RESUMEN
Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkin's lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins and undergoes phosphorylation by PKC and methylation by PRMT1. Previous characterization of its protein interaction profile by yeast two-hybrid screening showed that Ki-1/57 interacts with proteins of the SUMOylation machinery, the SUMO E2 conjugating enzyme UBC9 and the SUMO E3 ligase PIAS3, which suggested that Ki-1/57 could be involved with this process. Here we identified seven potential SUMO target sites (lysine residues) on Ki-1/57 sequence and observed that Ki-1/57 is modified by SUMO proteins in vitro and in vivo. We showed that SUMOylation of Ki-1/57 occurred on lysines 213, 276, and 336. In transfected cells expressing FLAG-Ki-1/57 wild-type, its paralog FLAG-CGI-55 wild-type, or their non-SUMOylated triple mutants, the number of PML-nuclear bodies (PML-NBs) is reduced compared with the control cells not expressing the constructs. More interestingly, after treating cells with arsenic trioxide (As2O3), the number of PML-NBs is no longer reduced when the non-SUMOylated triple mutant Ki-1/57 is expressed, suggesting that the SUMOylation of Ki-1/57 has a role in the control of As2O3-induced PML-NB formation. A proteome-wide analysis of Ki-1/57 partners in the presence of either SUMO-1 or SUMO-2 suggests that the involvement of Ki-1/57 with the regulation of gene expression is independent of the presence of either SUMO-1 or SUMO-2; however, the presence of SUMO-1 strongly influences the interaction of Ki-1/57 with proteins associated with cellular metabolism, maintenance, and cell cycle.
Asunto(s)
Factores Reguladores Miogénicos/metabolismo , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Trióxido de Arsénico , Arsenicales/farmacología , Ciclo Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina , Factores Reguladores Miogénicos/genética , Oligopéptidos/genética , Oligopéptidos/metabolismo , Óxidos/farmacología , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína SUMO-1/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Sumoilación , Transcripción GenéticaRESUMEN
Aliivibrio salmonicida causes "cold-water vibriosis" (or "Hitra disease") in fish, including marine-reared Atlantic salmon. During development of the disease the bacterium will encounter macrophages with antibacterial activities such as production of damaging reactive oxygen species (ROS). To defend itself the bacterium will presumably start producing detoxifying enzymes, reducing agents, and proteins involved in DNA and protein repair systems. Even though responses to oxidative stress are well studied for a few model bacteria, little work has been done in general to explain how important groups of pathogens, like members of the Vibrionaceae family, can survive at high levels of ROS. We have used bioinformatic tools and microarray to study how A. salmonicida responds to hydrogen peroxide (H(2)O(2)). First, we used the recently published genome sequence to predict potential binding sites for OxyR (H(2)O(2) response regulator). The computer-based search identified OxyR sites associated with 20 single genes and 8 operons, and these predictions were compared to experimental data from Northern blot analysis and microarray analysis. In general, OxyR binding site predictions and experimental results are in agreement. Up- and down regulated genes are distributed among all functional gene categories, but a striking number of ≥2 fold up regulated genes encode proteins involved in detoxification and DNA repair, are part of reduction systems, or are involved in carbon metabolism and regeneration of NADPH. Our predictions and -omics data corroborates well with findings from other model bacteria, but also suggest species-specific gene regulation.
RESUMEN
Acidithiobacillus ferrooxidans is a Gram-negative, chemolithoautotrophic bacterium involved in metal bioleaching. Using the RNA arbitrarily primed polymerase chain reaction (RAP-PCR), we have identified several cDNAs that were differentially expressed when A. ferrooxidans LR was submitted to potassium- and phosphate-limiting conditions. One of these cDNAs showed similarity with ribB. An analysis of the A. ferrooxidans ATCC 23270 genome, made available by The Institute for Genomic Research, showed that the ribB gene was not located in the rib operon, but a ribBA gene was present in this operon instead. The ribBA gene was isolated from A. ferrooxidans LR and expression of both ribB and ribBA was investigated. Transcript levels of both genes were enhanced in cells grown in the absence of K2HPO4, in the presence of zinc and copper sulfate and in different pHs. Transcript levels decreased upon exposure to a temperature higher than the ideal 30 degrees C and at pH 1.2. A comparative genomic analysis using the A. ferrooxidans ATCC 23270 genome revealed similar putative regulatory elements for both genes. Moreover, an RFN element was identified upstream from the ribB gene. Phylogenetic analysis of the distribution of RibB and RibBA in bacteria showed six different combinations. We suggest that the presence of duplicated riboflavin synthesis genes in bacteria must provide their host with some benefit in certain stressful situations.
