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SH2 domains are a class of protein–protein interaction modules with the function to recognize and bind sequences characterized by the presence of a phosphorylated tyrosine. SHP2 is a protein phosphatase involved in the Ras‐ERK1/2 signaling pathway that possess two SH2 domains, namely, N‐SH2 and C‐SH2, that mediate the interaction of SHP2 with various partners and determine the regulation of its catalytic activity. One of the main interactors of the SH2 domains of SHP2 is Gab2, a scaffolding protein with critical role in determining cell differentiation. Despite their key biological role and the importance of a correct native fold to ensure it, the mechanism of binding of SH2 domains with their ligands and the determinants of their stability have been poorly characterized. In this article, we present a comprehensive kinetic study of the folding of the C‐SH2 domain and the binding mechanism with a peptide mimicking a region of Gab2. Our data, obtained at different pH and ionic strength conditions and supported by site‐directed mutagenesis, highlight the role of electrostatic interactions in the early events of recognition. Interestingly, our results suggest a key role of a highly conserved histidine residue among SH2 family in the interaction with negative charges carried by the phosphotyrosine of Gab2. Moreover, the analysis of the equilibrium and kinetic folding data of C‐SH2 describes a complex mechanism implying a change in rate‐limiting step at high denaturant concentrations. Our data are discussed under the light of previous works on N‐SH2 domain of SHP2 and other SH2 domains.

Yeast genetics made it possible to derive the first fundamental insights into prion composition, conformation, and propagation. Fast-forward 30 years and the same model organism is now proving an extremely powerful tool to comprehensively explore the impact of mutations in prion sequences on their function, toxicity, and physical properties. Here, we provide an overview of novel multiplexed strategies where deep mutagenesis is combined to a range of tailored selection assays in yeast, which are particularly amenable for investigating prions and prion-like sequences. By mimicking evolution in a flask, these multiplexed approaches are revealing mechanistic insights on the consequences of prion self-assembly, while also reporting on the structure prion sequences adopt in vivo.

Albeit SH2 domains are abundant protein-protein interaction modules with fundamental roles in the regulation of several physiological and molecular pathways in the cell, the available information about the determinants of their thermodynamic stability and folding properties are still very limited. In this work, we provide a quantitative characterization of the folding pathway of the C-terminal SH2 domain of SHP2, conducted through a combination of site-directed mutagenesis and kinetic (un)folding experiments (Φ-value analysis). The energetic profile of the folding reaction of the C-SH2 domain is described by a three-state mechanism characterized by the presence of two transition states and a high-energy intermediate. The production of 29 site-directed variants allowed us to calculate the degree of native-like interactions occurring in the early and late events of the folding reaction. Data analysis highlights the presence of a hydrophobic folding nucleus surrounded by a lower degree of structure in the early events of folding, further consolidated as the reaction proceeds towards the native state. Interestingly, residues physically located in the functional region of the domain reported unusual Φ-values, a hallmark of the presence of transient misfolding. We compared our results with previous ones obtained for the N-terminal SH2 domain of SHP2. Notably, a conserved complex folding mechanism implying the presence of a folding intermediate arise from comparison, and the relative stability of such intermediate appears to be highly sequence dependent. Data are discussed under the light of previous works on SH2 domains.

Background: CRISPR base editors are powerful tools for large-scale mutagenesis studies. This kind of approach can elucidate the mechanism of action of compounds, a key process in drug discovery. Here, we explore the utility of base editors in an early drug discovery context, and we focus on G-protein coupled receptors.Results: We set up a pooled mutagenesis screening framework based on a modified version of the CRISPR-X base editor system. We determine optimized experimental conditions for mutagenesis where sgRNAs are delivered by cell transfection or viral infection over extended time periods (>14 days), resulting in high mutagenesis produced in a short region located at -4/+8 nucleotides with respect to the sgRNA match. We thus target the Beta 2 Adrenergic Receptor (B2AR) and employ a 6xCRE-mCherry reporter system to monitor its activity. The results of our screening indicate that residue 184 of B2AR is crucial for its activation. Based on our experience, we then outline the crucial points to consider when designing and performing CRISPR-based pooled mutagenesis screening, including the typical technical hurdles encountered when studying compound pharmacology. Conclusions: The base editing technology has a great potential to help deciphering the mechanism of action of drugs, and it is a very powerful tool in drug discovery. Here we show an application of pooled mutagenesis screening to study B2AR, and we provide a roadmap for successfully applying this approach to other target proteins.

Src Homology 2 (SH2) domains are a class of protein domains that present a conserved three-dimensional structure and possess a crucial role in mediating protein-protein interactions. Despite their importance and abundance in the proteome, knowledge about the folding properties of SH2 domain is limited. Here we present an extensive mutational analysis (Φ value analysis) of the folding pathway of the N-SH2 domain of the Src homology region 2 domain-containing phosphatase-2 (SHP2) protein, a 104 residues domain that presents the classical SH2 domain fold (two α-helices flanking a central β-sheet composed of 3-5 antiparallel β-strands), with a fundamental role in mediating the interaction of SHP2 with its substrates and triggering key metabolic pathways in the cell. By analysing folding kinetic data we demonstrated that the folding pathway of N-SH2 presents an obligatory on-pathway intermediate that accumulates during the folding reaction. The production of 24 conservative site-directed variants allowed us to perform a Φ value analysis, by which we could fully characterize the intermediate and the transition state native-like interactions, providing a detailed quantitative analysis of the folding pathway of N-SH2. Results highlight the presence of a hydrophobic nucleus that stabilizes the intermediate, leading to a higher degree of native-like interactions in the transition state. Data are discussed and compared with previous works on SH2 domains.

Somatic mutation signatures may represent footprints of genetic and environmental exposures that cause different cancer. Few studies have comprehensively examined their association with germline variants, and none in an indigenous African population. SomaticSignatures was employed to extract mutation signatures based on whole‐genome or whole‐exome sequencing data from female patients with breast cancer (TCGA, training set, n = 1,011; Nigerian samples, validation set, n = 170), and to estimate contributions of signatures in each sample. Association between somatic signatures and common single nucleotide polymorphisms (SNPs) or rare deleterious variants were examined using linear regression. Nine stable signatures were inferred, and four signatures (APOBEC C>T, APOBEC C>G, aging and homologous recombination deficiency) were highly similar to known COSMIC signatures and explained the majority (60–85%) of signature contributions. There were significant heritable components associated with APOBEC C>T signature (h 2 = 0.575, p = 0.010) and the combined APOBEC signatures (h 2 = 0.432, p = 0.042). In TCGA dataset, seven common SNPs within or near GNB5 were significantly associated with an increased proportion (beta = 0.33, 95% CI = 0.21–0.45) of APOBEC signature contribution at genome‐wide significance, while rare germline mutations in MTCL1 was also significantly associated with a higher contribution of this signature (p = 6.1 × 10−6). This is the first study to identify associations between germline variants and mutational patterns in breast cancer across diverse populations and geography. The findings provide evidence to substantiate causal links between germline genetic risk variants and carcinogenesis., What's new? Women of African ancestry are more likely to be diagnosed with clinically aggressive breast cancer than women of European or Asian ancestry. Here, the authors examined associations between germline variants and mutational signatures in breast cancers across different ethnicities, especially in a unique sample set of indigenous African women. They identified four stable mutational signatures that explained the majority of tumor mutations, leading to a better understanding of the complex interplay between germline genetics and somatic mutations across different ethnicities.

Ostreococcus tauri is a simple unicellular green alga representing an ecologically important group of phytoplankton in oceans worldwide. Modern molecular techniques must be developed in order to understand the mechanisms that permit adaptation of microalgae to their environment. We present for the first time in O. tauri a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration occurred randomly at a single locus in the genome and mainly as a single copy. Thus, we confirmed the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the O. tauri model remains to be elucidated, we clearly demonstrate by genome resequencing that the integration of the vector leads to frequent structural variations (deletions/insertions and duplications) and some chromosomal rearrangements in the genome at the insertion loci. Furthermore, we often observed variations in the vector sequence itself. From these observations, we speculate that a nonhomologous end-joining-like mechanism is employed during random insertion events, as described in plants and other freshwater algal models. PEG-mediated transformation is therefore a promising molecular biology tool, not only for functional genomic studies, but also for biotechnological research in this ecologically important marine alga. This research received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 824110—EASI-Genomics and the French National Research Agency project grant (ANR Algalvirus ANR-17-CE02-0012). We acknowledge the support of the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III and the 2014–2020 Smart Growth Operating Program, to the EMBL partnership and cofinancing with the European Regional Development Fund (MINECO/FEDER, BIO2015-71792-P). We also acknowledge the support of the Centro de Excelencia Severo Ochoa, and the Generalitat de Catalunya through the Departament de Salut, Departament d’Empresa i Coneixement and the CERCA Programme

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

[EU]Mikroorganismoek garrantzi handia dute osasunarentzat, eta baita aktibitate ekonomikoarentzat ere. Batzuetatik garrantzia ekonomiko handiko konposatuak erauzten ditugu (antibiotikoak, inmunosupresoreak edo entzimak, esaterako). Bestetan, bakterio edo onddo espezie patogenoek gaixotasunak eragiten dituzte uzta ala pertsonengan. Infekzio hauek leku berrietara zabaltzeko harizpi itxurako onddoek konidia izeneko esporak milioika sortzen dituzte. GrAL honetan ikasleak konidiak sortzeko beharrezkoa den gene baten eremu desberdinetan mutagenesi zuzendua burutuko du PCR teknikaren bidez. Ondoren mutazio horiek daramatzaten andui errekonbinanteak sortu eta fenotipikoki karakterizatuko ditu, mutazio bakoitzak konidien produkzioan duen eragina aztertzeko. Bereganatutako teknikek, geneetan informazioa nola antolatzen den ulertu eta informazio hori nola manipulatu daitekeen ezagutzeko aukera emango diote ikasleari. Bestetik, etorkizunean edozein zelula bizian horrelako experimentoak diseinatu eta gauzatu ahal izateko oinarrizko ezagutza ere jasoko du. [EN]The main aim of this Final Degree project has been the development of basic skills in stardard molecular biology techniques. The filamentous fungus Aspergillus nidulans and FlbE, a protein required for asexual development, have been used as models. Tryptophan in position 11 of FlbE has been replaced by an Alanine, and the effects of this replacement on the fungi’s development were studied, as well as the impact on subcellular localization and the interactions with other proteins. To proceed with the analysis, different molecular biology techniques were applied, such as PCR (Polymerase Chain Reaction), transformation, DNA extraction and detection, protein inmunodetection, protein-protein interaction assays and fluorescent microscopy. Overall, W11A substitution within FlbE causes an inhibition of its subcellular localization, interactions and the ability to induce development.

SH2 domains are common protein interaction domains able to recognize short aminoacidic sequences presenting a phosphorylated tyrosine (pY). In spite of their fundamental importance for cell physiology there is a lack of information about the mechanism by which these domains recognize and bind their natural ligands. The N-terminal SH2 (N-SH2) domain of PI3K mediates the interaction with different scaffolding proteins and is known to recognize a specific pY-X-X-M consensus sequence. These interactions are at the cross roads of different molecular pathways and play a key role for cell development and division. By combining mutagenesis, chemical kinetics and NMR, here we provide a complete characterization of the interaction between N-SH2 and a peptide mimicking the scaffolding protein Gab2. Our results highlight that N-SH2 is characterized by a remarkable structural plasticity, with the binding reaction being mediated by a diffused structural region and not solely by the residues located in the binding pocket. Furthermore, the analysis of kinetic data allow us to pinpoint an allosteric network involving residues far from the binding pocket involved in specificity. Results are discussed on the light of previous works on the binding properties of SH2 domains.

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed "templated folding," whereby the binding partner dictates distinct structural transitions en route to product, while ensuring a cooperative folding.

Noonan syndrome (NS) is a genetic disorder caused by the hyperactivation of the RAS-MAPK molecular pathway. About 50% of NS cases are caused by mutations affecting the SHP2 protein, a multi-domain phosphatase with a fundamental role in the regulation of the RAS-MAPK pathway. Most NS-causing mutations influence the stability of the inactive form of SHP2. However, one NS-causing mutation, namely T42A, occurs in the binding pocket of the N-SH2 domain of the protein. Here, we present a quantitative characterization of the effect of the T42A mutation on the binding of the N-terminal SH2 domain of SHP2 with a peptide mimicking Gab2, a fundamental interaction that triggers the activation of the phosphatase in the cellular environment. Our results show that whilst the T42A mutation does not affect the association rate constant with the ligand, it causes a dramatic increase of the affinity for Gab2. This effect is due to a remarkable decrease of the microscopic dissociation rate constant of over two orders of magnitudes. In an effort to investigate the molecular basis of the T42A mutation in causing Noonan syndrome, we also compare the experimental results with a more conservative variant, T42S. Our findings are discussed in the context of the structural data available on SHP2.

The first aim of this thesis was to tackle a core question in biology—to understand how large numbers of mutations combine together to influence phenotypes. In order to do so, we built a combinatorially-complete library of naturally occurring variants in a yeast tRNA. For the first time in any gene, we could quantify the extent of which both the effects of individual mutations and the interactions between pairs of mutations change across a large number of closely-related genotypes. We found that all mutations switch from beneficial to detrimental effects and all interactions switch from positive to negative in different backgrounds. Secondly, with the use of systematic mutagenesis, protein complementation assays and deep sequencing, we developed a new experimental methodology to map the interaction interfaces of physically interacting proteins at amino acid resolution. The approach works by quantifying the effects of mutations on both protein binding and stability, resulting in a high resolution map of an interaction interface. El primer objectiu d'aquesta tesi va ser abordar una qüestió fonamental en biologia: entendre com la combinació d’un gran nombre de mutacions poden produir canvis en el fenotip. Per tal d’investigar aquest problema, vam construir una col·lecció de totes les variants genètiques observades en l’evolució d’un ARNt del llevat. Per primera vegada en un anàlisi d’un gen complet, vam quantificar com els efectes de les mutacions individuals, així com les interaccions entre parelles de mutacions, canvien el fenotip. Els resultats mostren que en diferent contextos genètics, totes les mutacions poden ser beneficioses o deletèries. De la mateixa manera, les interactions entre parelles de mutacions exageren o atenuen els efectes de les mutacions individuals depenent del context genètic on ocorren. En segon lloc, utilitzant tècniques de mutagènesi sistemàtica, mètodes de complementació de proteïnes i seqüenciació d’ADN, hem desenvolupat una nova metodologia experimental per identificar a resolució d’aminoàcid la interfície de contacte entre dues proteïnes. La metodologia es basa en quantificar els efectes de mutacions que alteren la unió de les dues proteïnes, bé degut a que alteren l’estabilitat d’una d’elles, o bé perquè canvien l’afinitat de l’una per l’altre.

β(2)-Glycoprotein I (β(2)GPI) is an abundant plasma protein displaying phospholipid-binding properties. Because it binds phospholipids, it is a target of antiphospholipid antibodies (aPLs) in antiphospholipid syndrome (APS), a life-threatening autoimmune thrombotic disease. Indeed, aPLs prefer membrane-bound β(2)GPI to that in solution. β(2)GPI exists in two almost equally populated redox states: oxidized, in which all the disulfide bonds are formed, and reduced, in which one or more disulfide bonds are broken. Furthermore, β(2)GPI can adopt multiple conformations (i.e. J-elongated, S-twisted, and O-circular). While strong evidence indicates that the J-form is the structure bound to aPLs, which conformation exists and predominates in solution remains controversial, and so is the conformational pathway leading to the bound state. Here, we report that human recombinant β(2)GPI purified under native conditions is oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible conformation, not circular or twisted, in which the N-terminal domain I (DI) and the C-terminal domain V (DV) are exposed to the solvent. Consistent with this model, binding kinetics and mutagenesis experiments revealed that in solution the J-form interacts with negatively charged liposomes and with MBB2, a monoclonal anti-DI antibody that recapitulates most of the features of pathogenic aPLs. We conclude that the preferential binding of aPLs to phospholipid-bound β(2)GPI arises from the ability of its preexisting J-form to accumulate on the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of β(2)GPI provides a strategy to block pathogenic aPLs in APS.

Protein structure is tightly intertwined with function according to the laws of evolution. Understanding how structure determines function has been the aim of structural biology for decades. Here, we have wondered instead whether it is possible to exploit the function for which a protein was evolutionary selected to gain information on protein structure and on the landscape explored during the early stages of molecular and natural evolution. To answer to this question, we developed a new methodology, which we named CAMELS (Coupling Analysis by Molecular Evolution Library Sequencing), that is able to obtain the in vitro evolution of a protein from an artificial selection based on function. We were able to observe with CAMELS many features of the TEM-1 beta-lactamase local fold exclusively by generating and sequencing large libraries of mutational variants. We demonstrated that we can, whenever a functional phenotypic selection of a protein is available, sketch the structural and evolutionary landscape of a protein without utilizing purified proteins, collecting physical measurements, or relying on the pool of natural protein variants. Protein structure is tightly intertwined with function according to the laws of evolution. Understanding how structure determines function has been the aim of structural biology for decades. Here, we have wondered instead whether it is possible to exploit the function for which a protein was evolutionary selected to gain information on protein structure and on the landscape explored during the early stages of molecular and natural evolution. To answer to this question, we developed a new methodology, which we named CAMELS (Coupling Analysis by Molecular Evolution Library Sequencing), that is able to obtain the in vitro evolution of a protein from an artificial selection based on function. We were able to observe with CAMELS many features of the TEM-1 beta-lactamase local fold exclusively by generating and sequencing large libraries of mutational variants. We demonstrated that we can, whenever a functional phenotypic selection of a protein is available, sketch the structural and evolutionary landscape of a protein without utilizing purified proteins, collecting physical measurements, or relying on the pool of natural protein variants.

Pseudomonas donghuensis strain SVBP6, an isolate from an agricultural plot in Argentina, displays a broad-spectrum and diffusible antifungal activity, which requires a functional gacS gene but could not be ascribed yet to known secondary metabolites typical of Pseudomonas biocontrol species. Here, we report that Tn5 mutagenesis allowed the identification of a gene cluster involved in both the fungal antagonism and the production of a soluble tropolonoid compound. The ethyl acetate extract from culture supernatant showed a dose-dependent inhibitory effect against the phytopathogenic fungus Macrophomina phaseolina. The main compound present in the organic extract was identified by spectroscopic and X-ray analyses as 7-hydroxytropolone (7HT). Its structure and tautomerism was confirmed by preparing the two key derivatives 2,3-dimethoxy- and 2,7-dimethoxy-tropone. 7HT, but not 2,3- or 2,7-dimethoxy-tropone, mimicked the fungal inhibitory activity of the ethyl acetate extract from culture supernatant. The activity of 7HT, as well as its production, was barely affected by the presence of up to 50 μM added iron (Fe+2). To summarize, P. donghuensis SVBP6 produces 7HT under the positive control of the Gac-Rsm cascade and is the main active metabolite responsible for the broad-spectrum inhibition of different phytopathogenic fungi. Fil: Muzio, Federico Matías. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Centro de Bioquimica y Microbiologia de Suelos. Laboratorio de Fisiologia, Genetica de Bacterias Para Plantas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Agaras, Betina Cecilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Centro de Bioquimica y Microbiologia de Suelos. Laboratorio de Fisiologia, Genetica de Bacterias Para Plantas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Masi, Marco. Università degli Studi di Napoli Federico II; Italia Fil: Tuzi, Angela. Università degli Studi di Napoli Federico II; Italia Fil: Evidente, Antonio. Università degli Studi di Napoli Federico II; Italia Fil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Centro de Bioquimica y Microbiologia de Suelos. Laboratorio de Fisiologia, Genetica de Bacterias Para Plantas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

© 2019 The Authors., Somatic epigenetic inactivation of the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) is frequent in colorectal cancer (CRC); however, its involvement in CRC predisposition remains unexplored. We assessed the role and relevance of MGMT germline mutations and epimutations in familial and early-onset CRC. Mutation and promoter methylation screenings were performed in 473 familial and/or early-onset mismatch repair-proficient nonpolyposis CRC cases. No constitutional MGMT inactivation by promoter methylation was observed. Of six rare heterozygous germline variants identified, c.346C > T (p.H116Y) and c.476G > A (p.R159Q), detected in three and one families respectively, affected highly conserved residues and showed segregation with cancer in available family members. In vitro, neither p.H116Y nor p.R159Q caused statistically significant reduction of MGMT repair activity. No evidence of somatic second hits was found in the studied tumors. Case-control data showed over-representation of c.346C > T (p.H116Y) in familial CRC compared to controls, but no overall association of MGMT mutations with CRC predisposition. In conclusion, germline mutations and constitutional epimutations in MGMT are not major players in hereditary CRC. Nevertheless, the over-representation of c.346C > T (p.H116Y) in our familial CRC cohort warrants further research., This work was funded by the Spanish Ministry of Science, Innovation and Universities, co-funded by FEDER funds -a way to build Europe- [SAF2016-80888-R (LV), SAF2014-55000-R (ME), SAF2015-68016-R (GC/MP), Juan de la Cierva and Sara Borrell postdoctoral contracts (PM)]; Instituto de Salud Carlos III [DTS16/00153 (ME) and CIBERONC CB16/12/00234]; the Government of Catalonia [Pla Estratègic de Recerca i Innovació en Salut SLT002/16/0037, 2017SGR1282, 2017SGR1080, 2014SGR633 and 2009SGR1315]; and Fundación Olga Torres. We thank the CERCA/Generalitat de Catalunya Program for institutional support. This study has been enabled by COST Action CA17118.

Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours. Funding sources for this study include grants to the Pancreatic Cancer Sequencing Initiative program from the Ontario Institute for Cancer Research (OICR), through support from the Ontario Ministry of Research and Innovation, the Canada Foundation for Innovation; research award to F.N. from the OICR and the Canadian Institutes for Health Research (CIHR); Canadian Friends of the Hebrew University, the SMGS Family Foundation, NCI grant P50 CA102701 (Mayo Clinic SPORE in Pancreatic Cancer) and NCI grant R01 CA97075 (Pancreatic Cancer Genetic Epidemiology Consortium). F.N. is supported by a fellowship award from CIHR and is a recipient of a scholar’s research award from the Ontario Institute of Cancer Research (OICR), through support from the Ontario Ministry of Research and Innovation. G.Z. is a Clinician–Scientist of the Fonds de la Recherche en Sante du Quebec. P.J.C. is a Wellcome Trust Senior Clinical Fellow. T.J.H., L.D.S., J.D.M. and S.G. are recipients of Senior or Clinician–Scientist Awards from the Ontario Institute for Cancer Research

International audience; Over the past decades, membranotropic peptides such as positively charged cell-penetrating peptides (CPPs) or amphipathic antimicrobial peptides (AMPs) have received increasing interest in order to improve therapeutic agent cellular uptake.As far as we are concerned, we were interested in studying HCV fusion peptides as putative anchors. Two peptides, HCV6 and HCV7, were identified and conjugated to a fluorescent tag NBD and tested for their interaction with liposomes as model membranes. DSC and spectrofluorescence analyses demonstrate HCV7 propensity to insert or internalize in vesicles containing anionic lipids DMPG whereas no activity was observed with zwitterionic DMPC. This behavior could be explained by the peptide sequence containing a cationic arginine residue. On the contrary, HCV6 did not exhibit any membranotropic activity but was the only sequence able to induce liposomes' fusion or aggregation monitored by spectrofluorescence and DLS. This two peptides mild activity was related to their inefficient structuration in contact with membrane mimetics, which was demonstrated by CD and NMR experiments.Altogether, our data allowed us to identify two promising membrane-active peptides from E1 and E2 HCV viral proteins, one fusogenic (HCV6) and the other membranotropic (HCV7). The latter was also confirmed by fluorescence microscopy with CHO cells, indicating that HCV7 could cross the plasma membrane via an endocytosis process. Therefore, this study provides new evidences supporting the identification of HCV6 as the HCV fusion peptide as well as insights on a novel membranotropic peptide from the HCV-E2 viral protein.

International audience; The photochemical behavior of etodolac was investigated under various irradiation conditions. Kinetic data were obtained after irradiation of 10− 4 M aqueous solutions by UVB, UVA and direct exposure to sunlight. The Xenon lamp irradiation was used in order to determine the photodegradation quantum yield under sun-simulated condition (ϕsun). The value was determined to be = 0.10 ± 0.01. In order to obtain photoproducts and for mechanistic purposes, experiments were carried out on more concentrated solutions by exposure to sunlight and to UVA and UVB lamps. The drug underwent photooxidative processes following an initial oxygen addition to the double bond of the five membered ring and was mainly converted into a spiro compound and a macrolactam. Ecotoxicity tests were performed on etodolac, its photostable spiro derivative and its sunlight irradiation mixture on two different aquatic trophic levels, plants (algae) and invertebrates (rotifers and crustaceans). Mutagenesis and genotoxicity were detected on bacterial strains. The results showed that only etodolac had long term effects on rotifers although at concentrations far from environmental detection values. A mutagenic and genotoxic potential was found for its derivative.

Upon binding, ligands can chaperone their protein targets by preventing them from misfolding and aggregating. Thus, an organic molecule that works as folding chaperone for a protein might be its specific ligand, and, similarly, the chaperone potential could represent an alternative readout in a molecular screening campaign toward the identification of new hits. Here we show that small molecules selected for acting as pharmacological chaperones on a misfolded mutant of the Frizzled4 (Fz4) receptor bind and modulate wild-type Fz4, representing what are to our knowledge the first organic ligands of this until-now-undruggable GPCR. The novelty and the advantages of the screening platform, the allosteric binding site addressed by these new ligands and the mechanism they use to modulate Fz4 suggest new avenues for development of inhibitors of the Wnt-beta-catenin pathway and for drug discovery.

Gain-of-function somatic mutations introducing cysteines to either the extracellular or to the transmembrane domain (TMD) in interleukin-7 receptor α (IL7R) or cytokine receptor-like factor 2 (CRLF2) have been described in acute lymphoblastic leukemias. Here we report noncysteine in-frame mutations in IL7R and CRLF2 located in a region of the TMD closer to the cytosolic domain. Biochemical and functional assays showed that these are activating mutations conferring cytokine-independent growth of progenitor lymphoid cells in vitro and are transforming in vivo. Protein fragment complementation assays suggest that despite the absence of cysteines, the mechanism of activation is through ligand-independent dimerization. Mutagenesis experiments and ConSurf calculations suggest that the mutations stabilize the homodimeric conformation, positioning the cytosolic kinases in predefined orientation to each other, thereby inducing spontaneous receptor activation independently of external signals. Hence, type I cytokine receptors may be activated in leukemia through 2 types of transmembrane somatic dimerizing mutations.

SH2 domains are protein domains that modulate protein&ndash, protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the &beta, E and &beta, F strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.

The current world population is over 7.4 billion and expected to exceed 9 billion in 2040, causing a 70% increase in food demand. Global environmental degradation, in the form of salinization, pollution and global warming, has also reduced the availability of suitable arable land and water sources, contributing to promote crop improvement in order to increase the potential yields. Rice (Oryza sativa) is the most widely consumed staple food for a large proportion of the world population. Classical rice breeding programs use its natural variability to create new allelic combinations which are screened for selecting those presenting superior agronomic traits such as improved yield. Those improved lines are stabilized through inbreeding to maintain the phenotype in their progeny. Certified seed producers systematically select and propagate registered varieties year by year in order to maintain their uniformity and the original registered cultivar traits, since natural mutations, spontaneous breeding between varieties and alien grain contamination can introduce undesirable variability at this stage. Nowadays biotechnology is used to drive the improvement of rice traits such as increased yield and grain quality. Moreover it helps to rapidly bestow tolerance to biotic (diseases and insects) and abiotic (drought, salinity, cold temperatures, nutrients deficiency) factors. Some of the available biotechnological techniques applied for crop improvement are i) the genetic engineering, which allows the addition of foreign genes in the rice genome although being controversial due to the social and environmental concerns, ii) the anther culture, which fasten and improves the selection of new breeding lines, and iii) the Targeting Induced Local Lesions IN Genomes (TILLING) which combines the production of large mutant populations with the detection of mutants in genes of interest through molecular screening. The main aim of the thesis is to study different biotechnological tools and their applications to the improvement of Mediterranean rice varieties. To achieve this biotechnology is used to study the pollen dispersion of a genetically engineered rice line, to accelerate the stabilization process through anther culture technique and to introduce new variability using a mutagenesis protocol followed by molecular detection of mutants. In this thesis we first studied the pollen-mediated gene flow between wild rice, conventional rice and an herbicide resistant transgenic rice line in order to determine gene flow rates in relation to the distance and the prevailing wind speed and direction. Results showed that pollen dispersal is dramatically effected by the distances between rice plants and the speed and direction of the prevailing wind. Furthermore, the enhanced pollen dispersal capability of weedy rice can also play an important role in transgenic pollen dispersal, which unfortunately had been underestimated. Then, we adapted an anther culture protocol in order to efficiently obtain commercial dihaploid lines from a Mediterranean japonica variety. Furthermore, we described the greenhouse and field trials used to select the best lines for registration which are now being successfully commercialized. Finally, we developed a fast protocol for obtaining mutants with agronomic interest. This protocol is based on ethyl methanesulfonate mutagenesis of seed-derived calli. The in vitro regenerated mutant population plants were directly screened for senescence-related genes, allowing to shorten in more than eight months the common seed mutagenesis protocol. The molecular screening protocol was also optimized and several potential delayed senescence mutants were identified and tested.

The JAK/STAT pathway is an essential mediator of cytokine signaling, often upregulated in human diseases and therefore recognized as a relevant therapeutic target. We previously identified the synthetic chalcone α-bromo-2′,3,4,4′-tetramethoxychalcone (α-Br-TMC) as a novel JAK2/STAT5 inhibitor. We also found that treatment with α-Br-TMC resulted in a downward shift of STAT5 proteins in SDS-PAGE, suggesting a post-translational modification that might affect STAT5 function. In the present study, we show that a single cysteine within STAT5 is responsible for the α-Br-TMC-induced protein shift, and that this modification does not alter STAT5 transcriptional activity. We also compared the inhibitory activity of α-Br-TMC to that of another synthetic chalcone, α-trifluoromethyl-2′,3,4,4′-tetramethoxychalcone (α-CF3-TMC). We found that, like α-Br-TMC, α-CF3-TMC inhibits JAK2 and STAT5 phosphorylation in response to interleukin-3, however without altering STAT5 mobility in SDS-PAGE. Moreover, we demonstrate that both α-Br-TMC and α-CF3-TMC inhibit interferon-α-induced activation of STAT1 and STAT2, by inhibiting their phosphorylation and the expression of downstream interferon-stimulated genes. Together with the previous finding that α-Br-TMC and α-CF3-TMC inhibit the response to inflammation by inducing Nrf2 and blocking NF-κB activities, our data suggest that synthetic chalcones might be useful as anti-inflammatory, anti-cancer and immunomodulatory agents in the treatment of human diseases.

An elastic light-scattering-based method that makes use of phantom nanoparticles as a substrate organizer (see illustration) allowed the quantitative evaluation of the molecular recognition events between a series of inactivated mutants of phospholipase D and lysophosphatidylcholine. The results highlight the remarkable effects on binding capability caused by single amino acid substitutions.

A fluorescence-based screen was used to analyze 70 LAT transporter mutants and identify variants with improved stability and monodispersity., The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis l-serine/l-threonine exchanger is the best-known prokaryotic paradigm of the mammalian l–amino acid transporter (LAT) family. Unfortunately, SteT’s lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest.

[spa] Uno de los temas fundamentales en el estudio de la evolución es el papel que desempeña la modificación de la actividad genética en la aparición de nuevas morfologías. Con el fin de comprender dicha relación, hemos comparado el sistema respiratorio altamente derivado del díptero Drosophila melanogaster con el del coleóptero Tribolium castaneum, menos derivado. A diferencia de Drosophila, el sistema traqueal de Tribolium no ha sido prácticamente descrito, por lo que ha sido necesario definirlo previamente, tanto a nivel morfológico como molecular. Así, hemos comprobado que en ambos casos el sistema traqueal se origina a partir de 10 placodas de origen ectodérmico a cada lado del embrión, llamadas primordios traqueales (Manning and Krasnow, 1993). Una vez las células traqueales han sido determinadas, invaginan y comienzan a migrar hacia las zonas dorsal y ventral. Durante este proceso las células se alargan e intercalan, formando las futuras ramas traqueales. Por último, cada metámero traqueal acaba uniéndose al adyacente por medio de la fusión de las ramas laterales. Sin embargo, ambos sistemas presentan un diferente grado de organización durante sus etapas larvadas, adaptada al medio que habitan. Este se diferencia principalmente en la presencia en Drosophila y ausencia en Tribolium de dos tubos longitudinales llamados troncos dorsales (TD), así como en la diferente disposición a lo largo del cuerpo de los espiráculos, las estructuras a través de las cuales se conecta la tráquea con el exterior. En Tribolium las tráqueas conectan con el exterior en cada uno de los metámeros a través de los espiráculos laterales. En cambio en Drosophila, estos espiráculos laterales no se desarrollan y el sistema traqueal conecta con el exterior mediante los espiráculos anteriores y posteriores, que están conectados a los tubos dorsales. A continuación investigamos los mecanismos moleculares responsables de las citadas diferencias morfológicas. Así, durante nuestro estudio hemos comprobado que el factor de transcripción Spalt (Sal) no mantienen el mismo patrón de expresión en las células traqueales de ambas especies. De hecho sal, responsable de la formación del tronco dorsal en Drosophila, no se expresa en las células traqueales de Tribolium, sugiriendo que esta diferente expresión de sal en las células traqueales de Drosophila podría ser la responsable de la adquisición de los troncos dorsales en esta especie. Así mismo hemos descubierto que el factor de transcripción Cut es necesario para el desarrollo de los espiráculos. Sin embargo su activación es diferente en las dos especies. Mientras que en Drosophila cut únicamente se expresa en el último segmento abdominal, en Tribolium se expresa en todos los segmentos laterales. Además, hemos visto que Cut reprime la expresión de sal en Drosophila. Esta diferencia de expresión del gen cut en Drosophilla podría deberse a la separación de los primordios traqueal y espiracular en Drosophila. Los sistemas traqueal y espiracular de Drosophila se originan a partir de diferentes poblaciones celulares bajo el control de diferentes mecanismos génicos, la vía de JAK/STAT y el gen homeótico Abdominal-B, respectivamente., [eng] Evolution, through modification of morphogenesis, led animals to acquire the body shapes and organ systems that enabled them to enter, survive and reproduce in a vast number of different habitats. While it is undisputed that evolving genomes are ultimately responsible for morphological modifications, little is known about how major innovations can be conveyed by small genetic changes. We have addressed this issue by analyzing expression and function of regulatory genes in the developing tracheal systems of two insect species. The tracheal system of Drosophila is distinguished from the less derived tracheal system of the beetle Tribolium by two main features: First, the lateral spiracles, which in Tribolium connect the tracheal branches to the exterior in each segment, are not present in Drosophila. Instead it has only one pair of heavily derived posterior spiracles. Second, the dorsal trunks, two prominent branches that distribute air from the posterior spiracles and span longitudinally through the larvae, do not exist in Tribolium. Both innovations, while considered independent structures, are functionally dependent on each other and linked to habitat occupancy: half-buried Drosophila larvae in semi-liquid environments keep their posterior spiracles above the surface and distribute the gas to the body through the dorsal trunks. Conversely, the lateral spiracles of free-living Tribolium larvae provide sufficient airflow to all segments, so that no thick dorsal trunks are needed to distribute the oxygen. Here we show that changes in the domains of spalt and cut expression are associated with the acquisition of each innovation. Moreover, we show these two genetic modifications to be not only functionally but also genetically connected, providing an evolutionary scenario by which a single genetic event can contribute to the joint evolution of functionally inter-related organs.

The occurrence of pharmaceuticals in the environment is of great concern and only few data are available about the adverse effects of such molecules and their derivatives on non-target aquatic organisms. This study was designed to assess the toxic potential of Naproxen, a nonsteroidal anti-inflammatory, Naproxen Na, its freely water soluble sodium salt and their photoproducts in the aquatic environment. Bioassays were performed on algae, rotifers and microcrustaceans to assess acute and chronic toxicity. Furthermore, possible genotoxic effects of photoderivatives were investigated using SOS chromotest and Ames fluctuation test. The results showed that photoproducts were more toxic than the parent compounds both for acute and chronic values, while genotoxic and mutagenic effects were not found. These findings suggested the opportunity to consider derivatives in ecotoxicology assessment of drugs. © 2005 Elsevier B.V. All rights reserved.

The ecotoxicity of the following six antibiotics on aquatic organisms was investigated: Erythromycin, Oxytetracyclin, Sulfamethoxazole, Ofloxacin, Lincomycin and Clarithromycin. Bioassays were performed on bacteria, algae, rotifers, microcrustaceans and fish to assess acute and chronic toxicity, while SOS Chromotest and Ames test were used to detect the genotoxic potential of the investigated drugs. For risk assessment, the environmental impact was calculated by MEC/PNEC ratio using the available data from the literature regarding their occurrence in the aquatic environment and the toxicity data obtained from the bioassays performed. The ecotoxicological results showed that acute toxicity was in the order of mg/L while, for the chronic data the antibiotics were bioactive at concentrations in the order of μg/L, mainly for the algae. Drugs investigated were one or two order of magnitude less active against rotifers and crustaceans. Ofloxacin was the only genotoxic compound and Sulfamethoxazole, Ofloxacin and Lincomycin were mutagenic. As for environmental risk, the macrolides were found to be the most harmful for the aquatic environment. © 2004 Elsevier B.V. All rights reserved.

Collections of deletion mutants are important tools for functional genomics in crops. In order to establish an efficient physical γ-ray-based mutagenesis protocol and estimate the frequency of induced chromosome deletion events in apple, we mutagenized a total of 338 leaves of the apple cultivar ‘Golden Delicious’ with a range of irradiation doses (from 0 to 30 Gy). After in vitro culture and regeneration, a total of 248 plantlets were genotyped by a custom-made 384 SNP-based array suited to identify homozygous regions, corresponding to chromosome deletions produced by the irradiation and/or the in vitro culture treatments. With this approach, we identified nine multi-SNP chromosome deletions corresponding to 3.7 % of all regenerated plantlets and ranging from one to 44 cM (0.1–20.2 Mb). Chromosome deletions were also observed at mildly, not significantly lower frequency in plantlets regenerated from non-irradiated leaves. Our results confirm the possibility to generate apple plants with sizeable deletions of known dimension and location, opening the way to creating a collection of deletions spanning the apple genome useful for functional genomics. The high frequency of deletions apparently caused by somaclonal variation deserves further investigation.

Embryonic stem (ES) cells are derived from the inner cell mass of blastocysts, an early-stage of pre-implantation embryo, and are capable of unlimited, undifferentiated proliferation in vitro. They are pluripotent, meaning they are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. ES cells are key tools for genetic engineering, development of stem cell-based therapies and basic research on pluripotency and early lineage commitment. The use of stem cells as therapeutics to treat genetic defects depends on how efficient are the approaches to manipulate their genome. Traditional non-viral strategies are generally less efficient in delivering DNA and initiating gene expression, but they are safer, cheaper, producible easily in large quantities and have higher genetic material carrying capacity. Therefore, FuGENE® HD (Promega), SuperFect® (Qiagen), Lipofectamine® 2000 (Invitrogen) and also electroporation were used to transiently transfect fluorescently labelled expression vectors into an mES cell line in order to optimize a reliable and efficient protocol that could be applied for some of the new mutagenesis methods. In most of the new site-directed mutagenesis techniques, more than one plasmid has to be introduced into the cell. For this reason, co-transfection and single transfection efficiencies of plasmids encoding the mCherry fluorescent protein and the EGFP were simultaneously determined. Transfection and co-transfection efficiencies of 52-83% were found for FuGENE® HD transfection reagent, which was shown to be most efficient and reliable. In addition, in 90-93% of the co-transfected colonies both fluorescent proteins were co-expressed. This optimized transfection protocol was followed to successfully assess a new recombinase mediated cassette exchange (RMCE) system in an mES RMCE-in cell line as a more attractive alternative to the commercial Flp-in cell lines

Mutations in the KCNQ2 gene encoding for voltage-gated potassium channel subunits have been found in patients affected with early onset epilepsies with wide phenotypic heterogeneity, ranging from benign familial neonatal seizures (BFNS) to epileptic encephalopathy with cognitive impairment, drug resistance, and characteristic electroencephalography (EEG) and neuroradiologic features. By contrast, only few KCNQ3 mutations have been rarely described, mostly in patients with typical BFNS. We report clinical, genetic, and functional data from a family in which early onset epilepsy and neurocognitive deficits segregated with a novel mutation in KCNQ3 (c.989G>T; p.R330L). Electrophysiological studies in mammalian cells revealed that incorporation of KCNQ3 R330L mutant subunits impaired channel function, suggesting a pathogenetic role for such mutation. The degree of functional impairment of channels incorporating KCNQ3 R330L subunits was larger than that of channels carrying another KCNQ3 mutation affecting the same codon but leading to a different amino acid substitution (p.R330C), previously identified in two families with typical BFNS. These data suggest that mutations in KCNQ3, similarly to KCNQ2, can be found in patients with more severe phenotypes including intellectual disability, and that the degree of the functional impairment caused by mutations at position 330 in KCNQ3 may contribute to clinical disease severity.

The metallothionein (MT)3 gene is expressed predominantly in the brain and the organs of the reproductive system, and fails to respond to metal ions in vivo. A CTG repeat was proposed to function as a potential repressor element in nonpermissive cells, and a sequence similar to the JC virus silencer element was found to function as a negative element in permissive primary astrocytes. The objective of this study was to characterize further the mechanisms governing cell-type specific MT-3 gene transcription. We searched for a suitable cell line expressing the MT-3 gene to be used for determination of MT-3 promoter tissue specificity, and showed that MT-3 expression is activated during neuroectodermal differentiation of P 19 cells induced by retinoic acid to levels similar to those found in whole brain. Deletion of the CTG repeat or of the JC virus silencer did not promote MT-3 promoter activity in nonpermissive cells, or enhance expression in permissive cells. We identified MT-3 promoter sequences interacting with liver and brain nuclear proteins, as assayed by DNase I footprinting analyses and electrophoretic mobility shift assay, and assessed the role of these sequences in the regulation of MT-3 expression by cotransfection experiments. We generated stable transfectants in permissive C6 and nonpermissive NIH-3T3 cells, and analysed the methylation status of the MT-3 gene. These studies show that regulation of tissue-specific MT-3 gene expression does not appear to involve a repressor, and suggest that other mechanisms such as chromatin organization and epigenetic modifications could account for the absence of MT-3 gene transcription in nonpermissive cells.

The σ1 receptor is an integral membrane protein that shares no homology with other receptor systems, has no unequivocally identified natural ligands, but appears to play critical roles in a wide variety of cell functions. While the number of reports of the possible functions of the σ1 receptor is increasing, almost no information about the three-dimensional structure of the receptor and/or possible modes of interaction of the σ1 protein with its ligands have been described. Here we performed an in vitro/in silico investigation to analyze the molecular interactions of the σ1 receptor with its prototypical agonist (+)-pentazocine. Accordingly, 23 mutant σ1 isoforms were generated, and their interactions with (+)-pentazocine were determined experimentally. All direct and/or indirect effects exerted by the mutant residues on the receptor–agonist interactions were reproduced and rationalized in silico, thus shining new light on the three-dimensional structure of the σ1 receptor and its ligand binding site.

T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) contains the canonical phosphoprotein phosphatase 1 (PPP1) binding motif, composed by the amino acid sequence RVSF. We identified and validated the binding of TCTEX1D4 to PPP1 and demonstrated that indeed this protein is a novel PPP1 interacting protein. Analyses of twenty-one mammalian species available in public databases and seven Lagomorpha sequences obtained in this work showed that the PPP1 binding motif 90RVSF93 is present in all of them and is flanked by a palindromic sequence, PLGS, except in three species of pikas (Ochotona princeps, O. dauurica and O. pusilla). Furthermore, for the Ochotona species an extra glycosylation site, motif 96NLS98, and the loss of the palindromic sequence were observed. Comparison with other lagomorphs suggests that this event happened before the Ochotona radiation. The dN/dS for the sequence region comprising the PPP1 binding motif and the flanking palindrome highly supports the hypothesis that for Ochotona species this region has been evolving under positive selection. In addition, mutational screening shows that the ability of pikas TCTEX1D4 to bind to PPP1 is maintained, although the PPP1 binding motif is disrupted, and the N- and C-terminal surrounding residues are also abrogated. These observations suggest pika as an ideal model to study novel PPP1 complexes regulatory mechanisms. published

Chlorination experiments on two drugs (sildenafil and tadalafil) were performed mimicking the conditions of a typical wastewater treatment process. The main transformation products were isolated by chromatographic techniques (Thin Layer Chromatography (TLC), Column Chromatography (CC), High Performance Liquid Chromatography (HPLC)) and fully characterized employing Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) analyses. The environmental effects of the parent compounds and transformation products were evaluated using an overall toxicity approach that considered aquatic acute and chronic toxicity on Brachionus calyciflorus and Ceriodaphnia dubia as well as mutagenesis and genotoxicity on bacterial strains. The results revealed that both parent drugs did not show high acute and chronic toxicity for the organisms utilized in the bioassays while, chronic exposure to chlorine derivatives caused inhibition of growth population on rotifers and crustaceans. A mutagenic potential was found for all the compounds investigated. © 2013 Elsevier B.V.

[spa] La presente tesis se ha centrado en la mejora de lipasas de Pseudomonas sp. y en la evaluación de las lipasas microbianas para su aplicación en la síntesis de biodiésel. Diferentes aspectos de este proceso han sido investigados para promover la introducción de la biocatálisis enzimática en la nueva era de la industria, mediante el desarrollo de procesos rentables y sostenibles. Se aplicaron nuevas técnicas de mutagénesis basadas en el diseño racional para mejorar la termoestabilidad de una lipasa psicrófila de Pseudomonas sp. (LipC) con el fin de poderla aplicar en procesos con temperaturas más altas. La variante obtenida (LipCmut) y la proteína salvaje, además de otras lipasas de Pseudomonas sp., fueron eficientemente inmovilizadas en soportes hidrofóbicos. Se puso a punto un protocolo de inmovilización, basado en la adsorción de la enzima a soportes porosos, rápido, de fácil reproducción y de bajo coste, y, por lo tanto, adecuado para un eventual escalado del proceso. Las preparaciones enzimáticas fueron ensayadas en la transesterificación de trioleína como modelo a pequeña escala de síntesis de biodiésel. Cambiando el vector de expresión, mejorando el método de obtención de las lipasas y optimizando las condiciones de transesterificación, se incrementaron las bajas producciones obtenidas inicialmente. Se evaluó el contendido de agua como parámetro fundamental en la síntesis de biodiésel y se introdujo, con éxito, la novedosa alternativa de las lipasas solubles aplicadas en este proceso. Una nueva lipasa soluble desarrollada por Novozymes, Callera Trans L, se ensayó en la transesterificación de aceites crudos, como ejemplo de materia prima más barata. Finalmente, se desarrolló un sistema combinado donde los fosfolípidos (o gomas), presentes en los aceites crudos, son hidrolizados por unas fosfolipasas y los ácidos grasos liberados se esterifican con el metanol para la síntesis final de biodiésel conducida por una lipasa soluble. Esta combinación de desgomado y transesterificación lleva a un proceso de síntesis de biodiésel más barato, más respetuoso con el medio ambiente y, por lo tanto, más sostenible., [eng] This thesis is focused on the improvement and evaluation of microbial lipases for biodiesel synthesis. First, the thermal stability of LipC, lipase from Pseudomonas sp. (P. aeruginosa ) was improved. LipC is an enzyme with an interesting psychrophilic behavior and its biochemical characteristics make it a very promising enzyme for biotechnological applications, nevertheless it shows a low thermal stability. Based on the relation structure-function of the protein, a mutagenesis by rational design and saturation of different amino acid positions was performed. A mutant, showing a 7-fold increase in thermal stability at 60°C respect to the wild-type LipC, was obtained. Moreover, this variant kept its cold-adapted properties presenting an optimal activity between 4 and 20°C. Further, immobilization by adsorption of these lipases on different porous supports was carried out. The immobilization is a technique for enzyme stabilization when they have to be applied in industrial biocatalytic processes, such as synthesis of biodiesel. For the immobilization by adsorption, a very fast, efficient, low cost technique, therefore suitable for an eventual process scale, two polypropylene matrices and a silica were tested. Immobilization was performed from the previously obtained mutant (LipCmut) the wild-type (LipC ), its mesophilic homologous (LipA) and another lipase (LipI.3) from Pseudomonas CR611 (P. fluorescens). All lipases were successfully immobilized on the supports tested defining polypropylene Accurel MP1000 as the best carrier. Subsequently immobilized preparations were tested in transformation of triolein into FAMEs (Fatty Acid Metyl Esters) as a small scale model of biodiesel synthesis. LipA, LipCmut and LipC resulted positive for this reaction, but with moderate efficiency. LipCmut and LipC FAMEs production was improved changing their expression vector, increasing the level of protein production and recovery and optimizing the transesterification conditions, in terms of water and methanol content. in this manner, an enormous increase in available lipolytic activity was achieved, corresponding to a higher activity of the immobilized preparations, which were successfully applied for transesterification of soybean oil. The water content in the transesterification reactions was studied in collaboration with Novozymes (Denmark ), where was evaluated a new soluble lipase, Callera Trans L, in the production of biodiesel. Water was found to be an essential factor in the transesterification reaction and a particular reaction mechanism for this lipase, depending on the water and on the free fatty acids, was described. The possibility to apply soluble lipases in a process, so far been carried out only with immobilized enzymes, was an innovation tested also for LipC and LipCmut. The growth culture supernatant, where these extracellular lipases are secreted, was prepared and characterized for the main properties necessary for a transesterification reaction (temperature and methanol resistance). LipC and LipCmut in soluble form resulted to be able to form of FAMEs starting from soybean oil. Moreover, soluble Trans Callera L was tested in the transesterification of crude soybean oil, a substrate difficult for this reaction due to its high content of impurities (free fatty acids and phospholipids). Callera Trans L, showed to be extremely efficient also with this raw material. This result opened the way for the next research which aimed to combine the transesterification with an enzymatic degumming process to remove phospholipids from crude soybean oil. The degumming was carried out with phospholipases that hydrolize phospholipids present in the raw material and prepare it for an efficient transesterification carried out by the lipase. The two processes were performed in the same step, which means an increased sustainability and a substantial reduction in the costs of biodiesel production process.

Glycosynthases are engineered glycoside hydrolases that in suitable reaction conditions promote the synthesis of oligosaccharides with exquisite stereoselectivity and enhanced regioselectivity, if compared to traditional chemical methods. This approach was demonstrated to be successful in a number of cases including β-glycosynthases acting at the termini or within an oligosaccharide chain ( exo - and endo -glycosynthases, respectively) and, more recently, α-glycosynthases. This led to the production of a vast repertoire of products that include poly- and oligosaccharides, glycoconjugates, and glycopeptides. These molecules can be used as ligands of glycoside hydrolases, for the characterization of therapeutic enzymes, and as leads of drugs for the pharmaceutical industry. In this panorama, hyperthermophilic organisms, which thrive at temperatures as high as 80 °C, which usually impede the growth of other living forms, have been used in the development of interesting novel glycosynthases. In fact, the extreme stability of these catalysts to extremes of pH and high concentrations of organics has allowed the exploration of novel reaction conditions, revealing new avenues for enzyme-catalyzed oligosaccharide synthesis.

The dipeptidyl aminopeptidase-like protein 10 (DPP10) is a type II transmembrane protein homologue to the serine protease DPPIV/CD26 but enzymatically inactive. In the mammalian brain, DPP10 forms a complex with voltage-gated potassium channels of the Kv4 family, regulating their cell surface expression and biophysical properties. DPP10 is a glycoprotein containing eight predicted N-glycosylation sites in the extracellular domain. In this study we investigated the role of N-glycosylation on DPP10 trafficking and functional activity. Using site-directed mutagenesis (N to Q) we showed that N-glycosylation occured at six positions. Glycosylation at these specific residues was necessary for DPP10 trafficking to the plasma membrane as observed by flow cytometry. The surface expression levels of the substitutions N90Q, N119Q, N257Q and N342Q were reduced by more than 60%. Hence the interaction with the Kv4.3/KChIP2a channel complex was disrupted preventing the hastening effect of wild type DPP10 on current kinetics. Interestingly, N257 was crucial for this function and its substitution to glutamine completely blocked DPP10 sorting to the cell surface and prevented DPP10 dimerization. In summary, we demonstrated that glycosylation was necessary for both DPP10 trafficking to the cell surface and functional interaction with Kv4 channels.

The oxido-reductases are enzymes able to catalyze the electron removal from oxidizer to reducer. Laccase and tyrosinase, multicopper enzymes oxidizing phenolic compounds and aromatic diamines by using molecular oxygen as the electron acceptor, belong to the oxido-reductases group. They are enzymes commonly found in plants, fungi, bacteria and insects. Laccase substrates have large versatility and this makes these enzymes highly interesting for various biotechnological and industrial applications. Though laccases and tyrosinases can be directly purified from natural producers, protein recombinant expression in a suitable host organism is the most profitable procedure to obtain, industrially, these enzymes in high quantities and with high purity levels. The presence of oxidative enzyme activities has been also associated to microbial detoxification of olive mill wastewaters (OMWs) after their treatment with selected basidio- and asco-mycetes. The objectives of this study were to characterize genes coding for oxidoreductases in Basidiomycetes, in particular the ERY3 and ERY4 laccase genes of Pleurotus eryingii and the PPO2 gene coding for the Agaricus bisporus tyrosinase, and to select non-conventional yeast able to produce new oxidoreductase enzymes. The ERY3 gene, coding for a P. eryngii laccase, was isolated, cloned and transformed in Saccharomyces cerevisiae cells. The production of recombinant laccase was assayed with a colorimetric test in presence of ABTS, on SDS-PAGE and Western blot analysis. The recombinant yeasts cells were immobilized in calcium alginate beads and the immobilization parameters (inoculum, sodium alginate and CaCl2 concentration) were optimized. The ERY4 laccase gene of P. eryngii was expressed in S. cerevisiae and the recombinant laccase resulted to be not biologically active. This gene was thus modified in order to study the role of its N- and C-terminal regions in determining enzyme catalytic properties. ERY4 gene was subjected to a mutational analysis following these approaches: i) C-terminal progressive deletion to study the role of specific amino acid residues at the C-terminus of Ery4 protein, ii) site-directed mutagenesis of the C-terminal region, iii) chimerical laccases derived from the substitution of both its terminal regions with the corresponding regions of ERY3 gene. The fourteen recombinant genes were separately expressed in S. cerevisiae. 2 Almost all mutant recombinant isoforms showed that they possess enzymatic activity and affinities for different substrates. Genes encoding the laccase isoforms which showed the best enzymatic performances on different substrates, were expressed in S. cerevisiae by displaying the recombinant enzyme on yeast cell surface. The biochemical characterization, immunodetection and Western blot analysis of displayed proteins were performed. To our knowledge, this is the first example of a functional laccase immobilized on yeast surface. The PPO2 tyrosinase gene of Agaricus bisporus was expressed in S. cerevisiae cells and the produced protein was purified by using affinity chromatography and biochemically characterized. To our knowledge, this is the first time that an A. bisporus tyrosinase was expressed in heterologous host S. cerevisiae and in biologically active form. In order to select, from biotechnologically relevant habitat, microorganisms producing oxidoreductases, the effluents from 5 different local olive mills were collected. Three hundred isolates were identified according to their rDNA sequence. Twelve on the 300 identified isolates, belonging to Candida membranifaciens, C. tropicalis, Geotricum candidum, Pichia fermentans, P. holstii and S. cerevisiae species, demonstrated that they were able to use OMW as unique nutrient source for their growth. Phenolic compound concentration and antimicrobial activity was rapidly and significantly decreased by the G. candidum strains. The physiological properties of the described G. candidum isolates confirmed the potential of these yeasts to produce a wide range of extracellular enzymes. One of the selected G. candidum strains was chosen as a model to set up an immobilized system of viable cells for mill waste bioremediation. Concentration of G. candidum as free cells in the medium, the COD values, the concentration of phenolic compounds, the medium decolourization and the antimicrobial activity were measured at estabished interval times for both immobilized and free G. candidum biomasses. Indeed, the COD removal and phenolics degradation by calcium alginate entrapped cells were higher than those of free cells, because the immobilization dramatically reduces the extracellular protease(s) activity, thus increasing the stability of microbial secreted oxidases. L’avvento delle biotecnologie ha consentito di produrre grandi quantitativi di enzimi ad elevati livelli di purezza da utilizzare in diversi processi industriali. Questa potenzialità ha permesso di aumentare l’efficienza delle reazioni, ridurre i costi di produzione ed i prodotti reflui di lavorazione. Le ossidasi, in particolare laccasi e tirosinasi, sono enzimi ubiquitari che trovano applicazioni biotecnologiche in ambito biomedico, farmaceutico, cosmetico, industriale ed ambientale. Scopo di questo progetto di ricerca è l’isolamento, la caratterizzazione funzionale di laccasi e tirosinasi di origine fungina, l’espressione di tali proteine in forma ricombinante e lo sviluppo di loro potenziali applicazioni in ambito biotecnologico ed industriale. Per quanto riguarda le laccasi da basidiomiceti, il gene ERY3 codificante un’isoforma di laccasi, è stato isolato dal fungo basidiomicete Pleurotus eryngii, ed è stato espresso in forma eterologa e funzionalmente attiva nel lievito Saccharomyces cerevisiae. Inoltre, è stata ottimizzata una procedura di immobilizzazione delle cellule di lievito ricombinanti in sferette di calcio alginato. Il gene ERY4 di P. eryngii, codificante una laccasi, è stato espresso in forma ricombinante in S. cerevisiae, ma la proteina prodotta si è dimostrata non biologicamente attiva. Il gene ERY4 è stato sottoposto ad un’analisi mutazionale secondo i tre seguenti approcci: i) delezione progressiva della parte C-terminale della proteina codificata, ii) mutagenesi puntiforme della medesima regione, iii) sostituzione nelle porzioni N e C terminale della laccasi Ery4 con le equivalenti regioni dell’isoforma Ery3. I geni ricombinanti sono stati espressi in S. cerevisiae. Le laccasi ricombinanti prodotte hanno mostrato di possedere, nella quasi totalità, attività enzimatica e peculiari affinità per una serie di differenti substrati. Due delle isoforme mutanti prodotte sono state immobilizzate sulla superficie esterna delle cellule di lievito mediante fusione con proteine endogene di parete. Le proteine immobilizzate sono state caratterizzate da un punto di vista biochimico e visualizzate correttamente sulla superficie esterna delle cellule ricombinanti di lievito. La tirosinasi Ppo2 di Agaricus bisporus è stata prodotta nel lievito S. cerevisiae, purificata mediante cromatografia di affinità e caratterizzata biochimicamente. Secondo quanto riportato in letteratura, questa è la prima espressione di una tirosinasi di origine fungina nel lievito S. cerevisae in forma biologicamente attiva. 4 Allo scopo di isolare microorganismi produttori di ossido reduttasi da nicchie ecologiche di interese biotecnologico, sono stati prelevati dei campioni di acque reflue di lavorazione (OMW) da cinque differenti oleifici del Salento (Puglia). E’ stata isolata la popolazione microbica (lieviti e muffe) e un campione della stessa è stato identificato molecolarmente. I lieviti sono stati selezionati sulla base della loro capacità di usare le OMW come unica fonte di nutrimento. Un ceppo di Geotricum candidum, utilizzato per fermentare un campione di OMW, ha mostrato la capacità di ridurre significativamente la concentrazione dei composti fenolici e l’attività antimicrobica dello stesso. Il ceppo di G. candidum è stato utilizzato per la messa a punto di un sistema di immobilizzazione di cellule vive per la riduzione dell’impatto ambientale dei reflui oleari. La rimozione della COD e la degradazione dei fenoli risulta più elevata per le cellule immobilizzate rispetto alle stesse in forma libera, perché l’immobilizzazione riduce notevolmente la presenza di proteasi extracellulari, aumentando così la stabilità delle ossidasi microbiche. Dottorato di ricerca in Biotecnologie vegetali

[cat] Les miopaties miofibril•lars (MFM) són un grup heterogeni de malalties musculars progressives, que es caracteritzen morfològicament per la dissolució focal de les miofibril•les, l’acumulació dels productes que resulten de la degradació miofibril•lar i l’expressió ectòpica de múltiples proteïnes en forma d’agregats intracitoplasmàtics insolubles. Les MFM estan causades per mutacions a diferents gens, la majoria dels quals codifiquen per proteïnes sarcomèriques del disc Z o bé per proteïnes que resulten indispensables per mantenir-ne la seva integritat. Aquestes són la desmina, l’αB-cristal•lina, la miotilina, la ZASP (Z-band alternatively spliced PDZ motif-containing protein) i la filamina C. Altres gens que s’inclouen en classificacions més àmplies de les MFM són el gen del FHL1 (four-and-a-half LIM domain 1), el gen del BAG3 (Bcl-2- associated athanogene-3) o el gen de la plectina.