RESUMO
Metabolic studies and animal knockout models point to the critical role of polyunsaturated docosahexaenoic acid (22:6, DHA)-containing phospholipids (DHA-PLs) in physiology. Here, we investigated the impact of DHA-PLs on the dynamics of transendothelial cell macroapertures (TEMs) triggered by RhoA inhibition-associated cell spreading. Lipidomic analyses showed that human umbilical vein endothelial cells (HUVECs) subjected to a DHA diet undergo a 6-fold enrichment in DHA-PLs at the plasma membrane (PM) at the expense of monounsaturated oleic acid-containing PLs (OA-PLs). Consequently, DHA-PL enrichment at the PM induces a reduction in cell thickness and shifts cellular membranes towards a permissive mode of membrane fusion for transcellular tunnel initiation. We provide evidence that a global homeostatic control of membrane tension and cell cortex rigidity minimizes overall changes of TEM area through a decrease of TEM size and lifetime. Conversely, low DHA-PL levels at the PM lead to the opening of unstable and wider TEMs. Together, this provides evidence that variations of DHA-PL levels in membranes affect cell biomechanical properties.
Assuntos
Ácidos Docosa-Hexaenoicos , Fosfolipídeos , Animais , Membrana Celular/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Células Endoteliais/metabolismo , Humanos , Fusão de Membrana , Fosfolipídeos/metabolismoRESUMO
Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with ß-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase ß-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.
Assuntos
Suscetibilidade a Doenças , Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Ubiquitinas/metabolismo , Adesão Celular , Proteínas de Ciclo Celular/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Hippo , Humanos , Mecanotransdução Celular , Modelos Biológicos , Neoplasias/mortalidade , Neoplasias/patologia , Prognóstico , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The aim of this study was to identify the molecular signals produced in human endothelial cells (ECs) by the interaction of α5ß1 integrin with soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) present in the extracellular matrix. We generated a gene expression profile of ECs adhering to sVEGFR-1 or to fibronectin, the classic extracellular matrix ligand for α5ß1 integrin or in a nonadhering condition. Several biological pathways were differently modulated, 3 protein kinase C substrates [adducin, myristoylated alanine-rich protein kinase C substrate (MARCKS), and radixin] were differently expressed and phosphorylated when cells adhering to sVEGFR-1 were compared with those adhering to fibronectin. Rac1 activation and Gα13 protein involvement through the interaction with radixin were also detected after attachment to sVEGFR-1, and these responses depended on active VEGFR-2 signaling. On sVEGFR-1, ECs exhibited a motile phenotype that was consistent with the abundant presence of MARCKS, a stabilizer of dynamic adhesions. Moreover, ECs silenced for radixin expression no longer responded to the proangiogenic VEGFR-1-derived peptide 12. We propose that the presence of sVEGFR-1 in the EC microenvironment directs α5ß1 integrin signaling to generate a dynamic, motile phenotype. Our findings also provide new insights into the mechanism of action of proangiogenic peptide 12, relevant to a therapeutic perspective.
Assuntos
Adesão Celular/fisiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Western Blotting , Movimento Celular/genética , Movimento Celular/fisiologia , Células Cultivadas , Fibronectinas/metabolismo , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologiaRESUMO
In culture, cell confluence generates signals that commit actively growing keratinocytes to exit the cell cycle and differentiate to form a stratified epithelium. Using a comparative proteomic approach, we studied this 'confluence switch' and identified a new pathway triggered by cell confluence that regulates basement membrane (BM) protein composition by suppressing the uPA-uPAR-plasmin pathway. Indeed, confluence triggers adherens junction maturation and enhances TGF-ß and activin A activity, resulting in increased deposition of PAI-1 and perlecan in the BM. Extracellular matrix (ECM)-accumulated PAI-1 suppresses the uPA-uPAR-plasmin pathway and further enhances perlecan deposition by inhibiting its plasmin-dependent proteolysis. We show that perlecan deposition in the ECM strengthens cell adhesion, inhibits keratinocyte motility and promotes additional accumulation of PAI-1 in the ECM at confluence. In agreement, during wound-healing, perlecan concentrates at the wound-margin, where BM matures to stabilize keratinocyte adhesion. Our results demonstrate that confluence-dependent signaling orchestrates not only growth inhibition and differentiation, but also controls ECM proteolysis and BM formation. These data suggest that uncontrolled integration of confluence-dependent signaling, might favor skin disorders, including tumorigenesis, not only by promoting cell hyperproliferation, but also by altering protease activity and deposition of ECM components.
Assuntos
Matriz Extracelular/metabolismo , Fibrinolisina/metabolismo , Queratinócitos/metabolismo , Proteólise , Transdução de Sinais , Ativinas/metabolismo , Junções Aderentes/metabolismo , Animais , Membrana Basal/metabolismo , Adesão Celular , Diferenciação Celular , Movimento Celular , Proliferação de Células , Regulação para Baixo , Retroalimentação Fisiológica , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Queratinócitos/patologia , Camundongos , Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ligação Proteica , Proteômica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , CicatrizaçãoRESUMO
Intratumoral bacteria flexibly contribute to cellular and molecular tumor heterogeneity for supporting cancer recurrence through poorly understood mechanisms. Using spatial metabolomic profiling technologies and 16SrRNA sequencing, we herein report that right-sided colorectal tumors are predominantly populated with Colibactin-producing Escherichia coli (CoPEC) that are locally establishing a high-glycerophospholipid microenvironment with lowered immunogenicity. It coincided with a reduced infiltration of CD8+ T lymphocytes that produce the cytotoxic cytokines IFN-γ where invading bacteria have been geolocated. Mechanistically, the accumulation of lipid droplets in infected cancer cells relied on the production of colibactin as a measure to limit genotoxic stress to some extent. Such heightened phosphatidylcholine remodeling by the enzyme of the Land's cycle supplied CoPEC-infected cancer cells with sufficient energy for sustaining cell survival in response to chemotherapies. This accords with the lowered overall survival of colorectal patients at stage III-IV who were colonized by CoPEC when compared to patients at stage I-II. Accordingly, the sensitivity of CoPEC-infected cancer cells to chemotherapies was restored upon treatment with an acyl-CoA synthetase inhibitor. By contrast, such metabolic dysregulation leading to chemoresistance was not observed in human colon cancer cells that were infected with the mutant strain that did not produce colibactin (11G5∆ClbQ). This work revealed that CoPEC locally supports an energy trade-off lipid overload within tumors for lowering tumor immunogenicity. This may pave the way for improving chemoresistance and subsequently outcome of CRC patients who are colonized by CoPEC.
Assuntos
Neoplasias Colorretais , Microbioma Gastrointestinal , Peptídeos , Policetídeos , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Microambiente Tumoral , Resistencia a Medicamentos Antineoplásicos , Mutagênicos/metabolismo , Recidiva Local de Neoplasia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologia , Policetídeos/metabolismo , LipídeosRESUMO
Rho GTPases, which are master regulators of both the actin cytoskeleton and membrane trafficking, are often hijacked by pathogens to enable their invasion of host cells. Here we report that the cytotoxic necrotizing factor-1 (CNF1) toxin of uropathogenic Escherichia coli (UPEC) promotes Rac1-dependent entry of bacteria into host cells. Our screen for proteins involved in Rac1-dependent UPEC entry identifies the Toll-interacting protein (Tollip) as a new interacting protein of Rac1 and its ubiquitinated forms. We show that knockdown of Tollip reduces CNF1-induced Rac1-dependent UPEC entry. Tollip depletion also reduces the Rac1-dependent entry of Listeria monocytogenes expressing InlB invasion protein. Moreover, knockdown of Tollip, Tom1 and clathrin, decreases CNF1 and Rac1-dependent internalization of UPEC. Finally, we show that Tollip, Tom1 and clathrin associate with Rac1 and localize at the site of bacterial entry. Collectively, these findings reveal a new link between Rac1 and Tollip, Tom1 and clathrin membrane trafficking components hijacked by pathogenic bacteria to allow their efficient invasion of host cells.
Assuntos
Infecções Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Escherichia coli Uropatogênica/metabolismo , Escherichia coli Uropatogênica/patogenicidade , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Endocitose/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas/metabolismo , Escherichia coli Uropatogênica/citologia , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
RATIONALE: Integrins play a crucial role in controlling endothelial cell proliferation and migration during angiogenesis. The Delta-like 4 (Dll4)/Notch pathway establishes an adequate ratio between stalk and tip cell populations by restricting tip cell formation through "lateral inhibition" in response to a vascular endothelial growth factor gradient. Because angiogenesis requires a tight coordination of these cellular processes, we hypothesized that adhesion, vascular endothelial growth factor, and Notch signaling pathways are interconnected. OBJECTIVE: This study was aimed at characterizing the cross-talk between integrin and Notch signaling in endothelial cells. METHODS AND RESULTS: Adhesion of primary human endothelial cells to laminin-111 triggers Dll4 expression, leading to subsequent Notch pathway activation. SiRNA-mediated knockdown of α2ß1 and α6ß1 integrins abolishes Dll4 induction, which discloses a selective integrin signaling acting upstream of Notch pathway. The increase in Foxc2 transcription, triggered by α2ß1 binding to laminin, is required but not sufficient per se for Dll4 expression. Furthermore, vascular endothelial growth factor stimulates laminin γ1 deposition, which leads to integrin signaling and Dll4 induction. Interestingly, loss of integrins α2 or α6 mimics the effects of Dll4 silencing and induces excessive network branching in an in vitro sprouting angiogenesis assay on three-dimensional matrigel. CONCLUSIONS: We show that, in endothelial cells, ligation of α2ß1 and α6ß1 integrins induces the Notch pathway, and we disclose a novel role of basement membrane proteins in the processes controlling tip vs stalk cell selection.
Assuntos
Células Endoteliais/metabolismo , Integrina alfa2beta1/metabolismo , Integrina alfa6beta1/metabolismo , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Receptores Notch/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Membrana Basal , Proteínas de Ligação ao Cálcio , Adesão Celular , Células Cultivadas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Laminina/metabolismo , Proteínas de Membrana/fisiologia , Neovascularização Fisiológica , Receptor Cross-TalkRESUMO
How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin ß1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.
Assuntos
Membrana Basal/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Membrana Basal/ultraestrutura , Proteínas de Ligação ao Cálcio , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Integrinas/metabolismo , Laminina/deficiência , Laminina/metabolismo , Camundongos , Neovascularização Fisiológica , Receptores Notch/antagonistas & inibidoresRESUMO
Colorectal cancer (CRC) patients are frequently colonized by colibactin-producing Escherichia coli (CoPEC) (>40%), which enhances tumorigenesis in mouse models of CRC. We observed that 50% of CoPEC also contains the cnf1 gene, which encodes cytotoxic necrotizing factor-1 (CNF1), an enhancer of the eukaryotic cell cycle. The impact of its co-occurrence with colibactin (Clb) has not yet been investigated. We evaluated the impact of CNF1 on colorectal tumorigenesis using human colonic epithelial HT-29 cells and CRC-susceptible ApcMin/+ mice inoculated with the CoPEC 21F8 clinical strain (Clb+Cnf+) or 21F8 isogenic mutants (Clb+Cnf-, Clb-Cnf+ and Clb-Cnf-). Infection with the Clb+Cnf- strain induced higher levels of inflammatory cytokines and senescence markers both in vitro and in vivo compared to those induced by infection with the Clb+Cnf+ strain. In contrast, the Clb+Cnf- and Clb+Cnf+ strains generated similar levels of DNA damage in HT-29 cells and in colonic murine tissues. Furthermore, the ApcMin/+ mice inoculated with the Clb+Cnf- strain developed significantly more tumors than the mice inoculated with the Clb+Cnf+ strain or the isogenic mutants, and the composition of their microbiota was changed. Finally, rectal administration of the CNF1 protein in ApcMin/+ mice inoculated with the Clb+Cnf- strain significantly decreased tumorigenesis and inflammation. Overall, this study provides evidence that CNF1 decreases the carcinogenic effects of CoPEC in ApcMin/+ mice by decreasing CoPEC-induced cellular senescence and inflammation.
Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Microbioma Gastrointestinal , Camundongos , Humanos , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Colo , Carcinogênese , Transformação Celular Neoplásica , InflamaçãoRESUMO
Recently, an intestinal dysbiotic microbiota with enrichment in oral cavity bacteria has been described in colorectal cancer (CRC) patients. Here, we characterize and investigate one of these oral pathobionts, the Gram-positive anaerobic coccus Parvimonas micra. We identified two phylotypes (A and B) exhibiting different phenotypes and adhesion capabilities. We observed a strong association of phylotype A with CRC, with its higher abundance in feces and in tumoral tissue compared with the normal homologous colonic mucosa, which was associated with a distinct methylation status of patients. By developing an in vitro hypoxic co-culture system of human primary colonic cells with anaerobic bacteria, we show that P. micra phylotype A alters the DNA methylation profile promoters of key tumor-suppressor genes, oncogenes, and genes involved in epithelial-mesenchymal transition. In colonic mucosa of CRC patients carrying P. micra phylotype A, we found similar DNA methylation alterations, together with significant enrichment of differentially expressed genes in pathways involved in inflammation, cell adhesion, and regulation of actin cytoskeleton, providing evidence of P. micra's possible role in the carcinogenic process.
Assuntos
Neoplasias Colorretais , Microbioma Gastrointestinal , Humanos , Microbioma Gastrointestinal/genética , Firmicutes/genética , Bactérias , Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologiaRESUMO
Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that alpha2beta1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, alpha2beta1 engagement alters assembly of mature focal adhesions by alpha5beta1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of alpha5beta1 to signal for GTP loading of Rac is preserved, the joint engagement of alpha2beta1 interferes with membrane anchorage of Rac. Adapting the 'split-ubiquitin' sensor to screen for membrane-proximal alpha2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by alpha2beta1 integrin. Altogether, our data establish that alpha2beta1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.
Assuntos
Antígenos CD/metabolismo , Células Endoteliais/metabolismo , Integrina alfa2beta1/metabolismo , Laminina/farmacologia , Glicoproteínas de Membrana/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Antígenos CD/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibronectinas/farmacologia , Adesões Focais/genética , Adesões Focais/metabolismo , Humanos , Integrina alfa2beta1/agonistas , Integrina alfa5beta1/agonistas , Integrina alfa5beta1/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Tetraspanina 29RESUMO
Extracellular matrix (ECM) elasticity is perceived by cells via focal adhesion structures, which transduce mechanical cues into chemical signalling to conform cell behavior. Although the contribution of ECM compliance to the control of cell migration or division is extensively studied, little is reported regarding infectious processes. We study this phenomenon with the extraintestinal Escherichia coli pathogen UTI89. We show that UTI89 takes advantage, via its CNF1 toxin, of integrin mechanoactivation to trigger its invasion into cells. We identify the HACE1 E3 ligase-interacting protein Optineurin (OPTN) as a protein regulated by ECM stiffness. Functional analysis establishes a role of OPTN in bacterial invasion and integrin mechanical coupling and for stimulation of HACE1 E3 ligase activity towards the Rac1 GTPase. Consistent with a role of OPTN in cell mechanics, OPTN knockdown cells display defective integrin-mediated traction force buildup, associated with limited cellular invasion by UTI89. Nevertheless, OPTN knockdown cells display strong mechanochemical adhesion signalling, enhanced Rac1 activation and increased cyclin D1 translation, together with enhanced cell proliferation independent of ECM stiffness. Together, our data ascribe a new function to OPTN in mechanobiology.
Assuntos
Ciclina D1 , Integrinas , Divisão Celular , Ciclina D1/metabolismo , Integrinas/metabolismo , Mecanotransdução Celular/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by Escherichia coli, a commensal of the digestive tract and a source of urinary tract pathogens. Bioinformatics analyses of a large collection of E. coli genomes from EnteroBase, enriched in clinical isolates of worldwide origins, suggest the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, cnf1, is preferentially distributed in four common sequence types (ST) encompassing the pandemic E. coli MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment, with known enhanced capacities to colonize the gastrointestinal tract. Statistical projections based on this dataset point to a global expansion of cnf1-positive multidrug-resistant ST131 strains from subclade H30Rx/C2, accounting for a rising prevalence of cnf1-positive strains in ST131. Despite the absence of phylogeographical signals, cnf1-positive isolates segregated into clusters in the ST131-H30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same cnf1 allele. The suggested dominant expansion of cnf1-positive strains in ST131-H30Rx/C2 led us to uncover the competitive advantage conferred by cnf1 for gut colonization to the clinical strain EC131GY ST131-H30Rx/C2 versus cnf1-deleted isogenic strain. Complementation experiments showed that colon tissue invasion was compromised in the absence of deamidase activity on Rho GTPases by CNF1. Hence, gut colonization factor function of cnf1 was confirmed for another clinical strain ST131-H30Rx/C2. In addition, functional analysis of the cnf1-positive clinical strain EC131GY ST131-H30Rx/C2 and a cnf1-deleted isogenic strain showed no detectable impact of the CNF1 gene on bacterial fitness and inflammation during the acute phase of bladder monoinfection. Together these data argue for an absence of role of CNF1 in virulence during UTI, while enhancing gut colonization capacities of ST131-H30Rx/C2 and suggested expansion of cnf1-positive MDR isolates in subclade ST131-H30Rx/C2.
Assuntos
Toxinas Bacterianas , Infecções por Escherichia coli , Proteínas de Escherichia coli , Microbioma Gastrointestinal , Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Humanos , Fatores de Virulência/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo , Proteínas rho de Ligação ao GTPRESUMO
The development of anti-infectives against a large range of AB-like toxin-producing bacteria includes the identification of compounds disrupting toxin transport through both the endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of compounds blocking Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, which was followed by orthogonal screens against two toxins that hijack the endolysosomal (diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule C910 that induces the enlargement of EEA1-positive early endosomes associated with sorting defects of CNF1 and Shiga toxins to their trafficking pathways. C910 protects cells against eight bacterial AB toxins and the CNF1-mediated pathogenic Escherichia coli invasion. Interestingly, C910 reduces influenza A H1N1 and SARS-CoV-2 viral infection in vitro. Moreover, parenteral administration of C910 to mice resulted in its accumulation in lung tissues and a reduction in lethal influenza infection.
RESUMO
The GTPase RhoA is a major regulator of the assembly of actin stress fibers and the contractility of the actomyosin cytoskeleton. The epidermal cell differentiation inhibitor (EDIN) and EDIN-like ADP-ribosyltransferases of Staphylococcus aureus catalyze the inactivation of RhoA, producing actin cable disruption. We report that purified recombinant EDIN and EDIN-producing S. aureus provoke large transcellular tunnels in endothelial cells that we have named macroapertures (MAs). These structures open transiently, followed by the appearance of actin-containing membrane waves extending over the aperture. Disruption of actin cables, either directly or indirectly, through rhoA RNAi knockdown also triggers the formation of MAs. Intoxication of endothelial monolayers by EDIN produces a loss of barrier function and provides direct access of the endothelium basement membrane to S. aureus.
Assuntos
ADP Ribose Transferases/farmacologia , Proteínas de Bactérias/farmacologia , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , ADP Ribose Transferases/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Interferência de RNA/fisiologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Staphylococcus aureus/química , Staphylococcus aureus/enzimologia , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
We have investigated how Bacillus anthracis lethal toxin (LT) triggers caspase-3 activation and the formation of thick actin cables in human endothelial cells. By DNA array analysis we show that LT has a major impact on the cell transcriptome and we identify key host genes involved in LT cytotoxic effects. Indeed, upregulation of TRAIL and downregulation of XIAP both participate in LT-induced caspase-3 activation. LT induces a downregulation of the immediate early gene and master regulator of transcription egr1. Importantly, its re-expression in LT-intoxicated cells blocks caspase-3 activation. In parallel, we found that the formation of actin cables induced by LT occurs in the absence of direct activation of RhoA/ROCK signalling. We show that knock-down of cortactin and rhophilin-2 under conditions of calponin-1 expression defines the minimal set of genes regulated by LT for actin cable formation. Together our data establish that the modulation of the cell transcriptome by LT plays a key role in triggering human endothelial cell toxicity.
Assuntos
Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Caspase 3/genética , Linhagem Celular , Células Endoteliais/citologia , Regulação da Expressão Gênica/genética , Humanos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
The presence of aberrant number of centrioles is a recognized cause of aneuploidy and hallmark of cancer. Hence, centriole duplication needs to be tightly regulated. It has been proposed that centriole separation limits centrosome duplication. The mechanism driving centriole separation is poorly understood and little is known on how this is linked to centriole duplication. Here, we propose that actin-generated forces regulate centriole separation. By imposing geometric constraints via micropatterns, we were able to prove that precise acto-myosin force arrangements control direction, distance and time of centriole separation. Accordingly, inhibition of acto-myosin contractility impairs centriole separation. Alongside, we observed that organization of acto-myosin force modulates specifically the length of S-G2 phases of the cell cycle, PLK4 recruitment at the centrosome and centriole fidelity. These discoveries led us to suggest that acto-myosin forces might act in fundamental mechanisms of aneuploidy prevention.
Assuntos
Actinas/metabolismo , Ciclo Celular/fisiologia , Centríolos/metabolismo , Miosinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Actinas/fisiologia , Aneuploidia , Ciclo Celular/efeitos dos fármacos , Centríolos/fisiologia , Células HeLa , Humanos , Microscopia Intravital/métodos , Microscopia Confocal , Miosinas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Timidina/farmacologia , Imagem com Lapso de Tempo/métodosRESUMO
Alterations of the cellular proteome over time due to spontaneous or toxin-mediated enzymatic deamidation of glutamine (Gln) and asparagine (Asn) residues contribute to bacterial infection and might represent a source of aging-related diseases. Here, we put into perspective what is known about the mode of action of the CNF1 toxin from pathogenic Escherichia coli, a paradigm of bacterial deamidases that activate Rho GTPases, to illustrate the importance of determining whether exposure to these factors are risk factors in the etiology age-related diseases, such as cancer. In particular, through in silico analysis of the distribution of the CNF1-like deamidase active site Gly-Cys-(Xaa)n-His sequence motif in bacterial genomes, we unveil the wide distribution of the super-family of CNF-like toxins and CNF-like deamidase domains among members of the Enterobacteriacae and in association with a large variety of toxin delivery systems. We extent our discussion with recent findings concerning cellular systems that control activated Rac1 GTPase stability and provide protection against cancer. These findings point to the urgency for developing holistic approaches toward personalized medicine that include monitoring for asymptomatic carriage of pathogenic toxin-producing bacteria and that ultimately might lead to improved public health and increased lifespans.
Assuntos
Amidoidrolases/metabolismo , Toxinas Bacterianas/metabolismo , Enterobacteriaceae/enzimologia , Proteínas de Escherichia coli/metabolismo , Fatores Imunológicos/metabolismo , Fatores de Virulência/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Amidoidrolases/genética , Asparagina/metabolismo , Toxinas Bacterianas/genética , Biologia Computacional , Infecções por Enterobacteriaceae/complicações , Infecções por Enterobacteriaceae/patologia , Proteínas de Escherichia coli/genética , Glutamina/metabolismo , Neoplasias/etiologia , Neoplasias/fisiopatologia , Fatores de Virulência/genéticaRESUMO
The regulation of Rac1 by HACE1-mediated ubiquitination and proteasomal degradation is emerging as an essential element in the maintenance of cell homeostasis. However, how the E3 ubiquitin ligase activity of HACE1 is regulated remains undetermined. Using a proteomic approach, we identified serine 385 as a target of group-I PAK kinases downstream Rac1 activation by CNF1 toxin from pathogenic E. coli. Moreover, cell treatment with VEGF also promotes Ser-385 phosphorylation of HACE1. We have established in vitro that HACE1 is a direct target of PAK1 kinase activity. Mechanistically, we found that the phospho-mimetic mutant HACE1(S385E), as opposed to HACE1(S385A), displays a lower capacity to ubiquitinate Rac1 in cells. Concomitantly, phosphorylation of Ser-385 plays a pivotal role in controlling the oligomerization state of HACE1. Finally, Ser-385 phosphorylated form of HACE1 localizes in the cytosol away from its target Rac1. Together, our data point to a feedback inhibition of HACE1 ubiquitination activity on Rac1 by group-I PAK kinases.
Assuntos
Serina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Toxinas Bacterianas/farmacologia , Linhagem Celular , Proteínas de Escherichia coli/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Fosforilação , Multimerização Proteica , Proteômica , Ubiquitinação , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Finely tuned regulation of epithelial cell death maintains tissue integrity and homeostasis. At the cellular level, life and death decisions are controlled by environmental stimuli such as the activation of death receptors. We show that cell polarity and adherens junction formation prevent proapoptotic signals emanating from the Fas death receptor. Fas is sequestered in E-cadherin actin-based adhesion structures that are less able to induce downstream apoptosis signaling. Using a proteomic-based approach, we find that the polarity molecule Dlg1 interacts with the C-terminal PDZ-binding site in Fas and that this interaction decreases formation of the death-inducing complex upon engagement with Fas ligand (FasL), thus acting as an additional cell death protection mechanism. We propose that E-cadherin and Dlg1 inhibit FasL-induced cell death by two complementary but partially independent mechanisms that help to maintain epithelial homeostasis by protecting normal polarized epithelia from apoptosis. When polarity is lost, the Fas-cadherin-Dlg1 antiapoptotic complex is disrupted, and FasL can promote the elimination of compromised nonpolarized cells.