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Uncropped blots related to Fig. 2.

Uncropped blots related to Fig. 3.

Uncropped blots related to Fig. 5.

Primer sequences used in RT-qPCR analyses.

Uncropped blots related to Fig. 6.

Trabajo presentado en el 2nd International Insect Hormone Workshop, celebrado en Creta del 12 al 18 de julio de 2015., SUMOylation, the covalent binding of Small Ubiquitin-like Modifier (SUMO) to target proteins, is a posttranslational modification that regulates critical cellular processes in eukaryotes. In insects, SUMOylation has been studied in holometabolous species, particularly in the dipteran Drosophila melanogaster, which contains a single SUMO gene (smt3). This has led to the assumption that insects contain a single SUMO gene. However, the analysis of insect genomes shows that basal insects contain two SUMO genes, orthologous to vertebrate SUMO1 and SUMO2/3. Our phylogenetical analysis reveals that the SUMO gene has been duplicated giving rise to SUMO1 and SUMO 2/3 families early in Metazoan evolution, and that later in insect evolution the SUMO1 gene has been lost after the Hymenoptera divergence. To explore the consequences of this loss, we have examined the characteristics and different biological functions of the two SUMO genes (SUMO1 and SUMO3) in the hemimetabolous cockroach Blattella germanica and compared them with those of Drosophila Smt3. Here, we show that the metamorphic role of the SUMO genes (cell proliferation, ecdysone signalling response and proper molting) is evolutionary conserved in insects, although there has been a regulatory switch from SUMO1 in basal insects to SUMO3 in more derived ones. We also show that, unlike vertebrates, insect SUMO3 proteins cannot form polySUMO chains due to the loss of critical lysine residues within the N-terminal part of the protein. Furthermore, the formation of polySUMO chains by expression of ectopic human SUMO3 has a deleterious effect in Drosophila. These findings contribute to the understanding of the functional consequences of the evolution of SUMO genes.

Póster presentado en el Insect Hormones (19th Ecdysone) International Workshop, celebrado en Minneapolis del 21 al 26 de julio de 2013., SUMOylation is a highly conserved post-translational modification that modulates target protein activity in a great number of cellular processes during development. In the holometabolous insect Drosophila melanogaster, SUMOylation has been shown to be necessary during the metamorphic transition. Reduction of the single D. melanogaster SUMO homologue (smt3) expression in the prothoracic gland prevents the larval to pupal transition due to reduced lipid content in the gland and low ecdysone titer1. Unfortunately, our knowledge on the role of SUMOylation in insect development exclusively derives from studies in D. melanogaster, which shows a highly derived type of insect development that may not be representative in the insect class context. Our research, therefore, is devoted to establish the role of SUMOylation in the hemimetabolous insect Blattella germanica. In contrast to D. melanogaster, we have identified two B. germanica SUMO homologs, BgSumo1 and BgSumo3 that are ubiquitously expressed throughout development. By using RNAi in vivo experiments we have shown that, whereas BgSumo3 is dispensable for the correct development of B. germanica, reduction of BgSumo1 levels resulted in severe defects during the metamorphic transition, including a marked developmental delay due to impaired activation of the ecdysone-triggered signaling cascade. Furthermore, we have shown that all the proteins belonging to the ecdysone-dependent transcriptional cascade of nuclear hormone receptors (BgEcR, BgRXR, BgE75, BgHR3 and BgFTZ-F1) are SUMOylated in vitro. Finally, to test whether the functions of SUMO proteins are evolutionary conserved between hemimetabolous and holometabolous insects, we performed functional analysis in vivo of the two B. germanica Sumo homologues using D. melanogaster as model system. These experiments showed that BgSumo3 contains all the elements necessary to substitute functionally for D. melanogaster smt3, while BgSumo1 does not.

SUMOylation contributes to the regulation of many essential cellular factors. Diverse techniques have been used to explore the functional consequences of protein SUMOylation. Most approaches consider the identification of sequences on substrates, adaptors, or receptors regulating the SUMO conjugation, recognition, or deconjugation. The large majority of the studied SUMOylated proteins contain the sequence [IVL]KxE. SUMOylated proteins are recognized by at least 3 types of hydrophobic SUMO-interacting motifs (SIMs) that contribute to coordinate SUMO-dependent functions. Typically, SIMs are constituted by a hydrophobic core flanked by one or two clusters of negatively charged amino acid residues. Multiple SIMs can integrate SUMO binding domains (SBDs), optimizing binding, and control over SUMO-dependent processes. Here, we present a survey of the methodologies used to study SUMO-regulated functions and provide guidelines for the identification of cis and trans sequences controlling SUMOylation. Furthermore, an integrative analysis of known and putative SUMO substrates illustrates an updated landscape of several SUMO-regulated events. The strategies and analysis presented here should contribute to the understanding of SUMO-controlled functions and provide rational approach to identify biomarkers or choose possible targets for intervention in processes where SUMOylation plays a critical role.

International audience; ICOS ligation in concert with TCR stimulation results in strong PI3K activation in T lymphocytes. The ICOS cytoplasmic tail contains an YMFM motif that binds the p85alpha subunit of class IA PI3K, similar to the YMNM motif of CD28, suggesting a redundant function of the two receptors in PI3K signaling. However, ICOS costimulation shows greater PI3K activity than CD28 in T cells. We show in this report that ICOS expression in activated T cells triggers the participation of p50alpha, one of the regulatory subunits of class IA PI3Ks. Using different T-APC cell conjugate systems, we report that p50alpha accumulates at the immunological synapse in activated but not in resting T cells. Our results demonstrate that ICOS membrane expression is involved in this process and that p50alpha plasma membrane accumulation requires a functional YMFM Src homology 2 domain-binding motif in ICOS. We also show that ICOS triggering with its ligand, ICOSL, induces the recruitment of p50alpha at the synapse of T cell/APC conjugates. In association with the p110 catalytic subunit, p50alpha is known to carry a stronger lipid kinase activity compared with p85alpha. Accordingly, we observed that ICOS engagement results in a stronger activation of PI3K. Together, these findings provide evidence that p50alpha is likely a determining factor in ICOS-mediated PI3K activity in T cells. These results also suggest that a differential recruitment and activity of class IA PI3K subunits represents a novel mechanism in the control of PI3K signaling by costimulatory molecules.

Le contrôle de la quiescence lymphocytaire est un élément essentiel à l’homéostasie du système immunitaire. Cet état quiescent est activement maintenu par des facteurs transcriptionnels nucléaires, de la famille FoxO (Forkhead subgroup O) notamment. Cet équilibre est cependant rompu à la suite de la reconnaissance de l’antigène, ce qui déclenche la division cellulaire. Cet article fait le point des données récemment acquises sur les mécanismes moléculaires impliqués dans ce phénomène. Il décrit ainsi comment la synapse immunologique formée entre un lymphocyte T et une cellule présentant l’antigène constitue une plate-forme d’intégration au sein de laquelle il existe une production continue de 3’-phospho-inositides sous l’impulsion des phosphoinositides-3-kinases. En provoquant l’activation de la sérine-thréonine kinase Akt, ce processus stimule l’exclusion nucléaire des FoxO pour lever le frein permanent qu’ils exercent et permettre l’expansion clonale des cellules T en réponse à l’antigène., T cell clonal expansion contributing to host defense against pathogens is a tightly controlled process to maintain the homeostasis of the immune system. Our understanding of how T cell growth and proliferation are controlled following antigenic stimulation is therefore a major challenge. Antigen recognition occurs when a naive T lymphocyte contacts an antigen-presenting cell. A specialized junction enriched in T-cell receptors, costimulation molecules and signaling adaptors, called the immunological synapse, is then created for several hours between the two cell types. Recent discoveries now clarify the molecular mechanisms used by this organization to control T cell growth and proliferation. It has been established that the immunological synapse functions in fact as an integrative platform where class Ia phosphoinositide-3-kinases (PI3Ks) are recruited and activated to continuously produce high levels of 3’-phosphoinositides. These lipids regulate the localization and the activation of a wide range of PH-domain containing proteins, among which the serine-threonine kinase Akt, a downstream effector of PI3Ks, appears to be a key player. FoxO (Forkhead subgroup O) family members control in various cell systems genes implicated in apoptosis, stress resistance and cell cycle arrest, thereby contributing to maintain quiescence in unstimulated cells. In naïve T cells contacting antigen-presenting cells a rapid but also very prolonged nuclear exclusion of these transcription factors is observed downstream of Akt. Mainly, this compartmentalization process is mandatory to induce T cell growth triggered by the T cell/antigen-presenting cell interaction. These findings demonstrate that to initiate cell cycle progression the formation of the immunological synapse is an undemanding tactic used by primary T cells to securely maintain the 3’-phosphoinositide-dependent mitotic switch governed by the spatial control of FoxO transcription factors.