RESUMO
PMT is a protein toxin produced by Pasteurella multocida serotypes A and D. As causative agent of atrophic rhinitis in swine, it leads to rapid degradation of the nasal turbinate bone. The toxin acts as a deamidase to modify a crucial glutamine in heterotrimeric G proteins, which results in constitutive activation of the G proteins and permanent stimulation of numerous downstream signaling pathways. Using a lentiviral based genome wide CRISPR knockout screen in combination with a lethal toxin chimera, consisting of full length inactive PMT and the catalytic domain of diphtheria toxin, we identified the LRP1 gene encoding the Low-Density Lipoprotein Receptor-related protein 1 as a critical host factor for PMT function. Loss of LRP1 reduced PMT binding and abolished the cellular response and deamidation of heterotrimeric G proteins, confirming LRP1 to be crucial for PMT uptake. Expression of LRP1 or cluster 4 of LRP1 restored intoxication of the knockout cells. In summary our data demonstrate LRP1 as crucial host entry factor for PMT intoxication by acting as its primary cell surface receptor.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Pasteurella multocida , Animais , Suínos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas de Transporte/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Pasteurella multocida/genética , Pasteurella multocida/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
In the last decade, it was discovered that protein mucin-type O-glycosylation and O-GlcNAcylation modify Tyr residues besides the well explored Thr and Ser amino acids. Several glycoproteomic studies have identified α-GalNAc-O-Tyr modifications, and studies propose that ß-GlcNAc-O-Tyr also exists as a new group of posttranslational modifications (PTMs). Specific bacterial toxins have further been identified to modify host GTPases with α-GlcNAc-O-Tyr to promote bacterial virulence. Despite being identified on numerous proteins, the biological roles, biosynthesis and expression of GalNAc- and GlcNAc-O-Tyr modifications are poorly understood. A major obstacle is the lack of tools to specifically detect and identify proteins containing these modifications. With this in mind, we prepared vaccine constructs and raised antibodies to enable selective detection of proteins carrying these new PTMs. The obtained polyclonal antibody sera were evaluated using ELISA and glycopeptide microarrays and were found to be highly selective for GlcNAc- and GalNAc-O-Tyr glycopeptides over the corresponding Ser- and Thr-modifications. For microarray analysis, synthetic GlcNAc- and GalNAc-O-Tyr Fmoc-amino acids were prepared and applied in Fmoc-SPPS to obtain an extensive O-glycopeptide library. After affinity purification, the antibodies were applied in western blot analysis and showed specific detection of α-GlcNAc-O-Tyr modified RhoA GTPase.
Assuntos
Glicopeptídeos , Tirosina , Sequência de Aminoácidos , Tirosina/metabolismo , Glicopeptídeos/química , Glicosilação , Processamento de Proteína Pós-Traducional , Anticorpos/metabolismoRESUMO
The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron-electron double resonance, and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. Especially the C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to a hypothetical protein receptor may support the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface.
Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Linfócitos/metabolismo , Proteínas Recombinantes/metabolismo , Yersinia pseudotuberculosis/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Conformação Proteica , Proteínas Recombinantes/genéticaRESUMO
Photorhabdus luminescens Tc toxins are large tripartite ABC-type toxin complexes, composed of TcA, TcB and TcC proteins. Tc toxins are widespread and have shown a tropism for a variety of targets including insect, mammalian and human cells. However, their receptors and the specific mechanisms of uptake into target cells remain unknown. Here, we show that the TcA protein TcdA1 interacts with N-glycans, particularly Lewis X/Y antigens. This is confirmed using N-acetylglucosamine transferase I (Mgat1 gene product)-deficient Chinese hamster ovary (CHO) Lec1 cells, which are highly resistant to intoxication by the Tc toxin complex most likely due to the absence of complex N-glycans. Restoring Mgat1 gene activity, and hence complex N-glycan biosynthesis, recapitulated the sensitivity of these cells to the toxin. Exogenous addition of Lewis X trisaccharide partially inhibits intoxication in wild-type cells. Additionally, sialic acid also largely reduced binding of the Tc toxin. Moreover, proteolytic activation of TcdA1 alters glycan-binding and uptake into target cells. The data suggest that TcdA1-binding is most likely multivalent, and carbohydrates probably work cooperatively to facilitate binding and intoxication.
Assuntos
Toxinas Bacterianas , Photorhabdus , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , PolissacarídeosRESUMO
Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.
Assuntos
Movimento Celular/fisiologia , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Apoptose , Células HeLa , Humanos , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Peixe-ZebraRESUMO
The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin's action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore, we conclude that LRP and Lu/BCAM are both required for toxin action but with different functions.
Assuntos
Toxinas Bacterianas/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Sistema do Grupo Sanguíneo Lutheran/metabolismo , Fatores de Virulência/metabolismo , Toxinas Bacterianas/genética , Moléculas de Adesão Celular/imunologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Células HEK293 , Células HeLa , Humanos , Sistema do Grupo Sanguíneo Lutheran/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Virulência/genéticaRESUMO
The ADP-ribosyltransferases TccC3 and TccC5 are the biologically active TcC components of the tripartite Photorhabdus luminescens Tc toxin, which consist of TcA, TcB, and TcC components. TcA is the binding and membrane translocation component. TcB is a functional linker between TcC and TcA and also involved in the translocation of the toxin. While TccC3 ADP-ribosylates actin at threonine 148, TccC5 modifies Rho proteins at glutamine 61/63. Both modifications result in major alteration of the actin cytoskeleton. Here we discuss structure and function of the Tc toxin and compare its ADP-ribosyltransferase activities with other types of actin and Rho modifying toxins.
Assuntos
ADP Ribose Transferases/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Glutamina/metabolismo , Inseticidas/metabolismo , Photorhabdus/enzimologia , Treonina/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Inseticidas/química , Photorhabdus/química , Photorhabdus/genéticaRESUMO
PTC3 and PTC5 are tripartite Tc (toxin complex) toxins from Photorhabdus luminescens, which consist of the binding component TcdA1, the linker component TcdB2 and the enzyme components TccC3 and TccC5 respectively. While PTC5 adenosine diphosphate (ADP)-ribosylates Rho proteins at Gln61/63 resulting in constitutive activation of the GTPases, PTC3 ADP-ribosylates actin at Thr148 thereby inducing actin polymerization. Here, we identified amino acids involved in ADP-ribosyltransferase activity of TccC3 and TccC5 and analysed the substrate specificity of Rho-activating TccC5. We compared the time dependency of Rho protein activation by PTC5 in HeLa cells with the effects of Escherichia coli cytotoxic necrotizing factor 1, which activates Rho GTPases by deamidation of Gln61/63. Using a luciferase reporter assay, we show that PTC5 and PTC3 stimulated gene transcription via myocardin-related transcription factor A (also called MAL) and AP1. MAL activation by PTC5 involved Rho kinase and formins. Activation of AP1 by PTC5 occurred via two MAP kinase pathways involving extracellular signal-regulated kinase and Jun kinase respectively.
Assuntos
ADP Ribose Transferases/metabolismo , Actinas/metabolismo , Toxinas Bacterianas/metabolismo , Photorhabdus/enzimologia , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Genes Reporter , Células HeLa , Humanos , Luciferases/análise , Luciferases/genética , Processamento de Proteína Pós-Traducional , Transcrição GênicaRESUMO
Impaired wound healing is one of the main risk factors associated with diabetes mellitus. Few options are available to treat diabetic wounds, and therefore efficient remedies are urgently needed. An interesting option might be an extract of birch bark (TE) that has been clinically proven to accelerate acute wound healing. We investigated the effects of TE and its main components betulin and lupeol in cultured normal keratinocytes and dermal fibroblasts from diabetic and nondiabetic donors. These in vitro models can provide insights into possible beneficial effects in wound healing. TE and betulin treatment led to increased mRNA levels of chemokines, pro-inflammatory cytokines, and mediators important in wound healing, e.g., IL-6, TNFα, IL-8, and RANTES. We observed a pronounced upregulation of MIF, IL-8, and RANTES on the protein level. Furthermore, a shape change of the actin cytoskeleton was seen in keratinocytes and fibroblasts, and the Rho-GTPases and p38-MAPK were found to be activated in keratinocytes. On the basis of our results, TE is worthy of further study as a potential option to influence wound-healing processes under diabetic conditions. These first insights need to be confirmed by clinical studies with diabetic patients.
Assuntos
Betula/química , Diabetes Mellitus/tratamento farmacológico , Queratinócitos/efeitos dos fármacos , Triterpenos Pentacíclicos/farmacologia , Triterpenos/isolamento & purificação , Triterpenos/farmacologia , Citocinas/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Triterpenos Pentacíclicos/química , Triterpenos Pentacíclicos/isolamento & purificação , Casca de Planta/química , Triterpenos/química , Cicatrização/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The 146-kDa Pasteurella multocida toxin (PMT) is the main virulence factor to induce P. multocida-associated progressive atrophic rhinitis in various animals. PMT leads to a destruction of nasal turbinate bones implicating an effect of the toxin on osteoblasts and/or osteoclasts. The toxin induces constitutive activation of Gα proteins of the G(q/11)-, G12/13- and G(i)-family by deamidating an essential glutamine residue. To study the PMT effect on bone cells, we used primary osteoblasts derived from rat calvariae and stromal ST-2 cells as differentiation model. As marker of functional osteoblasts the expression and activity of alkaline phosphatase, formation of mineralization nodules or expression of specific transcription factors as osterix was determined. Here, we show that the toxin inhibits differentiation and/or function of osteoblasts by activation of Gα(q/11). Subsequently, Gα(q/11) activates RhoA via p63RhoGEF, which specifically interacts with Gα(q/11) but not with other G proteins like Gα12/13 and Gα(i). Activated RhoA transactivates the mitogen-activated protein (MAP) kinase cascade via Rho kinase, involving Ras, MEK and ERK, resulting in inhibition of osteoblast differentiation. PMT-induced inhibition of differentiation was selective for the osteoblast lineage as adipocyte-like differentiation of ST-2 cells was not hampered. The present work provides novel insights, how the bacterial toxin PMT can control osteoblastic development by activating heterotrimeric G proteins of the Gα(q/11)-family and is a molecular pathogenetic basis for understanding the role of the toxin in bone loss during progressive atrophic rhinitis induced by Pasteurella multocida.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Diferenciação Celular , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Sistema de Sinalização das MAP Quinases , Osteoblastos/metabolismo , Infecções por Pasteurella/metabolismo , Pasteurella multocida/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Ativação Transcricional , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Camundongos , Osteoblastos/patologia , Osteólise/metabolismo , Osteólise/patologia , Infecções por Pasteurella/patologia , Pasteurella multocida/patogenicidade , Ratos , Rinite Atrófica/metabolismo , Rinite Atrófica/patologia , Crânio/metabolismo , Crânio/patologia , Células Estromais/metabolismo , Células Estromais/patologia , Fatores de Virulência/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Kindlins are a family of integrin adapter and cell-matrix adhesion proteins causally linked to human genetic disorders. Kindlin-2 is a ubiquitously expressed protein with manifold functions and interactions. The contribution of kindlin-2 to integrin-based cell-matrix adhesions has been extensively explored, while other integrin-independent roles emerge. Because of the early involvement of kindlin-2 in development, no viable animal models with its constitutional knockout are available to study its physiological functions in adult skin. Here, we uncovered a critical physiological role of kindlin-2 in the epidermis by using a skin-equivalent model with shRNA-mediated knock-down of kindlin-2 in keratinocytes. Kindlin-2-deficient keratinocytes built stratified epidermal layers, but displayed impaired dermal-epidermal and intra-epidermal adhesion and barrier function. Co-immunoprecipitation studies demonstrated that kindlin-2 interacts with both integrin- and cadherin-based adhesions. In kindlin-2-deficient keratinocytes, reduced cell-cell adhesion was associated with abnormal cytoplasmic distribution of adherens junctions and desmosomal proteins, which was dependent on RhoA activation. Direct activation of RhoA with recombinant bacterial cytotoxic necrotizing factor y (CNFy) reverted the abnormal phenotype and barrier function of kindlin-2-deficient keratinocytes and skin equivalents. These findings have physiological and pathological significance, since kindlin-2 expression modulates the phenotype in Kindler syndrome, a skin fragility disorder caused by kindlin-1 deficiency. Our results suggest that pharmacological regulation of RhoGTPase activity may represent a therapeutic option for skin fragility.
Assuntos
Toxinas Bacterianas/farmacologia , Adesão Celular/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Epiderme/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Proteínas de Membrana/deficiência , Proteínas de Neoplasias/deficiência , Proteína rhoA de Ligação ao GTP/metabolismo , Linhagem Celular , Técnicas de Cocultura , Ativação Enzimática , Epiderme/enzimologia , Epiderme/patologia , Células Alimentadoras , Técnicas de Silenciamento de Genes , Genótipo , Humanos , Queratinócitos/enzimologia , Queratinócitos/patologia , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Permeabilidade , Fenótipo , Interferência de RNA , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co-expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive-like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co-expressed constitutively active Rop4, blocking the hypersensitive response caused by over-expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop-dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop-dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Toxinas Bacterianas/genética , Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/genética , Dados de Sequência Molecular , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Nicotiana/genética , Nicotiana/metabolismo , Proteínas rac de Ligação ao GTP/genéticaRESUMO
Pathogenic bacteria produce diverse protein toxins to disturb the host's defenses. This includes the opening of epithelial barriers to establish bacterial growth in deeper tissues of the host and to modulate immune cell functions. To achieve this, many toxins share the ability to enter mammalian cells, where they catalyze the modification of cellular proteins. The enzymatic activity is diverse and ranges from ribosyl- or glycosyl-transferase activity, the deamidation of proteins, and adenylate-cyclase activity to proteolytic cleavage. Protein toxins are highly active enzymes often with tight specificity for an intracellular protein or a protein family coupled with the intrinsic capability of entering mammalian cells. A broad understanding of their molecular mechanisms established bacterial toxins as powerful tools for cell biology. Both the enzymatic part and the pore-forming/protein transport capacity are currently used as tools engineered to study signaling pathways or to transport cargo like labeled compounds, nucleic acids, peptides, or proteins directly into the cytosol. Using several representative examples, this review is intended to provide a short overview of the state of the art in the use of bacterial toxins or parts thereof as tools.
Assuntos
Toxinas Bacterianas , Animais , Humanos , Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacologia , Toxinas Bacterianas/toxicidade , Transporte ProteicoRESUMO
RhoA is reportedly involved in signal transducers and activators of transcription (STAT)-dependent transcription. However, the pathway connecting the GTPase and STAT signaling has not been characterized. Here, we made use of bacterial toxins, which directly activate Rho GTPases to analyze this pathway. Cytotoxic necrotizing factors (CNFs) are produced by pathogenic Escherichia coli strains and by Yersinia pseudotuberculosis. They activate small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. We show that RhoA activation leads to phosphorylation and activation of STAT3 and identify signal proteins involved in this pathway. RhoA-dependent STAT3 stimulation requires ROCK and Jun kinase activation as well as AP1-induced protein synthesis. The secretion of one or more factors activates the JAK-STAT pathway in an auto/paracrine manner. We identify CCL1/I-309 as an essential cytokine, which is produced and secreted upon RhoA activation and which is able to activate STAT3-dependent signaling pathways.
Assuntos
Toxinas Bacterianas/farmacologia , Quimiocina CCL1/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Janus Quinases/metabolismo , Fator de Transcrição AP-1/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
The small GTPase Rac1 is involved in multiple cytosolic functions but recent data point out that Rac1 also translocates to the nucleus to regulate signalling pathways that control gene expression and progression through the cell cycle. Here, we identify the nuclear import receptor karyopherin alpha2 (KPNA2) as a direct interaction partner of Rac1. The C-terminal polybasic region of Rac1 contains a nuclear localization signal (NLS), whereas Rac2 and Rac3 lack a functional NLS and do not bind to KPNA2. The presence of the NLS in Rac1 determines the specificity of the interaction and is a prerequisite for the nuclear import. Although this interaction is independent of the Rac1 GDP/GTP loading, the induction of the translocation requires Rac1 activation. The activation of Rac1 via the cytotoxic necrotizing factor 1 and the concurrent inhibition of its proteasomal degradation are crucial for the nuclear accumulation of Rac1. Conversely, the reduction of KPNA2 expression inhibits the nuclear import of Rac1. For the first time, our results show a direct interaction between Rac1 and KPNA2 and argue for a KPNA2-dependent nuclear import of Rac1. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis revealed that nuclear Rac1 coimmunoprecipitates with numerous proteins. In the nucleus, Rac1 may participate in a variety of so far uncharacterized processes.
Assuntos
Núcleo Celular/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , alfa Carioferinas/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Linhagem Celular , Regulação para Baixo , Células HeLa , Humanos , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido , alfa Carioferinas/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Directional cell migration is crucially dependent on the spatiotemporal control of intracellular signalling events. These events regulate polarized actin dynamics, resulting in protrusion at the front of the cell and contraction at the rear. The actin cytoskeleton is regulated through signalling by Rho-like GTPases, such as RhoA, which stimulates myosin-based contractility, and CDC42 and Rac1, which promote actin polymerization and protrusion. Here, we show that Rac1 binds the adapter protein caveolin-1 (Cav1) and that Rac1 activity promotes Cav1 accumulation at Rac1-positive peripheral adhesions. Using Cav1-deficient mouse fibroblasts and depletion of Cav1 expression in human epithelial and endothelial cells mediated by small interfering RNA and short hairpin RNA, we show that loss of Cav1 induces an increase in Rac1 protein and its activated, GTP-bound form. Cav1 controls Rac1 protein levels by regulating ubiquitylation and degradation of activated Rac1 in an adhesion-dependent fashion. Finally, we show that Rac1 ubiquitylation is not required for effector binding, but regulates the dynamics of Rac1 at the periphery of the cell. These data extend the canonical model of Rac1 inactivation and uncover Cav1-regulated polyubiquitylation as an additional mechanism to control Rac1 signalling.
Assuntos
Caveolina 1/metabolismo , Extensões da Superfície Celular/metabolismo , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Caveolina 1/genética , Movimento Celular , Extensões da Superfície Celular/efeitos dos fármacos , Retroalimentação Fisiológica , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Adesões Focais/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Microscopia Confocal , Pironas/farmacologia , Quinolinas/farmacologia , RNA Interferente Pequeno/genética , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
The autoimmune blistering skin diseases pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are mainly caused by autoantibodies against desmosomal cadherins. In this study, we provide evidence that PV-immunoglobulin G (IgG) and PF-IgG induce skin blistering by interference with Rho A signaling. In vitro, pemphigus IgG caused typical hallmarks of pemphigus pathogenesis such as epidermal blistering in human skin, cell dissociation, and loss of desmoglein 1 (Dsg 1)-mediated binding probed by laser tweezers. These changes were accompanied by interference with Rho A activation and reduction of Rho A activity. Pemphigus IgG-triggered keratinocyte dissociation and Rho A inactivation were p38 mitogen-activated protein kinase dependent. Specific activation of Rho A by cytotoxic necrotizing factor-y abolished all pemphigus-triggered effects, including keratin retraction and release of Dsg 3 from the cytoskeleton. These data demonstrate that Rho A is involved in the regulation of desmosomal adhesion, at least in part by maintaining the cytoskeletal anchorage of desmosomal proteins. This may open the possibility of pemphigus treatment with the epidermal application of Rho A agonists.
Assuntos
Vesícula/etiologia , Imunoglobulina G/fisiologia , Pênfigo/etiologia , Pele/patologia , Proteína rhoA de Ligação ao GTP/metabolismo , Vesícula/enzimologia , Caderinas/metabolismo , Linhagem Celular , Desmogleína 1/metabolismo , Desmogleína 3/metabolismo , Desmossomos/enzimologia , Desmossomos/fisiologia , Ativação Enzimática , Humanos , Queratina-14/metabolismo , Queratinócitos/patologia , Pênfigo/enzimologia , Pênfigo/imunologia , Transdução de Sinais , Pele/metabolismoRESUMO
Current strategies for glioma treatment are only partly effective because of the poor selectivity for tumoral cells. Hence, the necessity to identify novel approaches is urgent. Recent studies highlighted the effectiveness of the bacterial protein cytotoxic necrotizing factor 1 (CNF1) in reducing tumoral mass, increasing survival of glioma-bearing mice and protecting peritumoral neural tissue from dysfunction. However, native CNF1 needs to be delivered into the brain, because of its incapacity to cross the blood-brain barrier (BBB) per se, thus hampering its clinical translation. To allow a non-invasive administration of CNF1, we here developed a chimeric protein (CTX-CNF1) conjugating CNF1 with chlorotoxin (CTX), a peptide already employed in clinics due to its ability of passing the BBB and selectively binding glioma cells. After systemic administration, we found that CTX-CNF1 is able to target glioma cells and significantly prolong survival of glioma-bearing mice. Our data point out the potentiality of CTX-CNF1 as a novel effective tool to treat gliomas.
Assuntos
Antineoplásicos/farmacologia , Toxinas Bacterianas/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Proteínas de Escherichia coli/farmacologia , Glioma/tratamento farmacológico , Venenos de Escorpião/farmacologia , Animais , Antineoplásicos/metabolismo , Toxinas Bacterianas/metabolismo , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Glioma/metabolismo , Glioma/patologia , Humanos , Injeções Intravenosas , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/farmacologia , Venenos de Escorpião/metabolismoRESUMO
Low molecular weight GTP-binding proteins of the Rho family control the organization of the actin cytoskeleton in eukaryotic cells. RhoA governs the formation of actin stress fibers and is responsible for the formation of the contractile ring in cytokinesis. Cytokinesis completion requires RhoA inactivation resulting in disassembly of the contractile ring. Cytokinesis thus requires switching of RhoA activity. This switch of RhoA activity is blocked by Rho-modifying bacterial protein toxins that either activate or inactivate RhoA by covalent modifications. Exoenzyme C3 from Clostridium limosum (C3-lim) and Clostridium difficile toxin B (TcdB) inactivate RhoA by mono-ADP-ribosylation and mono-glucosylation, respectively. Cytotoxic necrotizing factors (CNF), produced by either Yersinia pseudotuberculosis (CNFY) or uropathogenic strains of E. coli (CNF1), deamidate and thereby activate RhoA. This study provides evidence that RhoA-activating as well as RhoA-inactivating toxins cause inhibition of cytokinesis and cell division. The toxins' effects on cytokinesis were analyzed in Hela cells synchronized using the thymidine double block technique. Treatment of G2-phase cells with either the RhoA-activating CNFY or CNF1 or the RhoA-inactivating C3-lim or TcdB resulted in cytokinesis inhibition, as evidenced by the formation of a 4N population on flow cytometry, the inhibition of contractile ring formation, and the formation of bi-nucleated cells. While TcdB and CNF1 modify a broad-spectrum of Rho proteins, C3-lim and CNFY specifically target RhoA. Since C3-lim and CNFY both caused cytokinesis inhibition, our study re-inforces the critical role of RhoA (not Rac1 or Cdc42) in cytokinesis and cell division.
Assuntos
Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Citocinese/efeitos dos fármacos , Proteínas de Escherichia coli/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/fisiologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/patologia , Células HeLa , HumanosRESUMO
Rho GTPases are important regulators of many cellular functions like cell migration, adhesion and polarity. The molecular switches are often dysregulated in cancer. We detected Rho-dependent upregulation of the orphan seven-transmembrane receptor G-protein-coupled receptor family C group 5 member A (GPRC5A). GPRC5A is highly expressed in breast cancer whereas in lung cancer, it is often downregulated. Here, we analyzed the function of GPRC5A in breast epithelial and breast cancer cells. Activation or expression of RhoA/C led to GPRC5A-dependent inhibition of proliferation and reduction of the colony forming capacity of benign breast epithelial cells. This effect is based on an inhibition of EGFR signalling. Knockout of retinoic acid induced 3 (RAI3, the gene for GPRC5A) in breast cancer cells increased cell division, whereas Rho activation had no effect on proliferation. Knockout of RAI3 in benign breast epithelial cells led to decrease of EGFR expression and diminished proliferation.