally. In the absence of Ndfip1, the Nedd4 family member Itch can bind an E2 but cannot accept ubiquitin onto its catalytic cysteine. This is because Itch is autoinhibited by an intramolecular interaction between its HECT (homologous to the E6-AP carboxy terminus domain) and two central WW domains. Ndfip1 binds these WW domains to release the HECT, allowing trans-thiolation and Itch catalytic activity. This molecular switch also regulates the closely related family member WWP2. Importantly, multiple PY motifs are required for Ndfip1 to activate Itch, functionally distinguishing Ndfips from single PY-containing substrates. These data establish a novel mechanism for control of the function of a subfamily of Nedd4 E3 ligases at the level of E2-E3 trans-thiolation.

  4. Urinary intestinal fatty acid binding protein predicts necrotizing enterocolitis.

    PubMed

    Gregory, Katherine E; Winston, Abigail B; Yamamoto, Hidemi S; Dawood, Hassan Y; Fashemi, Titilayo; Fichorova, Raina N; Van Marter, Linda J

    2014-06-01

    Necrotizing enterocolitis, characterized by sudden onset and rapid progression, remains the most significant gastrointestinal disorder among premature infants. In seeking a predictive biomarker, we found intestinal fatty acid binding protein, an indicator of enterocyte damage, was substantially increased within three and seven days before the diagnosis of necrotizing enterocolitis.

  5. [Photochemistry and UV Spectroscopy of Proteins and Nucleic Acids].

    PubMed

    Wierzchowski, Kazimierz Lech

    2015-01-01

    The article presents a short history of David Shugar studies in the field of photochemistry and UV spectroscopy of proteins and nucleic acids, carried out since the late 1940s. to the beginning of the 1970s. of the 20th century, with some references to the state of related research in those days.

  6. Amino acid nutrition beyond methionine and lysine for milk protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Amino acids are involved in many important physiological processes affecting the production, health, and reproduction of high-producing dairy cows. Most research and recommendations for lactating dairy cows has focused on methionine and lysine for increasing milk protein yield. This is because these...

  7. Stimulation of protein synthesis by phosphatidic acid in rat cardiomyocytes.

    PubMed

    Xu, Y J; Yau, L; Yu, L P; Elimban, V; Zahradka, P; Dhalla, N S

    1996-12-13

    Phosphatidic acid (PA) was observed to stimulate protein synthesis in adult cardiomyocytes in a time- and concentration-dependent manner. The maximal stimulation in protein synthesis (142 +/- 12% vs 100% as the control) was achieved at 10 microM PA within 60 min and was inhibited by actinomycin D (107 +/- 4% of the control) or cycloheximide (105 +/- 6% of the control). The increase in protein synthesis due to PA was attenuated or abolished by preincubation of cardiomyocytes with a tyrosine kinase inhibitor, genistein (94 +/- 9% of the control), phospholipase C inhibitors 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate or carbon-odithioic acid O-(octahydro-4,7-methanol-1H-inden-5-yl (101 +/- 6 and 95 +/- 5% of the control, respectively), protein kinase C inhibitors staurosporine or polymyxin B (109 +/- 3 and 93 +/- 3% of the control), and chelators of extracellular and intracellular free Ca2+ EGTA or BAPTA/AM (103 +/- 6 and 95 +/- 6% of the control, respectively). PA at different concentrations (0.1 to 100 microM) also caused phosphorylation of a cell surface protein of approximately 24 kDa. In addition, mitogen-activated protein kinase was stimulated by PA in a concentration-dependent manner; maximal stimulation (217 +/- 6% of the control) was seen at 10 microM PA. These data suggest that PA increases protein synthesis in adult rat cardiomyocytes and thus may play an important role in the development of cardiac hypertrophy.

  8. Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining

    PubMed Central

    Lu, Guangqing; Duan, Jinzhi; Shu, Sheng; Wang, Xuxiang; Gao, Linlin; Guo, Jing; Zhang, Yu

    2016-01-01

    In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated. Surprisingly, we found that both Lig1- and Lig3-containing complexes could efficiently catalyze A-EJ for class switching recombination (CSR) in the IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes. However, only deletion of nuclear Lig3, but not Lig1, could significantly reduce the interchromosomal translocations in Lig4−/− cells, suggesting the unique role of Lig3 in catalyzing chromosome translocation. Additional sequence analysis of chromosome translocation junction microhomology revealed the specificity of different ligase-containing complexes. The data suggested the existence of multiple DNA ligase-containing complexes in A-EJ. PMID:26787905

  9. C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

    SciTech Connect

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

    2013-09-20

    Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

  10. Cullin-RING Ligases as Attractive Anti-cancer Targets

    PubMed Central

    Zhao, Yongchao; Sun, Yi

    2014-01-01

    The ubiquitin-proteasome system (UPS) promotes the timely degradation of short-lived proteins with key regulatory roles in a vast array of biological processes, such as cell cycle progression, oncogenesis and genome integrity. Thus, abnormal regulation of UPS disrupts the protein homeostasis and causes many human diseases, particularly cancer. Indeed, the FDA approval of bortezomib, the first class of general proteasome inhibitor, for the treatment of multiple myeloma, demonstrated that the UPS can be an attractive anti-cancer target. However, normal cell toxicity associated with bortezomib, resulting from global inhibition of protein degradation, promotes the focus of drug discovery efforts on targeting enzymes upstream of the proteasome for better specificity. E3 ubiquitin ligases, particularly those known to be activated in human cancer, become an attractive choice. Cullin-RING Ligases (CRLs) with multiple components are the largest family of E3 ubiquitin ligases and are responsible for ubiquitination of ~20% of cellular proteins degraded through UPS. Activity of CRLs is dynamically regulated and requires the RING component and cullin neddylation. In this review, we will introduce the UPS and CRL E3s and discuss the biological processes regulated by each of eight CRLs through substrate degradation. We will further discuss how cullin neddylation controls CRL activity, and how CRLs are being validated as the attractive cancer targets by abrogating the RING component through genetic means and by inhibiting cullin neddylation via MLN4924, a small molecule indirect inhibitor of CRLs, currently in several Phase I clinical trials. Finally, we will discuss current efforts and future perspectives on the development of additional inhibitors of CRLs by targeting E2 and/or E3 of cullin neddylation and CRL-mediated ubiquitination as potential anti-cancer agents. PMID:23151137

  11. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

    Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-06-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

  12. Kinetic study of sulphuric acid hydrolysis of protein feathers.

    PubMed

    Ben Hamad Bouhamed, Sana; Kechaou, Nabil

    2017-02-28

    Poultry feather keratin is the most important by-product from the poultry industry due to its abundance. Different methods have been still applied to process this by-product such as enzymatic hydrolysis which is expensive and inapplicable at the industrial level. This paper presents a study of acid hydrolysis of poultry feathers using different types of acids, sulphuric acid concentration, different temperatures and solid to liquid ratio to obtain a liquid product rich in peptides. The feathers analysis revealed a crude protein content of 88.83%. A maximum peptides production of 676 mg/g was reached using sulphuric acid, 1 molar acid concentration and 50 g/l solid to liquid ratio at a temperature of 90 °C after 300 min. A reaction scheme for protein aggregation and decomposition to polypeptides and amino acids was proposed and a kinetic model for peptides production was developed. The proposed kinetic model proved to be well adapted to the experimental data with R (2) = 0.99.

  13. Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay.

    PubMed

    Vashist, Sandeep Kumar; Dixit, Chandra Kumar

    2011-07-29

    We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu(2+) in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time-response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.

  14. The transcription factor Krox20 is an E3 ligase that sumoylates its Nab coregulators

    PubMed Central

    García-Gutiérrez, Pablo; Juárez-Vicente, Francisco; Gallardo-Chamizo, Francisco; Charnay, Patrick; García-Domínguez, Mario

    2011-01-01

    Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins regulates many processes in the eukaryotic cell. This reaction is similar to ubiquitination and usually requires an E3 ligase for substrate modification. However, only a few SUMO ligases have been described so far, which frequently facilitate sumoylation by bringing together the SUMO-conjugating enzyme Ubc9 and the target protein. Ubc9 is an interaction partner of the transcription factor Krox20, a key regulator of hindbrain development. Here, we show that Krox20 functions as a SUMO ligase for its coregulators—the Nab proteins—and that Nab sumoylation negatively modulates Krox20 transcriptional activity in vivo. PMID:21836637

  15. Structure of 5-formyltetrahydrofolate cyclo-ligase from Bacillus anthracis (BA4489)

    SciTech Connect

    Meier, Christoph; Carter, Lester G.; Winter, Graeme; Owens, Ray J.; Stuart, David I.; Esnouf, Robert M.

    2007-03-01

    The structure of 5-formyltetrahydrofolate cyclo-ligase from B. anthracis determined by X-ray crystallography at a resolution of 1.6 Å is described. Bacillus anthracis is a spore-forming bacterium and the causative agent of the disease anthrax. The Oxford Protein Production Facility has been targeting proteins from B. anthracis in order to develop high-throughput technologies within the Structural Proteomics in Europe project. As part of this work, the structure of 5-formyltetrahydrofolate cyclo-ligase (BA4489) has been determined by X-ray crystallography to 1.6 Å resolution. The structure, solved in complex with magnesium-ion-bound ADP and phosphate, gives a detailed picture of the proposed catalytic mechanism of the enzyme. Chemical differences from other cyclo-ligase structures close to the active site that could be exploited to design specific inhibitors are also highlighted.

  16. The α2 helix in the DNA ligase IV BRCT-1 domain is required for targeted degradation of ligase IV during adenovirus infection.

    PubMed

    Gilson, Timra; Greer, Amy E; Vindigni, Alessandro; Ketner, Gary; Hanakahi, Leslyn A

    2012-07-05

    In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.

  17. A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes.

    PubMed

    Araki, Kazuaki; Kawamura, Meiko; Suzuki, Toshiaki; Matsuda, Noriyuki; Kanbe, Daiji; Ishii, Kyoko; Ichikawa, Tomio; Kumanishi, Toshiro; Chiba, Tomoki; Tanaka, Keiji; Nawa, Hiroyuki

    2003-08-01

    Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).

  18. An allosteric inhibitor of substrate recognition by the SCF[superscript Cdc4] ubiquitin ligase

    SciTech Connect

    Orlicky, Stephen; Tang, Xiaojing; Neduva, Victor; Elowe, Nadine; Brown, Eric D.; Sicheri, Frank; Tyers, Mike

    2010-09-17

    The specificity of SCF ubiquitin ligase-mediated protein degradation is determined by F-box proteins. We identified a biplanar dicarboxylic acid compound, called SCF-I2, as an inhibitor of substrate recognition by the yeast F-box protein Cdc4 using a fluorescence polarization screen to monitor the displacement of a fluorescein-labeled phosphodegron peptide. SCF-I2 inhibits the binding and ubiquitination of full-length phosphorylated substrates by SCF{sup Cdc4}. A co-crystal structure reveals that SCF-I2 inserts itself between the {beta}-strands of blades 5 and 6 of the WD40 propeller domain of Cdc4 at a site that is 25 {angstrom} away from the substrate binding site. Long-range transmission of SCF-I2 interactions distorts the substrate binding pocket and impedes recognition of key determinants in the Cdc4 phosphodegron. Mutation of the SCF-I2 binding site abrogates its inhibitory effect and explains specificity in the allosteric inhibition mechanism. Mammalian WD40 domain proteins may exhibit similar allosteric responsiveness and hence represent an extensive class of druggable target.

  19. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins.

  20. A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement

    PubMed Central

    Valuchova, Sona; Fulnecek, Jaroslav; Petrov, Alexander P.; Tripsianes, Konstantinos; Riha, Karel

    2016-01-01

    Many fundamental biological processes depend on intricate networks of interactions between proteins and nucleic acids and a quantitative description of these interactions is important for understanding cellular mechanisms governing DNA replication, transcription, or translation. Here we present a versatile method for rapid and quantitative assessment of protein/nucleic acid (NA) interactions. This method is based on protein induced fluorescence enhancement (PIFE), a phenomenon whereby protein binding increases the fluorescence of Cy3-like dyes. PIFE has mainly been used in single molecule studies to detect protein association with DNA or RNA. Here we applied PIFE for steady state quantification of protein/NA interactions by using microwell plate fluorescence readers (mwPIFE). We demonstrate the general applicability of mwPIFE for examining various aspects of protein/DNA interactions with examples from the restriction enzyme BamHI, and the DNA repair complexes Ku and XPF/ERCC1. These include determination of sequence and structure binding specificities, dissociation constants, detection of weak interactions, and the ability of a protein to translocate along DNA. mwPIFE represents an easy and high throughput method that does not require protein labeling and can be applied to a wide range of applications involving protein/NA interactions. PMID:28008962

  1. Dietary protein's and dietary acid load's influence on bone health.

    PubMed

    Remer, Thomas; Krupp, Danika; Shi, Lijie

    2014-01-01

    A variety of genetic, mechano-response-related, endocrine-metabolic, and nutritional determinants impact bone health. Among the nutritional influences, protein intake and dietary acid load are two of the factors most controversially discussed. Although in the past high protein intake was often assumed to exert a primarily detrimental impact on bone mass and skeletal health, the majority of recent studies indicates the opposite and suggests a bone-anabolic influence. Studies examining the influence of alkalizing diets or alkalizing supplement provision on skeletal outcomes are less consistent, which raises doubts about the role of acid-base status in bone health. The present review critically evaluates relevant key issues such as acid-base terminology, influencing factors of intestinal calcium absorption, calcium balance, the endocrine-metabolic milieu related to metabolic acidosis, and some methodological aspects of dietary exposure and bone outcome examinations. It becomes apparent that for an adequate identification and characterization of either dietary acid load's or protein's impact on bone, the combined assessment of both nutritional influences is necessary.

  2. Secreted Protein Acidic and Rich in Cysteine in Ocular Tissue

    PubMed Central

    Scavelli, Kurt; Chatterjee, Ayan

    2015-01-01

    Abstract Secreted protein acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, is the prototypical matricellular protein. Matricellular proteins are nonstructural secreted proteins that provide an integration between cells and their surrounding extracellular matrix (ECM). Regulation of the ECM is important in maintaining the physiologic function of tissues. Elevated levels of SPARC have been identified in a variety of diseases involving pathologic tissue remodeling, such as hepatic fibrosis, systemic sclerosis, and certain carcinomas. Within the eye, SPARC has been identified in the trabecular meshwork, lens, and retina. Studies have begun to show the role of SPARC in these tissues and its possible role, specifically in primary open-angle glaucoma, cataracts, and proliferative vitreoretinopathy. SPARC may, therefore, be a therapeutic target in the treatment of certain ocular diseases. Further investigation into the mechanism of action of SPARC will be necessary in the development of SPARC-targeted therapy. PMID:26167673

  3. Saturated fatty acids modulate autophagy's proteins in the hypothalamus.

    PubMed

    Portovedo, Mariana; Ignacio-Souza, Letícia M; Bombassaro, Bruna; Coope, Andressa; Reginato, Andressa; Razolli, Daniela S; Torsoni, Márcio A; Torsoni, Adriana S; Leal, Raquel F; Velloso, Licio A; Milanski, Marciane

    2015-01-01

    Autophagy is an important process that regulates cellular homeostasis by degrading dysfunctional proteins, organelles and lipids. In this study, the hypothesis that obesity could lead to impairment in hypothalamic autophagy in mice was evaluated by examining the hypothalamic distribution and content of autophagic proteins in animal with obesity induced by 8 or 16 weeks high fat diet to induce obesity and in response to intracerebroventricular injections of palmitic acid. The results showed that chronic exposure to a high fat diet leads to an increased expression of inflammatory markers and downregulation of autophagic proteins. In obese mice, autophagic induction leads to the downregulation of proteins, such as JNK and Bax, which are involved in the stress pathways. In neuron cell-line, palmitate has a direct effect on autophagy even without inflammatory activity. Understanding the cellular and molecular bases of overnutrition is essential for identifying new diagnostic and therapeutic targets for obesity.

  4. Electricity-free, sequential nucleic acid and protein isolation.

    PubMed

    Pawlowski, David R; Karalus, Richard J

    2012-05-15

    Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable (1). The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment (2). The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters (3). CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation(4). By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification

  5. Circulating E3 ligases are novel and sensitive biomarkers for diagnosis of acute myocardial infarction.

    PubMed

    Han, Qiu-Yue; Wang, Hong-Xia; Liu, Xiao-Hong; Guo, Cai-Xia; Hua, Qi; Yu, Xiao-Hong; Li, Nan; Yang, Yan-Zong; Du, Jie; Xia, Yun-Long; Li, Hui-Hua

    2015-06-01

    Ubiquitin ligase (E3) is a decisive element of the ubiquitin-proteasome system (UPS), which is the main pathway for intracellular protein turnover. Recently, circulating E3 ligases have been increasingly considered as cancer biomarkers. In the present study, we aimed to determine if cardiac-specific E3 ligases in circulation can serve as novel predictors for early diagnosis of acute myocardial infarction (AMI). By screening and verifying their tissue expression patterns with microarray and real-time PCR analysis, six of 261 E3 ligases, including cardiac-specific Rnf207 and cardiac- and muscle-enriched Fbxo32/atrogin-1, Trim54/MuRF3, Trim63/MuRF1, Kbtbd10/KLHL41, Asb11 and Asb2 in mouse heart, were selected for the present study. In the AMI rats, the levels of five E3 ligases including Rnf207, Fbxo32, Trim54, Trim63 and Kbtbd10 in the plasma were significantly increased compared with control animals. Especially, the plasma levels of Rnf207 was markedly increased at 1 h, peaked at 3 h and decreased at 6-24 h after ligation. Further evaluation of E3 ligases in AMI patients confirmed that plasma Rnf207 level increased significantly compared with that in healthy people and patients without AMI, and showed a similar time course to that in AMI rats. Simultaneously, plasma level of cardiac troponin I (cTnI) was measured by ELISA assays. Finally, receiver operating characteristic (ROC) curve analysis indicated that Rnf207 showed a similar sensitivity and specificity to the classic biomarker troponin I for diagnosis of AMI. Increased cardiac-specific E3 ligase Rnf207 in plasma may be a novel and sensitive biomarkers for AMI in humans.

  6. Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

    PubMed Central

    Lutz, Lindy M.; Pace, Chandler R.

    2016-01-01

    ABSTRACT The rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or β-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or β-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity. IMPORTANCE The ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin

  7. A Ubiquitin Ligase Complex Regulates Caspase Activation During Sperm Differentiation in Drosophila

    PubMed Central

    Arama, Eli; Bader, Maya; Rieckhof, Gabrielle E; Steller, Hermann

    2007-01-01

    In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, we screened a large collection of sterile male flies for mutants that block effector caspase activation at the onset of spermatid individualization. Here, we describe the identification and characterization of a testis-specific, Cullin-3–dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3Testis), the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC) domain of Cul3Testis that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis–like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation. PMID:17880263

  8. Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay.

    PubMed

    Vashist, Sandeep Kumar; Zhang, BinBin; Zheng, Dan; Al-Rubeaan, Khalid; Luong, John H T; Sheu, Fwu-Shan

    2011-10-01

    This study revealed a major interference from sulfo-N-hydroxysuccinimide (sulfo-NHS) in the bicinchoninic acid (BCA) protein assay. Sulfo-NHS, a common reagent used in bioconjugation and analytical biochemistry, exhibited absorbance signals and absorbance peaks at 562 nm, comparable to bovine serum albumin (BSA). However, the combined absorbance of sulfo-NHS and BSA was not strictly additive. The sulfo-NHS interference was suggested to be caused by the reduction of Cu(2+) in the BCA Kit's reagent B (4% cupric sulfate) in a manner similar to that of the protein.

  9. Hypochlorous acid-mediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure?

    PubMed

    Pattison, David I; Hawkins, Clare L; Davies, Michael J

    2007-08-28

    Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.

  10. Polyunsaturated Branched-Chain Fatty Acid Geranylgeranoic Acid Induces Unfolded Protein Response in Human Hepatoma Cells

    PubMed Central

    Iwao, Chieko; Shidoji, Yoshihiro

    2015-01-01

    The acyclic diterpenoid acid geranylgeranoic acid (GGA) has been reported to induce autophagic cell death in several human hepatoma-derived cell lines; however, the molecular mechanism for this remains unknown. In the present study, several diterpenoids were examined for ability to induce XBP1 splicing and/or lipotoxicity for human hepatoma cell lines. Here we show that three groups of diterpenoids emerged: 1) GGA, 2,3-dihydro GGA and 9-cis retinoic acid induce cell death and XBP1 splicing; 2) all-trans retinoic acid induces XBP1 splicing but little cell death; and 3) phytanic acid, phytenic acid and geranylgeraniol induce neither cell death nor XBP1 splicing. GGA-induced ER stress/ unfolded protein response (UPR) and its lipotoxicity were both blocked by co-treatment with oleic acid. The blocking activity of oleic acid for GGA-induced XBP1 splicing was not attenuated by methylation of oleic acid. These findings strongly suggest that GGA at micromolar concentrations induces the so-called lipid-induced ER stress response/UPR, which is oleate-suppressive, and shows its lipotoxicity in human hepatoma cells. PMID:26186544

  11. The Ubiquitin Ligase Hul5 Promotes Proteasomal Processivity▿

    PubMed Central

    Aviram, Sharon; Kornitzer, Daniel

    2010-01-01

    The 26S proteasome is a large cytoplasmic protease that degrades polyubiquitinated proteins to short peptides in a processive manner. The proteasome 19S regulatory subcomplex tethers the target protein via its polyubiquitin adduct and unfolds the target polypeptide, which is then threaded into the proteolytic site-containing 20S subcomplex. Hul5 is a 19S subcomplex-associated ubiquitin ligase that elongates ubiquitin chains on proteasome-bound substrates. We isolated hul5Δ as a mutation with which fusions of an unstable cyclin to stable reporter proteins accumulate as partially processed products. These products appear transiently in the wild type but are strongly stabilized in 19S ATPase mutants and in the hul5Δ mutant, supporting a role for the ATPase subunits in the unfolding of proteasome substrates before insertion into the catalytic cavity and suggesting a role for Hul5 in the processive degradation of proteins that are stalled on the proteasome. PMID:20008553

  12. Exploring Peptide Ligase Orthologs in Actinobacteria-Discovery of Pseudopeptide Natural Products, Ketomemicins.

    PubMed

    Ogasawara, Yasushi; Kawata, Junpei; Noike, Motoyoshi; Satoh, Yasuharu; Furihata, Kazuo; Dairi, Tohru

    2016-06-17

    We recently identified a novel peptide ligase (PGM1), an ATP-grasp-ligase, that catalyzes amide bond formation between (S)-2-(3,5-dihydroxy-4-methoxyphenyl)-2-guanidinoacetic acid and ribosomally supplied oligopeptides in pheganomycin biosynthesis. This was the first example of an ATP-grasp-ligase utilizing peptides as nucleophiles. To explore the potential of this type of enzyme, we performed a BLAST search and identified many orthologs. The orthologs of Streptomyces mobaraensis, Salinispora tropica, and Micromonospora sp. were found in similar gene clusters consisting of six genes. To probe the functions of these genes, we heterologously expressed each of the clusters in Streptomyces lividans and detected novel and structurally similar pseudotripeptides in the broth of all transformants. Moreover, a recombinant PGM1 ortholog of Micromonospora sp. was demonstrated to be a novel dipeptide ligase catalyzing amide bond formation between amidino-arginine and dipeptides to yield tripeptides; this is the first report of a peptide ligase utilizing dipeptides as nucleophiles.

  13. Assembly of the Elongin A Ubiquitin Ligase Is Regulated by Genotoxic and Other Stresses*

    PubMed Central

    Weems, Juston C.; Slaughter, Brian D.; Unruh, Jay R.; Hall, Shawn M.; McLaird, Merry B.; Gilmore, Joshua M.; Washburn, Michael P.; Florens, Laurence; Yasukawa, Takashi; Aso, Teijiro; Conaway, Joan W.; Conaway, Ronald C.

    2015-01-01

    Elongin A performs dual functions in cells as a component of RNA polymerase II (Pol II) transcription elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that has been shown to target Pol II stalled at sites of DNA damage. Here we investigate the mechanism(s) governing conversion of the Elongin complex from its elongation factor to its ubiquitin ligase form. We report the discovery that assembly of the Elongin A ubiquitin ligase is a tightly regulated process. In unstressed cells, Elongin A is predominately present as part of Pol II elongation factor Elongin. Assembly of Elongin A into the ubiquitin ligase is strongly induced by genotoxic stress; by transcriptional stresses that lead to accumulation of stalled Pol II; and by other stimuli, including endoplasmic reticulum and nutrient stress and retinoic acid signaling, that activate Elongin A-dependent transcription. Taken together, our findings shed new light on mechanisms that control the Elongin A ubiquitin ligase and suggest that it may play a role in Elongin A-dependent transcription. PMID:25878247

  14. A novel ubiquitin ligase is deficient in Fanconi anemia.

    PubMed

    Meetei, Amom Ruhikanta; de Winter, Johan P; Medhurst, Annette L; Wallisch, Michael; Waisfisz, Quinten; van de Vrugt, Henri J; Oostra, Anneke B; Yan, Zhijiang; Ling, Chen; Bishop, Colin E; Hoatlin, Maureen E; Joenje, Hans; Wang, Weidong

    2003-10-01

    Fanconi anemia is a recessively inherited disease characterized by congenital defects, bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Fanconi anemia proteins function in a DNA damage response pathway involving breast cancer susceptibility gene products, BRCA1 and BRCA2 (refs. 1,2). A key step in this pathway is monoubiquitination of FANCD2, resulting in the redistribution of FANCD2 to nuclear foci containing BRCA1 (ref. 3). The underlying mechanism is unclear because the five Fanconi anemia proteins known to be required for this ubiquitination have no recognizable ubiquitin ligase motifs. Here we report a new component of a Fanconi anemia protein complex, called PHF9, which possesses E3 ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitination in vivo. Because PHF9 is defective in a cell line derived from an individual with Fanconi anemia, we conclude that PHF9 (also called FANCL) represents a novel Fanconi anemia complementation group (FA-L). Our data suggest that PHF9 has a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for monoubiquitination of FANCD2.

  15. An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

    PubMed

    Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

    2014-05-20

    Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies.

  16. Nucleic acid (cDNA) and amino acid sequences of the maize endosperm protein glutelin-2.

    PubMed Central

    Prat, S; Cortadas, J; Puigdomènech, P; Palau, J

    1985-01-01

    The cDNA coding for a glutelin-2 protein from maize endosperm has been cloned and the complete amino acid sequence of the protein derived for the first time. An immature maize endosperm cDNA bank was screened for the expression of a beta-lactamase:glutelin-2 (G2) fusion polypeptide by using antibodies against the purified 28 kd G2 protein. A clone corresponding to the 28 kd G2 protein was sequenced and the primary structure of this protein was derived. Five regions can be defined in the protein sequence: an 11 residue N-terminal part, a repeated region formed by eight units of the sequence Pro-Pro-Pro-Val-His-Leu, an alternating Pro-X stretch 21 residues long, a Cys rich domain and a C-terminal part rich in Gln. The protein sequence is preceded by 19 residues which have the characteristics of the signal peptide found in secreted proteins. Unlike zeins, the main maize storage proteins, 28 kd glutelin-2 has several homologous sequences in common with other cereal storage proteins. Images PMID:3839076

  17. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis.

    PubMed

    Nguyen, Giang K T; Wang, Shujing; Qiu, Yibo; Hemu, Xinya; Lian, Yilong; Tam, James P

    2014-09-01

    Proteases are ubiquitous in nature, whereas naturally occurring peptide ligases, enzymes catalyzing the reverse reactions of proteases, are rare occurrences. Here we describe the discovery of butelase 1, to our knowledge the first asparagine/aspartate (Asx) peptide ligase to be reported. This highly efficient enzyme was isolated from Clitoria ternatea, a cyclic peptide-producing medicinal plant. Butelase 1 shares 71% sequence identity and the same catalytic triad with legumain proteases but does not hydrolyze the protease substrate of legumain. Instead, butelase 1 cyclizes various peptides of plant and animal origin with yields greater than 95%. With Kcat values of up to 17 s(-1) and catalytic efficiencies as high as 542,000 M(-1) s(-1), butelase 1 is the fastest peptide ligase known. Notably, butelase 1 also displays broad specificity for the N-terminal amino acids of the peptide substrate, thus providing a new tool for C terminus-specific intermolecular peptide ligations.

  18. Hyperdimensional Analysis of Amino Acid Pair Distributions in Proteins

    PubMed Central

    Henriksen, Svend B.; Arnason, Omar; Söring, Jón; Petersen, Steffen B.

    2011-01-01

    Our manuscript presents a novel approach to protein structure analyses. We have organized an 8-dimensional data cube with protein 3D-structural information from 8706 high-resolution non-redundant protein-chains with the aim of identifying packing rules at the amino acid pair level. The cube contains information about amino acid type, solvent accessibility, spatial and sequence distance, secondary structure and sequence length. We are able to pose structural queries to the data cube using program ProPack. The response is a 1, 2 or 3D graph. Whereas the response is of a statistical nature, the user can obtain an instant list of all PDB-structures where such pair is found. The user may select a particular structure, which is displayed highlighting the pair in question. The user may pose millions of different queries and for each one he will receive the answer in a few seconds. In order to demonstrate the capabilities of the data cube as well as the programs, we have selected well known structural features, disulphide bridges and salt bridges, where we illustrate how the queries are posed, and how answers are given. Motifs involving cysteines such as disulphide bridges, zinc-fingers and iron-sulfur clusters are clearly identified and differentiated. ProPack also reveals that whereas pairs of Lys residues virtually never appear in close spatial proximity, pairs of Arg are abundant and appear at close spatial distance, contrasting the belief that electrostatic repulsion would prevent this juxtaposition and that Arg-Lys is perceived as a conservative mutation. The presented programs can find and visualize novel packing preferences in proteins structures allowing the user to unravel correlations between pairs of amino acids. The new tools allow the user to view statistical information and visualize instantly the structures that underpin the statistical information, which is far from trivial with most other SW tools for protein structure analysis. PMID:22174733

  19. Aminoacyl-coenzyme A synthesis catalyzed by a CoA ligase from Penicillium chrysogenum.

    PubMed

    Koetsier, Martijn J; Jekel, Peter A; Wijma, Hein J; Bovenberg, Roel A L; Janssen, Dick B

    2011-03-23

    Coenzyme A ligases play an important role in metabolism by catalyzing the activation of carboxylic acids. In this study we describe the synthesis of aminoacyl-coenzyme As (CoAs) catalyzed by a CoA ligase from Penicillium chrysogenum. The enzyme accepted medium-chain length fatty acids as the best substrates, but the proteinogenic amino acids L-phenylalanine and L-tyrosine, as well as the non-proteinogenic amino acids D-phenylalanine, D-tyrosine and (R)- and (S)-β-phenylalanine were also accepted. Of these amino acids, the highest activity was found for (R)-β-phenylalanine, forming (R)-β-phenylalanyl-CoA. Homology modeling suggested that alanine 312 is part of the active site cavity, and mutagenesis (A312G) yielded a variant that has an enhanced catalytic efficiency with β-phenylalanines and D-α-phenylalanine.

  20. 4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

    PubMed

    Rastogi, Shubhra; Kumar, Ritesh; Chanotiya, Chandan S; Shanker, Karuna; Gupta, Madan M; Nagegowda, Dinesh A; Shasany, Ajit K

    2013-08-01

    Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway.

  1. ATLs and BTLs, plant-specific and general eukaryotic structurally-related E3 ubiquitin ligases.

    PubMed

    Guzmán, Plinio

    2014-02-01

    Major components of the ubiquitin proteasome system are the enzymes that operate on the transfer of ubiquitin to selected target substrate, known as ubiquitin ligases. The RING finger is a domain that is present in key classes of ubiquitin ligases. This domain coordinates the interaction with a suitable E2 conjugase and the transfer of ubiquitin from the E2 to protein targets. Additional domains coupled to the same polypeptide are important for modulating the function of these ubiquitin ligases. Plants contain several types of E3 ubiquitin ligases that in many cases have expanded as multigene families. Some families are specific to the plant lineage, whereas others may have a common ancestor among plants and other eukaryotic lineages. Arabidopsis Tóxicos en Levadura (ATLs) and BCA2 zinc finger ATLs (BTLs) are two families of ubiquitin ligases that share some common structural features. These are intronless genes that encode a highly related RING finger domain, and yet during evolutionary history, their mode of gene expansion and function is rather different. In each of these two families, the co-occurrence of transmembrane helices or C2/C2 (BZF finger) domains with a selected variation on the RING finger has been subjected to strong selection pressure in order to preserve their unique domain architectures during evolution.

  2. The biological activities of protein/oleic acid complexes reside in the fatty acid.

    PubMed

    Fontana, Angelo; Spolaore, Barbara; Polverino de Laureto, Patrizia

    2013-06-01

    A complex formed by human α-lactalbumin (α-LA) and oleic acid (OA), named HAMLET, has been shown to have an apoptotic activity leading to the selective death of tumor cells. In numerous publications it has been reported that in the complex α-LA is monomeric and adopts a partly folded or "molten globule" state, leading to the idea that partly folded proteins can have "beneficial effects". The protein/OA molar ratio initially has been reported to be 1:1, while recent data have indicated that the OA-complex is given by an oligomeric protein capable of binding numerous OA molecules per protein monomer. Proteolytic fragments of α-LA, as well as other proteins unrelated to α-LA, can form OA-complexes with biological activities similar to those of HAMLET, thus indicating that a generic protein can form a cytotoxic complex under suitable experimental conditions. Moreover, even the selective tumoricidal activity of HAMLET-like complexes has been questioned. There is recent evidence that the biological activity of long chain unsaturated fatty acids, including OA, can be ascribed to their effect of perturbing the structure of biological membranes and consequently the function of membrane-bound proteins. In general, it has been observed that the cytotoxic effects exerted by HAMLET-like complexes are similar to those reported for OA alone. Overall, these findings can be interpreted by considering that the protein moiety does not have a toxic effect on its own, but merely acts as a solubilising agent for the inherently toxic fatty acid.

  3. Composition, Roles, and Regulation of Cullin-Based Ubiquitin E3 Ligases

    PubMed Central

    Choi, Christina M.; Gray, William M.; Mooney, Sutton; Hellmann, Hanjo

    2014-01-01

    Due to their sessile nature, plants depend on flexible regulatory systems that allow them to adequately regulate developmental and physiological processes in context with environmental cues. The ubiquitin proteasome pathway, which targets a great number of proteins for degradation, is cellular tool that provides the necessary flexibility to accomplish this task. Ubiquitin E3 ligases provide the needed specificity to the pathway by selectively binding to particular substrates and facilitating their ubiquitylation. The largest group of E3 ligases known in plants is represented by CULLIN-REALLY INTERESTING NEW GENE (RING) E3 ligases (CRLs). In recent years, a great amount of knowledge has been generated to reveal the critical roles of these enzymes across all aspects of plant life. This review provides an overview of the different classes of CRLs in plants, their specific complex compositions, the variety of biological processes they control, and the regulatory steps that can affect their activities. PMID:25505853

  4. Identification and Validation of Human DNA Ligase Inhibitors Using Computer-Aided Drug Design

    PubMed Central

    Zhong, Shijun; Chen, Xi; Zhu, Xiao; Dziegielewska, Barbara; Bachman, Kurtis E.; Ellenberger, Tom; Ballin, Jeff D.; Wilson, Gerald M.; Tomkinson, Alan E.; MacKerell, Alexander D.

    2009-01-01

    Linking together of DNA strands by DNA ligases is essential for DNA replication and repair. Since many therapies used to treat cancer act by causing DNA damage, there is growing interest in the development of DNA repair inhibitors. Accordingly, virtual database screening and experimental evaluation were applied to identify inhibitors of human DNA ligase I (hLigI). When a DNA binding site within the DNA binding domain (DBD) of hLigI was targeted, more than 1 million compounds were screened from which 192 were chosen for experimental evaluation. In DNA joining assays, 10 compounds specifically inhibited hLigI, 5 of which also inhibited the proliferation of cultured human cell lines. Analysis of the 10 active compounds revealed the utility of including multiple protein conformations and chemical clustering in the virtual screening procedure. The identified ligase inhibitors are structurally diverse and have druglike physical and molecular characteristics making them ideal for further drug development studies. PMID:18630893

  5. Disconnecting XRCC1 and DNA ligase III.

    PubMed

    Katyal, Sachin; McKinnon, Peter J

    2011-07-15

    DNA strand break repair is essential for the prevention of multiple human diseases, particularly those which feature neuropathology. To further understand the pathogenesis of these syndromes, we recently developed animal models in which the DNA single-strand break repair (SSBR) components, XRCC1 and DNA Ligase III (LIG3), were inactivated in the developing nervous system. Although biochemical evidence suggests that inactivation of XRCC1 and LIG3 should share common biological defects, we found profound phenotypic differences between these two models, implying distinct biological roles for XRCC1 and LIG3 during DNA repair. Rather than a key role in nuclear DNA repair, we found LIG3 function was central to mitochondrial DNA maintenance. Instead, our data indicate that DNA Ligase 1 is the main DNA ligase for XRCC1-mediated DNA repair. These studies refine our understanding of DNA SSBR and the etiology of neurological disease.

  6. Disconnecting XRCC1 and DNA ligase III

    PubMed Central

    Katyal, Sachin

    2011-01-01

    DNA strand break repair is essential for the prevention of multiple human diseases, particularly those which feature neuropathology. To further understand the pathogenesis of these syndromes, we recently developed animal models in which the DNA single-strand break repair (SSBR) components, XRCC1 and DNA Ligase III (LIG3), were inactivated in the developing nervous system. Although biochemical evidence suggests that inactivation of XRCC1 and LIG3 should share common biological defects, we found profound phenotypic differences between these two models, implying distinct biological roles for XRCC1 and LIG3 during DNA repair. Rather than a key role in nuclear DNA repair, we found LIG3 function was central to mitochondrial DNA maintenance. Instead, our data indicate that DNA Ligase 1 is the main DNA ligase for XRCC1-mediated DNA repair. These studies refine our understanding of DNA SSBR and the etiology of neurological disease. PMID:21636980

  7. Longitudinal evolution of true protein, amino acids and bioactive proteins in breast milk: a developmental perspective.

    PubMed

    Lönnerdal, Bo; Erdmann, Peter; Thakkar, Sagar K; Sauser, Julien; Destaillats, Frédéric

    2017-03-01

    The protein content of breast milk provides a foundation for estimating protein requirements of infants. Because it serves as a guideline for regulatory agencies issuing regulations for infant formula composition, it is critical that information on the protein content of breast milk is reliable. We have therefore carried out a meta-analysis of the protein and amino acid contents of breast milk and how they evolve during lactation. As several bioactive proteins are not completely digested in the infant and therefore represent "non-utilizable" protein, we evaluated the quantity, mechanism of action and digestive fate of several major breast milk proteins. A better knowledge of the development of the protein contents of breast milk and to what extent protein utilization changes with age of the infant will help improve understanding of protein needs in infancy. It is also essential when designing the composition of infant formulas, particularly when the formula uses a "staging" approach in which the composition of the formula is modified in stages to reflect changes in breast milk and changing requirements as the infant ages.

  8. Phytic acid reduction in soy protein improves zinc bioavailability

    SciTech Connect

    Zhou, J.R.; Wong, M.S.; Burns, R.A.; Erdman, J.W. Jr. Mead Johnson Research Center, Evansville, IN )

    1991-03-15

    The objective of this study was to confirm previous studies that have suggested that reduction of phytic acid in soy improved zinc bioavailability (BV). Two commercially-produced soybean isolates containing either a normal phytic acid level or a reduced level were formulated into diets so as to provide 6 or 9 ppm zinc. Control diets were egg white protein-based and contained 3, 6 or 9 ppm zinc from zinc carbonate. Weanling male rats were fed these diets for 21 days and food intake and weight gain monitored. Slope ratio analysis of total tibia zinc content compared to total zinc intake revealed that zinc BV from reduced phytic acid soy isolate-containing diets was indistinguishable from control egg white diets. In contrast, zinc BV from normal soy isolate diets was significantly reduced compared to reduced phytic acid and control diets. These results coupled with other results indicate that phytic acid is the inhibitory factor in soybean products that results in reduced zinc BV.

  9. Identification of HECT E3 ubiquitin ligase family genes involved in stem cell regulation and regeneration in planarians.

    PubMed

    Henderson, Jordana M; Nisperos, Sean V; Weeks, Joi; Ghulam, Mahjoobah; Marín, Ignacio; Zayas, Ricardo M

    2015-08-15

    E3 ubiquitin ligases constitute a large family of enzymes that modify specific proteins by covalently attaching ubiquitin polypeptides. This post-translational modification can serve to regulate protein function or longevity. In spite of their importance in cell physiology, the biological roles of most ubiquitin ligases remain poorly understood. Here, we analyzed the function of the HECT domain family of E3 ubiquitin ligases in stem cell biology and tissue regeneration in planarians. Using bioinformatic searches, we identified 17 HECT E3 genes that are expressed in the Schmidtea mediterranea genome. Whole-mount in situ hybridization experiments showed that HECT genes were expressed in diverse tissues and most were expressed in the stem cell population (neoblasts) or in their progeny. To investigate the function of all HECT E3 ligases, we inhibited their expression using RNA interference (RNAi) and determined that orthologs of huwe1, wwp1, and trip12 had roles in tissue regeneration. We show that huwe1 RNAi knockdown led to a significant expansion of the neoblast population and death by lysis. Further, our experiments showed that wwp1 was necessary for both neoblast and intestinal tissue homeostasis as well as uncovered an unexpected role of trip12 in posterior tissue specification. Taken together, our data provide insights into the roles of HECT E3 ligases in tissue regeneration and demonstrate that planarians will be a useful model to evaluate the functions of E3 ubiquitin ligases in stem cell regulation.

  10. Directed evolution of the substrate specificity of biotin ligase.

    PubMed

    Lu, Wei-Cheng; Levy, Matthew; Kincaid, Rodney; Ellington, Andrew D

    2014-06-01

    We have developed selection scheme for directing the evolution of Escherichia coli biotin protein ligase (BPL) via in vitro compartmentalization, and have used this scheme to alter the substrate specificity of the ligase towards the utilization of the biotin analogue desthiobiotin. In this scheme, a peptide substrate (BAP) was conjugated to a DNA library encoding BirA, emulsified such that there was a single template per compartment, and protein variants were transcribed and translated in vitro. Those variants that could efficiently desthiobiotinylate their corresponding peptide:DNA conjugate were subsequently captured and amplified. Following just six rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA6-40 and BirA6-47 , showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. While selected enzymes contained a number of substitutions, a single mutation, M157T, proved sufficient to provide much greater activity with desthiobiotin. Further characterization of BirA6-40 and the single substitution variant BirAM157T revealed that they had twoto threefold higher kcat values for desthiobiotin. These variants had also lost much of their ability to utilize biotin, resulting in orthogonal enzymes that in conjunction with streptavidin variants that can utilize desthiobiotin may prove to be of great use in developing additional, robust conjugation handles for a variety of biological and biotechnological applications.

  11. Retinoic acid binding protein in normal and neopolastic rat prostate.

    PubMed

    Gesell, M S; Brandes, M J; Arnold, E A; Isaacs, J T; Ueda, H; Millan, J C; Brandes, D

    1982-01-01

    Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (3H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen F1 rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5 alpha reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation.

  12. A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase.

    PubMed

    Low, Teck Yew; Peng, Mao; Magliozzi, Roberto; Mohammed, Shabaz; Guardavaccaro, Daniele; Heck, Albert J R

    2014-12-16

    Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases. Identification of substrates of ubiquitin ligases is crucial not only to unravel the molecular mechanisms by which the UPS controls protein degradation but also for drug discovery purposes because many established UPS substrates are implicated in disease. We developed a combined bioinformatics and affinity purification-mass spectrometry (AP-MS) workflow for the system-wide identification of substrates of SCF(βTrCP), a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are characterized by a multisubunit architecture typically consisting of the invariable subunits Rbx1, Cul1, and Skp1 and one of 69 F-box proteins. The F-box protein of this member of the family is βTrCP. SCF(βTrCP) binds, through the WD40 repeats of βTrCP, to the DpSGXX(X)pS diphosphorylated motif in its substrates. We recovered 27 previously reported SCF(βTrCP) substrates, of which 22 were verified by two independent statistical protocols, thereby confirming the reliability of this approach. In addition to known substrates, we identified 221 proteins that contained the DpSGXX(X)pS motif and also interacted specifically with the WD40 repeats of βTrCP. Thus, with SCF(βTrCP), as the example, we showed that integration of structural information, AP-MS, and degron motif mining constitutes an effective method to screen for substrates of ubiquitin ligases.

  13. Dynamics of palmitic acid complexed with rat intestinal fatty acid binding protein.

    PubMed

    Zhu, L; Kurian, E; Prendergast, F G; Kemple, M D

    1999-02-02

    Dynamics of palmitic acid (PA), isotopically enriched with 13C at the second, seventh, or terminal methyl position, were investigated by 13C NMR. Relaxation measurements were made on PA bound to recombinant rat intestinal fatty acid binding protein (I-FABP) at pH 5.5 and 23 degreesC, and, for comparison, on PA incorporated into 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC) micelles, and dissolved in methanol. The 13C relaxation data, T1, and steady-state nuclear Overhauser effect (NOE) obtained at two different magnetic fields were interpreted using the model-free approach [Lipari, G., and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The overall rotational correlation time of the fatty acid.protein complex was 2.5 +/- 0.4 ns, which is substantially less than the value expected for the protein itself (>6 ns). Order parameters (S2), which are a measure of the amplitude of the internal motion of individual C-H vectors with respect to the PA molecule, while largest for C-2 and smallest for the methyl carbon, were relatively small (<0.4) in the protein complex. S2 values for given C-H vectors also were smaller for PA in the MPPC micelles and in methanol than in the protein complex. Correlation times reflective of the time scale of the internal motion of the C-H vectors were in all cases <60 ps. These results support the view that the fatty acid is not rigidly anchored within the I-FABP binding pocket, but rather has considerable freedom to move within the pocket.

  14. An improved smaller biotin ligase for BioID proximity labeling

    PubMed Central

    Kim, Dae In; Jensen, Samuel C.; Noble, Kyle A.; KC, Birendra; Roux, Kenneth H.; Motamedchaboki, Khatereh; Roux, Kyle J.

    2016-01-01

    The BioID method uses a promiscuous biotin ligase to detect protein–protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein–protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius. PMID:26912792

  15. Minimal genome encoding proteins with constrained amino acid repertoire

    PubMed Central

    Tsoy, Olga; Yurieva, Marina; Kucharavy, Andrey; O'Reilly, Mary; Mushegian, Arcady

    2013-01-01

    Minimal bacterial gene set comprises the genetic elements needed for survival of engineered bacterium on a rich medium. This set is estimated to include 300–350 protein-coding genes. One way of simplifying an organism with such a minimal genome even further is to constrain the amino acid content of its proteins. In this study, comparative genomics approaches and the results of gene knockout experiments were used to extrapolate the minimal gene set of mollicutes, and bioinformatics combined with the knowledge-based analysis of the structure-function relationships in these proteins and their orthologs, paralogs and analogs was applied to examine the challenges of completely replacing the rarest residue, cysteine. Among several known functions of cysteine residues, their roles in the active centers of the enzymes responsible for deoxyribonucleoside synthesis and transfer RNA modification appear to be crucial, as no alternative chemistry is known for these reactions. Thus, drastic reduction of the content of the rarest amino acid in a minimal proteome appears to be possible, but its complete elimination is challenging. PMID:23873957

  16. USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase

    PubMed Central

    Harada, Kumi; Kato, Masako; Nakamura, Nobuhiro

    2016-01-01

    In the endoplasmic reticulum (ER), misfolded and unfolded proteins are eliminated by a process called ER-associated protein degradation (ERAD) in order to maintain cell homeostasis. In the ERAD pathway, several ER-localized E3 ubiquitin ligases target ERAD substrate proteins for ubiquitination and subsequent proteasomal degradation. However, little is known about how the functions of the ERAD ubiquitin ligases are regulated. Recently, USP19, an ER-anchored deubiquitinating enzyme (DUB), has been suggested to be involved in the regulation of ERAD. In this study, HRD1, an ERAD ubiquitin ligase, is shown to be a novel substrate for USP19. We demonstrate that USP19 rescues HRD1 from proteasomal degradation by deubiquitination of K48-linked ubiquitin chains. In addition, the altered expression of USP19 affects the steady-state levels of HRD1. These results suggest that USP19 regulates the stability of HRD1 and provide insight into the regulatory mechanism of the ERAD ubiquitin ligases. PMID:27827840

  17. Crystallization and preliminary crystallographic analysis of d-alanine-d-alanine ligase from Streptococcus mutans

    SciTech Connect

    Lu, Yong-Zhi; Sheng, Yu; Li, Lan-Fen; Tang, De-Wei; Liu, Xiang-Yu; Zhao, Xiaojun; Liang, Yu-He Su, Xiao-Dong

    2007-09-01

    A potential target for antibiotic drug design, d-alanine-d-alanine ligase from S. mutans, was expressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. d-Alanine-d-alanine ligase is encoded by the gene ddl (SMU-599) in Streptococcus mutans. This ligase plays a very important role in cell-wall biosynthesis and may be a potential target for drug design. To study the structure and function of this ligase, the gene ddl was amplified from S. mutans genomic DNA and cloned into the expression vector pET28a. The protein was expressed in soluble form in Escherichia coli strain BL21 (DE3). Homogeneous protein was obtained using a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Purified protein was crystallized and the cube-shaped crystal diffracted to 2.4 Å. The crystal belongs to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 79.50, c = 108.97 Å. There is one molecule per asymmetric unit.

  18. Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity.

    PubMed Central

    Dib-Hajj, F.; Khan, R.; Giedroc, D. P.

    1993-01-01

    The nucleocapsid protein (NC) is the major genomic RNA binding protein that plays integral roles in the structure and replication of all animal retroviruses. In this report, select biochemical properties of recombinant Mason-Pfizer monkey virus (MPMV) and HIV-1 NCs are compared. Evidence is presented that two types of saturated Zn2 NC-polynucleotide complexes can be formed under conditions of low [NaCl] that differ in apparent site-size (n = 8 vs. n = 14). The formation of one or the other complex appears dependent on the molar ratio of NC to RNA nucleotide with the putative low site-size mode apparently predominating under conditions of protein excess. Both MPMV and HIV-1 NCs kinetically facilitate the renaturation of two complementary DNA strands, suggesting that this is a general property of retroviral NCs. NC proteins increase the second-order rate constant for renaturation of a 149-bp DNA fragment by more than four orders of magnitude over that obtained in the absence of protein at 37 degrees C. The protein-assisted rate is 100-200-fold faster than that obtained at 68 degrees C, 1 M NaCl, solution conditions considered to be optimal for strand renaturation. Provided that sufficient NC is present to coat all strands, the presence of 400-1,000-fold excess nonhomologous DNA does not greatly affect the reaction rate. The HIV-1 NC-mediated renaturation reaction functions stoichiometrically, requiring a saturated strand of DNA nucleotide:NC ratio of about 7-8, rather than 14. Under conditions of less protein, the rate acceleration is not realized. The finding of significant nucleic acid strand renaturation activity may have important implications for various events of reverse transcription particularly in initiation and cDNA strand transfer. PMID:8443601

  19. Powdery Mildew Resistance Conferred by Loss of the ENHANCED DISEASE RESISTANCE1 Protein Kinase Is Suppressed by a Missense Mutation in KEEP ON GOING, a Regulator of Abscisic Acid Signaling1[W][OA

    PubMed Central

    Wawrzynska, Anna; Christiansen, Katy M.; Lan, Yinan; Rodibaugh, Natalie L.; Innes, Roger W.

    2008-01-01

    Loss-of-function mutations in the Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced resistance to infection by powdery mildew (Golovinomyces cichoracearum). EDR1 encodes a protein kinase, but its substrates and the pathways regulated by EDR1 are unknown. To identify components of the EDR1 signal transduction pathway(s), we conducted a forward genetic screen for mutations that suppressed edr1-mediated disease resistance. Genetic mapping and cloning of one of these suppressor mutations revealed a recessive missense mutation in the KEEP ON GOING gene (KEG; At5g13530), which we designated keg-4. KEG encodes a multidomain protein that includes a RING E3 ligase domain, a kinase domain, ankyrin repeats, and HERC2-like repeats. The KEG protein has previously been shown to have ubiquitin ligase activity and to negatively regulate protein levels of the transcription factor ABCISIC ACID INSENSITIVE5. KEG mRNA levels were found to be 3-fold higher in edr1 mutant plants compared to wild type. Loss-of-function mutations in KEG are seedling lethal and are hypersensitive to glucose and abscisic acid (ABA). The keg-4 mutation, in contrast, conferred resistance to 6% glucose and suppressed edr1-mediated hypersensitivity to ABA, suggesting that the keg-4 mutation suppresses ABA signaling by altering KEG function. Several ABA-responsive genes were found to be further up-regulated in the edr1 mutant following ABA treatment, and this up-regulation was suppressed by the keg-4 mutation. We conclude that edr1-mediated resistance to powdery mildew is mediated, in part, by enhanced ABA signaling. PMID:18815384

  20. Powdery mildew resistance conferred by loss of the ENHANCED DISEASE RESISTANCE1 protein kinase is suppressed by a missense mutation in KEEP ON GOING, a regulator of abscisic acid signaling.

    PubMed

    Wawrzynska, Anna; Christiansen, Katy M; Lan, Yinan; Rodibaugh, Natalie L; Innes, Roger W

    2008-11-01

    Loss-of-function mutations in the Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced resistance to infection by powdery mildew (Golovinomyces cichoracearum). EDR1 encodes a protein kinase, but its substrates and the pathways regulated by EDR1 are unknown. To identify components of the EDR1 signal transduction pathway(s), we conducted a forward genetic screen for mutations that suppressed edr1-mediated disease resistance. Genetic mapping and cloning of one of these suppressor mutations revealed a recessive missense mutation in the KEEP ON GOING gene (KEG; At5g13530), which we designated keg-4. KEG encodes a multidomain protein that includes a RING E3 ligase domain, a kinase domain, ankyrin repeats, and HERC2-like repeats. The KEG protein has previously been shown to have ubiquitin ligase activity and to negatively regulate protein levels of the transcription factor ABCISIC ACID INSENSITIVE5. KEG mRNA levels were found to be 3-fold higher in edr1 mutant plants compared to wild type. Loss-of-function mutations in KEG are seedling lethal and are hypersensitive to glucose and abscisic acid (ABA). The keg-4 mutation, in contrast, conferred resistance to 6% glucose and suppressed edr1-mediated hypersensitivity to ABA, suggesting that the keg-4 mutation suppresses ABA signaling by altering KEG function. Several ABA-responsive genes were found to be further up-regulated in the edr1 mutant following ABA treatment, and this up-regulation was suppressed by the keg-4 mutation. We conclude that edr1-mediated resistance to powdery mildew is mediated, in part, by enhanced ABA signaling.

  1. An amino acid code for irregular and mixed protein packing.

    PubMed

    Joo, Hyun; Chavan, Archana G; Fraga, Keith J; Tsai, Jerry

    2015-12-01

    To advance our understanding of protein tertiary structure, the development of the knob-socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob-socket model simplifies packing based on repeated patterns of two motifs: a three-residue socket for packing within secondary (2°) structure and a four-residue knob-socket for tertiary (3°) packing. For coil and turn secondary structure, knob-sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α-helices to tertiary packing. In irregular sockets, Gly, Pro, Asp, and Ser are favored, while in irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly. Cys, His,Met, and Trp are not favored in either. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β-sheet socket may potentially function to inhibit β-sheet extension. In addition, analysis of the preferred crossing angles for strands within a β-sheet and mixed α-helice/β-sheet identifies canonical packing patterns useful in protein design. Lastly, the knob-socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map.

  2. An Amino Acid Code for Irregular and Mixed Protein Packing

    PubMed Central

    Joo, Hyun; Chavan, Archana; Fraga, Keith; Tsai, Jerry

    2015-01-01

    To advance our understanding of protein tertiary structure, the development of the knob-socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob-socket model simplifies packing based on repeated patterns of 2 motifs: a 3 residue socket for packing within 2° structure and a 4 residue knob-socket for 3° packing. For coil and turn secondary structure, knob-sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α-helices to tertiary packing. Irregular secondary structure involves 3 residue cliques of consecutive contacting residues or XYZ sockets. In irregular sockets, Gly, Pro, Asp and Ser are favored, while Cys, His, Met and Trp are not. For irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly, while Cys, His, Met and Trp are not. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β-sheet socket may potentially function to inhibit β-sheet extension. In addition, analysis of the preferred crossing angles for strands within a β-sheet and mixed α-helices/β-sheets identifies canonical packing patterns useful in protein design. Lastly, the knob-socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map. PMID:26370334

  3. Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein

    PubMed Central

    Longo, Liam M.; Lee, Jihun; Blaber, Michael

    2013-01-01

    A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 “prebiotic” α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a “foldable set”—that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides? If so, what (if any) characteristic properties might such polypeptides exhibit? To investigate these questions, two “primitive” versions of an extant protein fold (the β-trefoil) were produced by top-down symmetric deconstruction, resulting in a reduced alphabet size of 12 or 13 amino acids and a percentage of prebiotic amino acids approaching 80%. These proteins show a substantial acidification of pI and require high salt concentrations for cooperative folding. The results suggest that the prebiotic amino acids do comprise a foldable set within the halophile environment. PMID:23341608

  4. Reversible lysine modification on proteins by using functionalized boronic acids.

    PubMed

    Cal, Pedro M S D; Frade, Raquel F M; Cordeiro, Carlos; Gois, Pedro M P

    2015-05-26

    Iminoboronates have been utilized to successfully install azide and alkyne bioorthogonal functions on proteins, which may then be further reacted with their bioorthogonal counterparts. These constructs were also used to add polyethylene glycol (PEG) to insulin, a modification which has been shown to be reversible in the presence of fructose. Finally, iminoboronates were used to assemble a folic acid/paclitaxel small-molecule/drug conjugate in situ with an IC50  value of 20.7 nM against NCI-H460 cancer cells and negligible cytotoxicity against the CRL-1502 noncancer cells.

  5. Photolabeling of brain membrane proteins by lysergic acid diethylamide

    SciTech Connect

    Mahon, A.C.; Hartig, P.R.

    1982-04-05

    /sup 3/H-Lysergic acid diethylamide (/sup 3/H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a sub-population of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. /sup 3/H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays.

  6. Acid-base chemistry of frustrated water at protein interfaces.

    PubMed

    Fernández, Ariel

    2016-01-01

    Water molecules at a protein interface are often frustrated in hydrogen-bonding opportunities due to subnanoscale confinement. As shown, this condition makes them behave as a general base that may titrate side-chain ammonium and guanidinium cations. Frustration-based chemistry is captured by a quantum mechanical treatment of proton transference and shown to remove same-charge uncompensated anticontacts at the interface found in the crystallographic record and in other spectroscopic information on the aqueous interface. Such observations are untenable within classical arguments, as hydronium is a stronger acid than ammonium or guanidinium. Frustration enables a directed Grotthuss mechanism for proton transference stabilizing same-charge anticontacts.

  7. Structure and function analysis of protein-nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein-nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  8. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

    PubMed Central

    Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael C.

    2014-01-01

    Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems (OTSs) has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications. PMID:24959531

  9. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

    NASA Astrophysics Data System (ADS)

    Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael

    2014-06-01

    Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.

  10. Photo-CIDNP NMR spectroscopy of amino acids and proteins.

    PubMed

    Kuhn, Lars T

    2013-01-01

    Photo-chemically induced dynamic nuclear polarization (CIDNP) is a nuclear magnetic resonance (NMR) phenomenon which, among other things, is exploited to extract information on biomolecular structure via probing solvent-accessibilities of tryptophan (Trp), tyrosine (Tyr), and histidine (His) amino acid side chains both in polypeptides and proteins in solution. The effect, normally triggered by a (laser) light-induced photochemical reaction in situ, yields both positive and/or negative signal enhancements in the resulting NMR spectra which reflect the solvent exposure of these residues both in equilibrium and during structural transformations in "real time". As such, the method can offer - qualitatively and, to a certain extent, quantitatively - residue-specific structural and kinetic information on both the native and, in particular, the non-native states of proteins which, often, is not readily available from more routine NMR techniques. In this review, basic experimental procedures of the photo-CIDNP technique as applied to amino acids and proteins are discussed, recent improvements to the method highlighted, and future perspectives presented. First, the basic principles of the phenomenon based on the theory of the radical pair mechanism (RPM) are outlined. Second, a description of standard photo-CIDNP applications is given and it is shown how the effect can be exploited to extract residue-specific structural information on the conformational space sampled by unfolded or partially folded proteins on their "path" to the natively folded form. Last, recent methodological advances in the field are highlighted, modern applications of photo-CIDNP in the context of biological NMR evaluated, and an outlook into future perspectives of the method is given.

  11. Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2.

    PubMed

    Heyne, Kristina; Förster, Juliane; Schüle, Roland; Roemer, Klaus

    2014-04-01

    NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator.

  12. Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase.

    PubMed

    Nohales, María-Ángeles; Flores, Ricardo; Daròs, José-Antonio

    2012-08-21

    Viroids are a unique class of noncoding RNAs: composed of only a circular, single-stranded molecule of 246-401 nt, they manage to replicate, move, circumvent host defenses, and frequently induce disease in higher plants. Viroids replicate through an RNA-to-RNA rolling-circle mechanism consisting of transcription of oligomeric viroid RNA intermediates, cleavage to unit-length strands, and circularization. Though the host RNA polymerase II (redirected to accept RNA templates) mediates RNA synthesis and a type-III RNase presumably cleavage of Potato spindle tuber viroid (PSTVd) and closely related members of the family Pospiviroidae, the host enzyme catalyzing the final circularization step, has remained elusive. In this study we propose that PSTVd subverts host DNA ligase 1, converting it to an RNA ligase, for the final step. To support this hypothesis, we show that the tomato (Solanum lycopersicum L.) DNA ligase 1 specifically and efficiently catalyzes circularization of the genuine PSTVd monomeric linear replication intermediate opened at position G95-G96 and containing 5'-phosphomonoester and 3'-hydroxyl terminal groups. Moreover, we also show a decreased PSTVd accumulation and a reduced ratio of monomeric circular to total monomeric PSTVd forms in Nicotiana benthamiana Domin plants in which the endogenous DNA ligase 1 was silenced. Thus, in a remarkable example of parasitic strategy, viroids reprogram for their replication the template and substrate specificity of a DNA-dependent RNA polymerase and a DNA ligase to act as RNA-dependent RNA polymerase and RNA ligase, respectively.

  13. Molecular evolution of monotreme and marsupial whey acidic protein genes.

    PubMed

    Sharp, Julie A; Lefèvre, Christophe; Nicholas, Kevin R

    2007-01-01

    Whey acidic protein (WAP), a major whey protein present in milk of a number of mammalian species has characteristic cysteine-rich domains known as four-disulfide cores (4-DSC). Eutherian WAP, expressed in the mammary gland throughout lactation, has two 4-DSC domains, (DI-DII) whereas marsupial WAP, expressed only during mid-late lactation, contains an additional 4-DSC (DIII), and has a DIII-D1-DII configuration. We report the expression and evolution of echidna (Tachyglossus aculeatus) and platypus (Onithorhynchus anatinus) WAP cDNAs. Predicted translation of monotreme cDNAs showed echidna WAP contains two 4-DSC domains corresponding to DIII-DII, whereas platypus WAP contains an additional domain at the C-terminus with homology to DII and has the configuration DIII-DII-DII. Both monotreme WAPs represent new WAP protein configurations. We propose models for evolution of the WAP gene in the mammalian lineage either through exon loss from an ancient ancestor or by rapid evolution via the process of exon shuffling. This evolutionary outcome may reflect differences in lactation strategy between marsupials, monotremes, and eutherians, and give insight to biological function of the gene products. WAP four-disulfide core domain 2 (WFDC2) proteins were also identified in echidna, platypus and tammar wallaby (Macropus eugenii) lactating mammary cells. WFDC2 proteins are secreted proteins not previously associated with lactation. Mammary gland expression of tammar WFDC2 during the course of lactation showed WFDC2 was elevated during pregnancy, reduced in early lactation and absent in mid-late lactation.

  14. A Family of Salmonella Virulence Factors Functions as a Distinct Class of Autoregulated E3 Ubiquitin Ligases

    SciTech Connect

    Quezada, C.; Hicks, S; Galan, J; Stebbins, C

    2009-01-01

    Processes as diverse as receptor binding and signaling, cytoskeletal dynamics, and programmed cell death are manipulated by mimics of host proteins encoded by pathogenic bacteria. We show here that the Salmonella virulence factor SspH2 belongs to a growing class of bacterial effector proteins that harness and subvert the eukaryotic ubiquitination pathway. This virulence protein possesses ubiquitination activity that depends on a conserved cysteine residue. A crystal structure of SspH2 reveals a canonical leucine-rich repeat (LRR) domain that interacts with a unique E{sub 3} ligase [which we have termed NEL for Novel E{sub 3} Ligase] C-terminal fold unrelated to previously observed HECT or RING-finger E{sub 3} ligases. Moreover, the LRR domain sequesters the catalytic cysteine residue contained in the NEL domain, and we suggest a mechanism for activation of the ligase requiring a substantial conformational change to release the catalytic domain for function. We also show that the N-terminal domain targets SspH2 to the apical plasma membrane of polarized epithelial cells and propose a model whereby binding of the LRR to proteins at the target site releases the ligase domain for site-specific function.

  15. Dependence of intestinal amino acid uptake on dietary protein or amino acid levels

    SciTech Connect

    Karasov, W.H.; Solberg, D.H.; Diamond, J.M.

    1987-05-01

    To understand how intestinal amino acid (AA) transport is regulated by dietary substrate levels, the authors measured uptake of seven radioactively-labelled AAs and glucose across the jejunal brush-border membrane of mice kept on one of three isocaloric rations differing in nitrogen content. In the high-protein ration, uptake increased by 77-81% for the nonessential, less toxic AAs, proline, and aspartate but only by 32-61% for the more toxic essential AAs tested. In the nitrogen-deficient ration, uptake decreased for the nonessential aspartate and proline but stayed constant or increased for essential AAs and for the nonessential alanine. These patterns imply independent regulation of the intestine's various AA transporters. With decreasing dietary AA (or protein), the imino acid and acidic AA private transporters are repressed, while activities of the basic AA transporter and the neutral AA public transporter decrease to an asymptote or else go through a minimum. These regulatory patterns can be understood as a compromise among conflicting constraints imposed by protein's multiple roles as a source of calories, nitrogen, and essential AAs and by the toxicity of essential AAs at high concentrations.

  16. Functional characterization of EI24-induced autophagy in the degradation of RING-domain E3 ligases

    PubMed Central

    Devkota, Sushil; Jeong, Hyobin; Kim, Yunmi; Ali, Muhammad; Roh, Jae-il; Hwang, Daehee; Lee, Han-Woong

    2016-01-01

    ABSTRACT Historically, the ubiquitin-proteasome system (UPS) and autophagy pathways were believed to be independent; however, recent data indicate that these pathways engage in crosstalk. To date, the players mediating this crosstalk have been elusive. Here, we show experimentally that EI24 (EI24, autophagy associated transmembrane protein), a key component of basal macroautophagy/autophagy, degrades 14 physiologically important E3 ligases with a RING (really interesting new gene) domain, whereas 5 other ligases were not degraded. Based on the degradation results, we built a statistical model that predicts the RING E3 ligases targeted by EI24 using partial least squares discriminant analysis. Of 381 RING E3 ligases examined computationally, our model predicted 161 EI24 targets. Those targets are primarily involved in transcription, proteolysis, cellular bioenergetics, and apoptosis and regulated by TP53 and MTOR signaling. Collectively, our work demonstrates that EI24 is an essential player in UPS-autophagy crosstalk via degradation of RING E3 ligases. These results indicate a paradigm shift regarding the fate of E3 ligases. PMID:27541728

  17. The natural non-protein amino acid N-β-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis.

    PubMed

    Glover, W Broc; Mash, Deborah C; Murch, Susan J

    2014-11-01

    N-β-methylamino-L-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC-MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding.

  18. Ribonucleic Acid Regulation in Permeabilized Cells of Escherichia coli Capable of Ribonucleic Acid and Protein Synthesis1

    PubMed Central

    Atherly, Alan G.

    1974-01-01

    A cell permeabilization procedure is described that reduces viability less than 10% and does not significantly reduce the rates of ribonucleic acid and protein synthesis when appropriately supplemented. Permeabilization abolishes the normal stringent coupling of protein and ribonucleic acid synthesis. PMID:4364330

  19. Building and remodelling Cullin–RING E3 ubiquitin ligases

    PubMed Central

    Lydeard, John R; Schulman, Brenda A; Harper, J Wade

    2013-01-01

    Cullin–RING E3 ubiquitin ligases (CRLs) control a plethora of biological pathways through targeted ubiquitylation of signalling proteins. These modular assemblies use substrate receptor modules to recruit specific targets. Recent efforts have focused on understanding the mechanisms that control the activity state of CRLs through dynamic alterations in CRL architecture. Central to these processes are cycles of cullin neddylation and deneddylation, as well as exchange of substrate receptor modules to re-sculpt the CRL landscape, thereby responding to the cellular requirements to turn over distinct proteins in different contexts. This review is focused on how CRLs are dynamically controlled with an emphasis on how cullin neddylation cycles are integrated with receptor exchange. PMID:24232186

  20. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay

    PubMed Central

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu1+-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu1+-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration. PMID:21625379

  1. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay.

    PubMed

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu(1+)-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu(1+)-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration.

  2. Shedding light on proteins, nucleic acids, cells, humans and fish

    NASA Technical Reports Server (NTRS)

    Setlow, Richard B.

    2002-01-01

    I was trained as a physicist in graduate school. Hence, when I decided to go into the field of biophysics, it was natural that I concentrated on the effects of light on relatively simple biological systems, such as proteins. The wavelengths absorbed by the amino acid subunits of proteins are in the ultraviolet (UV). The wavelengths that affect the biological activities, the action spectra, also are in the UV, but are not necessarily parallel to the absorption spectra. Understanding these differences led me to investigate the action spectra for affecting nucleic acids, and the effects of UV on viruses and cells. The latter studies led me to the discovery of the important molecular nature of the damages affecting DNA (cyclobutane pyrimidine dimers) and to the discovery of nucleotide excision repair. Individuals with the genetic disease xeroderma pigmentosum (XP) are extraordinarily sensitive to sunlight-induced skin cancer. The finding, by James Cleaver, that their skin cells were defective in DNA repair strongly suggested that DNA damage was a key step in carcinogenesis. Such information was important for estimating the wavelengths in sunlight responsible for human skin cancer and for predicting the effects of ozone depletion on the incidence of non-melanoma skin cancer. It took experiments with backcross hybrid fish to call attention to the probable role of the longer UV wavelengths not absorbed by DNA in the induction of melanoma. These reflections trace the biophysicist's path from molecules to melanoma.

  3. Specific high-affinity binding of fatty acids to epidermal cytosolic proteins

    SciTech Connect

    Raza, H.; Chung, W.L.; Mukhtar, H. )

    1991-08-01

    Cytosol from rat, mouse, and human skin or rat epidermis was incubated with (3H)arachidonic acid, (14C)retinoic acid, (14C)oleic acid, (3H)leukotriene A4, (3H)prostaglandin E2 (PGE2) or (3H) 15-hydroxyeicosatetraenoic acid (15-HETE), and protein-bound ligands were separated using Lipidex-1000 at 4C to assess the binding specificity. The binding of oleic acid and arachidonic acid with rat epidermal cytosol was rapid, saturable, and reversible. Binding of oleic acid was competed out with the simultaneous addition of other ligands and found to be in the following order: arachidonic acid greater than oleic acid greater than linoleic acid greater than lauric acid greater than leukotriene A4 greater than 15-HETE = PGE1 greater than PGE2 = PGF2. Scatchard analysis of the binding with arachidonic acid, oleic acid, and retinoic acid revealed high-affinity binding sites with the dissociation constant in the nM range. SDS-PAGE analysis of the oleic acid-bound epidermal cytosolic protein(s) revealed maximum binding at the 14.5 kDa region. The presence of the fatty acid-binding protein in epidermal cytosol and its binding to fatty acids and retinoic acid may be of significance both in the trafficking and the metabolism of fatty acids and retinoids across the skin.

  4. Detection of Protein-Protein Interaction Within an RNA-Protein Complex Via Unnatural-Amino-Acid-Mediated Photochemical Crosslinking.

    PubMed

    Yeh, Fu-Lung; Tung, Luh; Chang, Tien-Hsien

    2016-01-01

    Although DExD/H-box proteins are known to unwind RNA duplexes and modulate RNA structures in vitro, it is highly plausible that, in vivo, some may function to remodel RNA-protein complexes. Precisely how the latter is achieved remains a mystery. We investigated this critical issue by using yeast Prp28p, an evolutionarily conserved DExD/H-box splicing factor, as a model system. To probe how Prp28p interacts with spliceosome, we strategically placed p-benzoyl-phenylalanine (BPA), a photoactivatable unnatural amino acid, along the body of Prp28p in vivo. Extracts prepared from these engineered strains were then used to assemble in vitro splicing reactions for BPA-mediated protein-protein crosslinkings. This enabled us, for the first time, to "capture" Prp28p in action. This approach may be applicable to studying the roles of other DExD/H-box proteins functioning in diverse RNA-related pathways, as well as to investigating protein-protein contacts within an RNA-protein complex.

  5. Nucleic acid-binding specificity of human FUS protein

    PubMed Central

    Wang, Xueyin; Schwartz, Jacob C.; Cech, Thomas R.

    2015-01-01

    FUS, a nuclear RNA-binding protein, plays multiple roles in RNA processing. Five specific FUS-binding RNA sequence/structure motifs have been proposed, but their affinities for FUS have not been directly compared. Here we find that human FUS binds all these sequences with Kdapp values spanning a 10-fold range. Furthermore, some RNAs that do not contain any of these motifs bind FUS with similar affinity. FUS binds RNA in a length-dependent manner, consistent with a substantial non-specific component to binding. Finally, investigation of FUS binding to different nucleic acids shows that it binds single-stranded DNA with three-fold lower affinity than ssRNA of the same length and sequence, while binding to double-stranded nucleic acids is weaker. We conclude that FUS has quite general nucleic acid-binding activity, with the various proposed RNA motifs being neither necessary for FUS binding nor sufficient to explain its diverse binding partners. PMID:26150427

  6. Proximate composition, fatty acid analysis and protein digestibility-corrected amino acid score of three Mediterranean cephalopods.

    PubMed

    Zlatanos, Spiros; Laskaridis, Kostas; Feist, Christian; Sagredos, Angelos

    2006-10-01

    Proximate composition, fatty acid analysis and protein digestibility-corrected amino acid score (PDCAAS) in three commercially important cephalopods of the Mediterranean sea (cuttlefish, octopus and squid) were determined. The results of the proximate analysis showed that these species had very high protein:fat ratios similar to lean beef. Docosahexaenoic, palmitic and eicosipentaenoic acid were the most abundant fatty acids among analyzed species. The amount of n-3 fatty acids was higher than that of saturated, monounsaturated and n-6 fatty acids. Despite the fact that cephalopods contain small amounts of fat they were found quite rich in n-3 fatty acids. Finally, PDCAAS indicated that these organisms had a very good protein quality.

  7. Maternal folic acid supplementation to dams on marginal protein level alters brain fatty acid levels of their adult offspring.

    PubMed

    Rao, Shobha; Joshi, Sadhana; Kale, Anvita; Hegde, Mahabaleshwar; Mahadik, Sahebarao

    2006-05-01

    Studies on fetal programming of adult diseases have highlighted the importance of maternal nutrition during pregnancy. Folic acid and long-chain essential polyunsaturated fatty acids (LC-PUFAs) have independent effects on fetal growth. However, folic acid effects may also involve alteration of LC-PUFA metabolism. Because marginal deficiency of LC-PUFAs during critical periods of brain growth and development is associated with risks for adult diseases, it is highly relevant to investigate how maternal supplementation of such nutrients can alter brain fatty acid levels. We examined the impact of folic acid supplementation, conventionally used in maternal intervention, on brain essential fatty acid levels and plasma corticosterone concentrations in adult offspring at 11 months of age. Pregnant female rats from 4 groups (6 in each) were fed with casein diets either with 18 g protein/100 g diet (control diet) or treatment diets that were marginal in protein (MP), such as 12 g protein/100 g diet supplemented with 8 mg folic acid (FAS/MP), 12 g protein/100 g diet without folic acid (FAD/MP), or 12 g protein/100 g diet (MP) with 2 mg folic acid. Pups were weaned to a standard laboratory diet with 18 g protein/100 g diet. All male adult offspring in the FAS/MP group showed lower docosahexaenoic acid (P<.05) as compared with control adult offspring (6.04+/-2.28 vs 10.33+/-0.86 g/100 g fatty acids) and higher n-6/n-3 ratio (P<.05). Docosahexaenoic acid levels in FAS/MP adult offspring were also lower (P<.05) when compared with the MP group. Plasma corticosterone concentrations were higher (P<.05) in male adult offspring from the FAS/MP group compared with control as well as the MP adult offspring. Results suggest that maternal folic acid supplementation at MP intake decreased brain docosahexaenoic acid levels probably involving corticosterone increase.

  8. Assessing the Chemical Accuracy of Protein Structures via Peptide Acidity

    PubMed Central

    Anderson, Janet S.; Hernández, Griselda; LeMaster, David M.

    2012-01-01

    Although the protein native state is a Boltzmann conformational ensemble, practical applications often require a representative model from the most populated region of that distribution. The acidity of the backbone amides, as reflected in hydrogen exchange rates, is exquisitely sensitive to the surrounding charge and dielectric volume distribution. For each of four proteins, three independently determined X-ray structures of differing crystallographic resolution were used to predict exchange for the static solvent-exposed amide hydrogens. The average correlation coefficients range from 0.74 for ubiquitin to 0.93 for Pyrococcus furiosus rubredoxin, reflecting the larger range of experimental exchange rates exhibited by the latter protein. The exchange prediction errors modestly correlate with the crystallographic resolution. MODELLER 9v6-derived homology models at ~60% sequence identity (36% identity for chymotrypsin inhibitor CI2) yielded correlation coefficients that are ~0.1 smaller than for the cognate X-ray structures. The most recently deposited NOE-based ubiquitin structure and the original NMR structure of CI2 fail to provide statistically significant predictions of hydrogen exchange. However, the more recent RECOORD refinement study of CI2 yielded predictions comparable to the X-ray and homology model-based analyses. PMID:23182463

  9. Poly (ADP-ribose) polymerase (PARP) is essential for sulfur mustard-induced DNA damage repair, but has no role in DNA ligase activation.

    PubMed

    Bhat, K Ramachandra; Benton, Betty J; Ray, Radharaman

    2006-01-01

    Concurrent activation of poly (ADP-ribose) polymerase (PARP) and DNA ligase was observed in cultured human epidermal keratinocytes (HEK) exposed to the DNA alkylating compound sulfur mustard (SM), suggesting that DNA ligase activation could be due to its modification by PARP. Using HEK, intracellular 3H-labeled NAD+ (3H-adenine) was metabolically generated and then these cells were exposed to SM (1 mM). DNA ligase I isolated from these cells was not 3H-labeled, indicating that DNA ligase I is not a substrate for (ADP-ribosyl)ation by PARP. In HEK, when PARP was inhibited by 3-amino benzamide (3-AB, 2 mM), SM-activated DNA ligase had a half-life that was four-fold higher than that observed in the absence of 3-AB. These results suggest that DNA repair requires PARP, and that DNA ligase remains activated until DNA damage repair is complete. The results show that in SM-exposed HEK, DNA ligase I is activated by phosphorylation catalysed by DNA-dependent protein kinase (DNA-PK). Therefore, the role of PARP in DNA repair is other than that of DNA ligase I activation. By using the DNA ligase I phosphorylation assay and decreasing PARP chemically as well as by PARP anti-sense mRNA expression in the cells, it was confirmed that PARP does not modify DNA ligase I. In conclusion, it is proposed that PARP is essential for efficient DNA repair; however, PARP participates in DNA repair by altering the chromosomal structure to make the DNA damage site(s) accessible to the repair enzymes.

  10. Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK.

    PubMed

    Costantini, Silvia; Woodbine, Lisa; Andreoli, Lucia; Jeggo, Penny A; Vindigni, Alessandro

    2007-06-01

    DNA non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double-stranded (ds) breaks in mammalian cells. Here, we characterize the interaction between two key components of the NHEJ machinery, the Ku heterodimer and the DNA ligase IV/Xrcc4 complex. Our results demonstrate that Ku interacts with DNA ligase IV via its tandem BRCT domain and that this interaction is enhanced in the presence of Xrcc4 and dsDNA. Moreover, residues 644-748 of DNA ligase IV encompassing the first BRCT motif are necessary for binding. We show that Ku needs to be in its heterodimeric form to bind DNA ligase IV and that the C-terminal tail of Ku80, which mediates binding to DNA-PKcs, is dispensable for DNA ligase IV recognition. Although the interaction between Ku and DNA ligase IV/Xrcc4 occurs in the absence of DNA-PKcs, the presence of the catalytic subunit of DNA-PK kinase enhances complex formation. Previous studies have shown that DNA-PK kinase activity causes disassembly of DNA-PKcs from Ku at the DNA end. Here, we show that DNA-PK kinase activity also results in disassembly of the Ku/DNA ligase IV/Xrcc4 complex. Collectively, our findings provide novel information on the protein-protein interactions that regulate NHEJ in cells.

  11. Snake venom. The amino acid sequence of protein A from Dendroaspis polylepis polylepis (black mamba) venom.

    PubMed

    Joubert, F J; Strydom, D J

    1980-12-01

    Protein A from Dendroaspis polylepis polylepis venom comprises 81 amino acids, including ten half-cystine residues. The complete primary structures of protein A and its variant A' were elucidated. The sequences of proteins A and A', which differ in a single position, show no homology with various neurotoxins and non-neurotoxic proteins and represent a new type of elapid venom protein.

  12. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-06-23

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins.

  13. Thermophysical properties of starch and whey protein composite prepared in presence of organic acid and esters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously, we prepared starch and protein composite by reactive mixing in presence of various organic acids and found that use of these acid esters resulted in composites with good mechanical properties. In this study, concentration (% w/w) of acid citrates in the starch-protein composites were var...

  14. Defining meal requirements for protein to optimize metabolic roles of amino acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dietary protein provides essential amino acids (EAAs) for the synthesis of new proteins plus an array of other metabolic functions; many of these functions are sensitive to postprandial plasma and intracellular amino acid concentrations. Recent research has focused on amino acids as metabolic signal...

  15. Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid.

    PubMed

    Pan, Yijun; Scanlon, Martin J; Owada, Yuji; Yamamoto, Yui; Porter, Christopher J H; Nicolazzo, Joseph A

    2015-12-07

    The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of (14)C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (Kin) of (14)C-DHA between FABP5-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in (14)C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of (14)C-DHA (1 min)), the Kin of (14)C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5(+/+) mice, the Kin of (14)C-DHA decreased 36.7 ± 12.4% in FABP5(-/-) mice

  16. Exogenous amino acids stimulate net muscle protein synthesis in the elderly.

    PubMed Central

    Volpi, E; Ferrando, A A; Yeckel, C W; Tipton, K D; Wolfe, R R

    1998-01-01

    We have investigated the response of amino acid transport and protein synthesis in healthy elderly individuals (age 71+/-2 yr) to the stimulatory effect of increased amino acid availability. Muscle protein synthesis and breakdown, and amino acid transport were measured in the postabsorptive state and during the intravenous infusion of an amino acid mixture. Muscle-free amino acid kinetics were calculated by means of a three compartment model using data obtained by femoral arterio-venous catheterization and muscle biopsies from the vastus lateralis during the infusion of stable isotope tracers of amino acids. In addition, muscle protein fractional synthetic rate (FSR) was measured. Peripheral amino acid infusion significantly increased amino acid delivery to the leg, amino acid transport, and muscle protein synthesis when measured either with the three compartment model (P < 0.05) or with the traditional precursor-product approach (FSR increased from 0. 0474+/-0.0054 to 0.0940+/-0.0143%/h, P < 0.05). Because protein breakdown did not change during amino acid infusion, a positive net balance of amino acids across the muscle was achieved. We conclude that, although muscle mass is decreased in the elderly, muscle protein anabolism can nonetheless be stimulated by increased amino acid availability. We thus hypothesize that muscle mass could be better maintained with an increased intake of protein or amino acids. PMID:9576765

  17. Pepper CaREL1, a ubiquitin E3 ligase, regulates drought tolerance via the ABA-signalling pathway.

    PubMed

    Lim, Chae Woo; Park, Chanmi; Kim, Jung-Hyun; Joo, Hyunhee; Hong, Eunji; Lee, Sung Chul

    2017-03-28

    Drought stress conditions in soil or air hinder plant growth and development. Here, we report that the hot pepper (C apsicum a nnuum) RING type E3 Ligase 1 gene (CaREL1) is essential to the drought stress response. CaREL1 encodes a cytoplasmic- and nuclear-localized protein with E3 ligase activity. CaREL1 expression was induced by abscisic acid (ABA) and drought. CaREL1 contains a C3H2C3-type RING finger motif, which functions in ubiquitination of the target protein. We used CaREL1-silenced pepper plants and CaREL1-overexpressing (OX) transgenic Arabidopsis plants to evaluate the in vivo function of CaREL1 in response to drought stress and ABA treatment. CaREL1-silenced pepper plants displayed a drought-tolerant phenotype characterized by ABA hypersensitivity. In contrast, CaREL1-OX plants exhibited ABA hyposensitivity during the germination, seedling, and adult stages. In addition, plant growth was severely impaired under drought stress conditions, via a high level of transpirational water loss and decreased stomatal closure. Quantitative RT-PCR analyses revealed that ABA-related drought stress responsive genes were more weakly expressed in CaREL1-OX plants than in wild-type plants, indicating that CaREL1 functions in the drought stress response via the ABA-signalling pathway. Taken together, our results indicate that CaREL1 functions as a negative regulator of ABA-mediated drought stress tolerance.

  18. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta; Lital , Schultz; Peter G. , Zhang; Zhiwen

    2010-10-12

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  19. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2011-08-30

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  20. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  1. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2009-02-24

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  2. Interfacial inhibitors of protein-nucleic acid interactions.

    PubMed

    Pommier, Yves; Marchand, Christophe

    2005-07-01

    This essay develops the paradigm of "Interfacial Inhibitors" (Pommier and Cherfils, TiPS, 2005, 28: 136) for inhibitory drugs beside orthosteric (competitive or non-competitive) and allosteric inhibitors. Interfacial inhibitors bind with high selectivity to a binding site involving two or more macromolecules within macromolecular complexes undergoing conformational changes. Interfacial binding traps (generally reversibly) a transition state of the complex, resulting in kinetic inactivation. The exemplary case of interfacial inhibitor of protein-DNA interface is camptothecin and its clinical derivatives. We will also provide examples generalizing the interfacial inhibitor concept to inhibitors of topoisomerase II (anthracyclines, ellipticines, epipodophyllotoxins), gyrase (quinolones, ciprofloxacin, norfloxacin), RNA polymerases (alpha-amanitin and actinomycin D), and ribosomes (antibiotics such as streptomycin, hygromycin B, tetracycline, kirromycin, fusidic acid, thiostrepton, and possibly cycloheximide). We discuss the implications of the interfacial inhibitor concept for drug discovery.

  3. The EEL-1 ubiquitin ligase promotes DNA damage-induced germ cell apoptosis in C. elegans

    PubMed Central

    Ross, A J; Li, M; Yu, B; Gao, M X; Derry, W B

    2011-01-01

    E3 ubiquitin ligases target a growing number of pro- and anti-apoptotic proteins, including tumour suppressor p53, caspases, and the Bcl-2 family. The core apoptosis pathway is well conserved between mammals and Caenorhabditis elegans, but the extent to which ubiquitin ligases regulate apoptotic cell death is not known. To investigate the role of E3 ligases in apoptosis, we inhibited 108 of the 165 predicted E3 ubiquitin ligase genes by RNA interference and quantified apoptosis in the C. elegans germline after genotoxic stress. From this screen, we identified the homologous to E6-associated protein C terminus-domain E3 ligase EEL-1 as a positive regulator of apoptosis. Intriguingly, the human homologue of EEL-1, Huwe1/ARF-BP1/Mule/HectH9, has been reported to possess both pro- and anti-apoptotic functions through its ability to stimulate Mcl-1 and p53 degradation, respectively. Here, we demonstrate that eel-1 is required to promote DNA damage-induced germ cell apoptosis, but does not have a role in physiological germ cell apoptosis or developmental apoptosis in somatic tissue. Furthermore, eel-1 acts in parallel to the p53-like gene cep-1 and intersects the core apoptosis pathway upstream of the Bcl-2/Mcl-1 orthologue ced-9. Although ee1-1 mutants exhibit hypersensitivity to genotoxic stress they do not appear to be defective in DNA repair, suggesting a distinct role for EEL-1 in promoting damage-induced apoptosis in the germline. PMID:21233842

  4. The EEL-1 ubiquitin ligase promotes DNA damage-induced germ cell apoptosis in C. elegans.

    PubMed

    Ross, A J; Li, M; Yu, B; Gao, M X; Derry, W B

    2011-07-01

    E3 ubiquitin ligases target a growing number of pro- and anti-apoptotic proteins, including tumour suppressor p53, caspases, and the Bcl-2 family. The core apoptosis pathway is well conserved between mammals and Caenorhabditis elegans, but the extent to which ubiquitin ligases regulate apoptotic cell death is not known. To investigate the role of E3 ligases in apoptosis, we inhibited 108 of the 165 predicted E3 ubiquitin ligase genes by RNA interference and quantified apoptosis in the C. elegans germline after genotoxic stress. From this screen, we identified the homologous to E6-associated protein C terminus-domain E3 ligase EEL-1 as a positive regulator of apoptosis. Intriguingly, the human homologue of EEL-1, Huwe1/ARF-BP1/Mule/HectH9, has been reported to possess both pro- and anti-apoptotic functions through its ability to stimulate Mcl-1 and p53 degradation, respectively. Here, we demonstrate that eel-1 is required to promote DNA damage-induced germ cell apoptosis, but does not have a role in physiological germ cell apoptosis or developmental apoptosis in somatic tissue. Furthermore, eel-1 acts in parallel to the p53-like gene cep-1 and intersects the core apoptosis pathway upstream of the Bcl-2/Mcl-1 orthologue ced-9. Although ee1-1 mutants exhibit hypersensitivity to genotoxic stress they do not appear to be defective in DNA repair, suggesting a distinct role for EEL-1 in promoting damage-induced apoptosis in the germline.

  5. Identification of Arabidopsis MYB56 as a novel substrate for CRL3(BPM) E3 ligases.

    PubMed

    Chen, Liyuan; Bernhardt, Anne; Lee, JooHyun; Hellmann, Hanjo

    2015-02-01

    Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants.

  6. Parsing the life-shortening effects of dietary protein: effects of individual amino acids.

    PubMed

    Arganda, Sara; Bouchebti, Sofia; Bazazi, Sepideh; Le Hesran, Sophie; Puga, Camille; Latil, Gérard; Simpson, Stephen J; Dussutour, Audrey

    2017-01-11

    High-protein diets shorten lifespan in many organisms. Is it because protein digestion is energetically costly or because the final products (the amino acids) are harmful? To answer this question while circumventing the life-history trade-off between reproduction and longevity, we fed sterile ant workers on diets based on whole proteins or free amino acids. We found that (i) free amino acids shortened lifespan even more than proteins; (ii) the higher the amino acid-to-carbohydrate ratio, the shorter ants lived and the lower their lipid reserves; (iii) for the same amino acid-to-carbohydrate ratio, ants eating free amino acids had more lipid reserves than those eating whole proteins; and (iv) on whole protein diets, ants seem to regulate food intake by prioritizing sugar, while on free amino acid diets, they seem to prioritize amino acids. To test the effect of the amino acid profile, we tested diets containing proportions of each amino acid that matched the ant's exome; surprisingly, longevity was unaffected by this change. We further tested diets with all amino acids under-represented except one, finding that methionine, serine, threonine and phenylalanine are especially harmful. All together, our results show certain amino acids are key elements behind the high-protein diet reduction in lifespan.

  7. Clustering amino acid contents of protein domains: biochemical functions of proteins and implications for origin of biological macromolecules.

    PubMed

    Torshin, I Y

    2001-04-01

    Structural classes of protein domains correlate with their amino acid compositions. Several successful algorithms (that use only amino acid composition) have been elaborated for the prediction of structural class or potential biochemical significance. This work deals with dynamic classification (clustering) of the domains on the basis of their amino acid composition. Amino acid contents of domains from a non-redundant PDB set were clustered in 20-dimensional space of amino acid contents. Despite the variations of an empirical parameter and non-redundancy of the set, only one large cluster (tens-hundreds of proteins) surrounded by hundreds of small clusters (1-5 proteins), was identified. The core of the largest cluster contains at least 64% DNA (nucleotide)-interacting protein domains from various sources. About 90% of the proteins of the core are intracellular proteins. 83% of the DNA/nucleotide interacting domains in the core belong to the mixed alpha-beta folds (a+b, a/b), 14% are all-alpha (mostly helices) and all-beta (mostly beta-strands) proteins. At the same time, when core domains that belong to one organism (E.coli) are considered, over 80% of them prove to be DNA/nucleotide interacting proteins. The core is compact: amino acid contents of domains from the core lie in relatively narrow and specific ranges. The core also contains several Fe-S cluster-binding domains, amino acid contents of the core overlap with ferredoxin and CO-dehydrogenase clusters, the oldest known proteins. As Fe-S clusters are thought to be the first biocatalysts, the results are discussed in relation to contemporary experiments and models dealing with the origin of biological macromolecules. The origin of most primordial proteins is considered here to be a result of co-adsorption of nucleotides and amino acids on specific clays, followed by en-block polymerization of the adsorbed mixtures of amino acids.

  8. Thermodynamics of the interaction of globular proteins with powdered stearic acid in acid pH.

    PubMed

    Mitra, Atanu; Chattoraj, D K; Chakraborty, P

    2006-06-01

    Adsorption isotherms of different globular proteins and gelatin on strearic acid particles have been studied as a function of biopolymer concentration, ionic strength of the medium, and temperature. The effect of neutral salts including CaCl2, Na3PO4, and urea on the adsorption isotherms has been also investigated. It is observed that the extent of adsorption (Gamma2(1)) increases in two steps with the increase of biopolymer concentration (C2) in the bulk. Gamma2(1) increases with an increase of C2 until a steady maximum value Gamma2(m) is reached at a critical concentration C2(m). After initial saturation, Gamma2(1) again increases from Gamma2(m) without reaching any limiting value due to the surface aggregation of the protein. The values of the standard free energy change for adsorption have been calculated on the basis of the Gibbs equation. The standard entropy and enthalpy changes are also calculated.

  9. ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

    PubMed Central

    Gracheva, Ekaterina; Chitale, Shalaka; Wilhelm, Thomas; Rapp, Alexander; Byrne, Jonathan; Stadler, Jens; Medina, Rebeca; Cardoso, M. Cristina

    2016-01-01

    Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV–RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV–DDB–CUL4A E3 ligase complex (DDB1–DDB2–CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites. PMID:27091446

  10. The prolific ATL family of RING-H2 ubiquitin ligases.

    PubMed

    Guzmán, Plinio

    2012-08-01

    An abundant class of E3 ubiquitin ligases encodes the RING-finger domain. The RING finger binds to the E2 ubiquitin-conjugating enzyme and brings together both the E2 and substrate. It is predicted that 477 RING finger E3 ligases exist in Arabidopsis thaliana. A particular family among them, named Arabidopsis Tóxicos en Levadura (ATL), consists of 91 members that contain the RING-H2 variation and a hydrophobic domain located at the N-terminal end. Transmembrane E3 ligases are important in several biological processes. For instance, some transmembrane RING finger E3 ligases are main participants in the endoplasmic reticulum-associated degradation pathway that targets misfolded proteins. Functional analysis of a number of ATLs has shown that some of them regulate distinct pathways in plants. Several ATLs have been shown to participate in defense responses, while others play a role in the regulation of the carbon/nitrogen response during post-germinative seedling growth transition, in the regulation of cell death during root development, in endosperm development, or in the transition to flowering under short day conditions. The ATL family has also been instrumental in evolution studies for showing how gene families are expanded in plant genomes.

  11. The prolific ATL family of RING-H2 ubiquitin ligases

    PubMed Central

    Guzmán, Plinio

    2012-01-01

    An abundant class of E3 ubiquitin ligases encodes the RING-finger domain. The RING finger binds to the E2 ubiquitin-conjugating enzyme and brings together both the E2 and substrate. It is predicted that 477 RING finger E3 ligases exist in Arabidopsis thaliana. A particular family among them, named Arabidopsis Tóxicos en Levadura (ATL), consists of 91 members that contain the RING-H2 variation and a hydrophobic domain located at the N-terminal end. Transmembrane E3 ligases are important in several biological processes. For instance, some transmembrane RING finger E3 ligases are main participants in the endoplasmic reticulum-associated degradation pathway that targets misfolded proteins. Functional analysis of a number of ATLs has shown that some of them regulate distinct pathways in plants. Several ATLs have been shown to participate in defense responses, while others play a role in the regulation of the carbon/nitrogen response during post-germinative seedling growth transition, in the regulation of cell death during root development, in endosperm development, or in the transition to flowering under short day conditions. The ATL family has also been instrumental in evolution studies for showing how gene families are expanded in plant genomes. PMID:22827943

  12. DNA ligase IV syndrome; a review.

    PubMed

    Altmann, Thomas; Gennery, Andrew R

    2016-10-07

    DNA ligase IV deficiency is a rare primary immunodeficiency, LIG4 syndrome, often associated with other systemic features. DNA ligase IV is part of the non-homologous end joining mechanism, required to repair DNA double stranded breaks. Ubiquitously expressed, it is required to prevent mutagenesis and apoptosis, which can result from DNA double strand breakage caused by intracellular events such as DNA replication and meiosis or extracellular events including damage by reactive oxygen species and ionising radiation.Within developing lymphocytes, DNA ligase IV is required to repair programmed DNA double stranded breaks induced during lymphocyte receptor development.Patients with hypomorphic mutations in LIG4 present with a range of phenotypes, from normal to severe combined immunodeficiency. All, however, manifest sensitivity to ionising radiation. Commonly associated features include primordial growth failure with severe microcephaly and a spectrum of learning difficulties, marrow hypoplasia and a predisposition to lymphoid malignancy. Diagnostic investigations include immunophenotyping, and testing for radiosensitivity. Some patients present with microcephaly as a predominant feature, but seemingly normal immunity. Treatment is mainly supportive, although haematopoietic stem cell transplantation has been used in a few cases.

  13. Determination of the amount of protein and amino acids extracted from the microbial protein (SCP) of lignocellulosic wastes.

    PubMed

    Ahmadi, A R; Ghoorchian, H; Hajihosaini, R; Khanifar, J

    2010-04-15

    With the increasing world population, the use of lignocellulosic wastes for production of microbial protein as animal feed becomes a necessity of our time. In order to verify the most productive protein, the amount of protein and amino acid extracted from Single Cell Protein (SCP) needs to be determined by an effective method. In this study Microbial protein was produced by treatment of wheat straw with Pleurotus florida; with heat at 100 degrees C and NaOH 2% as substrate by solid state fermentation. Concentration of protein was 62.8% per 100 g of dried microbial protein. Then the extracted protein hydrolyzed with HCl 6 Normal for 48 h under 110 degrees C temperature condition. Then the amino acids analyzed by using A-200 Amino Nova analyzer. The results of this study indicated that the ratio of essential amino acids to total amino acids was 65.6%. The concentration of essnyial amino acids were: Lysine = 9.5, histidine = 19.8, threonine = 0.6, valine = 6.6, methionine = 2.1, isoleucine = 7.3, leucine = 6.8, phenylalanine = 4.3 and arginine = 8.3 g/100 g of extracted protein that indicated the obtained microbial protein can be a good or suitable substitute in the food program of animal feed.

  14. Identification and functional expression of the pepper RING type E3 ligase, CaDTR1, involved in drought stress tolerance via ABA-mediated signalling

    PubMed Central

    Joo, Hyunhee; Lim, Chae Woo; Lee, Sung Chul

    2016-01-01

    Drought negatively affects plant growth and development, thereby leading to loss of crop productivity. Several plant E3 ubiquitin ligases act as positive or negative regulators of abscisic acid (ABA) and thus play important roles in the drought stress response. Here, we show that the C3HC4-type RING finger E3 ligase, CaDTR1, regulates the drought stress response via ABA-mediated signalling. CaDTR1 contains an amino-terminal RING finger motif and two carboxyl-terminal hydrophobic regions; the RING finger motif functions during attachment of ubiquitins to the target proteins, and the carboxyl-terminal hydrophobic regions function during subcellular localisation. The expression of CaDTR1 was induced by ABA, drought, and NaCl treatments. CaDTR1 localised in the nucleus and displayed in vitro E3 ubiquitin ligase activity. CaDTR1-silenced pepper plants exhibited a drought-sensitive phenotype characterised by high levels of transpirational water loss. On the other hand, CaDTR1-overexpressing (OX) Arabidopsis plants exhibited an ABA-hypersensitive phenotype during the germinative and post-germinative growth stages. Moreover, in contrast to CaDTR1-silenced pepper plants, CaDTR1-OX plants exhibited a drought-tolerant phenotype characterised by low levels of transpirational water loss via increased stomatal closure and high leaf temperatures. Our data indicate that CaDTR1 functions as a positive regulator of the drought stress response via ABA-mediated signalling. PMID:27439598

  15. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    PubMed

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  16. Crystal growth of proteins, nucleic acids, and viruses in gels.

    PubMed

    Lorber, Bernard; Sauter, Claude; Théobald-Dietrich, Anne; Moreno, Abel; Schellenberger, Pascale; Robert, Marie-Claire; Capelle, Bernard; Sanglier, Sarah; Potier, Noëlle; Giegé, Richard

    2009-11-01

    Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.

  17. Small acid soluble proteins for rapid spore identification.

    SciTech Connect

    Branda, Steven S.; Lane, Todd W.; VanderNoot, Victoria A.; Jokerst, Amanda S.

    2006-12-01

    This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.

  18. The role and mechanism of CRL4 E3 ubiquitin ligase in cancer and its potential therapy implications

    PubMed Central

    Sang, Youzhou; Yan, Fan; Ren, Xiubao

    2015-01-01

    CRLs (Cullin-RING E3 ubiquitin ligases) are the largest E3 ligase family in eukaryotes, which ubiquitinate a wide range of substrates involved in cell cycle regulation, signal transduction, transcriptional regulation, DNA damage response, genomic integrity, tumor suppression and embryonic development. CRL4 E3 ubiquitin ligase, as one member of CRLs family, consists of a RING finger domain protein, cullin4 (CUL4) scaffold protein and DDB1–CUL4 associated substrate receptors. The CUL4 subfamily includes two members, CUL4A and CUL4B, which share extensively sequence identity and functional redundancy. Aberrant expression of CUL4 has been found in a majority of tumors. Given the significance of CUL4 in cancer, understanding its detailed aspects of pathogenesis of human malignancy would have significant value for the treatment of cancer. Here, the work provides an overview to address the role of CRL4 E3 ubiquitin ligase in cancer development and progression, and discuss the possible mechanisms of CRL4 ligase involving in many cellular processes associated with tumor. Finally, we discuss its potential value in cancer therapy. PMID:26460955

  19. Partial complementation of a DNA ligase I deficiency by DNA ligase III and its impact on cell survival and telomere stability in mammalian cells.

    PubMed

    Le Chalony, Catherine; Hoffschir, Françoise; Gauthier, Laurent R; Gross, Julia; Biard, Denis S; Boussin, François D; Pennaneach, Vincent

    2012-09-01

    DNA ligase I (LigI) plays a central role in the joining of strand interruptions during replication and repair. In our current study, we provide evidence that DNA ligase III (LigIII) and XRCC1, which form a complex that functions in single-strand break repair, are required for the proliferation of mammalian LigI-depleted cells. We show from our data that in cells with either dysfunctional LigI activity or depleted of this enzyme, both LigIII and XRCC1 are retained on the chromatin and accumulate at replication foci. We also demonstrate that the LigI and LigIII proteins cooperate to inhibit sister chromatid exchanges but that only LigI prevents telomere sister fusions. Taken together, these results suggest that in cells with dysfunctional LigI, LigIII contributes to the ligation of replication intermediates but not to the prevention of telomeric instability.

  20. Arabidopsis nitrate reductase activity is stimulated by the E3 SUMO ligase AtSIZ1

    PubMed Central

    Park, Bong Soo; Song, Jong Tae; Seo, Hak Soo

    2011-01-01

    Small ubiquitin-related modifier (SUMO) is a small polypeptide that modulates protein activity and regulates hormone signalling, abiotic and biotic responses in plants. Here we show that AtSIZ regulates nitrogen assimilation in Arabidopsis through its E3 SUMO ligase function. Dwarf plants of siz1-2 flower early, show abnormal seed development and have high salicylic acid content and enhanced resistance to bacterial pathogens. These mutant phenotypes are reverted to wild-type phenotypes by exogenous ammonium but not by nitrate, phosphate or potassium. Decreased nitrate reductase activity in siz1-2 plants resulted in low nitrogen concentrations, low nitric oxide production and high nitrate content in comparison with wild-type plants. The nitrate reductases, NIA1 and NIA2, are sumoylated by AtSIZ1, which dramatically increases their activity. Both sumoylated and non-sumoylated NIA1 and NIA2 can form dimers. Our results indicate that AtSIZ1 posit