Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25

Background: The ubiquitin-dependent protein degradation pathway is essential for the proteolysis of intracellular proteins and peptides. Deubiquitinating enzymes constitute a complex protein family involved in a multitude of cellular processes. The ubiquitin-specific proteases (UBP) are a group of enzymes whose predicted function is to reverse the ubiquitinating reaction by removing ubiquitin from a large variety of substrates. We have lately reported the characterization of human USP25, a specific-ubiquitin protease gene at 21q11.2, with a specific pattern of expression in murine fetal brains and adult testis. Results: Database homology searches at the DNA and protein levels and cDNA library screenings led to the identification of a new UBP member in the human genome, named USP28, at 11q23. This novel gene showed preferential expression in heart and muscle. Moreover, cDNA, expressed sequence tag and RT-PCR analyses provided evidence for alternatively spliced products and tissue-specific isoforms. Concerning function, USP25 overexpression in Down syndrome fetal brains was shown by real-time PCR. Conclusions: On the basis of the genomic and protein sequence as well as the functional data, USP28 and USP25 establish a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity. The overexpression of USP25 in Down syndrome fetal brains supports the gene-dosage effects suggested for other UBP members related to aneuploidy syndromes.

Expression of Glycogen Phosphorylase Isoforms in Cultured Muscle from Patients with McArdle´s Disease Carrying the p.R771PfsX33 PYGM Mutation

Mutations in the PYGM gene encoding skeletal muscle glycogen phosphorylase (GP) cause a metabolic disorder known as McArdle's disease. Previous studies in muscle biopsies and cultured muscle cells from McArdle patients have shown that PYGM mutations abolish GP activity in skeletal muscle, but that the enzyme activity reappears when muscle cells are in culture. The identification of the GP isoenzyme that accounts for this activity remains controversial.

L'autoetnografia com a memòria de trànsit

Projecte realitzat en el Departament de dibuix de la Facultat de Belles Arts, a la Universitat de Barcelona, durant el segon curs de Doctorat, en el seminari que a càrrec de Fernando Hernandez on treballem l’ Autoetnografia Narrativa. Aquesta és una investigació que no té un caràcter individual marcat tan sols per la meva veu, sinó que més aviat la configuren també tots els companys de Seminari, amb ells i a través d’ells he aconseguit aprendre molts factors que intento quedin reflectits en el texte que presento i que treballo des de l’Educació Artística i la Cultura Visual. Durant aquest any me n’he adonat de què l’aprenentatge es realitza i té molt a veure amb les vivències que cada un de nosaltres construeix en societat, en elles centro les següents pàgines que per mí són l’inici d’un recorregut cap a un nou coneixement. Intento en tot moment tenir present el grup amb el qual he tingut la possibilitat de treballar durant aquest any i des del qual he pogut treballar-me com a subjecte educadora. D’aquesta manera i des de mí presento la meva contribució a n’aquest construir aprenentatge en societat, a partir de la següent narració.

Bradykinin, but not muscarinic, inhibition of M-current in rat sympathetic ganglion neurons involves phospholipase C-β4

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (I-K(M)), which can be inhibited by activation of M-1 muscarinic receptors (M-1 mAChR) and bradykinin (BK) B-2 receptors. Inhibition by the M1 mAChR agonist oxotremorine methiodide (Oxo-M) is mediated, at least in part, by the pertussis toxin-insensitive G-protein G alpha (q) (Caulfield et al., 1994; Haley et al., 1998a), whereas BK inhibition involves G alpha (q) and/or G alpha (11) (Jones et al., 1995). G alpha (q) and G alpha (11) can stimulate phospholipase C-beta (PLC-beta), raising the possibility that PLC is involved in I-K(M) inhibition by Oxo-M and BK. RT-PCR and antibody staining confirmed the presence of PLC-beta1, - beta2, - beta3, and - beta4 in rat SCG. We have tested the role of two PLC isoforms (PLC-beta1 and PLC-beta4) using antisense-expression constructs. Antisense constructs, consisting of the cytomegalovirus promoter driving antisense cRNA corresponding to the 3'-untranslated regions of PLC-beta1 and PLC-beta4, were injected into the nucleus of dissociated SCG neurons. Injected cells showed reduced antibody staining for the relevant PLC-beta isoform when compared to uninjected cells 48 hr later. BK inhibition of I-K(M) was significantly reduced 48 hr after injection of the PLC-beta4, but not the PLC-beta1, antisense-encoding plasmid. Neither PLC-beta antisense altered M-1 mAChR inhibition by Oxo-M. These data support the conclusion of Cruzblanca et al. (1998) that BK, but not M-1 mAChR, inhibition of I-K(M) involves PLC and extends this finding by indicating that PLC-beta4 is involved.

Allele variants in functional MicroRNA target sites of the neurotrophin-3 receptor gene (NTRK3) as susceptibility factors for anxiety disorders

Genetic and functional data indicate that variation in the expression of the neurotrophin-3 receptor gene (NTRK3) may have an impact on neuronal plasticity, suggesting a role for NTRK3 in the pathophysiology of anxiety disorders. MicroRNA (miRNA) posttranscriptional gene regulators act by base-pairing to specific sequence sites, usually at the 3'UTR of the target mRNA. Variants at these sites might result in gene expression changes contributing to disease susceptibility. We investigated genetic variation in two different isoforms of NTRK3 as candidate susceptibility factors for anxiety by resequencing their 3'UTRs in patients with panic disorder (PD), obsessive-compulsive disorder (OCD), and in controls. We have found the C allele of rs28521337, located in a functional target site for miR-485-3p in the truncated isoform of NTRK3, to be significantly associated with the hoarding phenotype of OCD. We have also identified two new rare variants in the 3'UTR of NTRK3, ss102661458 and ss102661460, each present only in one chromosome of a patient with PD. The ss102661458 variant is located in a functional target site for miR-765, and the ss102661460 in functional target sites for two miRNAs, miR-509 and miR-128, the latter being a brain-enriched miRNA involved in neuronal differentiation and synaptic processing. Interestingly, these two variants significantly alter the miRNA-mediated regulation of NTRK3, resulting in recovery of gene expression. These data implicate miRNAs as key posttranscriptional regulators of NTRK3 and provide a framework for allele-specific miRNA regulation of NTRK3 in anxiety disorders.

Comparative analysis of cancer genes in the human and chimpanzee genomes

Background: Cancer is a major medical problem in modern societies. However, the incidence of this disease in non-human primates is very low. To study whether genetic differences between human and chimpanzee could contribute to their distinct cancer susceptibility, we have examined in the chimpanzee genome the orthologous genes of a set of 333 human cancer genes. Results: This analysis has revealed that all examined human cancer genes are present in chimpanzee, contain intact open reading frames and show a high degree of conservation between both species. However, detailed analysis of this set of genes has shown some differences in genes of special relevance for human cancer. Thus, the chimpanzee gene encoding p53 contains a Pro residue at codon 72, while this codon is polymorphic in humans and can code for Arg or Pro, generating isoforms with different ability to induce apoptosis or interact with p73. Moreover, sequencing of the BRCA1 gene has shown an 8 Kb deletion in the chimpanzee sequence that prematurely truncates the co-regulated NBR2 gene. Conclusion: These data suggest that small differences in cancer genes, as those found in tumor suppressor genes, might influence the differences in cancer susceptibility between human and chimpanzee. Nevertheless, further analysis will be required to determine the exact contribution of the genetic changes identified in this study to the different cancer incidence in non-human primates.

Differential regulation of RasGAPs in cancer

Ever since their discovery as cellular counterparts of viral oncogenes more than 25 years ago, much progress has been made in understanding the complex networks of signal transduction pathways activated by oncogenic Ras mutations in human cancers. The activity of Ras is regulated by nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), and much emphasis has been put into the biochemical and structural analysis of the Ras/GAP complex. The mechanisms by which GAPs catalyze Ras-GTP hydrolysis have been clarified and revealed that oncogenic Ras mutations confer resistance to GAPs and remain constitutively active. However, it is yet unclear how cells coordinate the large and divergent GAP protein family to promote Ras inactivation and ensure a certain biological response. Different domain arrangements in GAPs to create differential protein-protein and protein-lipid interactions are probably key factors determining the inactivation of the 3 Ras isoforms H-, K-, and N-Ras and their effector pathways. In recent years, in vitro as well as cell- and animal-based studies examining GAP activity, localization, interaction partners, and expression profiles have provided further insights into Ras inactivation and revealed characteristics of several GAPs to exert specific and distinct functions. This review aims to summarize knowledge on the cell biology of RasGAP proteins that potentially contributes to differential regulation of spatiotemporal Ras signaling.

Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions

Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB¿/¿ mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA¿/¿ MEFs, but not FlnB¿/¿ MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

Shaping mechanisms of metal specificity in a family of metazoan Metallothioneins: evolutionary differentiation of Mollusc MTs.

Background: The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu+ or Cd2+. They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order t o elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion. Results: HpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd2+, Zn2+ or Cu2+ and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd6-HpCdMT and Cu12-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions. Conclusion: The Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

Relacions especials entre germans

Aquest projecte tracta sobre la relació entre germans quan un d’ells és discapacitat. Podríem dir que la relació que s’estableix entre ells és “especial”? Aquesta ha estat la pregunta que m’ha portat a estudiar els diferents factors que influeixen en una relació d’aquest tipus. Per tal de poder comprovar el que diuen els estudis fets fins al moment, he analitzat dos casos, tots dos amb contextos molt diferenciats, i n’he tret unes conclusions amb la finalitat de respondre a la pregunta inicial. Quan hi ha un fill amb discapacitat a la família, l’atenció que aquest requereix és tan gran que sovint els germans tenen la sensació de sentir-se inferiors, culpables, etc. A tots aquests sentiments cal prestar-hi certa atenció de manera que la situació no els sigui negativa. Els tallers de germans, organitzats per associacions i escoles especialitzades amb el tema, contribueixen a treballar amb tots aquests germans, els sentiments i les emocions i així enfortir el vincle entre germans.

Do protein–protein interaction databases identify moonlighting proteins?

One of the most striking results of the human (and mammalian) genomes is the low number of protein-coding genes. To-date, the main molecular mechanism to increase the number of different protein isoforms and functions is alternative splicing. However, a less-known way to increase the number of protein functions is the existence of multifunctional, multitask, or ‘‘moonlighting’’, proteins. By and large, moonlighting proteins are experimentally disclosed by serendipity. Proteomics is becoming one of the very active areas of biomedical research, which permits researchers to identify previously unseen connections among proteins and pathways. In principle, protein–protein interaction (PPI) databases should contain information on moonlighting proteins and could provide suggestions to further analysis in order to prove the multifunctionality. As far as we know, nobody has verified whether PPI databases actually disclose moonlighting proteins. In the present work we check whether well-established moonlighting proteins present in PPI databases connect with their known partners and, therefore, a careful inspection of these databases could help to suggest their different functions. The results of our research suggest that PPI databases could be a valuable tool to suggest multifunctionality.

The redox state of cytochrome C modulates resistance to methotrexate in human MCF7 breast cancer cells

Background: Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells. Results: Cell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexateinduced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells. Conclusions: We conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.

Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions

Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB¿/¿ mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA¿/¿ MEFs, but not FlnB¿/¿ MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

The antilipolytic effect of insulin and epidermal growth factor in rat adipocytes are mediated by different mechanisms

Epidermal growth factor (EGF) and insulin induced similar effects in isolated rat adipocytes. To determine whether EGF and insulin produced similar effects through the same mechanisms, we focused on lipolysis. Insulin inhibited the lipolysis stimulated by isoproterenol, glucagon (either alone or in combination with adenosine deaminase), adenosine deaminase itself, or forskolin. In contrast, EGF did not inhibit the lipolysis stimulated by forskolin or by hormones when the cells were also incubated with adenosine deaminase. The effect of insulin, but not that of EGF, on isoproterenol-stimulated lipolysis disappeared when adipocytes were incubated with 1 microM wortmannin. These results indicate that EGF and insulin affected lipolysis through different mechanisms. We observed that EGF, but not insulin, increased cytosolic Ca2+. The effect of EGF, but not that of insulin, disappeared when the cells were incubated in a Ca2+-free medium. We suggest that EGF, but not insulin, mediate its antilipolytic effect through a Ca2+-dependent mechanism which, however, do not involve Ca2+-activated protein kinase C isoforms. This is based on the following: 1) phorbol 12-myristate 13-acetate affected lipolysis in an opposite way to that of EGF; and 2) the protein kinase C inhibitor bisindolylmaleimide GF 109203X did not affect the antilipolytic action of EGF. Our results indicate that the antilipolytic effect of EGF resembles more that of vasopressin than that of insulin.

CERKL Knockdown Causes Retinal Degeneration in Zebrafish

The human CERKL gene is responsible for common and severe forms of retinal dystrophies. Despite intense in vitro studies at the molecular and cellular level and in vivo analyses of the retina of murine knockout models, CERKL function remains unknown. In this study, we aimed to approach the developmental and functional features of cerkl in Danio rerio within an Evo-Devo framework. We show that gene expression increases from early developmental stages until the formation of the retina in the optic cup. Unlike the high mRNA-CERKL isoform multiplicity shown in mammals, the moderate transcriptional complexity in fish facilitates phenotypic studies derived from gene silencing. Moreover, of relevance to pathogenicity, teleost CERKL shares the two main human protein isoforms. Morpholino injection has been used to generate a cerkl knockdown zebrafish model. The morphant phenotype results in abnormal eye development with lamination defects, failure to develop photoreceptor outer segments, increased apoptosis of retinal cells and small eyes. Our data support that zebrafish Cerkl does not interfere with proliferation and neural differentiation during early developmental stages but is relevant for survival and protection of the retinal tissue. Overall, we propose that this zebrafish model is a powerful tool to unveil CERKL contribution to human retinal degeneration