In vitro modification of human centromere protein CENP-C fragments by small ubiquitin-like modifier (SUMO) protein: Definitive identification of the modification sites by tandem mass spectrometry analysis of the isopeptides

Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif {psi}KXE/D, where {psi} represents a large hydrophobic amino acid, and X represnts any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process

Engineering microparticles based on solidified stem cell secretome with an augmented pro-angiogenic factor portfolio for therapeutic angiogenesis

Tissue (re)vascularization strategies face various challenges, as therapeutic cells do not survive long enough in situ, while the administration of pro-angiogenic factors is hampered by fast clearance and insufficient ability to emulate complex spatiotemporal signaling. Here, we propose to address these limitations by engineering a functional biomaterial capable of capturing and concentrating the pro-angiogenic activities of mesenchymal stem cells (MSCs). In particular, dextran sulfate, a high molecular weight sulfated glucose polymer, supplemented to MSC cultures, interacts with MSC-derived extracellular matrix (ECM) components and facilitates their co-assembly and accumulation in the pericellular space. Upon decellularization, the resulting dextran sulfate-ECM hybrid material can be processed into MIcroparticles of SOlidified Secretome (MIPSOS). The insoluble format of MIPSOS protects protein components from degradation, while facilitating their sustained release. Proteomic analysis demonstrates that MIPSOS are highly enriched in pro-angiogenic factors, resulting in an enhanced pro-angiogenic bioactivity when compared to naïve MSC-derived ECM (cECM). Consequently, intravital microscopy of full-thickness skin wounds treated with MIPSOS demonstrates accelerated revascularization and healing, far superior to the therapeutic potential of cECM. Hence, the microparticle-based solidified stem cell secretome provides a promising platform to address major limitations of current therapeutic angiogenesis approaches

Étude de la régulation des sialo-conjugués au cours de l'embryogenèse du poisson zèbre

L'acide sialique joue un rôle primordial dans les phénomènes de reconnaissance inter-cellulaires. Au cours de ce travail, les fonctions biologiques des épitopes sialylés régulés au cours du développement, en relation avec la régulation spatiale et temporelle des a2-8 sialyltransferases (ST8sia) au cours de l'embryogenèse du poisson zèbre, ont été étudiées par une combinaison d'analyses chimiques et spectrométriques. Dans un premier temps, les glycolipides, N-glycannes et O-glycannes ont été séquentiellement purifiés d'extraits totaux de poissons zèbres et étudiés par une combinaison de spectrométrie de masse, de digestion enzymatique, de dérivation chimique et de résonance magnétique nucléaire (RMN). Nous avons ainsi mis en évidence l'existence de profils de glycosylation très particuliers dominés par des composés hautement sialylés. Dans un deuxième temps, le suivi de la glycosylation au cours du développement a révélé que la polysialylation des glycoconjugués était différentiellement régulée en fonction de leur nature. Sur ces bases structurales, nous avons suivi l'expression des gènes codant pour les ST8Sias du poisson zèbre par PCR quantitative. Nous émettons l'hypothèse que les glycoprotéines polysialylées observées dans l'embryon sont héritées de la mère, tandis que les glycolipides sialylés sont synthétisés de novo par l'embryon. Enfin, l'observation de la plupart des ST8Sias par hybridation in situ montre qu'elles sont exprimées dans le système nerveux central dés les premiers stades de développement embryonnaire. L'ensemble de nos travaux démontrent l'utilisation du poisson zèbre en tant que modèle animal d'étude cohérent de la glycosylation.The nine-carbon sugar, sialic acid, plays important roles as the recognition epitopes in cell-cell communications. ln this thesis work, the biological functions of developmentally regulated sialylation were investigated using a combination of chemical and mass spectrometry (MS) analyses. Glycomic survey mapping permitted to identify unusual structures typified by complex sialylation patterns. The analysis of sialyltransferase and sialidase activities along development led to a working model, which proposes that the oligosialic acids from glycoproteins are maternally inherited (synthesized before fertilization) and exocytosis out of the embryo into the perivitelline space where it undergoes catabolism, whereas oligosialic acids on the glycolipids are de novo synthesized by the increasingly expressed ST8sia in the embryos. Furthermore, through whole mount in-situ hybridization, most of the ST8sia were shown to be expressed in the nervous system during early embryogenesis, with different onset and locations. ln summary, the precise MS and MS/MS-based glycomic profiles, together with detailed structural determination, quantitative analysis of the developmentally regulated oligosialylation pattern, and the spatial and temporal expression profiling of ST8sia of zebrafish, collectively provides new sialoglycobiology insights for neuroscientists at the molecular level and highlighted the significance and complicated regulation of oligosialylation in early neuronal development.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

An embeddable molecular code for Lewis X modification through interaction with fucosyltransferase 9

A 29-amino acid sequence in the N-terminal domain of LAMP-1 promotes its Lewis X glycosylation and is embeddable to other proteins for Lewis X glycoengineering