Investigation of the effect of the adsorbent DAV131A on the propensity of moxifloxacin to induce simulated Clostridioides (Clostridium) difficile infection (CDI) in an in vitro human gut model.

BACKGROUND: Clostridioides difficile infection (CDI) remains a high burden worldwide. DAV131A, a novel adsorbent, reduces residual gut antimicrobial levels, reducing CDI risk in animal models. OBJECTIVES: We used a validated human gut model to investigate the efficacy of DAV131A in preventing moxifloxacin-induced CDI. METHODS: C. difficile (CD) spores were inoculated into two models populated with pooled human faeces. Moxifloxacin was instilled (43 mg/L, once daily, 7 days) alongside DAV131A (5 g in 18 mL PBS, three times daily, 14 days, Model A), or PBS (18 mL, three times daily, 14 days, Model B). Selected gut microbiota populations, CD total counts, spore counts, cytotoxin titre and antimicrobial concentrations (HPLC) were monitored daily. We monitored for reduced susceptibility of CD to moxifloxacin. Growth of CD in faecal filtrate and medium in the presence/absence of DAV131A, or in medium pre-treated with DAV131A, was also investigated. RESULTS: DAV131A instillation reduced active moxifloxacin levels to below the limit of detection (50 ng/mL), and prevented microbiota disruption, excepting Bacteroides fragilis group populations, which declined by ∼3 log10 cfu/mL. DAV131A delayed onset of simulated CDI by ∼2 weeks, but did not prevent CD germination and toxin production. DAV131A prevented emergence of reduced susceptibility of CD to moxifloxacin. In batch culture, DAV131A had minor effects on CD vegetative growth, but significantly reduced toxin/spores (P < 0.005). CONCLUSIONS: DAV131A reduced moxifloxacin-induced microbiota disruption and emergence of antibiotic-resistant CD. Delayed onset of CD germination and toxin production indicates further investigations are warranted to understand the clinical benefits of DAV131A in CDI prevention.

Spatiotemporal regulation of the Pak1 kinase.

Pak1 (p21-activated kinase 1) is a key regulator of the actin cytoskeleton, adhesion and cell motility. Such biological roles require a tight spatial and kinetic control of its localization and activity. We summarize here the current knowledge on Pak1 dynamics in vivo. Inactive dimeric Pak1 is mainly cytosolic. Localized interaction with the activators Cdc42-GTP and Rac1-GTP stimulates the kinase at the sites of cellular protrusions. Moreover, Pak1 is dynamically engaged into multiprotein complexes forming adhesions to the extracellular matrix. Cutting edge microscopy technologies on living cells are finally shedding light on the intricate spatiotemporal mechanisms regulating Pak1.

A novel group IIA phospholipase A2 interacts with v-Src oncoprotein from RSV-transformed hamster cells.

We have isolated a novel isoform of phospholipase A(2). This enzyme was designated srPLA(2) because it was discovered while analyzing the proteins interacting with different forms of the v-Src oncoproteins isolated from Rous sarcoma virus-transformed hamster cells. It contains all the functional regions of the PLA(2) group IIA proteins but differs at its C-terminal end where there is an additional stretch of 8 amino acids. The SrPLA(2) isoform was detected as a 17-kDa precursor in cells and as a mature 14-kDa form secreted in culture medium. A direct interaction of the 17-kDa precursor with the Src protein was observed in lysates of transformed cells. Both the 17- and 14-kDa forms were found to be phosphorylated on tyrosine. To our knowledge, this is the first report of a PLA(2) group II protein that is tyrosine phosphorylated. We surmise that srPLA(2) interacts with the Src protein at the cell membrane during the process of its maturation.

Thrombopoietin-mediated sustained activation of extracellular signal-regulated kinase in UT7-Mpl cells requires both Ras-Raf-1- and Rap1-B-Raf-dependent pathways.

Thrombopoietin (TPO) regulates growth and differentiation of megakaryocytes. We previously showed that extracellular signal-regulated kinases (ERKs) are required for TPO-mediated full megakaryocytic maturation in both normal progenitors and a megakaryoblastic cell line (UT7) expressing the TPO receptor (Mpl). In these cells, intensity and duration of TPO-induced ERK signal are controlled by several regions of the cytoplasmic domain of Mpl. In this study, we explored the signaling pathways involved in this control. We show that the small GTPases Ras and Rap1 contribute together to TPO-induced ERK activation in UT7-Mpl cells and that they do so by activating different Raf kinases as downstream effectors: a Ras-Raf-1 pathway is required to initiate ERK activation while Rap1 sustains this signal through B-Raf. Indeed, (i) in cells expressing wild-type or mutant Mpl, TPO-induced Ras and Rap1 activation correlates with early and sustained phases of ERK signal, respectively; (ii) interfering mutants of Ras and Rap1 both inhibit ERK kinase activity and ERK-dependent Elk1 transcriptional activation in response to TPO; (iii) the kinetics of activation of Raf-1 and B-Raf by TPO follow those of Ras and Rap1, respectively; (iv) RasV12-mediated Elk1 activation was modulated by the wild type or interfering mutants of Raf-1 but not those of B-Raf; (v) Elk1 activation mediated by a constitutively active mutant of Rap1 (Rap1V12) is potentiated by B-Raf and inhibited by an interfering mutant of this kinase. UT7-Mpl cells represent the second cellular model in which Ras and Rap1 act in concert to modulate the duration of ERK signal in response to a growth factor and thereby the differentiation program. This is also, to our knowledge, the first evidence suggesting that Rap1 may play an active role in megakaryocytic maturation.

RUN domains: a new family of domains involved in Ras-like GTPase signaling.

RUN domains are present in several proteins that are linked particularly to the functions of GTPases in the Rap and Rab families. They could hence play an important role in multiple Ras-like GTPase signaling pathways.

Mutation status of genes encoding RhoA, Rac1, and Cdc42 GTPases in a panel of invasive human colorectal and breast tumors.

PURPOSE: The constitutive activation of Ras proteins by point mutation is the most frequently observed oncogene activation in human malignancies. The goal of this study was to investigate whether the constitutive activation of RhoA, Rac1, and Cdc42 proteins by point mutations, which can lead to experimental transformation of cultured cells, actually occurred in a panel of invasive colorectal and breast tumors. METHODS: We performed denaturing gradient gel electrophoresis and sequencing of transcripts amplified by reverse transcription and PCR for RhoA; we used direct sequencing of PCR-amplified genomic DNA to search for mutations in coding exons of the Rac1 and Cdc42 genes. RESULTS: Although mutations of the Kras4B and the p53 genes were detected using these methods, no mutation was found in the coding sequences of RhoA, Rac1, and Cdc42 genes, in primary as well as in associated metastasis. CONCLUSIONS: Point mutations in the coding sequences of genes encoding RhoA, Rac1, and Cdc42 GTPases do not occur at high frequency in invasive breast and colorectal tumors.

RGS14, a GTPase-activating protein for Gialpha, attenuates Gialpha- and G13alpha-mediated signaling pathways.

Regulator of G protein signaling (RGS) proteins are a family of approximately 20 proteins that negatively regulate signaling through heterotrimeric G protein-coupled receptors. The RGS proteins act as GTPase-activating proteins (GAPs) for certain Galpha subunits and as effector antagonists for Gqalpha. Mouse RGS14 encodes a 547-amino-acid protein with an N-terminal RGS domain, which is highly expressed in lymphoid tissues. In this study, we demonstrate that RGS14 is a GAP for Gialpha subfamily members and it attenuates interleukin-8 receptor-mediated mitogen-activated protein kinase activation. However, RGS14 does not exhibit GAP activity toward Gsalpha or Gqalpha nor does it regulate Gsalpha- or Gqalpha-mediated signaling pathways. Although RGS14 does not act as a GAP for G12/13alpha, it impairs c-fos serum response element activation induced by either a constitutively active mutant of G13alpha (G13alphaQ226L) or by carbachol stimulation of muscarinic type 1 receptors. An RGS14 mutant (EN92/93AA), which does not block Gialpha-linked signaling, also inhibits serum response element activation. RGS14 localizes predominantly in the cytosol, but it can be recruited to membranes by expression of G13alphaQ226L. Although RGS14 is constitutively expressed in lymphoid cells, agents that activate B or T lymphocytes further enhance its levels. Taken together, our results suggest that signals generated after lymphocyte activation may via RGS14 directly impinge on Gialpha- or G13alpha-mediated cellular processes in lymphocytes, such as adhesion and migration.

Saethre-Chotzen mutations cause TWIST protein degradation or impaired nuclear location.

H-TWIST belongs to the family of basic helix-loop-helix (bHLH) transcription factors known to exert their activity through dimer formation. We have demonstrated recently that mutations in H-TWIST account for Saethre-Chotzen syndrome (SCS), an autosomal dominant craniosynostosis syndrome characterized by premature fusion of coronal sutures and limb abnormalities of variable severity. Although insertions, deletions, nonsense and missense mutations have been identified, no genotype-phenotype correlation could be found, suggesting that the gene alterations lead to a loss of protein function irrespective of the mutation. To assess this hypothesis, we studied stability, dimerization capacities and subcellular distribution of three types of TWIST mutant. Here, we show that: (i) nonsense mutations resulted in truncated protein instability; (ii) missense mutations involving the helical domains led to a complete loss of H-TWIST heterodimerization with the E12 bHLH protein in the two-hybrid system and dramatically altered the ability of the TWIST protein to localize in the nucleus of COS-transfected cells; and (iii) in-frame insertion or missense mutations within the loop significantly altered dimer formation but not the nuclear location of the protein. We conclude that at least two distinct mechanisms account for loss of TWIST protein function in SCS patients, namely protein degradation and subcellular mislocalization.

Identification and characterization of potential effector molecules of the Ras-related GTPase Rap2.

In search for effectors of the Ras-related GTPase Rap2, we used the yeast two-hybrid method and identified the C-terminal Ras/Rap interaction domain of the Ral exchange factors (RalGEFs) Ral GDP dissociation stimulator (RalGDS), RalGDS-like (RGL), and RalGDS-like factor (Rlf). These proteins, which also interact with activated Ras and Rap1, are effectors of Ras and mediate the activation of Ral in response to the activation of Ras. Here we show that the full-length RalGEFs interact with the GTP-bound form of Rap2 in the two-hybrid system as well as in vitro. When co-transfected in HeLa cells, an activated Rap2 mutant (Rap2Val-12) but not an inactive protein (Rap2Ala-35) co-immunoprecipitates with RalGDS and Rlf; moreover, Rap2-RalGEF complexes can be isolated from the particulate fraction of transfected cells and were localized by confocal microscopy to the resident compartment of Rap2, i.e. the endoplasmic reticulum. However, the overexpression of activated Rap2 neither leads to the activation of the Ral GTPase via RalGEFs nor inhibits Ras-dependent Ral activation in vivo. Several hypotheses that could explain these results, including compartmentalization of proteins involved in signal transduction, are discussed. Our results suggest that in cells, the interaction of Rap2 with RalGEFs might trigger other cellular responses than activation of the Ral GTPase.

Proteins of the Ras pathway as novel potential anticancer therapeutic targets.

Ras proteins are molecular switches that constitute a pivotal element in the control of cellular responses to many incoming signals, and in particular mitogenic stimulations. They act through multiple effector pathways that carry out the biological functions of Ras in cells. Since mutations that constitutively activate Ras proteins have been found in a high proportion of human malignancies and participate in oncogenesis, a number of therapeutic anticancer strategies aimed against the activity or action of Ras proteins have been developed. This paper reviews the principal aspects of the Ras signaling pathway and describes some of the attempts to develop antitumor drugs based on this concept.

Specific inhibition of phorbol ester-stimulated phospholipase D by Clostridium sordellii lethal toxin and Clostridium difficile toxin B-1470 in HEK-293 cells. Restoration by Ral GTPases.

Activation of m3 muscarinic acetylcholine receptor (mAChR), stably expressed in human embryonic kidney (HEK)-293 cells, leads to phospholipase D (PLD) stimulation, a process apparently involving Rho GTPases, as shown by studies with Clostridium botulinum C3 exoenzyme and Clostridium difficile toxin B (TcdB). Direct activation of protein kinase C (PKC) by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), also induces PLD stimulation, which is additive to the mAChR action and which is only poorly sensitive to inactivation of Rho proteins by TcdB. To study whether Ras-like GTPases are involved in PLD regulation, we studied the effects of the TcdB variant TcdB-1470 and Clostridium sordellii lethal toxin (TcsL), known to inactivate Rac and some members of the Ras protein family, on PLD activities. TcdB-1470 and TcsL did not affect basal PLD activity and PLD stimulation by mAChR or direct G protein activation. In contrast, PMA-induced PLD stimulation was inhibited by TcdB-1470 and TcsL in a time- and concentration-dependent manner, without alteration in immunologically detectable PKC isozyme levels. In membranes of HEK-293 cells pretreated with TcdB-1470 or TcsL, basal and stable GTP analog-stimulated PLD activities measured with exogenous phosphatidylcholine, in the presence or absence of phosphatidylinositol 4,5-bisphosphate, were not altered. In contrast, pretreatment with TcdB-1470 and TcsL, but not TcdB, strongly reduced PMA-stimulated PLD activity. The addition of recombinant Rac1, serving as glucosylation substrate for TcdB, TcsL, and TcdB-1470, did not restore PLD stimulation by PMA. Furthermore, PMA-stimulated PLD activity, suppressed by prior treatment with TcdB-1470 or TcsL, was not rescued by the addition of recombinant Ras (RasG12V) or Rap proteins, acting as glucosylation substrates for TcsL only (Ras) or TcdB-1470 and TcsL (Rap). In contrast, the addition of recombinant Ral proteins (RalA and RalB), glucosylation substrates for TscL and TcdB-1470, but not for TcdB, to membranes of TcdB-1470- or TcsL-treated cells fully restored PLD stimulation by PMA without altering the strict MgATP dependence of PMA-induced PLD stimulation. RalA-mediated restoration of PMA-stimulated PLD activity in membranes of TcsL-treated cells was not enhanced by coaddition of RasG12V. In conclusion, the data presented indicate that TcdB-1470 and TcsL selectively interfere with phorbol ester stimulation of PLD and suggest an essential role of Ral proteins in PKC signaling to PLD in HEK-293 cells.

Identification of a specific effector of the small GTP-binding protein Rap2.

Rap2 is a small GTP-binding protein that belongs to the Ras superfamily and whose function is still unknown. To elucidate Rap2 function, we searched for potential effectors by screening a mouse brain cDNA library in a yeast two-hybrid system using as a bait a Rap2A protein bearing a mutation of Gly to Val at position 12. This strategy lead to the identification of a protein that interacts specifically with Rap2A complexed with GTP, and requires an intact effector domain of Rap2A for interaction; we designated this protein Rap2-interacting protein 8 (RPIP8). Biochemical data obtained from in vitro studies with purified proteins confirmed the genetic results. Mouse RPIP8 consists of 446 amino acids, bears a coiled-coil domain between residues 265 and 313, and is expressed principally in brain. Its human counterpart, of 400 amino acids, exhibits 93.7% identity in their common region. A search for similar sequences in expressed-sequence-tags databanks revealed the presence in human and rodents of mRNAs encoding the 400-residue and 446-residue forms of RPIP8. Furthermore a doublet of 45-50 kDa, corresponding to the 400-residue and 446-residue forms of the protein, was detected by western blotting of mouse brain extracts and lysates from pheochromocytoma PC12 cells and the pancreatic beta-cell lines HIT-T15 and RIN-m5F. Using transient transfections of HIT-T15 cells it was possible to demonstrate that [Val12]Rap2 and wild-type Rap2 could be immunoprecipitated with RPIP8. These data therefore argue for RPIP8 being a specific effector of the Rap2 protein in cells exhibiting neuronal properties.

Exclusion of five subunits of cGMP phosphodiesterase in Leber's congenital amaurosis.

Leber's congenital amaurosis (LCA) is the earliest and most severe of all inherited retinal dystrophies. Recently, we mapped an LCA gene to chromosome 17p13.1 (LCA1) and ascribed the disease to mutations of the retinal guanylate cyclase (ret GC) gene in a subset of families of North African ancestry. Owing to the genetic heterogeneity of LCA and considering that LCA1 results from an impaired production of cGMP in the retina (with permanent closure of cGMP-gated cation channels), we hypothesized that the activation of the cGMP phosphodiesterase (PDE) could trigger the disease by lowering the intracellular cGMP level in the retina. The rod and cone cGMP-PDE inhibitory subunits were regarded therefore as candidate genes in LCA. Here, we report the exclusion of five rod and cone cGMP-PDE subunits in LCA families unlinked to chromosome 17p13.

Crystal structures of the small G protein Rap2A in complex with its substrate GTP, with GDP and with GTPgammaS.

The small G protein Rap2A has been crystallized in complex with GDP, GTP and GTPgammaS. The Rap2A-GTP complex is the first structure of a small G protein with its natural ligand GTP. It shows that the hydroxyl group of Tyr32 forms a hydrogen bond with the gamma-phosphate of GTP and with Gly13. This interaction does not exist in the Rap2A-GTPgammaS complex. Tyr32 is conserved in many small G proteins, which probably also form this hydrogen bond with GTP. In addition, Tyr32 is structurally equivalent to a conserved arginine that binds GTP in trimeric G proteins. The actual participation of Tyr32 in GTP hydrolysis is not yet clear, but several possible roles are discussed. The conformational changes between the GDP and GTP complexes are located essentially in the switch I and II regions as described for the related oncoprotein H-Ras. However, the mobile segments vary in length and in the amplitude of movement. This suggests that even though similar regions might be involved in the GDP-GTP cycle of small G proteins, the details of the changes will be different for each G protein and will ensure the specificity of its interaction with a given set of cellular proteins.

Getting more from the two-hybrid system: N-terminal fusions to LexA are efficient and sensitive baits for two-hybrid studies.

Two-hybrid methods detect interactions between two proteins fused at the C-termini of, respectively, a DNA-binding domain and the activation domain of a transcriptional activator. Thus the N-terminus of none of these proteins is available for interaction. We have tested whether a bait protein with a reverted polarity (i.e. N-bait-LexA-C) is suitable for two-hybrid interaction. We show that such constructs give a specific interaction signal, and document two cases where the sensitivity is dramatically increased. Such constructs might lead to the identification of partners missed during classical two-hybrid screens.

[Isoprenylated proteins and cell proliferation: regulators and effectors of Ras proteins]. / Protéines isoprénylées et prolifération cellulaire: régulateurs et effecteurs des protéines Ras.

Ras proteins play a central role in the control of cellular proliferation. They are 189 amino acid monomeric GTP-binding proteins that cycle between an inactive GDP-bound and the active GTP-bound state, and carry a slow intrinsic GTPase activity. Ras proteins are activated by growth promoting signals incoming from receptor tyrosine kinases via SH2 domain and SH3 domain containing adapter proteins and the Ras exchange factor Sos, as well as from serpentine receptors via the beta gamma subunits of heterotrimeric G proteins and the Ras exchange factor Ras-GRF (or Cdc25). Proteins that can stimulate the GTPase activity of Ras (GAPs) ensure that following mitogenic stimulations, they return to their inactive GDP-bound state; amongst these proteins are p120-GAP, neurofibomin (the product of the susceptibility gene to type I neurofibromatosis), as well as the inositol 1,3,4,5-tetrakisphosphate-dependent GAPIP4BF. Several effectors have been identified that mediate the biological effects of Ras. The serine/threonine kinase Raf-1, as well as the closely related protein B-Raf, elicit the ERK cascade of MAP kinases. Phosphatidylinositol-3-OH kinase is involved in the activation of the Rac/Rho family proteins that play a role in the control of actin polymerisation, as well as in growth control, RalGDS, RGL and Rlf, are responsible for the activation of the Ras-related protein Ral. Recent evidence, using effector domain mutants of Ras, demonstrates that these pathways cooperate to elicit the growth promoting effects of Ras proteins.

Expression of the TSC2 product tuberin and its target Rap1 in normal human tissues.

The tuberous sclerosis-2 (TSC2) gene is linked to tuberous sclerosis (TSC), a dominantly inherited genetic syndrome in which inactivation of the normal TSC2 allele is associated with the development of mostly benign tumors and focal dysplasias. TSC2 encodes the protein tuberin, which is a widely expressed 180-kd polypeptide that exhibits specific GTPase activating activity toward Rap1 in vitro and co-localizes with Rap1 in cultured cells. In this study, we have performed immunohistochemical analyses, using affinity-purified anti-tuberin antibodies, to study the distribution of tuberin in a panel of normal human organs that are commonly affected by TSC. Cryosections indicated that tuberin is widely expressed at low levels. More intense staining of tuberin, in the cryosections and in paraffin sections, was observed in the small blood vessels of many organs, including the kidney, skin, and adrenal gland. High levels of tuberin were also detected in cortical neurons and cerebellar Purkinje cells. These findings imply that loss-of-function mutations in TSC2 might lead to the development of highly vascularized tumors, subcortical tubers, and focal atrophy of the cerebellar cortex, which are features commonly associated with TSC. Moreover, Rap1 was also found to be highly expressed in many of the same cells that contained high levels of tuberin, suggesting a functional interaction between tuberin and Rap1 in these tissues.

Co-localization of the TSC2 product tuberin with its target Rap1 in the Golgi apparatus.

Tuberin is the protein product of the tuberous sclerosis-2 (TSC2) gene, which is associated with tuberous sclerosis (TSC), a human genetic syndrome characterized by the development of tumors in a variety of tissues. We have previously shown that tuberin is a widely expressed 180 kDa protein which exhibits specific GTPase activating activity in vitro towards the Ras-related Rap1 protein. In this study we have used affinity-purified antibodies against tuberin to analyse its expression in human and rat tissues and to examine its subcellular localization. Tuberin expression was detected in all adult human tissues tested, with the highest levels found in brain, heart and kidney, organs that are commonly affected in TSC patients. By contrast, in adult rats the highest levels of tuberin were found in brain, liver and testis. Indirect immunofluorescence of tuberin in various cultured cell lines revealed a punctate, mostly perinuclear staining pattern. Double-indirect immunofluorescence analysis with anti-tuberin sera and antisera against known Golgi markers (mannosidase-II and furin) revealed that the staining of tuberin was consistent with its localization in the stacks of the Golgi apparatus. In support of this, treatment of cells with brefeldin A, a drug known to cause disassembly of the Golgi apparatus, abolished the perinuclear staining of tuberin. Moreover, conventional and confocal immunofluorescence demonstrated co-localization of tuberin with Rap1, which has previously been localized to the Golgi apparatus. The co-localization of tuberin and Rap1 in vivo strengthens the likelihood that the in vitro catalytic activity of tuberin toward Rap1 plays a physiologically relevant role in the tumor suppressor function of tuberin.

Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation.

Lethal toxin (LT) from Clostridium sordellii is one of the high molecular mass clostridial cytotoxins. On cultured cells, it causes a rounding of cell bodies and a disruption of actin stress fibers. We demonstrate that LT is a glucosyltransferase that uses UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in vitro and in vivo. LT glucosylates Ras, Rap, and Rac. In Ras, threonine at position 35 was identified as the target amino acid glucosylated by LT. Other related members of the Ras GTPase superfamily, including RhoA, Cdc42, and Rab6, were not modified by LT. Incubation of serum-starved Swiss 3T3 cells with LT prevents the epidermal growth factor-induced phosphorylation of mitogen-activated protein kinases ERK1 and ERK2, indicating that the toxin blocks Ras function in vivo. We also demonstrate that LT acts inside the cell and that the glucosylation reaction is required to observe its dramatic effect on cell morphology. LT is thus a powerful tool to inhibit Ras function in vivo.