Compte M, Cuesta AM, Sánchez-Martín D, Camino VA, Vicario JL, Sanz L, Alvarez-Vallina L.

" Tumor Immunotherapy using Gene-Modified Human Mesenchymal Stem Cells Loaded into Synthetic Extracellular Matrix Scaffolds."

Más Información en http://stemcells.alphamedpress.org/cgi/r...

Sanz L, Cuesta AM, Salas C, Corbacho C, Bellas C, Alvarez-Vallina L.

"Differential transplantability of human endothelial cells in colorectal cancer and renal cell carcinoma primary xenografts."

Más Información en http://www.nature.com/labinvest/journal/...

Larsen M, Jensen KB, Christensen PA, Suarez E, Paris D, Sanz L, Ravn P, Sauce D, Saas P, Goletz S, Alvarez-Vallina L, Kristensen P.

"Functionally fused antibodies-A novel adjuvant fusion system. J Immunol Methods. 2008 Dec 31;339(2):220-7."

Más Información en http://www.sciencedirect.com/science?_ob...

Cuesta AM, Sánchez-Martín D, Sanz L, Alvarez-Vallina L.

"Production of multivalent protein binders using a self-trimerization collagen-like peptide scaffold"

Más Información en http://www.ncbi.nlm.nih.gov/pubmed/18827...

Sanz L, Santos-Valle P, Alonso-Camino V, Salas C, Serrano A, Vicario JL,Cuesta AM, Compte M, Sánchez-Martín D, Alvarez-Vallina L.

"Long-term in vivo imaging of human angiogenesis: critical role of bone marrow-derived mesenchymal stem cells for the generation of durable blood vessels."

Más Información en http://www.sciencedirect.com/science?_ob...

Martínez-Torrecuadrada JL, Cheung LH, López-Serra P, Barderas R, Cañamero M, Ferreiro S, Rosenblum MG, Casal JI.

"Antitumor activity of fibroblast growth factor receptor 3-specific immunotoxins in a xenograft mouse model of bladder carcinoma is mediated by apoptosis. Mol Cancer Ther. 2008 Apr;7(4):862-73. "

Barderas R, Shochat S, Timmerman P, Hollestelle MJ, Martínez-Torrecuadrada JL, Höppener JW, Altschuh D, Meloen R, Casal JI.

" Designing antibodies for the inhibition of gastrin activity in tumoral cell lines. Int J Cancer. 2008 May 15;122(10):2351-9. "

Martínez-Martínez S, Genescà L, Rodríguez A, Raya A, Salichs E, Were F, López-Maderuelo MD, Redondo JM, de la Luna S.

"The RCAN carboxyl-end mediates calcineurin docking-dependent inhibition via a site that dictates binding to substrates and regulators. Proc Natl Acad Sci U S A. 2009 Apr 14;106(15):6117-22. Epub 2009 Mar 30."

Specificity of signaling kinases and phosphatases toward their targets is usually mediated by docking interactions with substrates and regulatory proteins. Here, we characterize the motifs involved in the physical and functional interaction of the phosphatase calcineurin with one group of modulators, the RCAN protein family. Mutation of key residues within the hydrophobic docking-cleft of the calcineurin catalytic domain impairs binding to all human RCAN proteins and to the calcineurin interacting proteins Cabin1 and AKAP79. A valine-rich region within the RCAN carboxyl-region is essential for binding to the docking site in calcineurin. Although a peptide containing this sequence compromises NFAT signaling in living cells, it does not inhibit calcineurin catalytic activity directly. Instead, calcineurin catalytic activity is inhibited by a motif at the extreme C-terminal region of RCAN, which acts in cis with the docking motif. Our results therefore indicate that the inhibitory action of RCAN on calcineurin-NFAT signaling results not only from the inhibition of phosphatase activity, but also from competition between NFAT and RCAN for binding to the same docking site in calcineurin. Thus competition by substrates and modulators for a common docking site appears to be an essential mechanism in the regulation of Ca2+-calcineurin signaling.

Más Información en http://www.pnas.org/content/106/15/6117....

Rodríguez A, Roy J, Martínez-Martinez S, López-Maderuelo MD, Nińo-Moreno P, Ortí L, Pantoja-Uceda D, Pineda-Lucena A, Cyert MS, Redondo JM.

"A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants. Mol Cell. 2009 Mar 12; 33(5)"

The phosphatase calcineurin, target of the immunosuppressants cyclosporin A and FK506, dephosphorylates NFAT transcription factors to promote immune activation and development of the vascular and nervous systems. NFAT interacts with calcineurin through distinct binding motifs: the PxIXIT and LxVP sites. While many calcineurin substrates contain PxIxIT motifs, the generality of LxVP-mediated interactions is unclear. We define critical residues in the LxVP motif, and demonstrate its binding to a hydrophobic pocket at the interface of the two calcineurin subunits. Mutations in this region disrupt binding of mammalian calcineurin to NFATc1, and interaction of yeast calcineurin with substrates including Rcn1, which contains an LxVP motif. These mutations also interfere with calcineurin immunosuppressant binding, and an LxVP-based peptide competes with immunosuppressant-immunophilin complexes for binding to calcineurin. These studies suggest that LxVP-type sites are a common feature of calcineurin substrates and that immunosuppressant-immunophilin complexes inhibit calcineurin by interfering with this mode of substrate recognition.

Más Información en http://www.cell.com/molecular-cell/retri...

Bodelón G., Marín E., and Fernández L.A.

"Role of periplasmic chaperones and BamA (YaeT/Omp85) for folding and secretion of Intimin from enteropathogenic E. coli strains. Journal of Bacteriology, 2009, 191:5169-5179."

Intimin is a bacterial adhesin located on the surface of enteropathogenic Escherichia coli and other related bacteria that is believed to self-translocate across the outer membrane (OM), and therefore it has been regarded as a member of the type V secretion system (T5SS), which includes classical autotransporters (ATs). However, intimin has few structural similarities to classical ATs and an opposite topology with an OM-embedded N region and a secreted C region. Since the actual secretion mechanism of intimin is unknown, we investigated intimin biogenesis by analyzing its requirement of periplasmic chaperones (DsbA, SurA, Skp, and DegP) and of OM protein BamA (YaeT/Omp85) for folding, OM insertion, and translocation. Using full-length and truncated intimin polypeptides, we demonstrate that DsbA catalyzes the formation of a disulfide bond in the D3 lectin-like domain of intimin in the periplasm, indicating that this secreted C-terminal domain is at least partially folded prior to its translocation across the OM. We also show that SurA chaperone plays the major role for periplasmic transport and folding of the N region of intimin, whereas the parallel pathway made by Skp and DegP chaperones plays a secondary role in this process. Further, we demonstrate that BamA is essential for the insertion of the N region of intimin in the OM and that the protease activity of DegP participates in the degradation of misfolded intimin. The significance of these findings for a BamA-dependent secretion mechanism of intimin is discussed in the context of T5SSs.

Más Información en http://jb.asm.org/cgi/content/full/191/1...

Munera D., Palomino C., and Fernández L.A.

"Specific residues in the N-terminal domain of FimH stimulate type 1 fimbriae assembly in Escherichia coli following the initial binding of the adhesin to FimD usher. Molecular Microbiology, 2008, 69: 911-925"

Type 1 fimbriae are assembled by the chaperone-usher pathway where periplasmic protein complexes formed between fimbrial subunits and the FimC chaperone are recruited by the outer membrane protein FimD (the usher) for their ordered polymerization and export. FimH adhesin initiates and stimulates type 1 fimbriae polymerization by interacting with FimD. Previously we showed that the N-terminal lectin domain of FimH (N-FimH) is necessary for binding of the adhesin to FimD. In this work, we have selected mutants in N-FimH that reduce the levels of adhesin and type 1 fimbriae displayed in Escherichia coli without altering the levels of FimH in the periplasm. The selected mutations are mostly concentrated in residues G15, N46 and D47. In contrast to other mutations isolated that simply affect binding of FimH to FimD (e.g. C3Y), these variants associate to FimD and alter its susceptibility to trypsin digestion similarly to wild-type FimH. Importantly, their mutant phenotype is rescued when FimD is activated in vivo by the coexpression of wild-type FimH. Altogether, these data indicate that residues G15, N46 and D47 play an important role following initial binding of FimH to FimD for efficient type 1 fimbriae polymerization by this outer membrane usher.

Más Información en PMID: 18627459...

Demuth A, Aharonowitz Y, Bachmann TT, Blum-Oehler G, Buchrieser C, Covacci A, Dobrindt U, Emödy L, van der Ende A, Ewbank J, Fernández LA, et al.

"Pathogenomics: an updated European Research Agenda. Infection, Genetics and Evolution, 2008, 8: 386-393."

The emerging genomic technologies and bioinformatics provide novel opportunities for studying life-threatening human pathogens and to develop new applications for the improvement of human and animal health and the prevention, treatment, and diagnosis of infections. Based on the ecology and population biology of pathogens and related organisms and their connection to epidemiology, more accurate typing technologies and approaches will lead to better means of disease control. The analysis of the genome plasticity and gene pools of pathogenic bacteria including antigenic diversity and antigenic variation results in more effective vaccines and vaccine implementation programs. The study of newly identified and uncultivated microorganisms enables the identification of new threats. The scrutiny of the metabolism of the pathogen in the host allows the identification of new targets for anti-infectives and therapeutic approaches. The development of modulators of host responses and mediators of host damage will be facilitated by the research on interactions of microbes and hosts, including mechanisms of host damage, acute and chronic relationships as well as commensalisms. The study of multiple pathogenic and non-pathogenic microbes interacting in the host will improve the management of multiple infections and will allow probiotic and prebiotic interventions. Needless to iterate, the application of the results of improved prevention and treatment of infections into clinical tests will have a positive impact on the management of human and animal disease. The Pathogenomics Research Agenda draws on discussions with experts of the Network of Excellence "EuroPathoGenomics" at the management board meeting of the project held during 18-21 April 2007, in the Villa Vigoni, Menaggio, Italy. Based on a proposed European Research Agenda in the field of pathogenomics by the ERA-NET PathoGenoMics the meeting's participants updated the established list of topics as the research agenda for the future.

Más Información en PMID: 18321793...

Luque-García JL, Martínez-Torrecuadrada JL, Epifano C, Cañamero M, Babel I, Casal JI.

"Differential protein expression on the cell surface of colorectal cancer cells associated to tumor metastasis.Proteomics. 2010 Mar;10(5):940-52."

Progression to metastasis is the critical point in colorectal cancer (CRC) survival. However, the proteome associated to CRC metastasis is very poorly understood at the moment. In this study, we used stable isotope labeling by amino acids in cell culture to compare two CRC cell lines: KM12C and KM12SM, representing poorly versus highly metastatic potential, to find and quantify the differences in protein expression, mostly at the cell surface level. After biotinylation followed by affinity purification, membrane proteins were separated by SDS-PAGE and analyzed using nanoflow LC-ESI-LTQ. A total of 291 membrane and membrane-associated proteins were identified with a p value<0.01, from which 60 proteins were found to be differentially expressed by more than 1.5-fold. We identified a number of cell signaling, CDs, integrins and other cell adhesion molecules (cadherin 17, junction plakoglobin (JUP)) among the most deregulated proteins. They were validated by Western blot, confocal microscopy and flow cytometry analysis. Immunohistochemical analysis of paired tumoral samples confirmed that these differentially expressed proteins were also altered in human tumoral tissues. A good correlation with a major abundance in late tumor stages was observed for JUP and 17-beta-hydroxysteroid dehydrogenase type 8 (HSD17B8). Moreover, the combined increase in JUP, occludin and F11 receptor expression together with cadherin 17 expression could suggest a reversion to a more epithelial phenotype in highly metastatic cells. Relevant changes were observed also at the metabolic level in the pentose phosphate pathway and several amino acid transporters. In summary, the identified proteins provide us with a better understanding of the events involved in liver colonization and CRC metastasis.

Más Información en PMID: 20049862 [PubMed - indexed for MED...

Babel I, Barderas R, Díaz-Uriarte R, Martínez-Torrecuadrada JL, Sánchez-Carbayo M, Casal JI.

"Identification of tumor-associated autoantigens for the diagnosis of colorectal cancer in serum using high density protein microarrays.Mol Cell Proteomics. 2009 Oct;8(10):2382-95. Epub 2009 Jul 28."

There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers.

Más Información en PMID: 19638618 [PubMed - indexed for MED...

Casado-Vela J, Martínez-Torrecuadrada JL, Casal JI.

"Differential phosphorylation patterns between the Cyclin-A2/CDK2 complex and their monomers.Protein Expr Purif. 2009 Jul;66(1):15-21. Epub 2009 Feb 20."

The Cyclin-A2/CDK2 is a protein heterodimer with kinase activity that plays a key role in centrosome duplication and meiotic cell division. To check the suitability of the insect cells for the production and characterization of phosphorylated mammalian proteins, both proteins were expressed individually or as a complex using the baculovirus expression system. In this study, we used a linear ion trap mass spectrometer to identify the phosphorylated residues in mouse Cyclin-A2 and CDK2 recombinant proteins, after individual expression and after formation of the heterodimer complex, both in baculovirus. By using multi-protease digestion and data dependent neutral loss analysis, we identified a differential phosphorylation pattern before and after formation of the protein complex. The analysis of the monomeric proteins showed that Cyclin-A2 was phosphorylated on two Ser residues (Ser(14) and Ser(421)) and CDK2 on a single residue (Thr(160)). After heterodimer formation, Cyclin-A2 was phosphorylated only on Ser(14), whereas CDK2 contained two phosphorylated residues (Thr(39) and Thr(160)). These findings may clarify relevant aspects of the functionality of the Cyclin-A2/CDK2 protein complex and its role in cell cycle and support the efficiency of the baculovirus system for the production of phosphorylated proteins mimicking the

Más Información en PMID: 19233286 [PubMed - indexed for MED...

Kanellis G, Roncador G, Arribas A, Mollejo M, Montes-Moreno S, Maestre L, Campos-Martin Y, Ríos Gonzalez JL, Martinez-Torrecuadrada JL, Sanchez-Verde L, Pajares R, Cigudosa JC, Martin MC, Piris MA.

"Identification of MNDA as a new marker for nodal marginal zone lymphoma.Leukemia. 2009 Oct;23(10):1847-57. Epub 2009 May 28."

Clinical and biological studies on nodal marginal zone lymphoma (NMZL) are hampered by the lack of specific diagnostic markers and the low reproducibility of this diagnosis. A comparative expression-profiling study has shown a set of markers to be differentially expressed in NMZL compared with follicular lymphoma (FL), including myeloid cell nuclear differentiation antigen (MNDA), a nuclear protein expressed by myeloid cells and a subset of B-cells. The aim of this study was to characterize the expression of MNDA in normal and reactive human tissue, and in a large series of non-Hodgkin's B-cell lymphomas, with particular emphasis on NMZL and FL. Our results showed that MNDA is expressed in normal tissue by a subset of the marginal zone B cells. They also showed MNDA expression in subgroups of chronic lymphocytic leukemia, mantle-cell lymphoma, and diffuse large B-cell lymphoma, but MNDA was especially expressed by lymphomas derived from the marginal zone, such as mucosa-associated lymphoid-tissue lymphoma, splenic marginal-zone lymphoma and NMZL. MNDA expression was rarely observed in FL, a characteristic that is of potential value in distinguishing between NMZL and FL. MNDA expression is thus a useful tool for the recognition of NMZL.

Más Información en PMID: 19474799 [PubMed - indexed for MED...