Influence of the chirality of short peptide supramolecular hydrogels in protein crystallogenesis

For the first time the influence of the chirality of the gel fibers in protein crystallogenesis has been studied. Enantiomeric hydrogels 1 and 2 were tested with model proteins lysozyme and glucose isomerase and a formamidase from B. cereus. Crystallization behaviour and crystal quality of these proteins in both hydrogels are presented and compared.MICINN (Spain) projects BIO2010-16800 (JAG), CTQ-2011.22455 (LAC & JMC), CTQ2012-34778 (JJDM & ALG), “Factoría Española de Cristalización” Consolider-Ingenio 2010 (JAG & MCM) and EDRF Funds (JAG, LAC & JMC), P12-FQM-2721 (LAC) Junta de Andalucía.MINECO,Project No. FIS2013-41821-R

Regulación de la actividad de las proteínas c-Jun y p50 por estrés oxidativo y nitrosativo a través de S-glutationilación

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular, Fecha de lectura 22-02-200

Nitric oxide inhibits c-Jun DNA binding by specifically targeted S-glutathionylation

9 p.-8 fig.-1 tab.This study addresses potential molecular mechanisms underlying the inhibition of the transcription factor c-Jun by nitric oxide. We show that in the presence of the physiological sulfhydryl glutathione nitric oxide modifies the two cysteine residues contained in the DNA binding module of c-Jun in a selective and distinct way. Although nitric oxide induced the formation of an intermolecular disulfide bridge between cysteine residues in the leucine zipper site of c-Jun monomers, this same radical directed the covalent incorporation of stoichiometric amounts of glutathione to a single conserved cysteine residue in the DNA-binding site of the protein. We found that covalent dimerization of c-Jun apparently did not affect its DNA binding activity, whereas the formation of a mixed disulfide with glutathione correlated well with the inhibition of transcription factor binding to DNA. Furthermore, we provide experimental evidence that nitric oxide-induced S-glutathionylation and inhibition of c-Jun involves the formation of S-nitrosoglutathione. In conclusion, our results support the reversible formation of a mixed disulfide between glutathione and c-Jun as a potential mechanism by which nitrosative stress may be transduced into a functional response at the level of transcription.This work was supported by Biomed-2 grants from the European Community, Marie Curie Fellowship BMH4-CT98-5052 (to P. K.), Concerted Action Grant BMH4-CT96-0979 (to S. L.), Grant SAF 97-0035 from the Comisión Interministerial de Cienca y Tecnología (to S. L.),and a postgraduate fellowship of the Spanish Ministry of Education and Culture (to E. P. M.).Peer reviewe

15-deoxy-Δ12,14-prostaglandin J2 inhibition of NF-κB-DNA binding through covalent modification of the p50 subunit

Cyclopentenone prostaglandins display anti-inflammatory activities and interfere with the signaling pathway that leads to activation of transcription factor NF-ΚB. Here we explore the possibility that the NF-ΚB subunit p50 may be a target for the cyclopentenone 15-deoxy-Δ 12,14-prostaglandin J2 (15d-PGJ2). This prostaglandin inhibited the DNA binding ability of recombinant p50 in a dose-dependent manner. The inhibition required the cyclopentenone moiety and could be prevented but not reverted by glutathione and dithiothreitol. Moreover, a p50 mutant with a C62S mutation was resistant to inhibition, indicating that the effect of 15d-PGJ2 was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by 15d-PGJ2 was demonstrated by reverse-phase high pressure liquid chromatography and mass spectrometry analysis that showed an increase in retention time and in the molecular mass of 15d-PGJ2-treated p50, respectively. The interaction between p50 and 15d-PGJ2 was relevant in intact cells. 15d-PGJ2 effectively inhibited cytokine-elicited NF-κB activity in HeLa without reducing IκBα degradation or nuclear translocation of NF-κB subunits. 15d-PGJ2 reduced NF-κB DNA binding activity in isolated nuclear extracts, suggesting a direct effect on NF-κB proteins. Finally, treatment of HeLa with biotinylated-15d-PGJ2 resulted in the formation of a 15d-PGJ 2-p50 adduct as demonstrated by neutravidin binding and immunoprecipitation. These results clearly show that p50 is a target for covalent modification by 15d-PGJ2 that results in inhibition of DNA binding.Peer Reviewe

Administration of a sodium hypochlorite enema:: a patient safety incident in nursing practice

Adverse events related to flaws in administering enemas are usually related to dosage errors which may cause unintended harm to the patient, and studies documenting incidents and contributing factors are scare. Therefore, the objective of this study was to analyze a complaint-case, registered at the National Commission of Medical Arbitration, due to the erroneous administration of a sodium hypochlorite enema. We describe the clinical case of a 32-year-old female was scheduled for laparoscopic surgery in a private hospital. Before this procedure, the treating physician indicated the administration of a soap-based evacuating enema. The enema was administered by a nurse, who mistakenly administered an enema containing sodium hypochlorite which caused periproctitis and toxic colitis. Training, followup, and prevention in the application of routine procedures in clinical areas by nursing staff are key to avoiding adverse events.Los eventos adversos relacionados con fallas en la administración de enemas generalmente están relacionados con errores de dosificación que pueden causar daño no intencional al paciente, y los estudios que documentan incidentes y factores que contribuyen son alarmantes. Por lo tanto, el objetivo de este estudio fue analizar un caso de queja, registrado en la Comisión Nacional de Arbitraje Médico, debido a la administración errónea de un enema de hipoclorito sódico. Describimos el caso clínico de una mujer de 32 años que fue programada para una cirugía laparoscópica en un hospital privado. Antes de este procedimiento, el médico tratante indicó la administración de un enema de evacuación a base de jabón. El enema fue administrado por una enfermera, quién por error administró un enema que contenía hipoclorito de sodio que causó periproctitis y colitis tóxica. La capacitación, el seguimiento y la prevención en la aplicación de procedimientos rutinarios en áreas clínicas por parte del personal de enfermería son fundamentales para evitar eventos adverso

LmABCB3, an atypical mitochondrial ABC transporter essential for Leishmania major virulence, acts in heme and cytosolic iron/sulfur clusters biogenesis

Abstract Background Mitochondria play essential biological functions including the synthesis and trafficking of porphyrins and iron/sulfur clusters (ISC), processes that in mammals involve the mitochondrial ATP-Binding Cassette (ABC) transporters ABCB6 and ABCB7, respectively. The mitochondrion of pathogenic protozoan parasites such as Leishmania is a promising goal for new therapeutic approaches. Leishmania infects human macrophages producing the neglected tropical disease known as leishmaniasis. Like most trypanosomatid parasites, Leishmania is auxotrophous for heme and must acquire porphyrins from the host. Methods LmABCB3, a new Leishmania major protein with significant sequence similarity to human ABCB6/ABCB7, was identified and characterized using bioinformatic tools. Fluorescent microscopy was used to determine its cellular localization, and its level of expression was modulated by molecular genetic techniques. Intracellular in vitro assays were used to demonstrate its role in amastigotes replication, and an in vivo mouse model was used to analyze its role in virulence. Functional characterization of LmABCB3 was carried out in Leishmania promastigotes and Saccharomyces cerevisiae. Structural analysis of LmABCB3 was performed using molecular modeling software. Results LmABCB3 is an atypical ABC half-transporter that has a unique N-terminal extension not found in any other known ABC protein. This extension is required to target LmABCB3 to the mitochondrion and includes a potential metal-binding domain. We have shown that LmABCB3 interacts with porphyrins and is required for the mitochondrial synthesis of heme from a host precursor. We also present data supporting a role for LmABCB3 in the biogenesis of cytosolic ISC, essential cofactors for cell viability in all three kingdoms of life. LmABCB3 fully complemented the severe growth defect shown in yeast lacking ATM1, an orthologue of human ABCB7 involved in exporting from the mitochondria a gluthatione-containing compound required for the generation of cytosolic ISC. Indeed, docking analyzes performed with a LmABCB3 structural model using trypanothione, the main thiol in this parasite, as a ligand showed how both, LmABCB3 and yeast ATM1, contain a similar thiol-binding pocket. Additionally, we show solid evidence suggesting that LmABCB3 is an essential gene as dominant negative inhibition of LmABCB3 is lethal for the parasite. Moreover, the abrogation of only one allele of the gene did not impede promastigote growth in axenic culture but prevented the replication of intracellular amastigotes and the virulence of the parasites in a mouse model of cutaneous leishmaniasis. Conclusions Altogether our results present the previously undescribed LmABCB3 as an unusual mitochondrial ABC transporter essential for Leishmania survival through its role in the generation of heme and cytosolic ISC. Hence, LmABCB3 could represent a novel target to combat leishmaniasis. Keywords Heme trafficking and metabolism Iron/sulfur clusters Trypanosomatid parasites Mitochondrial ABC transporter LeishmaniaThis work was supported by grants from the Spanish Ministerio de Economía y Competitividad SAF2011-28215 (JMPV) and Junta de Andalucia BIO1786 (JMPV) and by FEDER funds from the EU to JMPV. MMG was recipient of a FPI fellowship from the Spanish Ministerio de Economía y Competitividad; MCD was recipient of a FPU fellowship from the Spanish Ministerio de Educación, Cultura y Deporte; SMC was recipient of a JAE-DOC from the Spanish CSIC (Ministerio de Economía y Competitividad), cofounded by the Fondo Social Europeo.Peer reviewe