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
The human disease fibrodysplasia ossificans progressiva (FOP) is a rare and highly disabling disorder of extensive heterotopic bone growth that is caused by a point mutation (R206H) in the activation domain of Alk2, a BMP (bone morphogenic protein) type 1 receptor. The mutation leads to extensive BMP-signaling induced by Activin A, which is normally an antagonist for wildtype receptors, resulting in excessive and uncontrolled bone formation. Here, we studied the effects of Pasteurella multocida toxin (PMT), which activates osteoclasts and inhibits osteoblast activity, in C2C12 myoblasts expressing the mutant Alk2(R206H) receptor as model of FOP. In our study, we mainly used alkaline phosphatase (ALP) activity as marker to determine osteoblast differentiation. BMP-4 stimulated an increase in ALP activity in C2C12-Alk2wt and C2C12-Alk2(R206H) cells. By contrast, Activin A only induced ALP activity in C2C12-Alk2(R206H) cells. In both cases, PMT acted as a potent inhibitor of ALP activity. PMT-induced inhibition of ALP activity was paralleled by a constitutive activation of the heterotrimeric Gq protein. Expression of a permanently active Gαq blocked Activin A/Alk2(R206H)-dependent increase in ALP activity. Inactivation of Gq by specific inhibitor FR900359 blocked the PMT effect. Similarly, canonical second messengers and effectors of Gαq (e.g. ionophore A23187-induced increase in intracellular Ca2+ and activation of PKC by PMA (phorbol 12-myristate 13-acetate)) inhibited Alk2(R206H)-mediated induction of ALP activity. Notably, Activin A-induced increase in ALP activity in C2C12-Alk2(R206H) cells was also inhibited by stimulation of the α1A-adrenoceptor, which couples to Gαq, by phenylephrine. PMT did not alter tail phosphorylation of the major downstream effectors of the Alk2 receptor, Smad1/5/9; neither did the toxin affect nuclear translocation of the Smad-complex. However, PMT diminished BMP responsive element-induced gene expression. The data indicate that PMT potently inhibits the induction of osteoblast markers in a FOP model via activation of G proteins. Moreover, our findings indicate that activation of G protein-coupled receptors and of G protein signaling might be a rationale for pharmacological therapy of FOP.
Assuntos
Ativinas/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Modelos Biológicos , Mioblastos/metabolismo , Miosite Ossificante/patologia , Osteoblastos/metabolismo , Transdução de Sinais , Fosfatase Alcalina/metabolismo , Animais , Proteína Morfogenética Óssea 4/metabolismo , Sinalização do Cálcio , Linhagem Celular , Camundongos , Proteínas Smad/metabolismoRESUMO
Pasteurella multocida toxin (PMT) causes progressive atrophic rhinitis with severe turbinate bone degradation in pigs. It has been reported that the toxin deamidates and activates heterotrimeric G proteins, resulting in increased differentiation of osteoclasts and blockade of osteoblast differentiation. So far, the action of PMT on osteocytes, which is the most abundant cell type in bone tissue, is not known. In MLO-Y4 osteocytes, PMT deamidated heterotrimeric G proteins, resulting in loss of osteocyte dendritic processes, stress fiber formation, cell spreading and activation of RhoC but not of RhoA. Moreover, the toxin caused processing of membrane-bound receptor activator of NF-κB ligand (RANKL) to release soluble RANKL and enhanced the secretion of osteoclastogenic TNF-α. In a co-culture model of osteocytes and bone marrow cells, PMT-induced osteoclastogenesis was largely increased as compared to the mono-culture model. The enhancement of osteoclastogenesis observed in the co-culture was blocked by sequestering RANKL with osteoprotegerin and by an antibody against TNF-α indicating involvement of release of the osteoclastogenic factors from osteocytes. Data support the crucial role of osteocytes in bone metabolism and osteoclastogenesis and identify osteocytes as important target cells of PMT in progressive atrophic rhinitis.
Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Osteócitos/efeitos dos fármacos , Animais , Linhagem Celular , Técnicas de Cocultura , Feminino , Proteínas de Ligação ao GTP/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos Endogâmicos C57BL , Osteócitos/fisiologia , Osteogênese/efeitos dos fármacos , Ligante RANK/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Salmonella enterica serotype Typhimurium (S. Typhimurium) is one of the most frequent causes of food-borne illness in humans and usually associated with acute self-limiting gastroenteritis. However, in immunocompromised patients, the pathogen can disseminate and lead to severe systemic diseases. S. Typhimurium are facultative intracellular bacteria. For uptake and intracellular life, Salmonella translocate numerous effector proteins into host cells using two type-III secretion systems (T3SS), which are encoded within Salmonella pathogenicity islands 1 (SPI-1) and 2 (SPI-2). While SPI-1 effectors mainly promote initial invasion, SPI-2 effectors control intracellular survival and proliferation. Here, we elucidate the mode of action of Salmonella SPI-2 effector SseI, which is involved in control of systemic dissemination of S. Typhimurium. SseI deamidates a specific glutamine residue of heterotrimeric G proteins of the Gαi family, resulting in persistent activation of the G protein. Gi activation inhibits cAMP production and stimulates PI3-kinase γ by Gαi-released Gßγ subunits, resulting in activation of survival pathways by phosphorylation of Akt and mTOR. Moreover, SseI-induced deamidation leads to non-polarized activation of Gαi and, thereby, to loss of directed migration of dendritic cells.
Assuntos
Proteínas de Bactérias/fisiologia , Quimiotaxia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Salmonella typhimurium , Sistemas de Secreção Tipo III/fisiologia , Animais , Proteínas de Bactérias/genética , Sobrevivência Celular/genética , Quimiotaxia/genética , Desaminação/genética , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Multimerização Proteica/genética , Processamento de Proteína Pós-Traducional/genética , Células RAW 264.7 , Infecções por Salmonella/metabolismo , Infecções por Salmonella/patologia , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismoRESUMO
Two-pore channels (TPCs) are localized in endo-lysosomal compartments and assumed to play an important role for vesicular fusion and endosomal trafficking. Recently, it has been shown that both TPC1 and 2 were required for host cell entry and pathogenicity of Ebola viruses. Here, we investigate the cellular function of TPC1 using protein toxins as model substrates for distinct endosomal processing routes. Toxin uptake and activation through early endosomes but not processing through other compartments were reduced in TPC1 knockout cells. Detailed co-localization studies with subcellular markers confirmed predominant localization of TPC1 to early and recycling endosomes. Proteomic analysis of native TPC1 channels finally identified direct interaction with a distinct set of syntaxins involved in fusion of intracellular vesicles. Together, our results demonstrate a general role of TPC1 for uptake and processing of proteins in early and recycling endosomes, likely by providing high local Ca2+ concentrations required for SNARE-mediated vesicle fusion.
Assuntos
Canais de Cálcio/metabolismo , Endossomos/metabolismo , Animais , Linhagem Celular , Cães , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Camundongos , Ligação Proteica , Transporte Proteico , Proteínas Qa-SNARE/metabolismoRESUMO
The AB-type protein toxin from Pasteurella multocida (PMT) contains a functionally important disulfide bond within its catalytic domain, which must be cleaved in the host cell cytosol to render the catalytic domain of PMT into its active conformation. Here, we found that the reductive potential of the cytosol of target cells, and more specifically, the activity of the thioredoxin reductase (TrxR) is crucial for this process. This was demonstrated by the strong inhibitory effect of the pharmacological TrxR inhibitor auranofin, which inhibited the intoxication of target cells with PMT, as determined by analyzing the PMT-catalyzed deamidation of GTP-binding proteins (G-proteins) in the cytosol of cells. The amount of endogenous substrate levels modified by PMT in cells pretreated with auranofin was reduced compared to cells treated with PMT alone. Auranofin had no inhibitory effect on the activity of the catalytic domain of constitutively active PMT in vitro, demonstrating that auranofin did not directly inhibit PMT activity, but interferes with the mode of action of PMT in cells. In conclusion, the results show that TrxR is crucial for the mode of action of PMT in mammalian cells, and that the drug auranofin can serve as an efficient inhibitor, which might be a starting point for novel therapeutic options against toxin-associated diseases.
Assuntos
Auranofina/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Pasteurella multocida/enzimologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Domínio Catalítico , Técnicas de Cultura de Células , Citosol/metabolismo , Células HeLa , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Pasteurella multocida/patogenicidade , VirulênciaRESUMO
B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs in trans within a Raf dimer. Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase.
Assuntos
Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Animais , Transformação Celular Neoplásica/química , Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Camundongos , Fosforilação , Proteômica , Transdução de Sinais/fisiologiaRESUMO
RATIONALE: In chronic heart failure, increased adrenergic activation contributes to structural remodeling and altered gene expression. Although adrenergic signaling alters histone modifications, it is unknown, whether it also affects other epigenetic processes, including DNA methylation and its recognition. OBJECTIVE: The aim of this study was to identify the mechanism of regulation of the methyl-CpG-binding protein 2 (MeCP2) and its functional significance during cardiac pressure overload and unloading. METHODS AND RESULTS: MeCP2 was identified as a reversibly repressed gene in mouse hearts after transverse aortic constriction and was normalized after removal of the constriction. Similarly, MeCP2 repression in human failing hearts resolved after unloading by a left ventricular assist device. The cluster miR-212/132 was upregulated after transverse aortic constriction or on activation of α1- and ß1-adrenoceptors and miR-212/132 led to repression of MeCP2. Prevention of MeCP2 repression by a cardiomyocyte-specific, doxycycline-regulatable transgenic mouse model aggravated cardiac hypertrophy, fibrosis, and contractile dysfunction after transverse aortic constriction. Ablation of MeCP2 in cardiomyocytes facilitated recovery of failing hearts after reversible transverse aortic constriction. Genome-wide expression analysis, chromatin immunoprecipitation experiments, and DNA methylation analysis identified mitochondrial genes and their transcriptional regulators as MeCP2 target genes. Coincident with its repression, MeCP2 was removed from its target genes, whereas DNA methylation of MeCP2 target genes remained stable during pressure overload. CONCLUSIONS: These data connect adrenergic activation with a microRNA-MeCP2 epigenetic pathway that is important for cardiac adaptation during the development and recovery from heart failure.
Assuntos
Adaptação Fisiológica/fisiologia , Epigênese Genética/fisiologia , Insuficiência Cardíaca/metabolismo , Proteína 2 de Ligação a Metil-CpG/biossíntese , Receptores Adrenérgicos/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Doença Crônica , Insuficiência Cardíaca/genética , Humanos , Proteína 2 de Ligação a Metil-CpG/antagonistas & inibidores , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Ratos , Receptores Adrenérgicos/genéticaRESUMO
Pasteurella multocida toxin (PMT) persistently activates heterotrimeric G proteins of the Gαq/11 , Gα12/13 and Gαi family without interaction with G protein-coupled receptors (GPCRs). We show that PMT acts on heart tissue in vivo and on cardiomyocytes and cardiac fibroblasts in vitro by deamidation of heterotrimeric G proteins. Increased normalized ventricle weights and fibrosis were detected after intraperitoneal administration of PMT in combination with the GPCR agonist phenylephrine. In neonatal rat cardiomyocytes, PMT stimulated the mitogen-activated protein kinase pathway, which is crucial for the development of cellular hypertrophy. The toxin induced phosphorylation of the canonical phosphorylation sites of the extracellular-regulated kinase 1/2 and, additionally, caused phosphorylation of the recently recognized autophosphorylation site, which appears to be important for the development of cellular hypertrophy. Moreover, PMT stimulated the small GTPases Rac1 and RhoA. Both switch proteins are involved in cardiomyocyte hypertrophy. In addition, PMT stimulated RhoA and Rac1 in neonatal rat cardiac fibroblasts. RhoA and Rac1 have been implicated in the regulation of connective tissue growth factor (CTGF) secretion and expression. Accordingly, we show that PMT treatment increased secretion and expression of CTGF in cardiac fibroblasts. Altogether, the data indicate that PMT is an inducer of pathological remodelling of cardiac cells and identifies the toxin as a promising tool for studying heterotrimeric G protein-dependent signalling in cardiac cells.
Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Cardiomegalia/patologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibrose/patologia , Animais , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Expressão Gênica/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Modelos Animais , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Fosforilação , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , RatosRESUMO
Bacterial components are recognized by the immune system through activation of the inflammasome, eventually causing processing of the proinflammatory cytokine interleukin-1? (IL-1?), a pleiotropic cytokine and one of the most important mediators of inflammation, through the protease caspase-1. Synthesis of the precursor protein and processing into its bioactive form are tightly regulated, given that disturbed control of IL-1? release can cause severe autoinflammatory diseases or contribute to cancer development. We show that the bacterial Pasteurella multocida toxin (PMT) triggers Il1b gene transcription in macrophages independently of Toll-like receptor signaling through RhoA/Rho-kinase-mediated NF-?? activation. Furthermore, PMT mediates signal transducer and activator of transcription (STAT) protein-controlled granzyme A (a serine protease) expression in macrophages. The exocytosed granzyme A enters target cells and mediates IL-1? maturation independently of caspase-1 and without inducing cytotoxicity. These findings show that macrophages can induce an IL-1?-initiated immune response independently of inflammasome activity.
Assuntos
Granzimas/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/biossíntese , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Caspase 1/metabolismo , Humanos , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Janus Quinases/metabolismo , Camundongos , NF-kappa B/metabolismo , Perforina/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Transcrição Gênica/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
UNLABELLED: Pasteurella multocida toxin (PMT) induces atrophic rhinitis in animals, which is characterized by a degradation of nasal turbinate bones, indicating an effect of the toxin on bone cells such as osteoblasts and osteoclasts. The underlying molecular mechanism of PMT was defined as a persistent activation of heterotrimeric G proteins by deamidation of a specific glutamine residue. Here, we show that PMT acts directly on osteoclast precursor cells such as bone marrow-derived CD14(+) monocytes and RAW246.7 cells to induce osteoclastogenesis as measured by expression of osteoclast-specific markers such as tartrate-resistant acid phosphatase and bone resorption activity. Treatment performed solely with PMT stimulates osteoclast differentiation, showing a receptor activator of nuclear factor-κB ligand (RANKL)-independent action of the toxin. The underlying signal transduction pathway was defined as activation of the heterotrimeric G proteins Gαq/11 leading to the transactivation of Ras and the mitogen-activated protein kinase pathway. Gαq/11 transactivates Ras via its effector phospholipase Cß-protein kinase C (PKC) involving proline-rich tyrosine kinase 2 (Pyk2). PMT-induced activation of the mitogen-activated protein kinase pathway results in stimulation of the osteoclastogenic transcription factors AP-1, NF-κB, and NFATc1. In addition, Ca(2+)-dependent calcineurin activation of NFAT is crucial for PMT-induced osteoclastogenesis. The data not only elucidate a rationale for PMT-dependent bone loss during atrophic rhinitis but also highlight a noncanonical, G-protein-dependent pathway toward bone resorption that is distinct from the RANKL-RANK pathway but mimics it. We define heterotrimeric G proteins as as-yet-underestimated entities/players in the maturation of osteoclasts which might be of pharmacological relevance. IMPORTANCE: Pasteurella multocida toxin (PMT) induces degradation of nasal turbinate bones, leading to the syndrome of atrophic rhinitis. Recently, the molecular mechanism and substrate specificity of PMT were identified. The toxin activates heterotrimeric G proteins by a covalent modification. However, the mechanism by which PMT induces bone degradation is poorly understood. Our report demonstrates a direct effect of PMT on osteoclast precursor cells, leading to maturation of bone-degrading osteoclasts. Interestingly, PMT stimulates osteoclastogenesis independently of the cytokine RANKL, which is a key factor in induction of osteoclast differentiation. This implicates a noncanonical osteoclastogenic signaling pathway induced by PMT. The elucidated Gαq/11-dependent osteoclastogenic signal transduction pathway ends in osteoclastogenic NFAT signaling. The noncanonical, heterotrimeric G protein-dependent osteoclast differentiation process may be of pharmacological relevance, as members of this pathway are highly druggable. In particular, modulation of G protein-coupled receptor activity in osteoclast progenitors by small molecules might be of specific interest.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Reabsorção Óssea , Proteínas de Ligação ao GTP/metabolismo , Interações Hospedeiro-Patógeno , Osteoclastos/metabolismo , Pasteurella multocida/fisiologia , Rinite Atrófica/patologia , Animais , Linhagem Celular , Macrófagos/efeitos dos fármacos , Camundongos , Osteoclastos/efeitos dos fármacos , Transdução de SinaisRESUMO
Entomopathogenic Photorhabdus asymbiotica is an emerging pathogen in humans. Here, we identified a P. asymbiotica protein toxin (PaTox), which contains a glycosyltransferase and a deamidase domain. PaTox mono-O-glycosylates Y32 (or Y34) of eukaryotic Rho GTPases by using UDP-N-acetylglucosamine (UDP-GlcNAc). Tyrosine glycosylation inhibits Rho activation and prevents interaction with downstream effectors, resulting in actin disassembly, inhibition of phagocytosis and toxicity toward insects and mammalian cells. The crystal structure of the PaTox glycosyltransferase domain in complex with UDP-GlcNAc determined at 1.8-Å resolution represents a canonical GT-A fold and is the smallest glycosyltransferase toxin known. (1)H-NMR analysis identifies PaTox as a retaining glycosyltransferase. The glutamine-deamidase domain of PaTox blocks GTP hydrolysis of heterotrimeric Gαq/11 and Gαi proteins, thereby activating RhoA. Thus, PaTox hijacks host GTPase signaling in a bidirectional manner by deamidation-induced activation and glycosylation-induced inactivation of GTPases.
Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Photorhabdus/enzimologia , Tirosina/metabolismo , Uridina Difosfato N-Acetilglicosamina/química , Uridina Difosfato N-Acetilglicosamina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Glicosilação , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
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
The protein toxin Pasteurella multocida toxin (PMT) is the causative agent of atrophic rhinitis in pigs, leading to atrophy of the nasal turbinate bones by affecting osteoblasts and osteoclasts. The mechanism of PMT-induced intoxication is a deamidation of α-subunits of heterotrimeric G proteins, including Gαq, Gα13, and Gαi, thereby causing persistent activation of the G proteins. Here we utilized PMT as a transporter of the non-cell-permeating A domain of diphtheria toxin (DTa). Fusion proteins of PMT and DTa ADP-ribosylated elongation factor 2, the natural target of diphtheria toxin, leading to cell toxicity. PMT-DTa effects were competed by PMT, indicating binding to the same cell surface receptor. Fluorescently labeled PMT-DTa and PMT colocalized with specific markers of early and late endosomes. Bafilomycin A, which inhibits vacuolar H(+)-ATPase, blocked PMT-DTa-induced intoxication of HEK-293 cells. By constructing various PMT-DTa chimeras, we identified a minimal region of PMT necessary for uptake of DTa. The data suggest that PMT is able to transport cargo proteins into eukaryotic cells by utilizing the PMT-specific uptake route.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Toxina Diftérica/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Biomarcadores/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular , Sobrevivência Celular , Clonagem Molecular , Citosol/metabolismo , Toxina Diftérica/genética , Endossomos/efeitos dos fármacos , Endossomos/genética , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Macrolídeos/farmacologia , Fragmentos de Peptídeos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/genéticaRESUMO
Pasteurella multocida is the causative agent of a number of epizootic and zoonotic diseases. Its major virulence factor associated with atrophic rhinitis in animals and dermonecrosis in bite wounds is P. multocida toxin (PMT). PMT stimulates signal transduction pathways downstream of heterotrimeric G proteins, leading to effects such as mitogenicity, blockade of apoptosis, or inhibition of osteoblast differentiation. On the basis of Gα(i2), it was demonstrated that the toxin deamidates an essential glutamine residue of the Gα(i2) subunit, leading to constitutive activation of the G protein. Here, we studied the specificity of PMT for its G-protein targets by mass spectrometric analyses and by utilizing a monoclonal antibody, which recognizes specifically G proteins deamidated by PMT. The studies revealed deamidation of 3 of 4 families of heterotrimeric G proteins (Gα(q/11), Gα(i1,2,3), and Gα(12/13) of mouse or human origin) by PMT but not by a catalytic inactive toxin mutant. With the use of G-protein fragments and chimeras of responsive or unresponsive G proteins, the structural basis for the discrimination of heterotrimeric G proteins was studied. Our results elucidate substrate specificity of PMT on the molecular level and provide evidence for the underlying structural reasons of substrate discrimination.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Pasteurella multocida/metabolismo , Pasteurella multocida/patogenicidade , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Sequência de Bases , Sítios de Ligação , Células Cultivadas , DNA Complementar/genética , Subunidades alfa de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Glutamina/química , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pasteurella multocida/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Especificidade por SubstratoAssuntos
Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Infecções por Pasteurella/imunologia , Pasteurella multocida/imunologia , Pasteurella multocida/patogenicidade , Animais , Especificidade de Hospedeiro , Humanos , Infecções por Pasteurella/microbiologia , Pasteurella multocida/genéticaRESUMO
Pasteurella multocida toxin (PMT) is the causative agent of progressive atrophic rhinitis in swine. The 146 kDa single-chain toxin harbours discrete domains important for receptor binding, internalisation and biological activity. The molecular basis of the toxin's activity is the deamidation of a specific glutamine residue in the α-subunit of heterotrimeric G proteins. This results in an inhibition of the inherent GTPase activity leading to a constitutively active phenotype of the G protein. Due to the ability of the toxin to act on various families of heterotrimeric G proteins, a large subset of signal transduction pathways is stimulated.
Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Infecções por Pasteurella/metabolismo , Pasteurella multocida/genética , Rinite Atrófica/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Glutamina/genética , Glutamina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Modelos Moleculares , Infecções por Pasteurella/genética , Infecções por Pasteurella/microbiologia , Pasteurella multocida/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Rinite Atrófica/genética , Rinite Atrófica/microbiologia , Transdução de Sinais/genética , Suínos , Regulação para CimaRESUMO
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
ADP-ribosyltransferases (ADP-RTs) use NAD(+) to transfer an ADP-ribosyl group to target proteins. Although some ADP-RTs are bacterial toxins only few inhibitors are known. Here we present the development of fluorescence-based assays and a focussed library screening using kinase inhibitors as a new approach towards inhibitors of ADP-RTs. Different screening setups were established using surrogate small molecule substrates or the quantitation of the cofactor NAD(+). Proof-of-principle screening experiments were performed using a kinase inhibitor library in order to target the NAD(+) binding pockets. This led to the discovery of structurally different lead inhibitors for the mono-ADP-ribosyltransferases Mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1, C3bot toxin from Clostridium botulinum and CDTa from Clostridium difficile. The interaction of the inhibitors with the toxin proteins was analyzed by means of docking and binding free energy calculations. Binding at the nicotinamide subpocket, which shows a significant difference in the three enzymes, is used to explain the selectivity of the identified inhibitors and offers an opportunity for further development of potent and selective inhibitors.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Inibidores de Proteínas Quinases/farmacologia , ADP Ribose Transferases/química , Bacillus/enzimologia , Clostridioides difficile/enzimologia , Clostridium botulinum/enzimologia , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Pierisin-like proteins comprise a growing family of ADP-ribosyltransferases expressed in various species of white butterflies. The prototype pierisin-1 from the cabbage butterfly, Pieris rapae, was identified as a potent apoptosis-inducing agent, acting on various types of carcinoma cell lines by mono-ADP-ribosylation of DNA. The characterization of pierisin-like proteins is hampered by its potent toxicity, which prevents its expression as a recombinant protein in Escherichia coli. Here we characterized a new member of the pierisin protein family named pierisin-1b, which was cloned from P. rapae. Pierisin-1b consists of 849 amino acids residues and shares 63%-91% identity with already described pierisins. For expression of pierisin-1b a novel in vitro translation system was utilized. Obtained protein exhibits specific ADP-ribosyltransferase activity on deoxyguanosine residues of DNA leading to induction of apoptosis and cell death.