Inhibition of Ca 2+ channel surface expression by mutant bestrophin‐1 in RPE cells

The BEST1 gene product bestrophin-1, a Ca2+-dependent anion channel, interacts with CaV1.3 Ca2+ channels in the retinal pigment epithelium (RPE). BEST1 mutations lead to Best vitelliform macular dystrophy. A common functional defect of these mutations is reduced trafficking of bestrophin-1 into the plasma membrane. We hypothesized that this defect affects the interaction partner CaV1.3 channel affecting Ca2+ signaling and altered RPE function. Thus, we investigated the protein interaction between CaV1.3 channels and bestrophin-1 by immunoprecipitation, CaV1.3 activity in the presence of mutant bestrophin-1 and intracellular trafficking of the interaction partners in confluent RPE monolayers. We selected four BEST1 mutations, each representing one mutational hotspot of the disease: T6P, F80L, R218C, and F305S. Heterologously expressed L-type channels and mutant bestrophin-1 showed reduced interaction, reduced CaV1.3 channel activity, and changes in surface expression. Transfection of polarized RPE (porcine primary cells, iPSC-RPE) that endogenously express CaV1.3 and wild-type bestrophin-1, with mutant bestrophin-1 confirmed reduction of CaV1.3 surface expression. For the four selected BEST1 mutations, presence of mutant bestrophin-1 led to reduced CaV1.3 activity by modulating pore-function or decreasing surface expression. Reduced CaV1.3 activity might open new ways to understand symptoms of Best vitelliform macular dystrophy such as reduced electro-oculogram, lipofuscin accumulation, and vision impairment

ATPase-Dependent Control of the Mms21 SUMO Ligase during DNA Repair

Modification of proteins by SUMO is essential for the maintenance of genome integrity. During DNA replication, the Mms21-branch of the SUMO pathway counteracts recombination intermediates at damaged replication forks, thus facilitating sister chromatid disjunction. The Mms21 SUMO ligase docks to the arm region of the Smc5 protein in the Smc5/6 complex; together, they cooperate during recombinational DNA repair. Yet how the activity of the SUMO ligase is controlled remains unknown. Here we show that the SUMO ligase and the chromosome disjunction functions of Mms21 depend on its docking to an intact and active Smc5/6 complex, indicating that the Smc5/6-Mms21 complex operates as a large SUMO ligase in vivo. In spite of the physical distance separating the E3 and the nucleotide-binding domains in Smc5/6, Mms21-dependent sumoylation requires binding of ATP to Smc5, a step that is part of the ligase mechanism that assists Ubc9 function. The communication is enabled by the presence of a conserved disruption in the coiled coil domain of Smc5, pointing to potential conformational changes for SUMO ligase activation. In accordance, scanning force microscopy of the Smc5-Mms21 heterodimer shows that the molecule is physically remodeled in an ATP-dependent manner. Our results demonstrate that the ATP-binding activity of the Smc5/6 complex is coordinated with its SUMO ligase, through the coiled coil domain of Smc5 and the physical remodeling of the molecule, to promote sumoylation and chromosome disjunction during DNA repair

A SUMO-dependent step during establishment of Sister Chromatid Cohesion

Els anells de cohesina, formats per les proteïnes Smc1, Smc3, Scc1 i Scc3, s’uneixen topològicament al DNA, mantenint les parelles de cromàtides germanes unides des de la duplicació del DNA fins al començament de l’anafase. Aquesta funció, coneguda com a Cohesió entre Cromàtides Germanes, permet la biorientació dels cromosomes en el fus mitòtic i, posteriorment, la seva correcta segregació. Es tracta per tant d’una funció fonamental per a la vida. La cohesió entre cromàtides germanes també té altres funcions, com ara afavorir la reparació del dany en el DNA a través de recombinació homòloga. És per aquests motius que la cohesina està sotmesa a diferents nivells de regulació al llarg del cicle cel·lular, a través de diversos factors reguladors i modificacions post-traduccionals. Per exemple, l’acetilació de la subunitat Smc3 és necessària per a que els anells es mantinguin establement units a cromatina. Alteracions en la molècula de cohesina o en la seva regulació poden provocar el desenvolupament de patologies i contribuir en la progressió tumoral. En aquest estudi, descrivim la sumoilació de la cohesina com una nova modificació post-traduccional necessària per la cohesió en Saccharomyces cerevisiae. La sumoilació de la cohesina depèn, en part, de la SUMO lligasa Nse2 i d’un complex Smc5/6 plenament funcional. Totes les subunitats del complex cohesina es sumoilen in vivo durant la replicació del DNA, després de la formació dels anells de cohesina i del seu reclutament a cromatina, en un procés depenent de la unió d’ATP a les subunitats SMC, i independent de l’acetilació de Smc3. Per tal d’alterar l’estat de sumoilació dels anells de cohesina i identificar la rellevància funcional d’aquesta modificació, hem dissenyat un nou sistema experimental que permet eliminar SUMO de totes les proteïnes del complex, basat en la fusió del domini SUMO peptidasa de Ulp1 (UD) a la proteïna Scc1. Les fusions Scc1-UD s’incorporen als anells de cohesina, es carreguen en la cromatina i es localitzen adequadament sobre els cromosomes de llevat. Tanmateix, la desumoilació dels anells de cohesina bloqueja la cohesió entre les cromàtides germanes, aturant el cicle cel·lular en G2/M i provocant la pèrdua de viabilitat de les cèl·lules. Aquests efectes són deguts a l’activitat del domini SUMO peptidasa, i no a problemes estructurals en la proteïna de fusió Scc1-UD, ja que la mutació puntual del centre catalític de UD restaura la cohesió i la viabilitat cel·lular. Experiments en paral·lel suggereixen que la sumoilació de la cohesina podria tenir funcions similars en cèl·lules humanes. Sorprenentment, els anells de cohesina continuen acetilats en absència de sumoilació. Donat que els models actuals proposen que els anells es tanquen de forma estable en ser acetilats, és probable que en absència de sumoilació la cohesina encercli una sola cromàtida. Per tant, proposem que la sumoilació de la cohesina seria necessària durant la replicació del DNA per atrapar les dues cromàtides germanes de forma estable en l’interior de l’anell.Los anillos de cohesina, formados por las proteínas Smc1, SMC3, Scc1 y Scc3, se unen topológicamente al DNA, manteniendo las parejas de cromátidas hermanas unidas desde la duplicación del DNA hasta el comienzo de la anafase. Esta función, conocida como Cohesión entre Cromátidas Hermanas, permite la biorientación de los cromosomas en el huso mitótico y, posteriormente, su correcta segregación. Se trata por lo tanto de una función fundamental para la vida. La cohesión entre cromátidas hermanas también tiene otras funciones, como favorecer la reparación del daño en el DNA a través de recombinación homóloga. Es por estos motivos que la cohesina está sometida a varios niveles de regulación a lo largo del ciclo celular, a través de diferentes factores reguladores y modificaciones post-traduccionales. Por ejemplo, la acetilación de la subunidad Smc3 es necesaria para que los anillos se mantengan establemente unidos a cromatina. Alteraciones en la molécula de cohesina y/o en su regulación pueden provocar el desarrollo de patologías y contribuir a la progresión tumoral. En este estudio, describimos la sumoilación de la cohesina como una nueva modificación post-traduccional necesaria para la cohesión en Saccharomyces cerevisiae. La sumoilación de la cohesina depende, en parte, de la SUMO ligasa Nse2 y de un complejo Smc5/6 plenamente funcional. Todas las subunidades del complejo cohesina se sumoilan in vivo durante la replicación del ADN, después de la formación de los anillos de cohesina y de su reclutamiento en cromatina, en un proceso dependiente de la unión de ATP a las subunidades SMC, e independiente de la acetilación de Smc3. Con el fin de alterar el estado de sumoilación de los anillos de cohesina e identificar la relevancia funcional de esta modificación, hemos diseñado una nueva aproximación experimental que permite eliminar SUMO de todas las proteínas del complejo, basado en la fusión del dominio SUMO peptidasa de Ulp1 (UD) a la proteína Scc1. Las fusiones Scc1-UD se incorporan a los anillos de cohesina, se cargan en la cromatina y se localizan adecuadamente sobre los cromosomas de levadura. Sin embargo, la desumoilación de los anillos de cohesina impide la cohesión entre las cromátidas hermanas, deteniendo el ciclo celular en G2/M y provocando la pérdida de viabilidad de las células. Estos efectos son debidos a la actividad del dominio SUMO peptidasa, y no a problemas estructurales en la proteína de fusión Scc1-UD, ya que la mutación puntual del centro catalítico de UD restaura la cohesión y la viabilidad celular. Experimentos en paralelo sugieren que la sumoilació de la cohesina podría tener funciones similares en células humanas. Sorprendentemente, los anillos de cohesina continúan acetilados en ausencia de sumoilación. Dado que los modelos actuales proponen que los anillos se cierran establemente al ser acetilados, es probable que en ausencia de sumoilación la cohesina se cierre en torno a una sola cromátida. En consecuencia, proponemos que la sumoilación de la cohesina sería necesaria durante la replicación del ADN para atrapar las dos cromátidas hermanas de forma estable en el interior del anillo.Cohesin rings composed of the Smc1, Smc3, Scc1 and Scc3 proteins topologically bind to DNA, keeping pairs of sister chromatids together from the time of DNA replication until the onset of anaphase. This feature, known as Sister Chromatid Cohesion (SCC), allows the biorientation of chromosomes on the mitotic spindle, and their subsequent segregation. Sister Chromatid Cohesion also has other roles, such as enabling repair of DNA damage through homologous recombination. Thus, it is not surprising that cohesin is subjected to multiple levels of control during the cell cycle by different regulatory factors and post-translational modifications. For example, acetylation of the Smc3 subunit is required to prevent the opening of cohesin rings, keeping them stably bound to chromatin. Alterations in the cohesin molecule itself and/or its regulation may lead to the development of serious pathologies and can contribute to tumor progression. In this study, we describe the sumoylation of cohesin as a new post-translational modification required for Sister Chromatid Cohesion in Saccharomyces cerevisiae. Sumoylation of cohesin is partially dependent on the Nse2 SUMO ligase and the Smc5/6 complex. All subunits of the cohesin complex are sumoylated in vivo during DNA replication, after the formation of cohesin rings and their recruitment onto chromatin, in a process dependent on the binding of ATP to the SMC subunits, and independent of Smc3 acetylation. In order to alter the sumoylation status of cohesin rings and to identify its functional relevance, we designed a new approach to remove SUMO from all cohesin subunits, based on the fusion of the SUMO peptidase domain of Ulp1 (UD) to the Scc1 protein. Scc1-UD fusions are properly incorporated into cohesin rings, loaded onto chromatin and located along yeast chromosomes. However, desumoylation of cohesin rings prevents Sister Chromatid Cohesion, arresting cells in G2/M and causing the loss of cell viability. These effects are due to the activity of the SUMO peptidase domain rather than structural problems in the Scc1-UD fusion, since mutation of the catalytic site in the UD restores cohesion and cell viability. Parallel experiments suggest that sumoylation of cohesin might have similar functions in human cells. Surprisingly, cohesin rings remain acetylated in the absence of sumoylation. Current models propose that cohesin rings are stably locked once they are acetylated. Therefore, it is likely that in the absence of sumoylation cohesin encircles a single chromatid. Consequently, we propose that sumoylation of cohesin is required during DNA replication to entrap the two sister chromatids inside its ring structure

Use of the 16S-23S ribosomal genes spacer region for the molecular typing of sphingomonads

The ability of sphingomonads in drinking water to cause community- and hospital-acquired opportunistic infections has raised the need to establish reproducible identification assays. In this study, a total of 129 isolates recovered from drinking water with yellow- to orange-pigmented colonies were distributed among 10 biotypes on the basis of colony morphology. Polymorphisms, based on the amplification and restriction digestion of the intergenic transcribed spacer (ITS) region within the 10 assigned biotypes and 18 ATCC reference strains, were used to investigate the ability of this approach to differentiate closely related sphingomonads. ITS size, which ranged between 400 and 1100 bp, did not vary enough among the different genera. However, 16 distinct banding patterns within the ATCC reference strains and 9 within the 10 biotypes were obtained through ITS restriction digestion, and the majority of the tested biotypes produced patterns similar to those generated by the ATCC strains. To our knowledge, this study is not only the first comprehensive record of the size of the ITS region in sphingomonads, it is also the first study that describes the use of ITS restriction digestion to subtype those isolates

A SUMO-Dependent Step during Establishment of Sister Chromatid Chohesion

Cohesin is a protein complex that ties sister DNA molecules from the time of DNA replication until the metaphase to anaphase transition. Current models propose that the association of the Smc1, Smc3, and Scc1/Mcd1 subunits creates a ring-shaped structure that entraps the two sister DNAs. Cohesin is essential for correct chromosome segregation and recombinational repair. Its activity is therefore controlled by several posttranslational modifications, including acetylation, phosphorylation, sumoylation, and site-specific proteolysis. Here we show that cohesin sumoylation occurs at the time of cohesion establishment, after cohesin loading and ATP binding, and independently from Eco1-mediated cohesin acetylation. In order to test the functional relevance of cohesin sumoylation, we have developed a novel approach in budding yeast to deplete SUMO from all subunits in the cohesin complex, based on fusion of the Scc1 subunit to a SUMO peptidase Ulp domain (UD). Downregulation of cohesin sumoylation is lethal, and the Scc1-UD chimeras have a failure in sister chromatid cohesion. Strikingly, the unsumoylated cohesin rings are acetylated. Our findings indicate that SUMO is a novel molecular determinant for the establishment of sister chromatid cohesion, and we propose that SUMO is required for the entrapment of sister chromatids during the acetylation-mediated closure of the cohesin ring

ATPase-Dependent Control of the Mms21 SUMO Ligase during DNA Repair

Modification of proteins by SUMO is essential for the maintenance of genome integrity. During DNA replication, the Mms21-branch of the SUMO pathway counteracts recombination intermediates at damaged replication forks, thus facilitating sister chromatid disjunction. The Mms21 SUMO ligase docks to the arm region of the Smc5 protein in the Smc5/6 complex; together, they cooperate during recombinational DNA repair. Yet how the activity of the SUMO ligase is controlled remains unknown. Here we show that the SUMO ligase and the chromosome disjunction functions of Mms21 depend on its docking to an intact and active Smc5/6 complex, indicating that the Smc5/6-Mms21 complex operates as a large SUMO ligase in vivo. In spite of the physical distance separating the E3 and the nucleotidebinding domains in Smc5/6, Mms21-dependent sumoylation requires binding of ATP to Smc5, a step that is part of the ligase mechanism that assists Ubc9 function. The communication is enabled by the presence of a conserved disruption in the coiled coil domain of Smc5, pointing to potential conformational changes for SUMO ligase activation. In accordance, scanning force microscopy of the Smc5-Mms21 heterodimer shows that the molecule is physically remodeled in an ATP-dependent manner. Our results demonstrate that the ATP-binding activity of the Smc5/6 complex is coordinated with its SUMO ligase, through the coiled coil domain of Smc5 and the physical remodeling of the molecule, to promote sumoylation and chromosome disjunction during DNA repair

ATPase-dependent control of the Mms21 SUMO ligase during DNA repair

Modification of proteins by SUMO is essential for the maintenance of genome integrity. During DNA replication, the Mms21-branch of the SUMO pathway counteracts recombination intermediates at damaged replication forks, thus facilitating sister chromatid disjunction. The Mms21 SUMO ligase docks to the arm region of the Smc5 protein in the Smc5/6 complex; together, they cooperate during recombinational DNA repair. Yet how the activity of the SUMO ligase is controlled remains unknown. Here we show that the SUMO ligase and the chromosome disjunction functions of Mms21 depend on its docking to an intact and active Smc5/6 complex, indicating that the Smc5/6-Mms21 complex operates as a large SUMO ligase in vivo. In spite of the physical distance separating the E3 and the nucleotide-binding domains in Smc5/6, Mms21-dependent sumoylation requires binding of ATP to Smc5, a step that is part of the ligase mechanism that assists Ubc9 function. The communication is enabled by the presence of a conserved disruption in the coiled coil domain of Smc5, pointing to potential conformational changes for SUMO ligase activation. In accordance, scanning force microscopy of the Smc5-Mms21 heterodimer shows that the molecule is physically remodeled in an ATP-dependent manner. Our results demonstrate that the ATP-binding activity of the Smc5/6 complex is coordinated with its SUMO ligase, through the coiled coil domain of Smc5 and the physical remodeling of the molecule, to promote sumoylation and chromosome disjunction during DNA repair

Binding of ATP to the ATPase head of Smc5 stimulates sumoylation in vitro.

<p><b>A</b>. Experimental outline for the purification of wild-type or K75I mutant Smc5/6-Mms21 complexes used in the reactions. <b>B</b>. In vitro sumoylation reactions on immunoprecipitated Smc5-9myc. Reactions were stopped after 1 h of incubation at 37°C with the human E1, E2, and SUMO enzymes, as described in Materials and Methods, and analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. <b>C</b>. Quantification of in vitro sumoylation rate in immunoprecipitated Smc5/6-Mms21 complexes, as described in Materials and Methods. Graph shows mean ± s.e.m.; <i>n</i> = 4; for each individual experiment, the rate of sumoylation for wild—type Smc5 was set to 1. <b>D</b>. In vitro sumoylation assay of the c-terminal domain (ct) of Nse4 (residues 246 to 402), using the Smc5-Mms21 heterodimer as the E3. Reactions were initiated by addition of ATP (time 0) and stopped at the indicated times. Samples were loaded in SDS-PAGE gels and stained with SYPRO-Ruby. <b>E</b>. Quantification of Nse4(ct) sumoylation rates, as described in Materials and Methods. Graph shows mean ± s.e.m.; <i>n</i> = 4; for each experiment, the rate of sumoylation using wild-type Smc5 was set to 1. wt = wild type; KI = <i>smc5(K75I)</i>. In B, asterisk marks unspecific band detected by the anti-SUMO2/3 antibody in immunoprecipitates.</p

Up-regulation of Mms21-dependent sumoylation through expression of an E3-E2 fusion suppresses the smc5-DLEL coiled coil mutant.

<p><b>A</b>. HF-SUMO pull-down analysis from wild-type or E3-E2 cells, expressing 9myc-tagged wild-type or <i>DLEL</i> mutant versions of the Smc5 protein form its endogenous location, as indicated. <b>B</b>. Growth test analysis of wild type, <i>E3-E2</i>, <i>smc5-DLEL</i>, and double <i>E3-E2 smc5-DLEL</i> mutant cells; plates were incubated at 30°C in the presence or absence of 0.01% MMS. In A, arrow points to unmodified Smc5; vertical bars are sumoylated forms.</p

The coiled coil domain of Smc5 participates in activation of the Mms21 SUMO ligase.

<p><b>A</b>. Coiled coil probability of the Smc5 protein sequence in different species (<i>Saccharomyces cerevisiae</i>, <i>Ashbya gossypii</i>, <i>Magnaporthe grisea</i>, <i>Kluyveromyces lactis</i>, <i>Schizosaccharomyces pombe</i>, <i>Arabidopsis thaliana</i>, <i>Oryza sativa</i>, <i>Drosophila melanogaster</i>, <i>Danio rerio</i>, <i>Xenopus laevis</i>, <i>Gallus gallus</i>, <i>Mus musculus</i>, <i>and Homo sapiens)</i>; sequences are aligned according to P393 position in budding yeast. Numerical values for coiled coil probability are colored as shown in the legend; small vertical lines mark position of proline residues, inverted arrowheads mark position of proline residues in coiled coils. <b>B</b>. HF-SUMO pull-down analysis from wild-type cells expressing the indicated <i>SMC5-9myc</i> alleles from a centromeric plasmid; <i>DLEL</i> mutant contains the H391D, P393E, and E394L mutations. <b>C</b>. Co-immunoprecipitation analysis of the Smc5-Mms21 interaction in wild-type and <i>smc5-DLEL</i> mutant cells. <i>GALp-SMC5</i> cells expressing wild-type <i>SMC5</i> or <i>smc5-DLEL</i> allele from a centromeric vector were shifted to glucose for 6 h before collection. Mms21-6HA was immunoprecipitated from protein extracts (input) with anti-HA beads (IP); samples were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. <b>D</b>. Growth test analysis of <i>GALp-SMC5</i> cells transformed with the indicated plasmids and plated in glucose-containing media at 30°C in the presence or absence of MMS 0.01%. <b>E</b>. Nuclear segregation defects in <i>smc5-DLEL</i> cells after DNA damage. Wild-type and <i>smc5-DLEL</i> cells were arrested in G1 with alpha factor, treated with MMS 0.01% for 30 min, and released into the cell cycle; samples were taken at the indicated times for microscopic analysis, as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002089#pbio.1002089.g001" target="_blank">Fig. 1E</a>. <b>F</b>. HF-SUMO pull-down analysis in <i>GALp-SMC5 SMC1-6HA</i> cells expressing the indicated <i>SMC5-9myc</i> alleles from a centromeric vector; cells were shifted from galactose to glucose 6 h before collection to switch off the <i>GAL</i> promoter. In B and F, arrow points to unmodified SMC proteins; vertical bars are sumoylated forms.</p