Alexandre Stella

Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins

Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA2-IVA, the calcium-independent iPLA2-VIA, and the secreted sPLA2-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPγS triggered activation of phospholipase A2 (PLA2)and PLD2. A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandinJ2 (15-d PGJ2), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell.

The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis

The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminal ACP domain of Pks13 (N-ACPPks13). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACPPks13) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce α-alkyl β-ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterial drug screening.

Lamellar Bodies of Human Epidermis Proteomics Characterization by High Throughput Mass Spectrometry and Possible Involvement of CLIP-170 in their Trafficking/Secretion

Lamellar bodies (LBs) are tubulovesicular secretory organelles of epithelial cells related to lysosomes. In the epidermis, they play a crucial role in permeability barrier homeostasis, secreting their contents, lipids, a variety of hydrolases, protease inhibitors, and antimicrobial peptides, in the upper keratinocyte layers. The identification of proteins transported in epidermal LBs is still far from complete, and the way their secretion is controlled unknown. In this study, we describe the first proteomics characterization by nano-LC-MS/MS of a fraction enriched in epidermal LBs. We identified 984 proteins, including proteins known or thought to be secreted by LBs. Moreover 31 proteins corresponded to lysosomal components further suggesting that LBs are a new class of secretory lysosomes. Many of the newly found proteins could play a role in the epidermal barrier and desquamation (one acid ceramidase-like protein, apolipoproteins, glycosidases, protease inhibitors, and peptidases) and in LB trafficking (e.g. Rab, Arf, and motor complex proteins). We focus here on CLIP-170/restin, a protein that mediates interactions between organelles and microtubules. Western blotting confirmed the presence of CLIP-170 and its known effectors IQGAP1 and Cdc42 in the LB-enriched fraction. We showed, by confocal microscopy analysis of skin cryosections, that CLIP-170 was expressed in differentiated keratinocytes, first at the periphery of the nucleus then with a granular cytoplasmic labeling evocative of LBs. It was preferentially co-localized with Cdc42 and with the known LB protein cathepsin D. CLIP-170 was also largely co-localized with Rab7. This study strongly suggests a new function for CLIP-170, its involvement together with Cdc42 and/or Rab7 in the intracellular trafficking of LBs, and provides evidence that nano-LC-MS/MS combined with monodimensional electrophoresis separation constitutes a powerful method for identifying proteins in a complex mixture such as subcellular structures.

Functional characterization of the Mycobacterium tuberculosis serine/threonine kinase PknJ

Eukaryotic-like Ser/Thr protein kinases (STPKs) are present in many bacterial species, where they control various physiological and virulence processes by enabling microbial adaptation to specific environmental signals. PknJ is the only member of the 11 STPKs identified in Mycobacterium tuberculosis that still awaits characterization. Here we report that PknJ is a functional kinase that forms dimers in vitro, and contains a single transmembrane domain. Using a high-density peptide-chip-based technology, multiple potential mycobacterial targets were identified for PknJ. We confirmed PknJ-dependent phosphorylation of four of these targets: PknJ itself, which autophosphorylates at Thr(168), Thr(171) and Thr(173) residues; the transcriptional regulator EmbR; the methyltransferase MmaA4/Hma involved in mycolic acid biosynthesis; and the dipeptidase PepE, whose encoding gene is located next to pknJ in the mycobacterial genome. Our results provide a number of candidate phospho-targets for PknJ and possibly other mycobacterial STPKs that could be studied to investigate the role of STPKs in M. tuberculosis physiology and virulence.

Proteomic Analysis Reveals Selective Impediment of Neuronal Remodeling upon Borna Disease Virus Infection

ABSTRACT The neurotropic virus Borna disease virus (BDV) persists in the central nervous systems of a wide variety of vertebrates and causes behavioral disorders. BDV represents an intriguing example of a virus whose persistence in neurons leads to altered brain function in the absence of overt cytolysis and inflammation. The bases of BDV-induced behavioral impairment remain largely unknown. To better characterize the neuronal response to BDV infection, we compared the proteomes of primary cultures of cortical neurons with and without BDV infection. We used two-dimensional liquid chromatography fractionation, followed by protein identification by nanoliquid chromatography-tandem mass spectrometry. This analysis revealed distinct changes in proteins implicated in neurotransmission, neurogenesis, cytoskeleton dynamics, and the regulation of gene expression and chromatin remodeling. We also demonstrated the selective interference of BDV with processes related to the adaptative response of neurons, i.e., defects in proteins regulating synaptic function, global rigidification of the cytoskeleton network, and altered expression of transcriptional and translational repressors. Thus, this work provides a global view of the neuronal changes induced by BDV infection together with new clues to understand the mechanisms underlying the selective interference with neuronal plasticity and remodeling that characterizes BDV persistence.

Label-free quantitative urinary proteomics identifies the arginase pathway as a new player in congenital obstructive nephropathy.

Obstructive nephropathy is a frequently encountered situation in newborns. In previous studies, the urinary peptidome has been analyzed for the identification of clinically useful biomarkers of obstructive nephropathy. However, the urinary proteome has not been explored yet and should allow additional insight into the pathophysiology of the disease. We have analyzed the urinary proteome of newborns (n = 5/group) with obstructive nephropathy using label free quantitative nanoLC-MS/MS allowing the identification and quantification of 970 urinary proteins. We next focused on proteins exclusively regulated in severe obstructive nephropathy and identified Arginase 1 as a potential candidate molecule involved in the development of obstructive nephropathy, located at the crossroad of pro- and antifibrotic pathways. The reduced urinary abundance of Arginase 1 in obstructive nephropathy was verified in independent clinical samples using both Western blot and MRM analysis. These data were confirmed in situ in kidneys obtained from a mouse obstructive nephropathy model. In addition, we also observed increased expression of Arginase 2 and increased total arginase activity in obstructed mouse kidneys. mRNA expression analysis of the related arginase pathways indicated that the pro-fibrotic arginase-related pathway is activated during obstructive nephropathy. Taken together we have identified a new actor in the development of obstructive nephropathy in newborns using quantitative urinary proteomics and shown its involvement in an in vivo model of disease. The present study demonstrates the relevance of such a quantitative urinary proteomics approach with clinical samples for a better understanding of the pathophysiology and for the discovery of potential therapeutic targets.

Substrates of the ASB2α E3 ubiquitin ligase in dendritic cells.

Conventional dendritic cells (cDCs) comprise distinct populations with specialized immune functions that are mediators of innate and adaptive immune responses. Transcriptomic and proteomic approaches have been used so far to identify transcripts and proteins that are differentially expressed in these subsets to understand the respective functions of cDCs subsets. Here, we showed that the Cullin 5-RING E3 ubiquitin ligase (E3) ASB2α, by driving degradation of filamin A (FLNa) and filamin B (FLNb), is responsible for the difference in FLNa and FLNb abundance in the different spleen cDC subsets. Importantly, the ability of these cDC subsets to migrate correlates with the level of FLNa. Furthermore, our results strongly point to CD4 positive and double negative cDCs as distinct populations. Finally, we develop quantitative global proteomic approaches to identify ASB2α substrates in DCs using ASB2 conditional knockout mice. As component of the ubiquitin-proteasome system (UPS) are amenable to pharmacological manipulation, these approaches aimed to the identification of E3 substrates in physiological relevant settings could potentially lead to novel targets for therapeutic strategies.

Asb2α-Filamin A Axis Is Essential for Actin Cytoskeleton Remodeling During Heart Development

Rationale: Heart development involves differentiation of cardiac progenitors and assembly of the contractile sarcomere apparatus of cardiomyocytes. However, little is known about the mechanisms that regulate actin cytoskeleton remodeling during cardiac cell differentiation. Objective: The Asb2&agr; (Ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2) CRL5 (cullin 5 RING E3 ubiquitin ligase) triggers polyubiquitylation and subsequent degradation by the proteasome of FLNs (filamins). Here, we investigate the role of Asb2&agr; in heart development and its mechanisms of action. Methods and Results: Using Asb2 knockout embryos, we show that Asb2 is an essential gene, critical to heart morphogenesis and function, although its loss does not interfere with the overall patterning of the embryonic heart tube. We show that the Asb2&agr; E3 ubiquitin ligase controls Flna stability in immature cardiomyocytes. Importantly, Asb2&agr;-mediated degradation of the actin-binding protein Flna marks a previously unrecognized intermediate step in cardiac cell differentiation characterized by cell shape changes and actin cytoskeleton remodeling. We further establish that in the absence of Asb2&agr;, myofibrils are disorganized and that heartbeats are inefficient, leading to embryonic lethality in mice. Conclusions: These findings identify Asb2&agr; as an unsuspected key regulator of cardiac cell differentiation and shed light on the molecular and cellular mechanisms determining the onset of myocardial cell architecture and its link with early cardiac function. Although Flna is known to play roles in cytoskeleton organization and to be required for heart function, this study now reveals that its degradation mediated by Asb2&agr; ensures essential functions in differentiating cardiac progenitors.