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1.  An ABC-transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa 
Environmental microbiology  2012;15(2):471-486.
Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SS). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Thr kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and export of Hcp1 and Tse1. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signaling pathway that promotes H1-T6SS activity under optimal environmental conditions.
doi:10.1111/j.1462-2920.2012.02816.x
PMCID: PMC3467343  PMID: 22765374
3.  Unique Biofilm Signature, Drug Susceptibility and Decreased Virulence in Drosophila through the Pseudomonas aeruginosa Two-Component System PprAB 
PLoS Pathogens  2012;8(11):e1003052.
Bacterial biofilm is considered as a particular lifestyle helping cells to survive hostile environments triggered by a variety of signals sensed and integrated through adequate regulatory pathways. Pseudomonas aeruginosa, a Gram-negative bacterium causing severe infections in humans, forms biofilms and is a fantastic example for fine-tuning of the transition between planktonic and community lifestyles through two-component systems (TCS). Here we decipher the regulon of the P. aeruginosa response regulator PprB of the TCS PprAB. We identified genes under the control of this TCS and once this pathway is activated, analyzed and dissected at the molecular level the PprB-dependent phenotypes in various models. The TCS PprAB triggers a hyper-biofilm phenotype with a unique adhesive signature made of BapA adhesin, a Type 1 secretion system (T1SS) substrate, CupE CU fimbriae, Flp Type IVb pili and eDNA without EPS involvement. This unique signature is associated with drug hyper-susceptibility, decreased virulence in acutely infected flies and cytotoxicity toward various cell types linked to decreased Type III secretion (T3SS). Moreover, once the PprB pathway is activated, decreased virulence in orally infected flies associated with enhanced biofilm formation and dissemination defect from the intestinal lumen toward the hemolymph compartment is reported. PprB may thus represent a key bacterial adaptation checkpoint of multicellular and aggregative behavior triggering the production of a unique matrix associated with peculiar antibiotic susceptibility and attenuated virulence, a particular interesting breach for therapeutic intervention to consider in view of possible eradication of P. aeruginosa biofilm-associated infections.
Author Summary
We unraveled that once the two-component system PprAB regulatory pathway is activated, Pseudomonas aeruginosa displays a unique hyper-biofilm phenotype due to a molecular signature combining a T1SS high molecular weight substrate, BapA, fimbriae of the chaperone-usher pathway, Type IVb pili and eDNA. Originally, this particular hyper-biofilm that is not strictly dependent on Psl or Pel exopolysaccharide (EPS) synthesis displays increased drug susceptibility, in contrary to previously reported biofilm lifestyle associated with increased resistance to antibiotics. PprB-dependent hyper-biofilm was also observed on intestinal mucosa of orally infected Drosophila flies in which it also displays a reduced capacity to cross the epithelial barrier from the intestinal lumen toward the hemolymph that consequently resulted in a reduced capacity to kill flies. Furthermore, constitutive activation of this PprB regulatory pathway triggers a reduced secretion of T3SS effectors which may account for the decreased virulence observed in epithelial and macrophage lineages and in acute Drosophila infections induced by septic injury. We appended in this study pieces of regulatory and molecular data that highlight the possibility to combat infections due to P. aeruginosa-biofilm with particular matrix.
doi:10.1371/journal.ppat.1003052
PMCID: PMC3510237  PMID: 23209420
4.  FYVE-Dependent Endosomal Targeting of an Arrestin-Related Protein in Amoeba 
PLoS ONE  2010;5(12):e15249.
Background
Visual and β-arrestins are scaffolding proteins involved in the regulation of receptor-dependent intracellular signaling and their trafficking. The arrestin superfamilly includes several arrestin domain-containing proteins and the structurally related protein Vps26. In Dictyostelium discoideum, the arrestin-domain containing proteins form a family of six members, namely AdcA to -F. In contrast to canonical arrestins, Dictyostelium Adc proteins show a more complex architecture, as they possess, in addition to the arrestin core, other domains, such as C2, FYVE, LIM, MIT and SAM, which potentially mediate selective interactions with either lipids or proteins.
Methodology and Principal Findings
A detailed analysis of AdcA has been performed. AdcA extends on both sides of the arrestin core, in particular by a FYVE domain which mediates selective interactions with PI(3)P, as disclosed by intrinsic fluorescence measurements and lipid overlay assays. Localization studies showed an enrichment of tagged- and endogenous AdcA on the rim of early macropinosomes and phagosomes. This vesicular distribution relies on a functional FYVE domain. Our data also show that the arrestin core binds the ADP-ribosylation factor ArfA, the unique amoebal Arf member, in its GDP-bound conformation.
Significance
This work describes one of the 6 arrestin domain-containing proteins of Dictyostelium, a novel and atypical member of the arrestin clan. It provides the basis for a better understanding of arrestin-related protein involvement in trafficking processes and for further studies on the expanding roles of arrestins in eukaryotes.
doi:10.1371/journal.pone.0015249
PMCID: PMC3001460  PMID: 21179207
5.  Pdro, a Protein Associated with Late Endosomes and Lysosomes and Implicated in Cellular Cholesterol Homeostasis 
PLoS ONE  2010;5(6):e10977.
Background
Cellular cholesterol is a vital component of the cell membrane. Its concentration is tightly controlled by mechanisms that remain only partially characterized. In this study, we describe a late endosome/lysosomes–associated protein whose expression level affects cellular free cholesterol content.
Methodology/Principal Findings
Using a restricted proteomic analysis of detergent-resistant membranes (DRMs), we have identified a protein encoded by gene C11orf59. It is mainly localized to late endosome/lysosome (LE/LY) compartment through N-terminal myristoylation and palmitoylation. We named it Pdro for protein associated with DRMs and endosomes. Very recently, three studies have reported on the same protein under two other names: the human p27RF-Rho that regulates RhoA activation and actin dynamics, and its rodent orthologue p18 that controls both LE/LY dynamics through the MERK-ERK pathway and the lysosomal activation of mammalian target of rapamycin complex 1 by amino acids. We found that, consistent with the presence of sterol-responsive element consensus sequences in the promoter region of C11orf59, Pdro mRNA and protein expression levels are regulated positively by cellular cholesterol depletion and negatively by cellular cholesterol loading. Conversely, Pdro is involved in the regulation of cholesterol homeostasis, since its depletion by siRNA increases cellular free cholesterol content that is accompanied by an increased cholesterol efflux from cells. On the other hand, cells stably overexpressing Pdro display reduced cellular free cholesterol content. Pdro depletion-mediated excess cholesterol results, at least in part, from a stimulated low-density lipoprotein (LDL) uptake and an increased cholesterol egress from LE/LY.
Conclusions/Significance
LDL-derived cholesterol release involves LE/LY motility that is linked to actin dynamics. Because Pdro regulates these two processes, we propose that modulation of Pdro expression in response to sterol levels regulates LDL-derived cholesterol through both LDL uptake and LE/LY dynamics, to ultimately control free cholesterol homeostasis.
doi:10.1371/journal.pone.0010977
PMCID: PMC2882324  PMID: 20544018
6.  The Twin Spot Generator for differential Drosophila lineage analysis 
Nature methods  2009;6(8):600-602.
In Drosophila, widely-used mitotic recombination-based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the Twin Spot Generator technique (TSG) and demonstrate that through mitotic recombination, TSG generates green and red twin spots in internal fly tissues, visible even as single cells. We discuss the wide applications of TSG to lineage and genetic mosaic studies.
doi:10.1038/nmeth.1349
PMCID: PMC2720837  PMID: 19633664
7.  Maurocalcine and domain A of the II-III loop of the dihydropyridine receptor Cav1.1 subunit share common binding sites on the skeletal ryanodine receptor 
The Journal of Biological Chemistry  2004;280(6):4013-4016.
Maurocalcine is a scorpion venom toxin of 33 amino acid residues that bears a striking resemblance to the domain A. This domain belongs to the II-III loop of Cav1.1 which is implicated in excitation-contraction coupling. Besides the structural homology, maurocalcine also modulates RyR1 channel activity in a manner akin to a synthetic peptide of domain A. Owing to these similarities, we hypothesized that maurocalcine and domain A may bind onto an identical region(s) of RyR1. Using a set of RyR1 fragments, we demonstrate that peptide A and maurocalcine bind onto two discrete RyR1 regions: fragments 3 and 7 encompassing amino acid residues 1021-1631 and 3201-3661, respectively. The binding onto fragment 7 is of greater importance and was thus further investigated. We found that the amino acid region 3350-3501 of RyR1 (fragment 7.2) is sufficient for these interactions. Proof that peptide A and maurocalcine bind onto the same site is provided by competition experiments in which binding of fragment 7.2 to peptide A is inhibited by preincubation with maurocalcine. At the functional level, deletion of fragment 7 abolishes the maurocalcine induced stimulation of [3H]-ryanodine binding onto microsomes of COS-7 cells transfected with RyR1 without affecting the caffeine response.
doi:10.1074/jbc.C400433200
PMCID: PMC2712624  PMID: 15591063
Adenosine Triphosphate; chemistry; Animals; Binding Sites; Binding, Competitive; COS Cells; Calcium Channels, L-Type; chemistry; Caveolin 1; Caveolins; chemistry; Chromatography; Cloning, Molecular; Cryoelectron Microscopy; Microscopy, Fluorescence; Muscle, Skeletal; metabolism; Peptides; chemistry; Plasmids; metabolism; Protein Binding; Protein Structure, Tertiary; Recombinant Fusion Proteins; chemistry; Ryanodine; chemistry; Ryanodine Receptor Calcium Release Channel; chemistry; Scorpion Venoms; chemistry; pharmacology; Transfection
8.  Transduction of the scorpion toxin maurocalcine into cells. Evidence that the toxin crosses the plasma membrane 
The Journal of Biological Chemistry  2005;280(13):12833-12839.
Maurocalcine (MCa) is a 33 amino acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. External application of MCa to cultured myotubes is known to produce Ca2+ release from intracellular stores. MCa binds directly to the skeletal muscle isoform of the ryanodine receptor, an intracellular channel target of the endoplasmic reticulum, and induces long-lasting channel openings in a mode of smaller conductance. Here, we investigated the way MCa proceeds to cross biological membranes in order to reach its target. A biotinylated derivative of MCa was produced (MCab) and complexed with a fluorescent indicator (streptavidine-cyanine 3) in order to follow the cell penetration of the toxin. The toxin complex efficiently penetrated in various cell types without requiring metabolic energy (low temperature) or implicating an endocytosis mechanism. MCa appeared to share the same features as the so-called Cell-Penetrating Peptides (CPP). Our results provide evidence that MCa has the ability to act as a molecular carrier and to cross cell membranes in a rapid manner (1–2 min) making this toxin the first demonstrated example of a scorpion toxin that translocates into cells.
doi:10.1074/jbc.M412521200
PMCID: PMC2713311  PMID: 15653689
Amino Acid Sequence; Biological Transport; Biotinylation; Calcium; chemistry; Carrier Proteins; chemistry; Cell Differentiation; Cell Line; Cell Membrane; drug effects; metabolism; Endocytosis; Endoplasmic Reticulum; metabolism; Gene Products, tat; metabolism; Humans; Kinetics; Microscopy, Confocal; Models, Molecular; Molecular Sequence Data; Muscle, Skeletal; cytology; metabolism; Peptides; chemistry; Protein Conformation; Protein Isoforms; Protein Transport; Ryanodine; metabolism; Ryanodine Receptor Calcium Release Channel; chemistry; Sarcoplasmic Reticulum; metabolism; Scorpion Venoms; metabolism; pharmacokinetics; Signal Transduction; Temperature; Time Factors
9.  Transcription Factor IIB (TFIIB)-Related Protein (pBrp), a Plant-Specific Member of the TFIIB-Related Protein Family 
Molecular and Cellular Biology  2003;23(9):3274-3286.
Although it is now well documented that metazoans have evolved general transcription factor (GTF) variants to regulate their complex patterns of gene expression, there is so far no information regarding the existence of specific GTFs in plants. Here we report the characterization of a ubiquitously expressed gene that encodes a bona fide novel transcription factor IIB (TFIIB)-related protein in Arabidopsis thaliana. We have shown that this protein is the founding member of a plant-specific TFIIB-related protein family named pBrp (for plant-specific TFIIB-related protein). Surprisingly, in contrast to common GTFs that are localized in the nucleus, the bulk of pBrp proteins are bound to the cytoplasmic face of the plastid envelope, suggesting an organelle-specific function for this novel class of TFIIB-related protein. We show that pBrp proteins harbor conditional proteolytic signals that can target these proteins for rapid turnover by the proteasome-mediated protein degradation pathway. Interestingly, under conditions of proteasome inhibition, pBrp proteins accumulate in the nucleus. Together, our results suggest a possible involvement of these proteins in an intracellular signaling pathway between plastids and the nucleus. Our data provide the first evidence for an organelle-related evolution of the eukaryotic general transcription machinery.
doi:10.1128/MCB.23.9.3274-3286.2003
PMCID: PMC153204  PMID: 12697827

Results 1-9 (9)