Bombardier Enables Delivery of Short-Form Bomanins in the Drosophila Toll Response.

Toll mediates a robust and effective innate immune response across vertebrates and invertebrates. In Drosophila melanogaster, activation of Toll by systemic infection drives the accumulation of a rich repertoire of immune effectors in hemolymph, including the recently characterized Bomanins, as well as the classical antimicrobial peptides (AMPs). Here we report the functional characterization of a Toll-induced hemolymph protein encoded by the bombardier (CG18067) gene. Using the CRISPR/Cas9 system to generate a precise deletion of the bombardier transcriptional unit, we found that Bombardier is required for Toll-mediated defense against fungi and Gram-positive bacteria. Assaying cell-free hemolymph, we found that the Bomanin-dependent candidacidal activity is also dependent on Bombardier, but is independent of the antifungal AMPs Drosomycin and Metchnikowin. Using mass spectrometry, we demonstrated that deletion of bombardier results in the specific absence of short-form Bomanins from hemolymph. In addition, flies lacking Bombardier exhibited a defect in pathogen tolerance that we trace to an aberrant condition triggered by Toll activation. These results lead us to a model in which the presence of Bombardier in wild-type flies enables the proper folding, secretion, or intermolecular associations of short-form Bomanins, and the absence of Bombardier disrupts one or more of these steps, resulting in defects in both immune resistance and tolerance

EDF1 coordinates cellular responses to ribosome collisions

Translation of aberrant mRNAs induces ribosomal collisions, thereby triggering pathways for mRNA and nascent peptide degradation and ribosomal rescue. Here we use sucrose gradient fractionation combined with quantitative proteomics to systematically identify proteins associated with collided ribosomes. This approach identified Endothelial differentiation-related factor 1 (EDF1) as a novel protein recruited to collided ribosomes during translational distress. Cryo-electron microscopic analyses of EDF1 and its yeast homolog Mbf1 revealed a conserved 40S ribosomal subunit binding site at the mRNA entry channel near the collision interface. EDF1 recruits the translational repressors GIGYF2 and EIF4E2 to collided ribosomes to initiate a negative-feedback loop that prevents new ribosomes from translating defective mRNAs. Further, EDF1 regulates an immediate-early transcriptional response to ribosomal collisions. Our results uncover mechanisms through which EDF1 coordinates multiple responses of the ribosome-mediated quality control pathway and provide novel insights into the intersection of ribosome-mediated quality control with global transcriptional regulation

Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i) motile ii) rod shaped iii) non spore forming iv) catalase and amylase positive v) able to grow in presence of 10 % NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7) exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98% sequence similarity, 1201 bp). The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 % activity at 80 0C after 4 h and more than 70 % activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 % active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability

Bombardier Enables Delivery of Short-Form Bomanins in the Drosophila Toll Response.

Toll mediates a robust and effective innate immune response across vertebrates and invertebrates. In Drosophila melanogaster, activation of Toll by systemic infection drives the accumulation of a rich repertoire of immune effectors in hemolymph, including the recently characterized Bomanins, as well as the classical antimicrobial peptides (AMPs). Here we report the functional characterization of a Toll-induced hemolymph protein encoded by the bombardier (CG18067) gene. Using the CRISPR/Cas9 system to generate a precise deletion of the bombardier transcriptional unit, we found that Bombardier is required for Toll-mediated defense against fungi and Gram-positive bacteria. Assaying cell-free hemolymph, we found that the Bomanin-dependent candidacidal activity is also dependent on Bombardier, but is independent of the antifungal AMPs Drosomycin and Metchnikowin. Using mass spectrometry, we demonstrated that deletion of bombardier results in the specific absence of short-form Bomanins from hemolymph. In addition, flies lacking Bombardier exhibited a defect in pathogen tolerance that we trace to an aberrant condition triggered by Toll activation. These results lead us to a model in which the presence of Bombardier in wild-type flies enables the proper folding, secretion, or intermolecular associations of short-form Bomanins, and the absence of Bombardier disrupts one or more of these steps, resulting in defects in both immune resistance and tolerance

Active Protein Neddylation or Ubiquitylation Is Dispensable for Stress Granule Dynamics.

Stress granule (SG) formation is frequently accompanied by ubiquitin proteasome system (UPS) impairment and ubiquitylated protein accumulation. SGs, ubiquitin, and UPS components co-localize, but the relationship between the ubiquitin pathway and SGs has not been systematically characterized. We utilize pharmacological inhibition of either the ubiquitin- or NEDD8-activating enzyme (UAE or NAE) to probe whether active ubiquitylation or neddylation modulate SG dynamics. We show that UAE inhibition results in rapid loss of global protein ubiquitylation using ubiquitin-specific proteomics. Critically, inhibiting neither UAE nor NAE significantly affected SG formation or disassembly, indicating that active protein ubiquitylation or neddylation is dispensable for SG dynamics. Using antibodies with varying preference for free ubiquitin or polyubiquitin and fluorescently tagged ubiquitin variants in combination with UAE inhibition, we show that SGs co-localize primarily with unconjugated ubiquitin rather than polyubiquitylated proteins. These findings clarify the role of ubiquitin in SG biology and suggest that free ubiquitin may alter SG protein interactions

Svensk Botanisk Tidskrift : Volym 109: Häfte 6, 2015

INNEHÅLLSFÖRTECKNING. J. ELVELAND: Våtslåttermarker i Norrland - förr och nu. P. TORÄNG: Biffekter. M. LIDÉN: Hemligheten är att vara hemlig… G. MATTIASSON: Om fyra nya Skånearter. B. OLDHAMMER: Bomb i bageriet. Böcker: I. BACKÉUS: Ett etnobotaniskt livsverk; I. LARSSON: Norske klosterplanter; I. LARSSON: 1600-talets växtnamn. Föreningsnytt: Ledare: Satsningarna fortsätter; Kallelse till SBF:s årsmöte; Föreningskonferens 5-6 mars i Uppsala; Botanisk resa till Jämtland; Tidskriftsval; Ny webbutik

Identification of a Tissue-Restricted Isoform of SIRT1 Defines a Regulatory Domain that Encodes Specificity

The conserved NAD+-dependent deacylase SIRT1 plays pivotal, sometimes contrasting, roles in diverse physiological and pathophysiological conditions. In this study, we uncover a tissue-restricted isoform of SIRT1 (SIRT1-ΔE2) that lacks exon 2 (E2). Candidate-based screening of SIRT1 substrates demonstrated that the domain encoded by this exon plays a key role in specifying SIRT1 protein-protein interactions. The E2 domain of SIRT1 was both necessary and sufficient for PGC1α binding, enhanced interaction with p53, and thus downstream functions. Since SIRT1-FL and SIRT1-ΔE2 were found to have similar intrinsic catalytic activities, we propose that the E2 domain tethers specific substrate proteins. Given the absence of SIRT1-ΔE2 in liver, our findings provide insight into the role of the E2 domain in specifying “metabolic functions” of SIRT1-FL. Identification of SIRT1-ΔE2 and the conserved specificity domain will enhance our understanding of SIRT1 and guide the development of therapeutic interventions

Mechanistic convergence of the TIGIT and PD-1 inhibitory pathways necessitates co-blockade to optimize anti-tumor CD8+ T cell responses.

Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic

Allosteric Regulation of Cyclin-B Binding by the Charge State of Catalytic Lysine in CDK1 Is Essential for Cell-Cycle Progression

International audienceCyclin-dependent kinase 1 (CDK1) is essential for cell-cycle progression. While dependence of CDK activity on cyclin levels is well established, molecular mechanisms that regulate their binding are less understood. Here, we report for the first time that CDK1:cyclin-B binding is not default but rather determined by the evolutionarily conserved catalytic residue, lysine-33 in CDK1. We demonstrate that the charge state of this lysine allosterically remodels the CDK1:cyclin-B interface. Cell cycle-dependent acetylation of lysine-33 or its mutation to glutamine, which mimics acetylation, abrogates cyclin-B binding. Using biochemical approaches and atomistic molecular dynamics simulations, we have uncovered both short-range and long-range effects of perturbing the charged state of the catalytic lysine, which lead to inhibition of kinase activity. Specifically, although loss of the charge state of catalytic lysine did not impact ATP binding significantly, it altered its orientation in the active site. In addition, the catalytic lysine also acts as an intra-molecular electrostatic tether at the active site to orient structural elements interfacing with cyclin-B. Physiologically, opposing activities of SIRT1 and P300 regulate acetylation and thus control the charge state of lysine-33. Importantly, cells expressing acetylation mimic mutant of Cdc2/CDK1 in yeast are arrested in G2 and fail to divide, indicating the requirement of the deacetylated state of the catalytic lysine for cell division. Thus, by illustrating the molecular role of the catalytic lysine and cell cycle-dependent deacetylation as a determinant of CDK1:cyclin-B interaction, our results redefine the current model of CDK1 activation and cell-cycle progression