RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans.

Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.

Skeletal muscle protein content of lipolytic inhibitor G(0)/G(1) switch gene-2 protein: the effect of endurance training

The first and rate-limiting step of lipolysis is the removal of the first fatty acid from a triglyceride molecule; it is catalyzed by adipose triglyceride lipase (ATGL). ATGL is co-activated by comparative gene identification-58 (CGI-58) and inhibited by the G(0)/G(1) switch gene-2 protein (G0S2). G0S2 has also recently been identified as a positive regulator of oxidative phosphorylation within the mitochondria. Previous research has demonstrated in cell culture, a dose dependent mechanism for inhibition by G0S2 on ATGL. However our data is not consistent with this hypothesis. There was no change in G0S2 protein content during an acute lipolytic inducing set of contractions in both whole muscle, and isolated mitochondria yet both ATGL and G0S2 increase following endurance training, in spite of the fact that there should be increased reliance on intramuscular lipolysis. Therefore, inhibition of ATGL by G0S2 appears to be regulated through more complicated intracellular or post-translation regulation.

Characterization of the Sterile20 kinase Slik : A regulator of growth in Drosophila

La prolifération cellulaire et la croissance tissulaire sont étroitement contrôlées au cours du développement. Chez la Drosophila melanogaster, ces processus sont régulés en partie par la kinase stérile-20 Slik (SLK et LOK chez les mammifères) et le suppresseur de tumeur Hippo (Hpo, MST1/2 chez les mammifères) dans les cellules épithéliales. La surexpression de la kinase Slik augmente la taille des tissus chez les mouches adultes. Cependant, les mutants slik-/- meurent avant d'avoir terminé leur développement. Lorsqu’elle est surexprimée dans les cellules épithéliales des ailes en voie de développement, cette protéine favorise la prolifération cellulaire. En outre, l'expression de Slik dans une population de cellules conduit à une surprolifération des cellules voisines, même quand elles sont physiquement séparées. Ceci est probablement dû à la sécrétion de facteurs de croissance qui stimulent la prolifération de manière paracrine. En utilisant des méthodes génétiques et transcriptomiques, nous essayons de déterminer les molécules et les mécanismes impliqués. Contrairement à ce qui a été publié, nous avons constaté que Slik ne transmet pas de signal prolifératif en inhibant le suppresseur de tumeur Merlin (Mer, NF2 chez les mammifères), un composant en amont de la voie Hippo. Plutôt, elle favorise la prolifération non-autonome et la croissance des tissus en signalisation par la kinase dRaf (la seule kinase de la famille Raf chez la drosophile). Nous prouvons que dRaf est nécessaire chez les cellules voisines pour conduire la prolifération chez ces cellules. De plus, nous avons utilisé le séquençage du transcriptome pour identifier de nouveaux effecteurs en aval de Slik. Ce qui permettra de mieux comprendre les effets de SLK et LOK chez les humains.

Overproduction, purification and preliminary X-ray diffraction analysis of YncE, an iron-regulated Sec-dependent periplasmic protein from Escherichia coli

The yncE gene of Escherichia coli encodes a predicted periplasmic protein of unknown function. The gene is de-repressed under iron restriction through the action of the global iron regulator Fur. This suggests a role in iron acquisition, which is supported by the presence of the adjacent yncD gene encoding a potential TonB-dependent outer-membrane transporter. Here, the preliminary crystallographic structure of YncE is reported, revealing that it consists of a seven-bladed beta-propeller which resembles the corresponding domain of the `surface-layer protein' of Methanosarcina mazei. A full structure determination is under way in order to provide insight into the function of this protein.

The structure of echovirus type 12 bound to a two-domain fragment of its cellular attachment protein decay-accelerating factor (CD 55)

Echovirus type 12 (EV12), an enterovirus of the Picornaviridae family, uses the complement regulator, decay-accelerating factor (DAF, CD55) as a cellular receptor. We have calculated a three-dimensional reconstruction of EV12 bound to a fragment of DAF, consisting of short consensus repeat domains 3 and 4, from cryo-negative stain electron microscopy data (EMD #1057). This shows that, as for an earlier reconstruction of the related echovirus type 7 bound to DAF, attachment is not within the viral canyon but occurs close to the two-fold symmetry axes. Despite this general similarity, our reconstruction reveals a receptor interaction that is quite different from that observed for EV7. Fitting of the crystallographic co-ordinates for DAF34 and EV11 into the reconstruction shows a close agreement between the crystal structure of the receptor fragment and the density for the virus-bound receptor, allowing unambiguous positioning of the receptor with respect to the virion (PDB #1UPN). Our finding that the mode of virus-receptor interaction in EV12 is distinct from that seen for EV7 raises interesting questions regarding the evolution and biological significance of the DAF-binding phenotype in these viruses.

Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells

Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule-1 (PECAM-1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcR-chain signaling via recruitment of SHP-2 to phosphorylated immunoreceptor tyrosine-based inhibitory motifs in PECAM-1. Objective: To investigate the possibility that PECAM-1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI-mediated platelet activation in platelets. Methods: The ability of PECAM-1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling. We therefore propose that PECAM-1-mediated inhibition of GPVI-dependent platelet responses result, at least in part, from recruitment of SHP-2–p85 complexes to tyrosine-phosphorylated PECAM-1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.

Transcriptional regulators of the GAD acid stress island are carried by effector protein-encoding prophages and indirectly control type III secretion in enterohemorrhagic Escherichia coli O157:H7

P>Type III secretion (T3S) plays a pivotal role in the colonization of ruminant hosts by Enterohemorrhagic Escherichia coli (EHEC). The T3S system translocates effector proteins into host cells to promote bacterial attachment and persistence. The repertoire and variation in prophage regions underpins differences in the pathogenesis and epidemiology of EHEC strains. In this study, we have used a collection of deletions in cryptic prophages and EHEC O157 O-islands to screen for novel regulators of T3S. Using this approach we have identified a family of homologous AraC-like regulators that indirectly repress T3S. These prophage-encoded secretion regulator genes (psr) are found exclusively on prophages and are associated with effector loci and the T3S activating Pch family of regulators. Transcriptional profiling, mutagenesis and DNA binding studies were used to show that these regulators usurp the conserved GAD acid stress resistance system to regulate T3S by increasing the expression of GadE (YhiE) and YhiF and that this regulation follows attachment to bovine epithelial cells. We further demonstrate that PsrA and effectors encoded within cryptic prophage CP933-N are required for persistence in a ruminant model of colonization.

The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183–3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRΔGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRΔGly mutants than in the wt strain, indicative of the CtsRΔGly protein being inactive. Further evidence that the CtsRΔGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRΔGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRΔGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.

Endosomal endothelin-converting enzyme-1: a regulator of beta-arrestin-dependent ERK signaling

Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and beta-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. beta-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that endothelin-converting enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, beta-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls beta-arrestin-dependent signaling of endocytosed GPCRs.

Stigma/style cell cycle inhibitor 1 (SCI1), a tissue-specific cell cycle regulator that controls upper pistil development

P>A cDNA encoding a small lysine-rich protein of unknown function was identified in a tobacco (Nicotiana tabacum) stigma/style suppression subtractive hybridization cDNA library. After its characterization, the corresponding gene was designated stigma/style cell cycle inhibitor 1 (SCI1). Fluorescence microscopy with an SCI1-GFP protein fusion demonstrated its nuclear localization, which was confined to the interchromatic region. Real-time RT-PCR and in situ hybridization experiments showed that SCI1 is stigma/style-specific and developmentally regulated. SCI1 RNAi knockdown and overexpression plants had stigmas/styles with remarkably enlarged and reduced areas, respectively, which was attributable to differences in cell numbers. These results indicate that SCI1 is a tissue-specific negative cell cycle regulator. The differences in cell division had an effect on the timing of the differentiation of the stigmatic papillar cells, suggesting that their differentiation is coupled to stigma cell divisions. This is consistent with a role for SCI1 in triggering differentiation through cell proliferation control. Our results revealed that SCI1 is a novel tissue-specific gene that controls cell proliferation/differentiation, probably as a component of a developmental signal transduction pathway.

Immune Evasion of Leptospira Species by Acquisition of Human Complement Regulator C4BP

Leptospirosis is a spirochetal zoonotic disease of global distribution with a high incidence in tropical regions. In the last 15 years it has been recognized as an important emerging infectious disease due to the occurrence of large outbreaks in warm-climate countries and, occasionally, in temperate regions. Pathogenic leptospires efficiently colonize target organs after penetrating the host. Their invasiveness is attributed to the ability to multiply in blood, adhere to host cells, and penetrate into tissues. Therefore, they must be able to evade the innate host defense. The main purpose of the present study was to evaluate how several Leptospira strains evade the protective function of the complement system. The serum resistance of six Leptospira strains was analyzed. We demonstrate that the pathogenic strain isolated from infected hamsters avoids serum bactericidal activity more efficiently than the culture-attenuated or the nonpathogenic Leptospira strains. Moreover, both the alternative and the classical pathways of complement seem to be responsible for the killing of leptospires. Serum-resistant and serum-intermediate strains are able to bind C4BP, whereas the serum-sensitive strain Patoc I is not. Surface-bound C4BP promotes factor I-mediated cleavage of C4b. Accordingly, we found that pathogenic strains displayed reduced deposition of the late complement components C5 to C9 upon exposure to serum. We conclude that binding of C4BP contributes to leptospiral serum resistance against host complement.

CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. beta-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

SpdR, a Response Regulator Required for Stationary-Phase Induction of Caulobacter crescentus cspD

The cold shock protein (CSP) family includes small polypeptides that are induced upon temperature downshift and stationary phase. The genome of the alphaproteobacterium Caulobacter crescentus encodes four CSPs, with two being induced by cold shock and two at the onset of stationary phase. In order to identify the environmental signals and cell factors that are involved in cspD expression at stationary phase, we have analyzed cspD transcription during growth under several nutrient conditions. The results showed that expression of cspD was affected by the medium composition and was inversely proportional to the growth rate. The maximum levels of expression were decreased in a spoT mutant, indicating that ppGpp may be involved in the signalization for carbon starvation induction of cspD. A Tn5 mutant library was screened for mutants with reduced cspD expression, and 10 clones that showed at least a 50% reduction in expression were identified. Among these, a strain with a transposon insertion into a response regulator of a two-component system showed no induction of cspD at stationary phase. This protein (SpdR) was able to acquire a phosphate group from its cognate histidine kinase, and gel mobility shift assay and DNase I footprinting experiments showed that it binds to an inverted repeat sequence of the cspD regulatory region. A mutated SpdR with a substitution of the conserved aspartyl residue that is the probable phosphorylation site is unable to bind to the cspD regulatory region and to complement the spdR mutant phenotype.

Pseudomonas aeruginosa PA14 cupD transcription is activated by the RcsB response regulator, but repressed by its putative cognate sensor RcsC

The opportunistic pathogen Pseudomonas aeruginosa PA14 possesses four fimbrial cup clusters, which may confer the ability to adapt to different environments. cupD lies in the pathogenicity island PAPI-1 next to genes coding for a putative phosphorelay system composed of the hybrid histidine kinase RcsC and the response regulator RcsB. The main focus of this work was the regulation of cupD at the mRNA level. It was found that the HN-S-like protein MvaT does not exert a strong influence on cupD transcript levels, as it does for cupA. cupD transcription is higher in cultures grown at 28 degrees C, which agrees with a cupD mutant presenting attenuated virulence only in a plant model, but not in a mouse model of infection. Whereas an rcsC in-frame deletion mutant presented higher levels of cupD mRNA, rcsB deletion had the opposite effect. Accordingly, overexpression of RcsB increased the levels of cupD transcription, and promoted biofilm formation and the appearance of fimbriae. A single transcription start site was determined for cupD and transcription from this site was induced by RcsB. A motif similar to the enterobacterial RcsB/RcsA-binding site was detected adjacent to the -35 region, suggesting that this could be the RcsB-binding site. Comparison of P. aeruginosa and Escherichia coli Rcs may provide insights into how similar systems can be used by different bacteria to control gene expression and to adapt to various environmental conditions.

SIRT1 Deacetylase Activity and the Maintenance of Protein Homeostasis in Response to Stress: An Overview

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)