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Initiation and maintenance of mitosis require the activation of protein kinase cyclin B-Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate-regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts.

Chromogranin A (CgA) is the precursor of several antimicrobial peptides, such as Catestatin (Cts, bovine CgA344-364), initially described as a potent inhibitor of catecholamines. This peptide displays direct antimicrobial activities and contributes to immune system regulation. The aim of the present study is to investigate a designed peptide based on Cts to fight infections against superbugs and more particularly Staphylococcus aureus . In addition to Cateslytin (Ctl, bovine CgA344-358), the active domain of Catestatin, several peptides including dimers, D-isomer and the new designed peptide DOPA-K-DOPA-K-DOPA-TLRGGE-RSMRLSFRARGYGFR (Dopa 5 T-Ctl) were prepared and tested. Cateslytin is resistant to bacterial degradation and does not induce bacterial resistance. The interaction of Catestatin with immune dermal cells (dendritic cells DC1a, dermal macrophages CD14 and macrophages) was analyzed by using confocal microscopy and cytokine release assay. The dimers and D-isomer of Ctl were tested against a large variety of bacteria showing the potent antibacterial activity of the D-isomer. The peptide Dopa 5 T-Ctl is able to induce the self-killing of S. aureus after release of Ctl by the endoprotease Glu-C produced by this pathogen. It permits localized on-demand delivery of the antimicrobial drug directly at the infectious site.

Ψ-1,4-naphthoquinones (Ψ-NQ) are non-quinoid compounds in which aromaticity—found in 1,4-naphthoquinones—is broken by the introduction of an angular methyl at C-4a or -8a. This series was designed to act as prodrugs of 1,4-naphthoquinones in an oxidative environment. Furthermore, from a medicinal chemistry point of view, the loss of planarity of the scaffold might lead to an improved solubility and circumvent the bad reputation of quinones in the pharmaceutical industry. In this work, we illustrated the concept by the synthesis of Ψ -plasmodione regioisomers as prodrugs of the antimalarial plasmodione. The presence of a chiral center introduces a new degree of freedom to be controlled by enantioselectivity and regioselectivity of the cycloaddition in the Diels–Alder reaction. The first strategy that was followed was based on the use of a chiral enantiopure sulfoxide to govern the stereoselective formation of (+)Ψ-NQ or (−)Ψ-NQ, depending on the chirality of the sulfoxide (R or S). New sulfinylquinones were synthesized but were found to be ineffective in undergoing cycloaddition with different dienes under a wide range of conditions (thermal, Lewis acid). The second strategy was based on the use of boronic acid-substituted benzoquinones as auxiliaries to control the regioselectivity. Using this methodology to prepare the (±)Ψ-NQ racemates, promising results (very fast cycloaddition time: ~2 h) were obtained with boronic acid-based quinones 25 and 27 in the presence of 1-methoxy-1,3-butadiene, to generate the 4a- and the 8a-Ψ-plasmodione regioisomers 1 and 2 (synthesized in six steps with a total yield of 10.5% and 4.1%, respectively. As the expected prodrug effect can only be revealed if the molecule undergoes an oxidation of the angular methyl, e.g., in blood-feeding parasites that digest hemoglobin from the host, the antimalarial and the antischistosomal properties of both (±)Ψ-NQ regioisomers were determined in drug assays with Plasmodium falciparum and Schistosoma mansoni. Metabolic studies under quasi-physiological conditions and LC-MS analyses were undertaken to reveal the generation of plasmodione from both the 4a- and the 8a-Ψ-plasmodione regioisomers.

The Syk tyrosine kinase acts downstream of several immune receptors such as the FcγR. Syk owns two SH2 domains that interact with biphosphorylated ITAMs of the FcγR upon phagocytosis. This results in the activation of Syk by autophosphorylation, triggering phosphorylation of several downstream targets, F-actin polymerization, and phagocytosis of the IgG-opsonized target. We found that Syk is S-acylated upon phagocytosis by macrophages. Palmitoylation is performed on a single Syk-Cys by the DHHC5 enzyme that specifically associates with Syk upon phagocytosis. Syk palmitoylation is important for Syk localization to the phagocytic cup, phosphorylation, and phagocytosis. We observed that another Syk-Cys residue, within a redox motif, is modified by sulfenylation. Nevertheless, Syk desulfenylation seems to occur during phagocytosis, when H 2 O 2 production at the cup decreases, after 3.5 min of phagocytosis. Molecular dynamics studies indicated that desulfenylation increased the exposure of a loop within the Syk interdomain B. This could facilitate phosphorylation of key Syk-Tyr residues by upstream kinases. We thus propose an updated model for Syk activation during FcγR-mediated phagocytosis that involves both Syk palmitoylation and desulfenylation.

Most HIV-1 Tat is unconventionally secreted by infected cells following Tat interaction with phosphatidylinositol (4,5) bisphosphate (PI(4,5)P 2 ) at the plasma membrane. Extracellular Tat is endocytosed by uninfected cells before escaping from endosomes to reach the cytosol and bind PI(4,5)P 2 . It is not clear whether and how incoming Tat concentrates in uninfected cells. Here we show that, in uninfected cells, the S-acyl transferase DHHC-20 together with the prolylisomerases cyclophilin A (CypA) and FKBP12 palmitoylate Tat on Cys31 thereby increasing Tat affinity for PI(4,5)P 2 . In infected cells, CypA is bound by HIV-1 Gag, resulting in its encapsidation and CypA depletion from cells. Because of the lack of this essential cofactor, Tat is not palmitoylated in infected cells but strongly secreted. Hence, Tat palmitoylation specifically takes place in uninfected cells. Moreover, palmitoylation is required for Tat to accumulate at the plasma membrane and affect PI(4,5)P 2 -dependent membrane traffic such as phagocytosis and neurosecretion. It is not clear whether and how incoming HIV-1 Tat accumulates in uninfected cells. Here, Chopard et al. show that, in uninfected cells, incoming Tat is palmitoylated on Cys31 by DHHC-20, which increases its affinity for PI(4,5)P 2 and results in its accumulation at the plasma membrane.

Titanium dioxide and copper oxide nanoparticles are more and more widely used because of their catalytic properties, of their light absorbing properties (titanium dioxide) or of their biocidal properties (copper oxide), increasing the risk of adverse health effects. In this frame, the responses of mouse macrophages were studied. Both proteomic and targeted analyses were performed to investigate several parameters, such as phagocytic capacity, cytokine release, copper release, and response at sub toxic doses. Besides titanium dioxide and copper oxide nanoparticles, copper ions were used as controls. We also showed that the overall copper release in the cell does not explain per se the toxicity observed with copper oxide nanoparticles. In addition, both copper ion and copper oxide nanoparticles, but not titanium oxide, induced DNA strands breaks in macrophages. As to functional responses, the phagocytic capacity was not hampered by any of the treatments at non-toxic doses, while copper ion decreased the lipopolysaccharide-induced cytokine and nitric oxide productions. The proteomic analyses highlighted very few changes induced by titanium dioxide nanoparticles, but an induction of heme oxygenase, an increase of glutathione synthesis and a decrease of tetrahydrobiopterin in response to copper oxide nanoparticles. Subsequent targeted analyses demonstrated that the increase in glutathione biosynthesis and the induction of heme oxygenase (e.g. by lovastatin/monacolin K) are critical for macrophages to survive a copper challenge, and that the intermediates of the catecholamine pathway induce a strong cross toxicity with copper oxide nanoparticles and copper ions.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body. We reveal the 3D structures of the free and Rtt106p-bound forms of Bcd1p using nuclear magnetic resonance and X-ray crystallography. The interaction is also studied by a combination of biophysical and proteomic techniques. Bcd1p interacts with a region that is distinct from the interaction interface between the histone chaperone and histone H3. Our results are evidence for a protein interaction interface for Rtt106p that controls its transcription-associated activity. Biogenesis of small nucleolar RNAs ribonucleoproteins (snoRNPs) requires dedicated assembly machinery. Here, the authors show that a subset of snoRNP assembly factors interacts, genetically or directly, with factors modulating chromatin architecture, suggesting a link between ribosome formation and chromatin functions.

Background: Whether therapeutic albumin (ThHSA) can serve as a defense tool in Candida species (spp.) infections is still a matter of debate, although many physicians are in the habit of infusing ThHSA to restore the physiological concentration of endogenous human serum albumin (HSA). Given the need for innovative anti-Candida strategies, we assessed in vitro the role of ThHSA alone or in combination with voriconazole (VCZ) in combating Candida spp. growth and the role of bovine serum albumin (BSA)—used as a substitute for HSA—with two endogenous bovine antimicrobial peptides in combating C. albicans and other microbes. Results: The combination of ThHSA with VCZ enhanced the antifungal effect on C. albicans, sensitive C. tropicalis, sensitive C. glabrata, and C. lusitaniae. However, for resistant C. tropicalis, the combination of ThHSA with VCZ promoted yeast growth, and VCZ tended to suppress the antimicrobial effect of ThHSA on resistant C. glabrata. As to the possible transposition of ThHSA-type properties to BSA (as regards the growth inhibition of other pathogens), we tested combinations of BSA with two physiological chromogranin A-derived antimicrobial peptides (catestatin and cateslytin). BSA enhanced significantly the activity of catestatin (but not cateslytin) in combating C. albicans, A. fumigatus, and M. luteus, but was inactive against S. aureus and E. coli. Conclusions: Our experiments support the fact that albumins display intrinsic antimicrobial properties, with an unpredictable growth inhibitory effect on various microbes. ThHSA can thus be an adjunctive tool for more efficient care of some, though not all, infections. The interaction of BSA with catestatin and cateslytin is related to their structure, with BSA significantly enhancing the effect of catestatin but not that of cateslytin.

Tissue injury causes activation of mesenchymal lineage cells into wound-repairing myofibroblasts (MFs), whose uncontrolled activity ultimately leads to fibrosis. Although this process is triggered by deep metabolic and transcriptional reprogramming, functional links between these two key events are not yet understood. Here, we report that the metabolic sensor post-translational modification O -linked β-D-N-acetylglucosaminylation ( O -GlcNAcylation) is increased and required for myofibroblastic activation. Inhibition of protein O -GlcNAcylation impairs archetypal myofibloblast cellular activities including extracellular matrix gene expression and collagen secretion/deposition as defined in vitro and using ex vivo and in vivo murine liver injury models. Mechanistically, a multi-omics approach combining proteomic, epigenomic, and transcriptomic data mining revealed that O- GlcNAcylation controls the MF transcriptional program by targeting the transcription factors Basonuclin 2 (BNC2) and TEA domain transcription factor 4 (TEAD4) together with the Yes-associated protein 1 (YAP1) co-activator. Indeed, inhibition of protein O -GlcNAcylation impedes their stability leading to decreased functionality of the BNC2/TEAD4/YAP1 complex towards promoting activation of the MF transcriptional regulatory landscape. We found that this involves O -GlcNAcylation of BNC2 at Thr 455 and Ser 490 and of TEAD4 at Ser 69 and Ser 99 . Altogether, this study unravels protein O- GlcNAcylation as a key determinant of myofibroblastic activation and identifies its inhibition as an avenue to intervene with fibrogenic processes.

Proteomic analysis of mature sugarbeet seeds led to the identification of 759 proteins and their specific tissue expression in root, cotyledons, and perisperm. In particular, the proteome of the perispermic storage tissue found in many seeds of the Caryophyllales is described here. The data allowed us to reconstruct in detail the metabolism of the seeds toward recapitulating facets of seed development and provided insights into complex behaviors such as germination. The seed appears to be well prepared to mobilize the major classes of reserves (the proteins, triglycerides, phytate, and starch) during germination, indicating that the preparation of the seed for germination is mainly achieved during its maturation on the mother plant. Furthermore, the data revealed several pathways that can contribute to seed vigor, an important agronomic trait defined as the potential to produce vigorous seedlings, such as glycine betaine accumulation in seeds. This study also identified several proteins that, to our knowledge, have not previously been described in seeds. For example, the data revealed that the sugarbeet seed can initiate translation either through the traditional cap-dependent mechanism or by a cap-independent process. The study of the tissue specificity of the seed proteome demonstrated a compartmentalization of metabolic activity between the roots, cotyledons, and perisperm, indicating a division of metabolic tasks between the various tissues. Furthermore, the perisperm, although it is known as a dead tissue, appears to be very active biochemically, playing multiple roles in distributing sugars and various metabolites to other tissues of the embryo.