المؤلفون: Sakin, Volkan¹, Richter, Sebastian M.¹, He-Hsuan Hsiao², Urlaub, Henning^2,3, Melchior, Frauke¹ f.melchior@zmbh.uni-heidelberg.de

المصدر: Journal of Biological Chemistry. 9/25/2015, Vol. 290 Issue 39, p23589-23602. 14p.

مصطلحات موضوعية: *GUANOSINE triphosphatase, *LIGASES, *CYTOPLASMIC filaments, *NUCLEOCYTOPLASMIC interactions, *ISOTHERMAL titration calorimetry

مستخلص: The SUMO E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9 localizes at cytoplasmic nuclear pore complex (NPC) filaments and is a docking site in nucleocytoplasmic transport. RanBP2 has four Ran binding domains (RBDs), two of which flank RanBP2's E3 ligase region. We thus wondered whether the small GTPase Ran is a target for RanBP2-dependent sumoylation. Indeed, Ran is sumoylated both by a reconstituted and the endogenous RanBP2 complex in semi-permeabilized cells. Generic inhibition of SUMO isopeptidases or depletion of the SUMO isopeptidase SENP1 enhances sumoylation of Ran in semi-permeabilized cells. As Ran is typically associated with transport receptors, we tested the influence of Crm1, Imp β, Transportin, and NTF2 on Ran sumoylation. Surprisingly, all inhibited Ran sumoylation. Mapping Ran sumoylation sites revealed that transport receptors may simply block access of the E2-conjugating enzyme Ubc9, however the acceptor lysines are perfectly accessible in Ran/NTF2 complexes. Isothermal titration calorimetry revealed that NTF2 prevents sumoylation by reducing RanGDP's affinity to RanBP2's RBDs to undetectable levels. Taken together, our findings indicate that RanGDP and not RanGTP is the physiological target for the RanBP2 SUMO E3 ligase complex. Recognition requires interaction of Ran with RanBP2's RBDs, which is prevented by the transport factor NTF2. [ABSTRACT FROM AUTHOR]

المؤلفون: Nijman, Sebastian M. B.¹, Hijmans, E. Marielle¹, El Messaoudi, Selma², Van Dongen, Miranda M. W.¹, Sardet, Claude², Bernards, René¹ r.bernards@nki.nl

المصدر: Journal of Biological Chemistry. 8/4/2006, Vol. 281 Issue 31, p21582-21587. 6p. 4 Diagrams.

مصطلحات موضوعية: *GENETIC testing, *HUMAN chromosome abnormality diagnosis, *MEDICAL screening, *RETINOBLASTOMA, *NEUROBLASTOMA, *TRANSFORMING growth factors

مستخلص: The helix-loop-helix transcription factor TFE3 has been suggested to play a role in the control of cell growth by acting as a binding partner of transcriptional regulators such as E2F3, SMAD3, and LEF-1 (1-4). Furthermore, translocations/TFE3 fusions have been directly implicated in tumorigenesis (5-7). Surprisingly, however, a direct functional role for TFE3 in the regulation of proliferation has not been reported. By screening retroviral cDNA expression libraries to identify cDNAs that confer resistance to a pRB-induced proliferation arrest, we have found that TFE3 overrides a growth arrest in Rat1 cells induced by pRB and its upstream regulator p16INK4A. In addition, TFE3 expression blocks the anti-mitogenic effects of TGF-β in rodent and human cells. We provide data supporting a role for endogenous TFE3 in the direct regulation of CYCLINE expression in an E2F3-dependent manner. These observations establish TFE3 as a functional regulator of proliferation and offer a potential mechanism for its involvement in cancer. [ABSTRACT FROM AUTHOR]

المؤلفون: Klaus, Sebastian M. J.¹, Kunji, Edmund R. S.², Bozzo, Gale G.¹, Noiriel, Alexandre¹, de la Garza, Rocío Díaz¹, Basset, Gilles J. C.¹, Ravanel, Stéphane³, Rébeillé, Fabrice³, Gregory III, Jesse F.⁴, Hanson, Andrew D.¹ adha@mail.ifas.ufl.edu

المصدر: Journal of Biological Chemistry. 11/18/2005, Vol. 280 Issue 46, p38457-38463. 7p. 3 Diagrams, 3 Graphs.

مصطلحات موضوعية: *PLASTIDS, *CYANOBACTERIA, *FOLIC acid, *ORGANELLES, *PLANT cells & tissues, *GREEN fluorescent protein, *ARABIDOPSIS, *GENOMICS

مستخلص: Cyanobacterial and plant genomes encode proteins with some similarity to the folate and biopterin transporters of the trypanosomatid parasite Leishmania. The Synechocystis slr0642 gene product and its closest Arabidopsis homolog, the At2g32040 gene product, are representative examples. Both have 12 probable transmembrane domains, and the At2g32040 protein has a predicted chloroplast transit peptide. When expressed in Escherichia coli pabA pabB or folE, mutants, which are unable to produce or take up folates, the slr0642 protein and a modified At2g32040 protein (truncated and fused to the N terminus of slr0642) enabled growth on 5-formyltetrahydrofolate or folic acid but not on 5-formyltetrahydrofolate triglutamate, demonstrating that both proteins mediate folate monoglutamate transport. Both proteins also mediate transport of the antifolate analogs methotrexate and aminopterin, as evidenced by their ability to greatly increase the sensitivity of E. coli to these inhibitors. The full-length At2g32040 polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to the envelope of Arabidopsis chloroplasts in transient expression experiments. At2g32040 transcripts were present at similar levels in roots and aerial organs, indicating that the protein occurs in non-green plastids as well as chloroplasts. Insertional inactivation of At2g32040 significantly raised the total folate content of chloroplasts and lowered the proportion of 5-methyltetrahydrofolate but did not discernibly affect growth. These findings establish conservation of function among folate and biopterin transporter family proteins from three kingdoms of life. [ABSTRACT FROM AUTHOR]

المؤلفون: Klaus, Sebastian M. J.¹, Wegkamp, Arno², Sybesma, Wilbert², Hugenholtz, Jeroen¹, Gregory III, Jesse F.³, Hanson, Andrew D.¹ adha@mail.ifas.ufl.edu

المصدر: Journal of Biological Chemistry. 2/18/2005, Vol. 280 Issue 7, p5274-5280. 7p. 4 Diagrams, 6 Charts, 9 Graphs.

مصطلحات موضوعية: *ENZYMES, *PYROPHOSPHATES, *FOLIC acid, *BACTERIA, *PLANTS, *PTERIDINES

مستخلص: Removal of pyrophosphate from dihydroneopterin triphosphate (DHNTP) is the second step in the pterin branch of the folate synthesis pathway. There has been controversy over whether this reaction requires a specific pyrophosphohydrolase or is a metal ion-dependent chemical process. The genome of Lactococcus lactis has a multicistronic folate synthesis operon that includes an open reading frame (ylgG) specifying a putative Nudix hydrolase. Because many Nudix enzymes are pyrophosphohydrolases, YlgG was expressed in Escherichia coli and characterized. The recombinant protein showed high DHNTP pyrophosphohydrolase activity with a Km value of 2 µM, had no detectable activity against deoxynucleoside triphosphates or other typical Nudix hydrolase substrates, required a physiological level (∼1 mM) of Mg2+, and was active as a monomer. Essentially no reaction occurred without enzyme at 1 mM Mg2+. Inactivation of ylgG in L. lactis resulted in DHNTP accumulation and folate depletion, confirming that YlgG functions in folate biosynthesis. We therefore propose that ylgG be redesignated as folQ. The closest Arabidopsis homolog of YlgG (encoded by Nudix gene At1g68760) was expressed in E. coli and shown to have Mg2+-dependent DHNTP pyrophosphohydrolase activity. This protein (AtNUDT1) was reported previously to have NADH pyrophosphatase activity in the presence of 5 mM Mn2+ (Dobrzanska, M., Szurmak, B., Wyslouch-Cieszynska, A., and Kraszewska, E. (2002) J. Biol. Chem. 277, 50482-50486). However, we found that this activity is negligible at physiological levels of Mn2+ and that, with 1 mM Mg2+, AtNUDT1 prefers DHNTP and (deoxy) nucleoside triphosphates. [ABSTRACT FROM AUTHOR]

المؤلفون: Kulicke, Corinna A.^1,2 kulicke@ohsu.edu, De Zan, Erica³, Hein, Zeynep⁴, Gonzalez-Lopez, Claudia¹, Ghanwat, Swapnil⁴, Veerapen, Natacha⁵, Besra, Gurdyal S.⁵, Klenerman, Paul^6,7, Christianson, John C.⁸, Springer, Sebastian⁴, Nijman, Sebastian M.^3,9, Cerundolo, Vincenzo¹, Salio, Mariolina¹ mariolina.salio@imm.ox.ac.uk

المصدر: Journal of Biological Chemistry. Feb2022, Vol. 298 Issue 2, p1-22. 22p.

مصطلحات موضوعية: *MAJOR histocompatibility complex, *ANTIGEN presentation, *ADENOSINE triphosphatase, *HAPLOIDY, *PROTEINS, *ENDOPLASMIC reticulum, *T cells

مستخلص: The monomorphic antigen-presenting molecule major his-tocompatibility complex-I-related protein 1 (MR1) presents small-molecule metabolites to mucosal-associated invariant T (MAIT) cells. The MR1-MAIT cell axis has been implicated in a variety of infectious and noncommunicable diseases, and recent studies have begun to develop an understanding of the molecular mechanisms underlying this specialized antigen presentation pathway. However, proteins regulating MR1 folding, loading, stability, and surface expression remain to be identified. Here, we performed a gene trap screen to discover novel modulators of MR1 surface expression through insertional mutagenesis of an MR1-overexpressing clone derived from the near-haploid human cell line HAP1 (HAP1.MR1). The most significant positive regulators identified included β2-microglobulin, a known regulator of MR1 surface expression, and ATP13A1, a P5-type ATPase in the endoplasmic reticulum (ER) not previously known to be associated with MR1-mediated antigen presentation. CRISPR/Cas9-mediated knockout of ATP13A1 in both HAP1.MR1 and THP-1 cell lines revealed a profound reduction in MR1 protein levels and a concomitant functional defect specific to MR1-mediated anti-gen presentation. Collectively, these data are consistent with the ER-resident ATP13A1 being a key posttranscriptional determinant of MR1 surface expression. [ABSTRACT FROM AUTHOR]

المؤلفون: Eudes, Aymerick¹, Kunji, Edmund R. S.², Noiriel, Alexandre¹, Klaus, Sebastian M. J.¹, Vickers, Tim J.³, Beverley, Stephen M.³, Gregory III, Jesse F.⁴, Hanson, Andrew D.¹ adha@ufl.edu

المصدر: Journal of Biological Chemistry. 1/22/2010, Vol. 285 Issue 4, p2867-2875. 16p.

مصطلحات موضوعية: *SYNECHOCYSTIS, *ARABIDOPSIS thaliana, *GENE expression, *ESCHERICHIA coli, *CARRIER proteins

مستخلص: The Synechocystis Slr0642 protein and its plastidial Arabidopsis (Arabidopsis thaliana) ortholog At2g32040 belong to the folate-biopterin transporter (FBT) family within the major facilitator superfamily. Both proteins transport folates when expressed in Escherichia coli. Because the structural requirements for transport activity are not known for any FBT protein, we applied mutational analysis to identify residues that are critical to transport and interpreted the results using a comparative structural model based on E. coli lactose permease. Folate transport was assessed via the growth of an E. coli pabA abgT strain, which cannot synthesize or take up folates or p-aminobenzoylglutamate. In total, 47 residues were replaced with Cys or Ala. Mutations at 22 positions abolished folate uptake without affecting Slr0642 expression in membranes, whereas other mutations had no effect. Residues important for function mostly line the predicted central cavity and are concentrated in the core α-helices H1, H4, H7, and H10. The essential residue locations are consistent with a folate-binding site lying roughly equidistant from both faces of the transporter. Arabidopsis has eight FBT proteins besides At2g32040, often lacking conserved critical residues. When six of these proteins were expressed in E. coli or in Leishmania folate or pterin transporter mutants, none showed evidence of folate or pterin transport activity, and only At2g32040 was isolated by functional screening of Arabidopsis cDNA libraries in E. coli. Such negative data could reflect roles in transport of other substrates. These studies provide the first insights into the native structure and catalytic mechanism of FBT family carriers. [ABSTRACT FROM AUTHOR]