Proteome analysis of grape skins during ripening

International audienceThe characterization of proteins isolated from skin tissue is apparently an essential parameter for understanding grape ripening as this tissue contains the key compounds for wine quality. It has been particularly difficult to extract proteins from skins for analysis by two-dimensional electrophoresis gels and, therefore, a protocol for this purpose has been adapted. The focus was on the evolution of the proteome profile of grape skin during maturation. Proteome maps obtained at three stages of ripening were compared to assess the extent to which protein distribution differs in grape skin during ripening. The comparative analysis shows that numerous soluble skin proteins evolve during ripening and reveal specific distributions at different stages. Proteins involved in photosynthesis, carbohydrate metabolisms, and stress response are identified as being over-expressed at the beginning of colour-change. The end of colour-change is characterized by the over-expression of proteins involved in anthocyanin synthesis and, at harvest, the dominant proteins are involved in defence mechanisms. In particular, increases in the abundance of different chitinase and ß-1,3-glucanase isoforms were found as the berry ripens. This observation can be correlated with the increase of the activities of both of these enzymes during skin ripening. The differences observed in proteome maps clearly show that significant metabolic changes occur in grape skin during this crucial phase of ripening. This comparative analysis provides more detailed characterization of the fruit ripening process

Analysis of the Flavobacterium psychrophilum outer-membrane subproteome and identification of new antigenic targets for vaccine by immunomics

International audienceFlavobacterium psychrophilum is an important infectious Gram-negative bacterium causing cold-water disease (CWD) and rainbow trout fry syndrome. Outer-membrane proteins (OMPs) are key molecules with regard to the interface between the cell and its environment. Therefore, we sought to define the outer-membrane (OM) subproteome of F. psychrophilum in order to gain insight into the biology and pathogenesis of this bacterium and to identify the dominant antigens targeted by the rainbow trout (Oncorhynchus mykiss) immune system during infection. First, OMs were prepared from a cell-envelope suspension by differential Sarkosyl (sodium lauryl sarcosinate) solubility. We then isolated the OMPs and identified 36 proteins from 34 spots resolved by two-dimensional electrophoresis and LC-MS/MS. An immunoproteomic approach using antibodies from CWD-convalescent rainbow trout was then used to identify 25 immunoreactive F. psychrophilum antigens that may be relevant in pathogenesis and diagnosis. These included the previously characterized surface-exposed OMPs OmpA, OmpH/P18 and FspA, as well as newly described antigenic proteins. This study provides a number of novel candidate proteins for developing vaccine(s) against flavobacteriosis infection in aquaculture

Integrase-LEDGF/p75 complex triggers the formation of biomolecular condensates that modulate HIV-1 integration efficiency in vitro

International audienceThe pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-PhaseSeparation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS in vitro andif IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the in vitro IN activities. While the 3' processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes

In Silico, In Vitro and In Cellulo Models for Monitoring SARS-CoV-2 Spike/Human ACE2 Complex, Viral Entry and Cell Fusion

International audienceSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent responsible for the recent coronavirus disease 2019 (COVID-19) pandemic. Productive SARS-CoV-2 infection relies on viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). Indeed, viral entry into cells is mostly mediated by the early interaction between the viral spike protein S and its ACE2 receptor. The S/ACE2 complex is, thus, the first contact point between the incoming virus and its cellular target; consequently, it has been considered an attractive therapeutic target. To further characterize this interaction and the cellular processes engaged in the entry step of the virus, we set up various in silico, in vitro and in cellulo approaches that allowed us to specifically monitor the S/ACE2 association. We report here a computational model of the SARS-CoV-2 S/ACE2 complex, as well as its biochemical and biophysical monitoring using pulldown, AlphaLISA and biolayer interferometry (BLI) binding assays. This led us to determine the kinetic parameters of the S/ACE2 association and dissociation steps. In parallel to these in vitro approaches, we developed in cellulo transduction assays using SARS-CoV-2 pseudotyped lentiviral vectors and HEK293T-ACE2 cell lines generated in-house. This allowed us to recapitulate the early replication stage of the infection mediated by the S/ACE2 interaction and to detect cell fusion induced by the interaction. Finally, a cell imaging system was set up to directly monitor the S/ACE2 interaction in a cellular context and a flow cytometry assay was developed to quantify this association at the cell surface. Together, these different approaches are available for both basic and clinical research, aiming to characterize the entry step of the original SARS-CoV-2 strain and its variants as well as to investigate the possible chemical modulation of this interaction. All these models will help in identifying new antiviral agents and new chemical tools for dissecting the virus entry step

Cassette recruitment in the chromosomal Integron of Vibrio cholerae

Posté sur BioRxiv le 24 novembre 2020Integrons are genetic systems conferring to bacteria a rapid adaptation capability. The integron integrase is able to capture, stockpile and shuffle novel functions embedded in cassettes. This involves the recognition of both substrates, the attI site, and the cassette associated attC sites. Integrons can be sedentary and chromosomally located (SCI) or, carried by conjugative plasmids (Mobile Integron, MI), hence favoring their dissemination among bacteria. Here, for the first time, we investigate the cassette recruitment in the Vibrio cholerae SCI during conjugation and natural transformation. We demonstrated that horizontally transferred cassette can be recruited inside the chromosomal integron. The endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of single-stranded cassettes during conjugation and natural transformation, to mediate significant cassette insertion. We demonstrate that the attIA insertion is preferential, despite the presence of 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to adaptive selection. We also show that the RecA protein is critical for cassette recruitment in V. cholerae SCI but not in MIs. Moreover, a contrario to MIs, the V. cholerae SCI is not active in others bacterial hosts. MIs might have evolved from the SCIs by overcoming host factors, which would explain their large dissemination in bacteria and their role in the antibioresistance expansion

Development of a method for the extraction and analysis of grape skin proteins strongly bound to cell walls

Aim: To better understand the protein composition of grape skin cell walls, we have developed a method to analyse the strongly bound cell wall proteins. Methods and results: The protocol was developed with grape skins at full maturity. The critical steps of this protocol were : (i) the elimination of cellular aggregates, (ii) the elimination of soluble proteins, and (iii) the localization of the identified proteins within the cell wall. To verify whether these three conditions were met, the decrease in the quantity of cellular aggregates was followed by optical microscopy, the removal of soluble proteins was measured by chemical assay, and the presence of proteins located in cell walls was demonstrated by extensive bioinformatic analysis. The process made it possible to obtain a four-fold reduction in the amount of cellular aggregates, a reduction in the concentration of soluble proteins below the method detection limit, and a high proportion of proteins predicted to be secreted (79 %). Conclusion: The protocol described in this paper constitutes the first method to analyse proteins strongly bound to cell walls in grape skins. However, this method excludes the identification of labile proteins or proteins weakly bound to the cell wall. Significance and impact of the study: This protocol can be used for studying the role that strongly bound cell wall proteins play in development and defense processes in grape skins

Two-long terminal repeat (LTR) DNA circles are a substrate for HIV-1 integrase

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