The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His 6 tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We The small ubiquitin-like modifier (SUMO) 1 proteins are structurally similar to ubiquitin, although they share less than 20% sequence identity (1). Like ubiquitylation, protein SUMOylation is regulated by a cascade of reactions involving SUMO-activating enzymes (SAE1/SAE2), -conjugating enzymes (Ubc9), and one of several SUMO E3 ligases (e.g. PIAS1, PIAS3, PIASx␣, PIASx␤, PIASy, RanBP2, and Pc2) that covalently attach SUMO to specific protein substrates (2, 3). SUMO proteins are expressed as an immature proform that comprises an invariant Gly-Gly motif followed by a Cterminal stretch of variable length (2-11 amino acids). Removal of this C-terminal extension by sentrin-specific proteases (SENPs) to expose the diglycine motif is necessary for the conjugation of SUMO to protein targets. These SUMO proteases are able to cleave both a peptide bond during the formation of mature SUMO and an isopeptide bond to deconjugate modified protein substrates (4). This covalent modification arises from the formation of an isopeptide bond between the -amino group of a lysine within the protein substrate and the C terminus carboxyl group of the SUMO glycine residue. SUMO conjugation frequently occurs at the lysine residue within the consensus motif KXE (where is an aliphatic residue and X is any amino acid) that is recognized by Ubc9 (5, 6). Recent studies have also identified a phosphorylation-dependent motif (⌿KXEXXpSP where pS is phosphoserine) (7) and a negatively charged amino acid-dependent motif (8) that harbor negative charges next to the basic SUMO consensus site to enhance protein SUMOylation. However, several other SUMOylated proteins including proliferating cell nuclear antigen, E2-25K, Daxx (death domainassociated protein), and USP25 are modified at non-consensus sites (9 -11). Whether these types of sites are rare 1 The abbreviations used are: SUMO, small ubiquitin-related modifier; As 2 O 3 , arsenic trioxide; E2-25K, E2 ubiquitin ligase, 25 kilodalto...

Small ubiquitin-related modifiers (SUMO) are evolutionarily conserved ubiquitin-like proteins that regulate several cellular processes including cell cycle progression, intracellular trafficking, protein degradation and apoptosis. Despite the importance of protein SUMOylation in different biological pathways, the global identification of acceptor sites in complex cell extracts remains a challenge. Here we generate a monoclonal antibody that enriches for peptides containing SUMO remnant chains following tryptic digestion. We identify 954 SUMO3-modified lysine residues on 538 proteins and profile by quantitative proteomics the dynamic changes of protein SUMOylation following proteasome inhibition. More than 86% of these SUMOylation sites have not been reported previously, including 5 sites on the tumour suppressor parafibromin (CDC73). The modification of CDC73 at K136 affects its nuclear retention within PML nuclear bodies on proteasome inhibition. In contrast, a CDC73 K136R mutant translocates to the cytoplasm under the same conditions, further demonstrating the effectiveness of our method to characterize the dynamics of lysine SUMOylation.

Phosphatidylinositol-4-kinase III␣ (PI4KIII␣) is an essential host cell factor for hepatitis C virus (HCV)replication. An N-terminally truncated 130-kDa form was used to reconstitute an in vitro biochemical lipid kinase assay that was optimized for smallmolecule compound screening and identified potent and specific inhibitors. Cell culture studies with PI4KIII␣ inhibitors demonstrated that the kinase activity was essential for HCV RNA replication. Two PI4KIII␣ inhibitors were used to select cell lines harboring HCV replicon mutants with a 20-fold loss in sensitivity to the compounds. Reverse genetic mapping isolated an NS4B-NS5A segment that rescued HCV RNA replication in PIK4III␣-deficient cells. HCV RNA replication occurs on specialized membranous webs, and this study with PIK4III␣ inhibitor-resistant mutants provides a genetic link between NS4B/NS5A functions and PI4-phosphate lipid metabolism. A comprehensive assessment of PI4KIII␣ as a drug target included its evaluation for pharmacologic intervention in vivo through conditional transgenic murine lines that mimic target-specific inhibition in adult mice. Homozygotes that induce a knockout of the kinase domain or knock in a single amino acid substitution, kinase-defective PI4KIII␣, displayed a lethal phenotype with a fairly widespread mucosal epithelial degeneration of the gastrointestinal tract. This essential host physiologic role raises doubt about the pursuit of PI4KIII␣ inhibitors for treatment of chronic HCV infection.

Many studies have shown that speciation can be facilitated when a trait under divergent selection also causes assortative mating. In Müllerian mimetic butterflies, a change in wing colour pattern can cause reproductive isolation. However, colour pattern divergence does not always lead to reproductive isolation. Understanding how divergent selection affects speciation requires identifying the mechanisms that promote mate preference and/or choosiness. This study addresses whether shifts in wing colour pattern drives mate preference and reproductive isolation in the tropical butterfly genus Melinaea (Nymphalidae: Ithomiini), and focuses on five taxa that form a speciation continuum, from subspecies to fully recognized species. Using genetic markers, wing colour pattern quantification, male pheromone characterization and behavioural assays of mating preference, we characterize the extent of genetic and phenotypic differentiation between taxa and compare it to the level of reproductive isolation. We show strong premating isolation between the closely related species M. satevis and M. marsaeus, in addition to genetic and phenotypic (colour pattern and pheromones) differentiation. By contrast, M. menophilus and M. marsaeus consist of pairs of subspecies that differ for colour pattern but that cannot be differentiated genetically. Pheromonal differentiation of subspecies was significant only for M. marsaeus, although most individuals were indistinguishable. Melinaea menophilus and M. marsaeus also differ in the strength of assortative mating, suggesting that mate preference has evolved only in M. marsaeus, consistent with selection against maladaptive offspring, as subspecific ‘hybrids’ of M. marsaeus have intermediate, non‐mimetic colour patterns, unlike those of M. menophilus which display either parental phenotypes. We conclude that a shift in colour pattern per se is not sufficient for reproductive isolation, but rather, the evolution of assortative mating may be caused by selection against maladaptive intermediate phenotypes. This study suggests that mate preference and assortative mating evolve when adaptive, and that even in the early stages of divergence, reproductive isolation can be nearly complete due to mating preferences.

Conclusion: The current results add to the growing body of evidence of endocannabinoid alterations in schizophrenia. The strong elevation of plasma anandamide levels in schizophrenia patients assessed in the psychiatric emergency setting suggests that anandamide and OEA area potential biomarkers of the psychological turmoil associated with this context.

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