A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and non-specific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL system a powerful complement to existing strategies for studying this important mode of protein regulation

Pattern of prescriptions and prudent use of antimicrobial in horse practice at a Veterinary Teaching Hospital

Antimicrobial prescriptions of the University of Pisa and their compliance with prudent use recommendations were investigated over 11 years (2011-2021). At least one antimicrobial was always prescribed in surgical prophylaxis for the suture of wounds and in 33% of horses with signs of disease of a body system. Antimicrobials were administered in monotherapy (48%) in fixed dose combinations (21%) and empirical combinations (31%). Antimicrobials were mostly (63%) administered by parenteral route, while oral and topical antimicrobials accounted for 14% and 23% of prescriptions, respectively. Gentamicin, benzylpenicillin and ceftiofur were the most prescribed antimicrobials; aminoglycosides, penicillins and cephalosporins were the most common class of antimicrobial prescribed. Protected antimicrobials (WHO HPCIA and rifampicin) represented 24% of antimicrobial dispensations. The pattern of classes of antimicrobial used by body system was broad and included up to eight different pharmaceutical classes. The heterogeneity of antimicrobial use was confirmed by the estimate of the prescription diversity index. Antimicrobial prescriptions were in compliance with prudent use recommendations in terms of availability of diagnosis, respect of the dose range and duration of treatment On the contrary, principles of appropriate antimicrobial use have only been partially observed in relation to off-label use, use of antimicrobials in empirical combination, use of antimicrobial susceptibility tests and use of protected antimicrobials, suggesting that additional interventions are required to improve the responsible use of antimicrobials use in our equine practice

Evolution of SUMO protein functions in insects

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.Peer Reviewe

In vivo analysis of SUMOylated proteins from Drosophila melanogaster using improved strategies for expression, purification, and identification of conjugation sites

Póster presentado en el 7th Inproteolys Meeting (Special meeting on “Protein quality control and ubiquitin systems in Health and desease), celebrado en Kusadasi (Turquía) del 14 al 16 de noviembre de 2012.-- Título del póster: Analysis of sumoylated proteins in vivoSUMOylation is a post-translational modification characterized by the covalent and reversible binding of the Small Ubiquitin-like Modifier ( SUMO) to a target protein. SUMOylation regulates many cellular processes, including transcription, DNA damage repair, protein-protein interactions, protein localization and trafficking. We are interested on studying the in vivo role of Drosophila SUMO (Smt3) implicated in the steroid biosynthetic pathway and required for metamorphosis in flies. We are developing a novel strategy to isolate tissue specific SUMOylated proteins, based on the in vivo biotinylation of a variant of Smt3 (bioSUMO).Peer Reviewe

Evolution of SUMO function and chain formation in insects

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.N

Molecular organization of the cullin E3 ligase adaptor KCTD11

The family of human proteins containing a potassium channel tetramerization domain (KCTD) includes 21 members whose function is largely unknown. Recent reports have however suggested that these proteins are implicated in very important biological processes. KCTD11/REN, the best-characterized member of the family to date, plays a crucial role in the ubiquitination of HDAC1 by acting, in complex with Cullin3, as an E3 ubiquitin ligase. By combining bioinformatics and mutagenesis analyses, here we show that the protein is expressed in two alternative variants: a short previously characterized form (sKCTD11) composed by 232 amino acids and a longer variant (IKCTD11) which contains an N-terminal extension of 39 residues. Interestingly, we demonstrate that IKCTD11 starts with a non-canonical AUU codon. Although both sKCTD11 and IKCTD11 bear a POZ/BTB domain in their N-terminal region, this domain is complete only in the long form. Indeed, sKCTD11 presents an incomplete POZ/BTB domain. Nonetheless, sKCTD11 is still able to bind Cul3, although to much lesser extent than IKCTD11, and to perform its biological activity. The heterologous expression of sKCTD11 and IKCTD11 and their individual domains in Escherichia coil yielded soluble products as fusion proteins only for the longer form. In contrast to the closely related KCTD5 which is pentameric, the characterization of both IKCTD11 and its POZ/BTB domain by gel filtration and light scattering indicates that the protein likely forms stable tetramers. In line with this result, experiments conducted in cells show that the active protein is not monomeric. Based on these findings, homology-based models were built for IKCTD11 BIB and for its complex with Cul3. These analyses indicate that a stable IKCTD11 BTB-Cul3 three-dimensional model with a 4:4 stoichiometry can be generated. Moreover, these models provide insights into the determinants of the tetramer stability and into the regions involved in IKCTD11-Cul3 recognition. (C) 2011 Elsevier Masson SAS. All rights reserved

Cullin3-BTB interface: a novel target for stapled peptides.

Cullin3 (Cul3), a key factor of protein ubiquitination, is able to interact with dozens of different proteins containing a BTB (Bric-a-brac, Tramtrack and Broad Complex) domain. We here targeted the Cul3-BTB interface by using the intriguing approach of stabilizing the α-helical conformation of Cul3-based peptides through the "stapling" with a hydrocarbon cross-linker. In particular, by combining theoretical and experimental techniques, we designed and characterized stapled Cul3-based peptides embedding the helix 2 of the protein (residues 49-68). Intriguingly, CD and NMR experiments demonstrate that these stapled peptides were able to adopt the helical structure that the fragment assumes in the parent protein. We also show that some of these peptides were able to bind to the BTB of the tetrameric KCTD11, a substrate adaptor involved in HDAC1 degradation, with high affinity (~ 300-600 nM). Cul3-derived staple peptides are also able to bind the BTB of the pentameric KCTD5. Interestingly, the affinity of these peptides is of the same order of magnitude of that reported for the interaction of full-length Cul3 with some BTB containing proteins. Moreover, present data indicate that stapling endows these peptides with an increased serum stability. Altogether, these findings indicate that the designed stapled peptides can efficiently mimic protein-protein interactions and are potentially able to modulate fundamental biological processes involving Cul3

Scavenger Receptors Mediate the Role of SUMO and Ftz-f1 in <i>Drosophila</i> Steroidogenesis

<div><p>SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in <i>Drosophila melanogaster</i>. Silencing the <i>Drosophila</i> SUMO homologue <i>smt3</i> in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval–pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the <i>smt3</i> knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by <i>in vitro</i> and <i>in vivo</i> experiments that Ftz-f1 is SUMOylated. RNAi–mediated knockdown of <i>ftz-f1</i> phenocopies that of <i>smt3</i> at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. <i>smt3</i> knockdown, as well as <i>ftz-f1</i> or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that <i>Drosophila</i> Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.</p></div

DDDR-32. A NEW IMMUNOMODULATORY FUNCTION OF PYRIDO-PYRIMIDINE DERIVATIVES TO IMPAIR METASTATIC GROUP 3 MEDULLOBLASTOMA IN VIVO

Medulloblastoma (MB) is an embryonal tumor of the cerebellum consti- tuting ~ 20% of pediatric brain tumors. To date, four MB molecular groups (further stratified in twelve subtypes) have been described. Among them, Groups 3 and Group 4 MB have the poorest prognosis due to their high metastatic potential. Recently, we have reported a metastatic axis driven by Prune1 overexpression in MB Group3 characterized by canonical TGF-β signaling enhancement and epithelial-mesenchymal transition. Here, we have developed a new not toxic pyrido-pyrimidine derivative with the ability to impair Prune-1-driven-axis, thus ameliorating the survival rate of a murine model of metastatic MB Group3 characterized by overexpression of human Prune1 gene in the cerebellum (under the control of MATH1 promoter). Of importance, this small molecule also is showing immunomodulatory functions thus inhibiting the conversion of tumor-infiltrating T lymphocytes (TILs) to immunosuppressive regulatory T cells (Tregs) in vivo via impairing the secretion of inflammatory cytokines from MB cells. Furthermore, this molecule can also act synergistically with the currently used modified- intensity chemotherapy (e.g. in PNET5 use of Vincristine) or potential in the combination with epigenetics drugs (e.g., LSD1/KDM1A inhibitors). Altogether these results are of importance for future targeted therapies of high-risk metastatic MB