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8 páginas, 2 figuras, 1 tabla., Ribosome biogenesis is an emerging therapeutic target. It has been proposed that cancer cells are addicted to ribosome production which is therefore considered a druggable pathway in cancer therapy. Cancer cells have been shown to be more sensitive to inhibition of the ribosome production than healthy cells. Initial attempts of inhibiting ribosome biogenesis have been focused on the inhibition of transcription by targeting RNA Pol I. Despite being a promising field of research, several limitations have been identified during the development of RNA Pol I inhibitors, like the lack of specificity or acquired resistance. Ribosome biogenesis is a multistep process and additional points of intervention, downstream the very initial stage, could be investigated. Eukaryotic ribosome maturation involves the participation of more than 200 essential assembly factors that will not be part of the final mature ribosome and frequently require protein-protein interactions to exert their biological action. Using mutagenesis, we have previously shown that alteration of the complex interface between assembly factors impairs proper ribosome maturation in yeast. As a first step toward the developing of ribosome biogenesis inhibitory tools, we have used our previously solved crystal structure of the Chaetomium thermophilum complex between the assembly factors Erb1 and Ytm1 to perform a structure-guided selection of interference peptides. The peptides have been assayed in vitro for their ability to bind their cellular partner using biophysical techniques., Funding was obtained from the Spanish Ministry of Economy, Industry and Competitiveness SAF2017-89901-R and Generalitat Valenciana PROMETEO/2018/055. Open access publication fees have been partially funded by URICI from Consejo Superior de Investigaciones Científicas (CSIC). Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311

11 páginas, 5 figuras, 1 tabla. Contiene 5 figuras en material suplementario, en este fichero., Human Drg1, a guanine nucleotide binding protein conserved in archaea and eukaryotes, is regulated by Lerepo4. Together they form a complex which interacts with translating ribosomes. Here we have purified and characterized the GTPase activity of Drg1 and three variants, a shortened mutant depleted of the TGS domain, a phosphomimicking mutant and a construct with the two combined mutations. Our data reveal that potassium strongly stimulates the GTPase activity, without changing the monomeric status of Drg1 and that this activity is notably reduced in the mutants. The nature of Lerepo4 association has also been investigated. Dissecting the role of the different domains revealed that Dfrp domain is the sole responsible for the Drg1 increase in thermal stability and the four fold stimulation over its catalytic activity. Lerepo4 action leaves Drg1 affinity for nucleotides unaffected, feasibly favoring a switch I reorientation, mainly via the TGS domain. Drg1 displayed a high temperature optimum of activity at 42°C, suggesting the ability of being active under possible heat stress conditions., This work was supported by Ministerio de Ciencia e Innovación (SAF2009-10667, SAF2012-31405) and Generalitat Valenciana (Prometeo/2012/061), Spain.

17 páginas, 8 figuras, 1 tabla. Este fichero contiene 5 figuras y 3 tablas de material suplementario, The CD2AP (CD2-associated protein) and CIN85 (Cbl-interacting protein of 85 kDa) adaptor proteins each employ three Src homology 3 (SH3) domains to cluster protein partners and ensure efficient signal transduction and down-regulation of tyrosine kinase receptors. Using NMR, isothermal titration calorimetry and small-angle X-ray scattering methods, we have characterized several binding modes of the N-terminal SH3 domain (SH3A) of CD2AP and CIN85 with two natural atypical proline-rich regions in CD2 (cluster of differentiation 2) and Cbl-b (Casitas B-lineage lymphoma), and compared these data with previous studies and published crystal structures. Our experiments show that the CD2AP-SH3A domain forms a type II dimer with CD2 and both type I and type II dimeric complexes with Cbl-b. Like CD2AP, the CIN85-SH3A domain forms a type II complex with CD2, but a trimeric complex with Cbl-b, whereby the type I and II interactions take place at the same time. Together, these results explain how multiple interactions among similar SH3 domains and ligands produce a high degree of diversity in tyrosine kinase, cell adhesion or T-cell signaling pathways., This research is funded by grant FQM02838 from the Junta de Andalucía. M.A.C. is supported by an FPU grant from the Spanish Ministry of Education (MEC). A.G-P is an FWO post-doc fellow. J.L.OR is a FEBS post-doc fellow. Work in NvN laboratory is supported by the VIB and the Flanders Hercules Foundation. Work in JB laboratory was supported by Ministerio de Ciencia e Innovación (SAF2009-10667, SAF2012-31405) and Generalitat Valenciana ACOMP/2012/024.

9 Pages, 4 Figures, 1 Table and 2 Supplemental Figures, Sorting nexin 6 (SNX6), a predominantly cytoplasmic protein involved in intracellular trafficking of membrane receptors, was identified as a TGF-β family interactor. However, apart from being a component of the Retromer, little is known about SNX6 cellular functions. Pim-1-dependent SNX6 nuclear translocation has been reported suggesting a putative nuclear role for SNX6. Here, we describe a previously non-reported association of SNX6 with breast cancer metastasis suppressor 1 (BRMS1) protein detected by a yeast two-hybrid screening. The interaction can be reconstituted in vitro and further FRET analysis confirmed the novel interaction. Additionally, we identified their coiled-coil domains as the minimal binding motives required for interaction. Since BRMS1 has been shown to repress transcription, we sought the ability of SNX6 to interfere with this nuclear activity. Using a standard gene reporter assay, we observed that SNX6 increases BRMS1-dependent transcriptional repression. Moreover, over-expression of SNX6 was capable of diminishing trans-activation in a dose-dependent manner., Grant sponsor: MCyT; Grant number: SAF2006-10269. Grant sponsor: MICINN; Grant number: SAF2008-04048-E, SAF2009-10667. Grant sponsor: CSIC; Grant number: 200820I020. Grant sponsor: Fundación-Mutua-Madrileña. Grant sponsor: Conselleria de Sanitat, Generalitat valenciana; Grant number: AP001/10.

8 páginas, 5 figuras, 2 tablas, The ubiquitin ligases c-Cbl and Cbl-b play a crucial role in receptor downregulation by mediating multiple monoubiquitination of receptors and promoting their sorting for lysosomal degradation. Their function is modulated through interactions with regulatory proteins including CIN85 and PIX, which recognize a proline-arginine motif in Cbl and thus promote or inhibit receptor endocytosis. We report the structures of SH3 domains of CIN85 and beta-PIX in complex with a proline-arginine peptide from Cbl-b. Both structures reveal a heterotrimeric complex containing two SH3 domains held together by a single peptide. Trimerization also occurs in solution and is facilitated by the pseudo-symmetrical peptide sequence. Moreover, ternary complexes of CIN85 and Cbl are formed in vivo and are important for the ability of Cbl to promote epidermal growth factor receptor (EGFR) downregulation. These results provide molecular explanations for a novel mechanism by which Cbl controls receptor downregulation., Research on CIN85 was supported by grant SAF2003-03860 of the Ministerio de Ciencia y Tecnología, Spain and N.C. received a fellowship from the Ramón Areces Foundation. D.J. and K.R. are funded by the Medical Research Council, UK. Work by Y.L.D, D.H. and I.D. are supported by grants from Deutsche Forschungsgemeinschaft, Germany.