Fernando Roncal

Identification of amino acid residues crucial for chemokine receptor dimerization.

Chemokines coordinate leukocyte trafficking by promoting oligomerization and signaling by G protein–coupled receptors; however, it is not known which amino acid residues of the receptors participate in this process. Bioinformatic analysis predicted that Ile52 in transmembrane region-1 (TM1) and Val150 in TM4 of the chemokine receptor CCR5 are key residues in the interaction surface between CCR5 molecules. Mutation of these residues generated nonfunctional receptors that could not dimerize or trigger signaling. In vitro and in vivo studies in human cell lines and primary T cells showed that synthetic peptides containing these residues blocked responses induced by the CCR5 ligand CCL5. Fluorescence resonance energy transfer showed the presence of preformed, ligand-stabilized chemokine receptor oligomers. This is the first description of the residues involved in chemokine receptor dimerization, and indicates a potential target for the modification of chemokine responses.

Molecular cloning, functional characterization and mRNA expression analysis of the murine chemokine receptor CCR6 and its specific ligand MIP‐3α1

We have cloned the murine CCR6 receptor and its ligand, the β‐chemokine mMIP‐3α. Calcium mobilization assays performed with mCCR6 transfectants showed significant responses upon addition of mMIP‐3α. Murine MIP‐3α RNA is expressed in thymus, small intestine and colon, whereas mCCR6 RNA is expressed in spleen and lymph nodes. RT‐PCR analysis of FACS‐sorted lymphoid and antigen presenting cell subsets showed mCCR6 expression mainly in B cells, CD8− splenic dendritic cells and CD4+ T cells. The cloning and functional characterization of the mCCR6 and mMIP‐3α will allow the study of the role of these proteins in mouse models of inflammation and immunity.

Identification of novel cellular proteins that bind to the LC8 dynein light chain using a pepscan technique

Dynein is a minus end‐directed microtubule motor that serves multiple cellular functions. We have performed a fine mapping of the 8 kDa dynein light chain (LC8) binding sites throughout the development of a library of consecutive synthetic dodecapeptides covering the amino acid sequences of the various proteins known to interact with this dynein member according to the yeast two hybrid system. Two different consensus sequences were identified: GIQVD present in nNOS, in DNA cytosine methyl transferase and also in GKAP, where it is present twice in the protein sequence. The other LC8 binding motif is KSTQT, present in Bim, dynein heavy chain, Kid‐1, protein 4 and also in swallow. Interestingly, this KSTQT motif is also present in several viruses known to associate with microtubules during retrograde transport from the plasma membrane to the nucleus during viral infection.

STAG2 and Rad21 mammalian mitotic cohesins are implicated in meiosis

STAG/SA proteins are specific cohesin complex subunits that maintain sister chromatid cohesion in mitosis and meiosis. Two members of this family, STAG1/SA1 and STAG2/SA2, ‡ are classified as mitotic cohesins, as they are found in human somatic cells and in Xenopus laevis as components of the cohesinSA1 and cohesinSA2 complexes, in which the shared subunits are Rad21/SCC1, SMC1 and SMC3 proteins. A recently reported third family member, STAG3, is germinal cell‐specific and is a subunit of the meiotic cohesin complex. To date, the meiosis‐specific cohesin complex has been considered to be responsible for sister chromatid cohesion during meiosis. We studied replacement of the mitotic by the meiotic cohesin complex during mouse germinal cell maturation, and we show that mammalian STAG2 and Rad21 are also involved in several meiosis stages. Immunofluorescence results suggest that a cohesin complex containing Rad21 and STAG2 cooperates with a STAG3‐specific complex to maintain sister chromatid cohesion during the diplotene stage of meiosis.

Mammalian SGO2 appears at the inner centromere domain and redistributes depending on tension across centromeres during meiosis II and mitosis

Shugoshin (SGO) is a family of proteins that protect centromeric cohesin complexes from release during mitotic prophase and from degradation during meiosis I. Two mammalian SGO paralogues—SGO1 and SGO2—have been identified, but their distribution and function during mammalian meiosis have not been reported. Here, we analysed the expression of SGO2 during male mouse meiosis and mitosis. During meiosis I, SGO2 accumulates at centromeres during diplotene, and colocalizes differentially with the cohesin subunits RAD21 and REC8 at metaphase I centromeres. However, SGO2 and RAD21 change their relative distributions during telophase I when sister‐kinetochore association is lost. During meiosis II, SGO2 shows a striking tension‐dependent redistribution within centromeres throughout chromosome congression during prometaphase II, as it does during mitosis. We propose a model by which the redistribution of SGO2 would unmask cohesive centromere proteins, which would be then released or cleaved by separase, to trigger chromatid segregation to opposite poles.

Bcl-2 Targets Protein Phosphatase 1α to Bad

The diverse forms of protein phosphatase 1 (PP1) in vivo result from the association of the catalytic subunit with different regulatory subunits. We recently have described that PP1α is a Ras-activated Bad phosphatase that regulates IL-2 deprivation-induced apoptosis. With the yeast two-hybrid system, GST fusion proteins, indirect immunofluorescence, and coimmunoprecipitation, we found that Bcl-2 interacts with PP1α and Bad. In contrast, Bad did not interact with 14-3-3 protein. Bcl-2 depletion decreased phosphatase activity and association of PP1α to Bad. Bcl-2 contains the RIVAF motif, analogous to the well characterized R/KXV/IXF consensus motif shared by most PP1-interacting proteins. This sequence is involved in the binding of Bcl-2 to PP1α. Disruption of Bcl-2/PP1α association strongly decreased Bcl-2 and Bad-associated phosphatase activity and formation of the trimolecular complex. These results suggest that Bcl-2 targets PP1α to Bad.

Recognition of novel viral sequences that associate with the dynein light chain LC8 identified through a pepscan technique

Recent data from multiple laboratories indicate that upon infection, many different families of viruses hijack the dynein motor machinery and become transported in a retrograde manner towards the cell nucleus. In certain cases, one of the dynein light chains, LC8, is involved in this interaction. Using a library of overlapping dodecapeptides synthesized on a cellulose membrane (pepscan technique) we have analyzed the interaction of the dynein light chain LC8 with 17 polypeptides of viral origin. We demonstrate the strong binding of two herpesvirus polypeptides, the human adenovirus protease, vaccinia virus polymerase, human papillomavirus E4 protein, yam mosaic virus polyprotein, human respiratory syncytial virus attachment glycoprotein, human coxsackievirus capsid protein and the product of the AMV179 gene of an insect poxvirus to LC8. Our data corroborate the manipulation of the dynein macromolecular complex of the cell during viral infection and point towards the light chain LC8 as one of the most frequently used targets of virus manipulation.

Modulation at multiple anchor positions of the peptide specificity of HLA–B27 subtypes differentially associated with ankylosing spondylitis

OBJECTIVE To investigate the rules governing peptide binding to HLA-B*2705, and to B*2704 and B*2706, which are 2 subtypes differentially associated with ankylosing spondylitis. METHODS Poly-Ala analogs carrying the HLA-B27 motif Arg-2, and substitutions at anchor positions P1, P3, or Pomega, were used to determine a binding score for each residue at each position. Binding was assessed in a quantitative epitope stabilization assay, where the cell surface expression of HLA-B27 was measured by flow cytometry as a function of peptide concentration. RESULTS Peptide anchor residues contributed additively to B*2705 binding. About 15% of the natural B*2705 ligands used a deficient P3 or Pomega anchor, but never both, indicating that detrimental anchoring at one of these positions is always compensated by a good anchor at the other one. About 50% of the B*2705 ligands used suboptimal P1 residues. However, this was compensated with optimal P3 and/or Pomega anchoring. Peptides that were longer than decamers used good anchor residues at the 3 positions, suggesting more stringent binding requirements. B*2704 and B*2706 differed in their residue specificity at P1, P3, and Pomega. The rules derived for B*2705 also applied to the known ligands of these 2 subtypes. CONCLUSION The B*2705, B*2704, and B*2706 peptide repertoires are limited by the allowed residue combinations described in this study. The differential association of B*2704 and B*2706 with spondylarthropathy correlates with differences in their peptide specificity at multiple anchor positions. However, it is now possible to predict the peptide features that determine this differential binding to both subtypes.

An optimized amphiphilic cationic peptide as an efficient non-viral gene delivery vector

Due to their chemical definition and reduced size, the use of peptides as gene delivery systems is gaining interest as compared to the more common polymeric non‐viral vectors. To achieve gene transfer efficiencies that would make peptides a realistic alternative to existing methods, we have evaluated and attempted to concert those properties with a direct impact on the activity of the system. These considerations have led to the design, synthesis and characterization of a 23‐residue cationic peptide which we term RAWA.

Direct interaction between the reductase domain of endothelial nitric oxide synthase and the ryanodine receptor

We have performed the recombinant expression and purification of the reductase domain of endothelial nitric oxide synthase (eNOS) and used it as a bait in search for interacting proteins present in endothelial cells. Using mass spectrometry of the bound proteins run in a PAGE–SDS gel, we were able to identify the ryanodine receptor (RyR) as a novel eNOS‐binding partner. This interaction was confirmed through immunoprecipitation of both RyR and eNOS from endothelial cells and cardiac myocytes. Immunofluorescence data indicated that a subpopulation of eNOS associates with RyR in perinuclear regions of the cell, where eNOS might be responsible for the known nitrosylation of RyR.