7 research outputs found
Total synthesis of SUMO-2/3 dimers
Les progrĂšs du gĂ©nie gĂ©nĂ©tique ont permis lâexpression dâun nombre considĂ©rable de protĂ©ines dans des cellules pour Ă©tudier les mĂ©canismes du vivant, dĂ©velopper des approches thĂ©rapeutiques et de biotechnologie. Ces protĂ©ines, dites recombinantes, prĂ©sentent certaines limitations associĂ©es Ă leur mode de production, notamment dans le choix des acides aminĂ©s de la sĂ©quence qui est limitĂ© aux vingt acides aminĂ©s naturellement codĂ©s par le gĂ©nome. Une autre limitation de ces mĂ©thodes est la difficultĂ© dâincorporer des acides aminĂ©s porteurs de modifications post-traductionnelles (MPT). Ces limitations ont motivĂ© les chimistes organiciens Ă dĂ©velopper de nouvelles mĂ©thodologies de synthĂšse afin dâaccĂ©der Ă ces macromolĂ©cules dâintĂ©rĂȘt biologique. Si la synthĂšse chimique sâest clairement imposĂ©e depuis quelques annĂ©es comme une alternative crĂ©dible Ă lâutilisation de mĂ©thodes biologiques pour la synthĂšse de protĂ©ines, la synthĂšse chimique de protĂ©ines de grande taille (au-delĂ de 100 acides aminĂ©s) reste un vĂ©ritable dĂ©fi.SUMO est une MPT apparentĂ©e Ă lâubiquitine constituĂ©e dâenviron 90 acides aminĂ©s. Cinq isoformes diffĂ©rentes de SUMO ont Ă©tĂ© identifiĂ©es Ă ce jour chez lâHomme. Parmi celles-ci, SUMO-2 et SUMO-3 sont exprimĂ©es par tous les types cellulaires et similaires Ă 97% au niveau de leur sĂ©quence. Lâobjectif de ce projet de thĂšse a Ă©tĂ© dâĂ©tudier la conformation et les propriĂ©tĂ©s biochimiques de conjuguĂ©s SUMO-2 et SUMO-3, ainsi que de rĂ©aliser la synthĂšse totale des diffĂ©rentes combinaisons de dimĂšres SUMO-2 / SUMO-3. La taille de ces constructions molĂ©culaires se situe Ă la limite haute des protĂ©ines pouvant ĂȘtre produite par synthĂšse chimique. Pour atteindre ces objectifs, nous avons dĂ©veloppĂ© des stratĂ©gies de synthĂšse performantes en one-pot et des rĂ©actions de dĂ©sulfurisation sĂ©lectives permettant dâaccĂ©der aux protĂ©ines cibles Ă lâĂ©chelle du milligramme.Nous montrons au cours de ses recherches que les protĂ©ines SUMO-2 et SUMO-3 ont des propriĂ©tĂ©s structurelles et biochimiques spĂ©cifiques, et ne doivent pas ĂȘtre considĂ©rĂ©es comme redondantes. Nous dĂ©crivons Ă©galement pour la premiĂšre fois la synthĂšse totale de dimĂšres SUMO-2/3. Ce travail devrait aider Ă mieux comprendre le rĂŽle spĂ©cifique des modifications SUMO-2 et/ou SUMO-3 sur la structure et la fonction des protĂ©ines qui sont la cible de ces MPT.Advances in genetic engineering enabled the expression of a huge number of proteins in cells to study biological mechanisms and to develop novel therapeutic and biotechnological approaches. Nevertheless, recombinant strategy of protein production has inherent limitations. In particular, the sequence composition is usually limited to the twenty naturally encoded amino acids.Moreover, the incorporation of amino acids carrying post-translational modifications (PTM) appears difficult. This second limitation motivated organic chemists to develop new synthetic methodologies to access biologically relevant macromolecules. Although chemical synthesis clearly emerged in recent years as a useful alternative to biological methods, the chemical synthesis of large proteins (beyond 100 amino acids) remains a real challenge.SUMO is an ubiquitin-like PTM consisting of approximately 90 amino acids. Five different SUMO isoforms have been identified in humans so far. Among them SUMO-2 and SUMO-3 are expressed by all cell types and are 97% similar in sequence. The aim of this thesis was to study the conformation and the biochemical properties of SUMO-2 and SUMO-3 conjugates. The total synthesis of the different combinations of SUMO-2 / SUMO-3 dimers was also carried out. The size of these molecules coincides with the upper limit of the proteins that can be produced by chemical synthesis. Considering this, we developed powerful one-pot synthetic strategies and selective desulfurization reactions to access the target proteins on a milligram scale.During this research, we show that the SUMO-2 and SUMO-3 proteins have specific structural and biochemical properties and should not be considered redundant. We also described the first total synthesis of SUMO-2/3 dimers. This work will help to better understand the specific role of SUMO-2 and/or SUMO-3 modifications on the structure and function of the SUMOylated proteins
SynthĂšse totale de dimĂšres SUMO-2/3
Advances in genetic engineering enabled the expression of a huge number of proteins in cells to study biological mechanisms and to develop novel therapeutic and biotechnological approaches. Nevertheless, recombinant strategy of protein production has inherent limitations. In particular, the sequence composition is usually limited to the twenty naturally encoded amino acids.Moreover, the incorporation of amino acids carrying post-translational modifications (PTM) appears difficult. This second limitation motivated organic chemists to develop new synthetic methodologies to access biologically relevant macromolecules. Although chemical synthesis clearly emerged in recent years as a useful alternative to biological methods, the chemical synthesis of large proteins (beyond 100 amino acids) remains a real challenge.SUMO is an ubiquitin-like PTM consisting of approximately 90 amino acids. Five different SUMO isoforms have been identified in humans so far. Among them SUMO-2 and SUMO-3 are expressed by all cell types and are 97% similar in sequence. The aim of this thesis was to study the conformation and the biochemical properties of SUMO-2 and SUMO-3 conjugates. The total synthesis of the different combinations of SUMO-2 / SUMO-3 dimers was also carried out. The size of these molecules coincides with the upper limit of the proteins that can be produced by chemical synthesis. Considering this, we developed powerful one-pot synthetic strategies and selective desulfurization reactions to access the target proteins on a milligram scale.During this research, we show that the SUMO-2 and SUMO-3 proteins have specific structural and biochemical properties and should not be considered redundant. We also described the first total synthesis of SUMO-2/3 dimers. This work will help to better understand the specific role of SUMO-2 and/or SUMO-3 modifications on the structure and function of the SUMOylated proteins.Les progrĂšs du gĂ©nie gĂ©nĂ©tique ont permis lâexpression dâun nombre considĂ©rable de protĂ©ines dans des cellules pour Ă©tudier les mĂ©canismes du vivant, dĂ©velopper des approches thĂ©rapeutiques et de biotechnologie. Ces protĂ©ines, dites recombinantes, prĂ©sentent certaines limitations associĂ©es Ă leur mode de production, notamment dans le choix des acides aminĂ©s de la sĂ©quence qui est limitĂ© aux vingt acides aminĂ©s naturellement codĂ©s par le gĂ©nome. Une autre limitation de ces mĂ©thodes est la difficultĂ© dâincorporer des acides aminĂ©s porteurs de modifications post-traductionnelles (MPT). Ces limitations ont motivĂ© les chimistes organiciens Ă dĂ©velopper de nouvelles mĂ©thodologies de synthĂšse afin dâaccĂ©der Ă ces macromolĂ©cules dâintĂ©rĂȘt biologique. Si la synthĂšse chimique sâest clairement imposĂ©e depuis quelques annĂ©es comme une alternative crĂ©dible Ă lâutilisation de mĂ©thodes biologiques pour la synthĂšse de protĂ©ines, la synthĂšse chimique de protĂ©ines de grande taille (au-delĂ de 100 acides aminĂ©s) reste un vĂ©ritable dĂ©fi.SUMO est une MPT apparentĂ©e Ă lâubiquitine constituĂ©e dâenviron 90 acides aminĂ©s. Cinq isoformes diffĂ©rentes de SUMO ont Ă©tĂ© identifiĂ©es Ă ce jour chez lâHomme. Parmi celles-ci, SUMO-2 et SUMO-3 sont exprimĂ©es par tous les types cellulaires et similaires Ă 97% au niveau de leur sĂ©quence. Lâobjectif de ce projet de thĂšse a Ă©tĂ© dâĂ©tudier la conformation et les propriĂ©tĂ©s biochimiques de conjuguĂ©s SUMO-2 et SUMO-3, ainsi que de rĂ©aliser la synthĂšse totale des diffĂ©rentes combinaisons de dimĂšres SUMO-2 / SUMO-3. La taille de ces constructions molĂ©culaires se situe Ă la limite haute des protĂ©ines pouvant ĂȘtre produite par synthĂšse chimique. Pour atteindre ces objectifs, nous avons dĂ©veloppĂ© des stratĂ©gies de synthĂšse performantes en one-pot et des rĂ©actions de dĂ©sulfurisation sĂ©lectives permettant dâaccĂ©der aux protĂ©ines cibles Ă lâĂ©chelle du milligramme.Nous montrons au cours de ses recherches que les protĂ©ines SUMO-2 et SUMO-3 ont des propriĂ©tĂ©s structurelles et biochimiques spĂ©cifiques, et ne doivent pas ĂȘtre considĂ©rĂ©es comme redondantes. Nous dĂ©crivons Ă©galement pour la premiĂšre fois la synthĂšse totale de dimĂšres SUMO-2/3. Ce travail devrait aider Ă mieux comprendre le rĂŽle spĂ©cifique des modifications SUMO-2 et/ou SUMO-3 sur la structure et la fonction des protĂ©ines qui sont la cible de ces MPT
Comment on âN-terminal Protein Tail Acts as Aggregation Protective Entropic Bristles: The SUMO Caseâ
International audienceSUMO-2 protein, SUMO-2 core domain, and the tail peptide corresponding to the first 14 residues were produced by chemical synthesis, and their secondary structures were analyzed by circular dichroism. The CD spectra of SUMO-2 and SUMO-2 core domain show distinct features and α-helical contents. In particular, the presence of the disordered tail in SUMO-2 lowers the α-helical content of the protein compared with SUMO-2 core domain and also explains the shift in the position of the minimum around 208 nm
Comment on « N-terminal Protein Tail Acts as Aggregation Protective Entropic Bristles : The SUMO Case »
We provide a robust protocol for the far-UV CD analysis of SUMO-2 protein and SUMO-2 core domain as well as all the data as supplementary files for those interested by the study of SUMO protein conformation.These analyses were performed after having read the report published in 2014 by Grana-Montes, R. et al. (Grana-Montes, R.; Marinelli, P.; Reverter, D.; Ventura, S., N-terminal protein tails act as aggregation protective entropic bristles: the SUMO case. Biomacromolecules 2014, 15 (4), 1194-203. DOI: 10.1021/bm401776z).<br /
The Problem of Aspartimide Formation During Protein Chemical Synthesis Using SEA-Mediated Ligation
Aspartimide formation often complicates the solid phase synthesis of peptides. Much less discussed is the potential occurrence of this side-reaction during the coupling of peptide segments using chemoselective peptide bond forming reactions such as the native chemical ligation and extended methods. Here we describe how to manage this problem using bis(2-sulfenylethyl)amido (SEA)-mediated ligation and SUMO-2/SUMO-3 as protein targets.<br /
Total Chemical Synthesis of All SUMO-2/3 Dimer Combinations
International audienceOne hallmark of protein chemical synthesis is its capacity to access proteins that living systems can hardly produce. This is typically the case for proteins harboring post-translational modifications such as ubiquitin or ubiquitin-like modifiers. Various methods have been developed for accessing polyubiquitin conjugates by semi- or total synthesis. Comparatively, the preparation of small-ubiquitin-like modifier (SUMO) conjugates, and more particularly of polySUMO scaffolds, is much less developed. We describe hereinafter a synthetic strategy for accessing all SUMO-2/3 dimer combinations