Proteins of Diverse Function and Subcellular Location Are Lysine Acetylated in Arabidopsis

Acetylation of theε-amino group of lysine (Lys) is a reversible posttranslational modification recently discovered to be widespread, occurring on proteins outside the nucleus, in most subcellular locations in mammalian cells. Almost nothing is known about this modification in plants beyond the well-studied acetylation of histone proteins in the nucleus. Here, we report that Lys acetylation in plants also occurs on organellar and cytosolic proteins. We identified 91 Lys-acetylated sites on 74 proteins of diverse functional classes. Furthermore, our study suggests that Lys acetylation may be an important posttrans-lational modification in the chloroplast, since four Calvin cycle enzymes were acetylated. The plastid-encoded large subunit of Rubisco stands out because of the large number of acetylated sites occurring at important Lys residues that are involved in Rubisco tertiary structure formation and catalytic function. Using the human recombinant deacetylase sirtuin 3, it was demonstrated that Lys deacetylation significantly affects Rubisco activity as well as the activities of other central metabolic enzymes, such as the Calvin cycle enzyme phosphoglycerate kinase, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase, and the tricarboxylic acid cycle enzyme malate dehydrogenase. Our results demonstrate that Lys acetylation also occurs on proteins outside the nucleus in Arabidopsis (Arabidopsis thaliana) and that Lys acetylation could be important in the regulation of key metabolic enzymes.</p

The phosphoproteome of Arabidopsis plants lacking the oxidative signal-inducible1 (OXI1) protein kinase

The AGC protein kinase OXI1 is a key protein in plant responses to oxidative signals, and is important for two oxidative burst-mediated processes: basal resistance to microbial pathogens and root hair growth. To identify possible components of the OXI1 signalling pathway, phosphoproteomic techniques were used to detect alterations in the abundance of phosphorylated proteins and peptides in an oxi1 null mutant of Arabidopsis thaliana. The relative abundance of phosphorylated proteins was assessed either using two-dimensional gel electrophoresis and staining with the phosphoprotein stain Pro-Q Diamond or by the identification and quantification, by mass spectrometry, of stable-isotope labelled phosphopeptides. A number of proteins show altered phosphorylation in the oxi1 mutant. Five proteins, including a putative F-box and 3-phosphoinositide-dependent kinase 1, show reduced phosphorylation in the oxi1 mutant, and may be direct or indirect targets of OXI1. Four proteins, including ethylene insensitive 2 and phospholipase d-gamma, show increased phosphorylation in the oxi1 mutant. This study has identified a range of candidate proteins from the OXI1 signalling pathway. The diverse activities of these proteins, including protein degradation and hormone signalling, may suggest crosstalk between OXI1 and other signal transduction cascades.</p

Thermal Antibubbles: When Thermalization of Encapsulated Leidenfrost Drops Matters

peer reviewedInter-university Attraction Pole Progra

BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo

International audienceA chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. Reproducing a proliferative stimulation ex vivo is critical to decipher the mechanisms of leukemogenesis in these malignancies. However, functional studies of CLL cells remains limited since current ex vivo B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells, pointing out the need of mandatory BCR co-factors in this process. Here, we investigated benefits of several BCR co-stimulatory molecules (IL-2, IL-4, IL-15, IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand, IL-4 and IL-21 stimulation allowed CLL cells proliferation ex vivo. In addition, we established a proliferative advantage for ZAP70 positive CLL cells, associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover, the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This ex vivo model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies. Like in most mature lymphoproliferative malignancies, an antigenic stimulation is believed to drive the leukemo-genic process in chronic lymphocytic leukemia (CLL) 1-3. A restricted use of IGHV genes and the existence of ste-reotypic B cell receptor (BCR) on CLL cells 4-6 provides evidence in favor of antigenic stimulation where different microbial antigens, as well as auto-antigens, have been suspected as actors of this chronic stimulation 7. In addition , a chronic BCR self-activation has been shown in subtypes of CLL cells 8. Moreover, several signaling aberrations have been described downstream of the BCR, notably in aggressive CLL with unmutated IGHV (UM-CLL), in which the expression of ZAP70 reinforces BCR responsiveness 9-12. BCR activation, which is essential for the physiological development of lymphocytes 13 would also be indispensable for the survival and proliferation of CLL cells in vivo 2. Accordingly, withdrawal of this stimulation is believed to be responsible for the rapid spontaneous apoptosis of CLL cells ex vivo 14. The cellular consequences of this BCR activation has been extensively studied an

TLRs1-10 Protein Expression in Circulating Human White Blood Cells during Bacterial and COVID-19 Infections

Introduction: Toll-like receptors play crucial roles in the sepsis-induced systemic inflammatory response. Septic shock mortality correlates with overexpression of neutrophilic TLR2 and TLR9, while the role of TLR4 overexpression remains a debate. In addition, TLRs are involved in the pathogenesis of viral infections such as COVID-19, where the single-stranded RNA of SARS-CoV-2 is recognized by TLR7 and TLR8, and the spike protein activates TLR4. Methods: In this study, we conducted a comprehensive analysis of TLRs 1–10 expressions in white blood cells from 71 patients with bacterial and viral infections. Patients were divided into 4 groups based on disease type and severity (sepsis, septic shock, moderate, and severe COVID-19) and compared to 7 healthy volunteers. Results: We observed a significant reduction in the expression of TLR4 and its co-receptor CD14 in septic shock neutrophils compared to the control group (p &lt; 0.001). Severe COVID-19 patients exhibited a significant increase in TLR3 and TLR7 levels in neutrophils compared to controls (p &lt; 0.05). Septic shock patients also showed a similar increase in TLR7 in neutrophils along with elevated intermediate monocytes (CD14+CD16+) compared to the control group (p &lt; 0.005 and p &lt; 0.001, respectively). However, TLR expression remained unchanged in lymphocytes. Conclusion: This study provides further insights into the mechanisms of TLR activation in various infectious conditions. Additional analysis is needed to assess their correlation with patient outcome and to evaluate the impact of TLR-pathway modulation during septic shock and severe COVID-19

New methodologies in mass spectrometry for proteomic analysis,application to the discovery of new biomarkers in leukaemia

Cette thèse porte sur le développement de nouvelles méthodologies pour l'étude des protéines membranaires par spectrométrie de masse afin de trouver de nouveaux biomarqueurs dans le cadre des leucémies. Pour cela, nous avons élaboré une stratégie combinanThis thesis deals with the development of new mass spectrometry strategies in order to study plasma membrane proteins and to find new specific biomarkers in various form of leukaemia. In this aim, we have developed an approach combining plasma membranes

New methodologies in mass spectrometry for proteomic analysis,application to the discovery of new biomarkers in leukaemia

Cette thèse porte sur le développement de nouvelles méthodologies pour l'étude des protéines membranaires par spectrométrie de masse afin de trouver de nouveaux biomarqueurs dans le cadre des leucémies. Pour cela, nous avons élaboré une stratégie combinant la séparation de mélanges de protéines membranaires issues de microparticules sur gel d'électrophorèse à une dimension, découpe systématique et analyse par nanoLC-MS/MS. A partir des résultats présentés dans cette thèse, nous avons pu mettre en évidence des protéines membranaires exprimées de façon différentielle entre les différents cas de leucémies.Dans un deuxième temps, nous avons appliqué la stratégie SELDI-TOF à des sérums de différents patients. Cette approche nous a permis de mettre en évidence trois protéines exprimées de façon différentielle entre le groupe témoin et le groupe malade. L'expression de ces protéines de discriminer de manière non ambiguë les témoins des patients.This thesis deals with the development of new mass spectrometry strategies in order to study plasma membrane proteins and to find new specific biomarkers in various form of leukaemia. In this aim, we have developed an approach combining plasma membranes proteins originating from microparticles, separated on 1D gel electrophoresis, cut each 2mm and nanoLC-MS/MS analysis. From the results presented in this thesis, we have found various plasma membranes proteins as potential biomarkers expressed differentially in function of the different leukemia.In a second time, we have applied the SELDI-TOF strategy on serums originating from various patients. This approach has permitted the identification of three different proteins over expressed in the patient group. The quantification of these proteins allows the classification of the patients versus the controls

Logiques sociales et TICES : l’approche par les procédés d’écriture

National audienc