RESUMO
ADP-ribosylation has primarily been known as post-translational modification of proteins. As signalling strategy conserved in all domains of life, it modulates substrate activity, localisation, stability or interactions, thereby regulating a variety of cellular processes and microbial pathogenicity. Yet over the last years, there is increasing evidence of non-canonical forms of ADP-ribosylation that are catalysed by certain members of the ADP-ribosyltransferase family and go beyond traditional protein ADP-ribosylation signalling. New macromolecular targets such as nucleic acids and new ADP-ribose derivatives have been established, notably extending the repertoire of ADP-ribosylation signalling. Based on the physiological relevance known so far, non-canonical ADP-ribosylation deserves its recognition next to the traditional protein ADP-ribosylation modification and which we therefore review in the following.
Assuntos
ADP-Ribosilação/fisiologia , ADP Ribose Transferases/química , ADP Ribose Transferases/classificação , ADP Ribose Transferases/fisiologia , Difosfato de Adenosina/metabolismo , Guanosina/metabolismo , N-Glicosil Hidrolases/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Sequências Reguladoras de Ácido Ribonucleico , Transdução de Sinais , Relação Estrutura-Atividade , Timidina/metabolismo , Ubiquitina/metabolismoRESUMO
Many Gram-negative pathogens use a type III secretion system (T3SS) to promote disease by injecting effector proteins into host cells. Common to many T3SSs is that injection of effector proteins is feedback inhibited. The mechanism of feedback inhibition and its role in pathogenesis are unclear. In the case of P. aeruginosa, the effector protein ExoS is central to limiting effector injection. ExoS is bifunctional, with an amino-terminal RhoGAP and a carboxy-terminal ADP-ribosyltransferase domain. We demonstrate that both domains are required to fully feedback inhibit effector injection. The RhoGAP-, but not the ADP-ribosyltransferase domain of the related effector protein ExoT also participates. Feedback inhibition does not involve translocator insertion nor pore-formation. Instead, feedback inhibition is due, in part, to a loss of the activating trigger for effector injection, and likely also decreased translocon stability. Surprisingly, feedback inhibition is abrogated in phagocytic cells. The lack of feedback inhibition in these cells requires phagocytic uptake of the bacteria, but cannot be explained through acidification of the phagosome or calcium limitation. Given that phagocytes are crucial for controlling P. aeruginosa infections, our data suggest that feedback inhibition allows P. aeruginosa to direct its effector arsenal against the cell types most damaging to its survival.
Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo III/metabolismo , ADP Ribose Transferases/genética , ADP Ribose Transferases/fisiologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Células Epiteliais/microbiologia , Retroalimentação Fisiológica/fisiologia , Proteínas Ativadoras de GTPase , Infecções por Pseudomonas/microbiologia , Sistemas de Secreção Tipo III/fisiologiaRESUMO
Bacteria respond to stressful growth conditions through a conserved phenomenon of stringent response mediated by synthesis of stress alarmones ppGpp and pppGpp [referred to as (p)ppGpp]. (p)ppGpp synthesis is known to occur by ribosome-associated RelA. In addition, a dual-function protein, SpoT (with both synthetase and hydrolase activities), maintains (p)ppGpp homeostasis. The presence of (p)ppGpp is also known to contribute to antibiotic resistance in bacteria. Mycobacterium smegmatis possesses Arr, which inactivates rifampin by its ADP ribosylation. Arr has been shown to be upregulated in response to stress. However, the roles Arr might play during growth have remained unclear. We show that Arr confers growth fitness advantage to M. smegmatis even in the absence of rifampin. Arr deficiency in M. smegmatis resulted in deficiency of biofilm formation. Further, we show that while Arr does not interact with the wild-type Escherichia coli ribosomes, it interacts with them when the E. coli ribosomal protein L11 (a stringent response regulator) is replaced with its homolog from M. smegmatis The Arr interaction with E. coli ribosomes occurs even when the N-terminal 33 amino acids of its L11 protein were replaced with the corresponding sequence of M. smegmatis L11 (Msm-EcoL11 chimeric protein). Interestingly, Arr interaction with the E. coli ribosomes harboring M. smegmatis L11 or Msm-EcoL11 results in the synthesis of ppGpp in vivo Our study shows a novel role of antibiotic resistance gene arr in stress response.IMPORTANCEMycobacterium smegmatis, like many other bacteria, possesses an ADP-ribosyltransferase, Arr, which confers resistance to the first-line antituberculosis drug, rifampin, by its ADP ribosylation. In this report, we show that in addition to its known property of conferring resistance to rifampin, Arr confers growth fitness advantage to M. smegmatis even when there is no rifampin in the growth medium. We then show that Arr establishes species-specific interactions with ribosomes through the N-terminal sequence of ribosomal protein L11 (a stringent response regulator) and results in ppGpp (stress alarmone) synthesis. Deficiency of Arr in M. smegmatis results in deficiency of biofilm formation. Arr protein is physiologically important both in conferring antibiotic resistance as well as in mediating stringent response.
Assuntos
ADP Ribose Transferases/genética , ADP Ribose Transferases/fisiologia , Proteínas de Bactérias/genética , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , ADP Ribose Transferases/deficiência , ADP-Ribosilação , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Metabolômica , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/crescimento & desenvolvimento , Ligação Proteica , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/fisiologia , Rifampina/farmacologia , Análise de Sequência de RNA , Especificidade da Espécie , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologiaRESUMO
ADP-ribosyltransferases promote repair of DNA single strand breaks and disruption of this pathway by Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) is toxic to cells with defects in homologous recombination (HR). Here, we show that this relationship is conserved in the simple eukaryote Dictyostelium and exploit this organism to define mechanisms that drive resistance of the HR-deficient cells to PARPi. Dictyostelium cells disrupted in exonuclease I, a critical factor for HR, are sensitive to PARPi. Deletion of exo1 prevents the accumulation of Rad51 in chromatin induced by PARPi, resulting in DNA damage being channelled through repair by non-homologous end-joining (NHEJ). Inactivation of NHEJ supresses the sensitivity of exo1- cells to PARPi, indicating this pathway drives synthetic lethality and that in its absence alternative repair mechanisms promote cell survival. This resistance is independent of alternate-NHEJ and is instead achieved by re-activation of HR. Moreover, inhibitors of Mre11 restore sensitivity of dnapkcs-exo1- cells to PARPi, indicating redundancy between nucleases that initiate HR can drive PARPi resistance. These data inform on mechanism of PARPi resistance in HR-deficient cells and present Dictyostelium as a convenient genetic model to characterize these pathways.
Assuntos
ADP Ribose Transferases/fisiologia , Dictyostelium/enzimologia , Resistência a Medicamentos/fisiologia , Recombinação Homóloga/fisiologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/fisiologia , Proteínas de Protozoários/fisiologia , Benzamidas/farmacologia , Células Clonais , Quinase 8 Dependente de Ciclina/deficiência , Quinase 8 Dependente de Ciclina/genética , Quinase 8 Dependente de Ciclina/fisiologia , Dano ao DNA , Dictyostelium/efeitos dos fármacos , Dictyostelium/genética , Exodesoxirribonucleases/deficiência , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/fisiologia , Deleção de Genes , Indóis/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Quinazolinas/farmacologia , Rad51 Recombinase/deficiência , Rad51 Recombinase/fisiologia , Proteínas Recombinantes/metabolismoRESUMO
Triple-negative breast cancers (TNBCs) are defined by lack of expressions of estrogen, progesterone, and ERBB2 receptors. Because biology of TNBC is poorly understood, no targeted therapy has been developed for this breast cancer subtype and chemotherapy is its only systemic treatment modality. In this study, we firstly determined that the expression of human ecto-ADP-ribosyltransferase 3 (ART3) is significantly associated with the basal-like breast cancer subgroup, which is largely overlapped with TNBC, through analyzing published data sets. We also found that ART3 protein is significantly overexpressed in human TNBC tumors tissue and cell lines through using immunohistochemistry and immunoblotting. Overexpression of ART3 in MDA-MB-231 breast cancer cells increased cell proliferation, invasion, and survival in vitro and growth of xenograft tumors. Conversely, knockdown of ART3 in breast cancer cells inhibited cell proliferation and invasion. In addition, we showed that ART 3 overexpression activated AKT and ERK in vitro and in xenograft tumors. Together, our findings demonstrate that ART3 is a critical TNBC marker with functional significance.
Assuntos
ADP Ribose Transferases/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Neoplasias de Mama Triplo Negativas/patologia , ADP Ribose Transferases/análise , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Proteínas Ligadas por GPI/análise , Proteínas Ligadas por GPI/fisiologia , Humanos , Camundongos , Invasividade Neoplásica , Neoplasias de Mama Triplo Negativas/mortalidade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The cytolethal distending toxin (CDT) is a well characterized bacterial genotoxin encoded by several Gram-negative bacteria, including Salmonella enterica (S. enterica). The CDT produced by Salmonella (S-CDT) differs from the CDT produced by other bacteria, as it utilizes subunits with homology to the pertussis and subtilase toxins, in place of the traditional CdtA and CdtC subunits. Previously, S-CDT was thought to be a unique virulence factor of S. enterica subspecies enterica serotype Typhi, lending to its classification as the "typhoid toxin." Recently, this important virulence factor has been identified and characterized in multiple nontyphoidal Salmonella (NTS) serotypes as well. The significance of S-CDT in salmonellosis with regards to the: (i) distribution of S-CDT encoding genes among NTS serotypes, (ii) contributions to pathogenicity, (iii) regulation of S-CDT expression, and (iv) the public health implication of S-CDT as it relates to disease severity, are reviewed here.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas , Desoxirribonucleases , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , ADP Ribose Transferases/fisiologia , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Desoxirribonucleases/química , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Desoxirribonucleases/fisiologia , Humanos , Salmonella/genética , Salmonella/metabolismo , Salmonella/fisiologia , VirulênciaRESUMO
Embryonic stem (ES) cells are in a dynamic equilibrium of distinct functional states, characterized by the heterogeneous expression of critical pluripotency factors and regulated by a spectrum of reversible histone modifications. Maintenance of this equilibrium is a hallmark of pluripotency. Here we find that the ADP-ribosyltransferases Parp1 and Parp7 play a critical role in safeguarding this state by occupying key pluripotency genes, notably Nanog, Pou5f1, Sox2, Stella, Tet1 and Zfp42, thereby protecting them from progressive epigenetic repression. In the absence of either Parp1 or Parp7, or upon inhibition of the ADP-ribosylating activity, ES cells exhibit a decrease in ground state pluripotency as they cannot maintain the typical heterogeneity characteristic of the metastable state. As a consequence, they display a higher propensity to differentiate. These findings place Parp1 and Parp7 at the genetic-epigenetic interface of pluripotency networks, fine-tuning the transcriptional heterogeneity and thereby determining the developmental plasticity of ES cells.
Assuntos
ADP Ribose Transferases/fisiologia , Células-Tronco Embrionárias/enzimologia , Células-Tronco Pluripotentes/enzimologia , Poli(ADP-Ribose) Polimerases/fisiologia , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Cromatina/química , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Metilação , Camundongos , Mutação , Células-Tronco Pluripotentes/citologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Fatores de Transcrição/metabolismo , Trofoblastos/citologia , Trofoblastos/enzimologiaRESUMO
Cytokine-induced killer (CIK) cells, one of the feasible and effective methods of adoptive immunotherapy, have shown anti-leukemia activity in vivo and in vitro. But the strategy exhibits limited cytotoxic activity in clinical studies. In this study, CIK cells were transfected with an interleukin-3/Pseudomonas exotoxin gene (IL3PE38KDEL). RT-PCR and ELISA were used to verify the expression of IL3PE38KDEL in the transfected CIK cells. These cells released 1,186.7 ± 149.6 pg IL3PE38KDEL/10(4) cells over 48 h into the medium and the culture supernatant selectively killed IL3 receptor(IL3R)-positive HL60 cells, but not IL3R-negative K562 cells. Moreover, IL3PE38KDEL transfection did not influence phenotypes and cytokine production of CIK cells. Co-cultured with leukemia cells, IL3PE38KDEL transfected CIK cells showed enhanced cytotoxicity against IL3R-positive HL60 cells at all effector-to-target (E:T) ratios, but exerted a basal anti-leukemia activity against IL3R-negative K562 cells. Our findings demonstrate that IL3PE38KDEL gene transfection may be a novel strategy for improving anti-leukemia activity of CIK cells.
Assuntos
ADP Ribose Transferases/fisiologia , Células Matadoras Induzidas por Citocinas/imunologia , Exotoxinas/fisiologia , Interleucina-3/fisiologia , Leucemia Mieloide Aguda/patologia , Pseudomonas aeruginosa/genética , Transfecção , Fatores de Virulência/fisiologia , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Técnicas de Cocultura , Citotoxicidade Imunológica , Exotoxinas/genética , Genes Sintéticos , Células HL-60 , Humanos , Imunofenotipagem , Imunoterapia , Interferon gama/análise , Interleucina-3/genética , Células K562 , Mutação , Estrutura Terciária de Proteína , Fator de Necrose Tumoral alfa/análise , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosaRESUMO
Cigarette smoking is an important risk factor for chronic obstructive pulmonary disease (COPD), yet its pathogenic mechanisms are not yet fully understood. Endothelial dysfunction is known to be involved in the pathogenesis of COPD. A detailed understanding of the mechanism involved in its progression would have a substantial impact on the optimization and development of treatment strategies. Here, we report that the expression of SIRT4, a mitochondrial sirtuin, is markedly down-regulated in cigarette smoke extract (CSE)-treated human pulmonary microvascular endothelial cells (HPMECs). Overexpression of SIRT4 significantly inhibits CSE-induced mononuclear cell adhesion to HPMECs. Consistently, we found that overexpression of SIRT4 attenuates the induction of vascular cell adhesion molecule 1 (VCAM-1) and E-selectin. Importantly, SIRT4 was found to negatively regulate CSE-induced NF-κB activation via inhibiting the degradation of IκBα. Moreover, we also found that proinflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and IL-6, the downstream target genes of NF-κB, are also inhibited by overexpression of SIRT4. These results suggest that SIRT4 protects HPMECs exposed to CSE stress via a mechanism that may involve the NF-κB pathway. Strategies based on the enhancement of SIRT4 may prove to be beneficial in the treatment of cigarette smoking caused COPD.
Assuntos
Células Endoteliais/fisiologia , Leucócitos Mononucleares/fisiologia , Proteínas Mitocondriais/fisiologia , NF-kappa B/fisiologia , Nicotiana/efeitos adversos , Doença Pulmonar Obstrutiva Crônica/etiologia , Sirtuínas/fisiologia , Fumaça/efeitos adversos , ADP Ribose Transferases/fisiologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Selectina E/análise , Humanos , Quinase I-kappa B/metabolismo , Molécula 1 de Adesão de Célula Vascular/análiseRESUMO
Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylates multiple eukaryotic targets to promote cytopathology and bacterial colonization. ADP-ribosylation of the small GTPase Rab5 has previously been shown to block fluid-phase endocytosis and trafficking of plasma membrane receptors to the early endosomes as well as inhibit phagocytosis of the bacterium. In this study, ExoS is shown to be capable of ADP-ribosylating 6 candidate arginine residues that are located in the effector binding region or in the C terminus of Rab5. Two Rab5 derivatives were engineered, which contained ArgâAla mutations at four Arg residues within the effector binding region (EF) or two Arg residues within the C-terminal tail (TL). Expression of Rab5(TL) does not affect the ability of ExoS to modify intracellular trafficking, while expression of Rab5(EF) rescued the ability of ExoS to inhibit intracellular trafficking. ADP-ribosylation of effector arginines likely uncouples Rab5 signaling to downstream effectors. This is a different mechanism for inhibition than observed for the ADP-ribosylation of Ras by ExoS, where ADP-ribosylated Ras loses the ability to bind guanine nucleotide exchange factor (GEF). Other experiments showed that expression of dominant negative Rab5(Ser34Asn) does not inhibit ExoS trafficking to the perinuclear region of intoxicated cells. This study provides insight into a mechanism for how ExoS ADP-ribosylation of Rab5 inhibits Rab5 function.
Assuntos
ADP Ribose Transferases/metabolismo , Adenosina Difosfato Ribose/metabolismo , Endocitose/fisiologia , Pseudomonas aeruginosa/enzimologia , Proteínas rab5 de Ligação ao GTP/metabolismo , ADP Ribose Transferases/genética , ADP Ribose Transferases/fisiologia , Arginina/genética , Toxinas Bacterianas , Transporte Biológico , Células Cultivadas , Células HeLa , Humanos , Transporte Proteico , Proteínas Recombinantes/metabolismo , Proteínas rab5 de Ligação ao GTP/fisiologiaRESUMO
Clostridium difficile infection is the main cause of healthcare-acquired diarrhea in the developed world. In addition to the main virulence factors toxin A and B, epidemic, PCR Ribotype 027 strains, such as R20291, produce a third toxin, CDT. To develop effective medical countermeasures, it is important to understand the importance of each toxin. Accordingly, we created all possible combinations of isogenic toxin mutants of R20291 and assessed their virulence. We demonstrated that either toxin A or toxin B alone can cause fulminant disease in the hamster infection model and present tantalizing data that C. difficile toxin may also contribute to virulence.
Assuntos
ADP Ribose Transferases/fisiologia , Proteínas de Bactérias/fisiologia , Clostridioides difficile/patogenicidade , Infecções por Clostridium/microbiologia , Enterotoxinas/fisiologia , ADP Ribose Transferases/genética , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Morte Celular , Clostridioides difficile/genética , Infecções por Clostridium/patologia , Cricetinae , Enterotoxinas/genética , Feminino , Células HT29 , Humanos , Mesocricetus , Virulência/genética , Virulência/fisiologiaRESUMO
During the development, progression and dissemination of neoplastic lesions, cancer cells can hijack normal pathways and mechanisms. This includes the control of the function of cellular proteins through reversible post-translational modifications, such as ADP-ribosylation, phosphorylation, and acetylation. In the case of mono-ADP-ribosylation and poly-ADP-ribosylation, the addition of one or several units of ADP-ribose to target proteins occurs via two families of enzymes that can generate ADP-ribosylated proteins: the diphtheria toxin-like ADP-ribosyltransferase (ARTD) family, comprising 17 different proteins that are either poly-ADP-ribosyltransferases or mono-ADP-ribosyltransferases or inactive enzymes; and the clostridial toxin-like ADP-ribosyltransferase family, with four human members, two of which are active mono-ADP-ribosyltransferases, and two of which are enzymatically inactive. In line with a central role for poly-ADP-ribose polymerase 1 in response to DNA damage, specific inhibitors of this enzyme have been developed as anticancer therapeutics and evaluated in several clinical trials. Recently, in combination with the discovery of a large number of enzymes that can catalyse mono-ADP-ribosylation, the role of this modification has been linked to human diseases, such as inflammation, diabetes, neurodegeneration, and cancer, thus revealing the need for the development of specific ARTD inhibitors. This will provide a better understanding of the roles of these enzymes in human physiology and pathology, so that they can be targeted in the future to generate new and efficacious drugs. This review summarizes our present knowledge of the ARTD enzymes that are involved in mono-ADP-ribosylation reactions and that have roles in cancer biology. In particular, the well-documented role of macro-containing ARTD8 in lymphoma and the putative role of ARTD15 in cancer are discussed.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Neoplasias/tratamento farmacológico , Processamento de Proteína Pós-Traducional , ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/fisiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Terapia de Alvo Molecular , Neoplasias/enzimologiaRESUMO
ADP ribosylation has been recently recognised as an important posttranslational modification regulating numerous cellular processes. This enzymatic activity is shared by two major families of enzymes, the extracellular ADP-ribosyl-transferases, or ecto-ARTS and the poly-ADP-ribosyltranferases, whose denomination derives from the capacity of its founding member, PARP1, to synthesise large linear or branched polymers of ADP-ribose on target proteins. This latter post-translational modification has recently attracted much interest based on its role in the cellular response to genotoxic and oxidative stress. Accordingly, a series of PARP-specific pharmacological inhibitors have demonstrated cell survival and anti-inflammatory properties in vivo, promoting a renewed interest in the potential immunoregulatory role of this gene family. More recently, the role of ADP-ribosylation in regulating several aspects of intracellular signalling and gene transcription has been uncovered, in particular within cells of the immune system, revealing the potential immunomodulatory role of several members of this family in addition to PARP1. We review herein the experimental evidence illustrating the complex role played by this gene family in regulating multiple aspects of the immune response, including cell survival, cytokine gene transcription and antiviral innate defences. In particular, the unexpected potential anti-inflammatory role of members of this family (including in particular PARP5a, 5b and PARP14) will be briefly discussed, raising some concern on the use of pan-specific PARP inhibitors to treat chronic inflammatory diseases.
Assuntos
Imunidade Adaptativa/genética , Inflamação/enzimologia , Inflamação/imunologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/fisiologia , ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/genética , ADP Ribose Transferases/fisiologia , Animais , Morte Celular/genética , Morte Celular/imunologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/patologia , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Bacterial C3 exoenzymes are widely used tools to investigate cellular events influenced by small GTPases of the Rho subfamily. In this respect they have gained increasing interest in addressing questions dealing with the neuronal morphogenic program during development and after lesion of the mature nervous system. Since central neurons display only very limited capacity to re-grow their axons after injury, successful strategies to improve regeneration are much sought-after. For a long time exclusively considered to be Rho-inhibiting exoenzymes, there is now accumulating evidence that C3 proteins of clostridial sources exhibit their often beneficial effects on neurite outgrowth by other means than ADP-ribosylation. The current review will outline previous attempts to foster neuronal cell growth by the use of C3 transferases and highlight the more recent approaches to improve regenerative axon outgrowth using enzyme-deficient C3 preparations.
Assuntos
ADP Ribose Transferases/farmacologia , Toxinas Botulínicas/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , ADP Ribose Transferases/fisiologia , Animais , Axônios/fisiologia , Toxinas Botulínicas/fisiologia , Sistema Nervoso Central/enzimologia , Sistema Nervoso Central/fisiologia , Humanos , Camundongos , Atividade Motora/fisiologia , Regeneração Nervosa/fisiologia , Ratos , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/fisiopatologia , Proteínas rho de Ligação ao GTP/fisiologiaRESUMO
Streptococcus pyogenes is an important human pathogen with an expansive repertoire of verified and putative virulence factors. Here we demonstrate that a mutant deficient in the production of the streptococcal ADP-ribosyltransferase SpyA generates lesions of reduced size in a subcutaneous mouse infection model. At early stages of infection, when the difference in lesion size is first established, inflamed tissue isolated from lesions of mice infected with spyA mutant bacteria has higher levels of mRNA encoding the chemokines CXCL1 and CCL2 than does tissue isolated from mice infected with wild-type bacteria. In addition, at these early times, the mRNA levels for the gene encoding the intermediate filament vimentin are higher in the mutant-infected tissue. As wound resolution progresses, mRNA levels of the gene encoding matrix metallopeptidase 2 are lower in mutant-infected tissue. Furthermore, we demonstrate that the spyA mutant is internalized more efficiently than wild-type bacteria by HeLa cells. We conclude that SpyA contributes to streptococcal pathogenesis in the mouse subcutaneous infection model. Our observations suggest that the presence of SpyA delays wound healing in this model.
Assuntos
ADP Ribose Transferases/fisiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/fisiologia , ADP Ribose Transferases/metabolismo , Animais , Western Blotting , Quimiocina CCL2/fisiologia , Quimiocina CXCL1/fisiologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Feminino , Células HeLa , Humanos , Camundongos , Neutrófilos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Streptococcus pyogenes/enzimologia , Fatores de Virulência/metabolismoRESUMO
CD4(+)CD25(+)FoxP3(+) regulatory T cells (T reg cells) play a major role in the control of immune responses but the factors controlling their homeostasis and function remain poorly characterized. Nicotinamide adenine dinucleotide (NAD(+)) released during cell damage or inflammation results in ART2.2-mediated ADP-ribosylation of the cytolytic P2X7 receptor on T cells. We show that T reg cells express the ART2.2 enzyme and high levels of P2X7 and that T reg cells can be depleted by intravenous injection of NAD(+). Moreover, lower T reg cell numbers are found in mice deficient for the NAD-hydrolase CD38 than in wild-type, P2X7-deficient, or ART2-deficient mice, indicating a role for extracellular NAD(+) in T reg cell homeostasis. Even routine cell preparation leads to release of NAD(+) in sufficient quantities to profoundly affect T reg cell viability, phenotype, and function. We demonstrate that T reg cells can be protected from the deleterious effects of NAD(+) by an inhibitory ART2.2-specific single domain antibody. Furthermore, selective depletion of T reg cells by systemic administration of NAD(+) can be used to promote an antitumor response in several mouse tumor models. Collectively, our data demonstrate that NAD(+) influences survival, phenotype, and function of T reg cells and provide proof of principle that acting on the ART2-P2X7 pathway represents a new strategy to manipulate T reg cells in vivo.
Assuntos
ADP Ribose Transferases/fisiologia , Fatores de Transcrição Forkhead/análise , NAD/fisiologia , Receptores Purinérgicos P2X7/fisiologia , Linfócitos T Reguladores/fisiologia , Animais , Apoptose , Selectina L/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , NAD/análise , Fosfatidilserinas/metabolismo , Transdução de SinaisRESUMO
Accumulating evidence suggests that the dichotomy between tolerance and active IgA immunity in mucosal immune responses is regulated at the APC level. Therefore, immunomodulation of the APC could be an effective mechanism to control the two response patterns. In this study, we demonstrate that ADP-ribosylation controls the outcome of tolerance or active effector T cell immunity to an internal peptide p323-339 from OVA inserted into the cholera toxin (CT)-derived CTA1-OVA-DD adjuvant. We found that a single point mutation, CTA1R7K-OVA-DD, resulting in lack of enzymatic activity, promoted peptide-specific tolerance in TCR transgenic CD4(+) T cells following a single intranasal (i.n.) treatment. The CTA1R7K-OVA-DD-induced tolerance was strong, long-lasting, and impaired the ability of adoptively transferred naive peptide-specific CD4(+) T cells to respond to Ag-challenge, irrespective if this was given i.p or i.n. The tolerance correlated with induction of regulatory T cells of the regulatory T type 1 characterized by CD25(-)Foxp3(-)CD4(+) T cells producing IL-10. In contrast, in IL-10-deficient mice, no peptide-specific tolerance was observed, and these mice exhibited unimpaired CD4(+) T cell responsiveness to recall Ag irrespective of if they were untreated (PBS) or treated i.n. with CTA1R7K-OVA-DD. Thus, for the first time, we can provide unequivocal proof that ADP-ribosylation can control the outcome of mucosal Ag exposure from tolerance to an enhanced effector CD4(+) T cell response. The exploitation of this system for clinical treatment of autoimmune diseases is discussed.
Assuntos
ADP Ribose Transferases/metabolismo , Tolerância Imunológica , Imunidade nas Mucosas , Mucosa Nasal/imunologia , ADP Ribose Transferases/fisiologia , Administração Intranasal , Animais , Células Cultivadas , Feminino , Tolerância Imunológica/genética , Imunidade nas Mucosas/genética , Interleucina-10/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/genética , Ovalbumina/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
OBJECTIVE: The aim of this study was to explore the possibility of creating a toxin, C-CPE-ETA', by fusing C-terminal high affinity binding domain of CPE (C-CPE) with a truncated form of Pseudomonas aeruginosa exotoxin A (ETA') and to examine whether C-CPE-ETA' could specifically target CLDN-3, 4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer. METHODS: CLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell lines and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA' into E. coli BL21 (DE3) plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells. RESULTS: Quantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3, OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0% (18/20) and 60.0% (12/20) of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA' was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA' was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA' was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC(50) was 7.364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22.340 ng/ml for SKOV3 cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA' at a concentration up to 10 µg/ml. CONCLUSIONS: The C-CPE-ETA' protein exhibits remarkably specific cytotoxicity for CLDN-3,4-overexpressing ovarian cancer cells. Its therapeutic potential warrants further development for ovarian cancer molecular targeted therapy.
Assuntos
ADP Ribose Transferases/metabolismo , Apoptose , Toxinas Bacterianas/metabolismo , Claudinas/metabolismo , Enterotoxinas/metabolismo , Exotoxinas/metabolismo , Neoplasias Ovarianas/patologia , Fatores de Virulência/metabolismo , ADP Ribose Transferases/fisiologia , Linhagem Celular Tumoral , Claudina-3 , Claudina-4 , Claudinas/genética , Enterotoxinas/fisiologia , Exotoxinas/fisiologia , Feminino , Humanos , Imunotoxinas/metabolismo , Neoplasias Ovarianas/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Fatores de Virulência/fisiologia , Exotoxina A de Pseudomonas aeruginosaRESUMO
Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by production of the Rho GTPase-glucosylating toxins A and B. Recently emerging hypervirulent Clostridium difficile strains additionally produce the binary ADP-ribosyltransferase toxin CDT (Clostridium difficile transferase), which ADP-ribosylates actin and inhibits actin polymerization. Thus far, the role of CDT as a virulence factor is not understood. Here we report by using time-lapse- and immunofluorescence microscopy that CDT and other binary actin-ADP-ribosylating toxins, including Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin, induce redistribution of microtubules and formation of long (up to >150 microm) microtubule-based protrusions at the surface of intestinal epithelial cells. The toxins increase the length of decoration of microtubule plus-ends by EB1/3, CLIP-170 and CLIP-115 proteins and cause redistribution of the capture proteins CLASP2 and ACF7 from microtubules at the cell cortex into the cell interior. The CDT-induced microtubule protrusions form a dense meshwork at the cell surface, which wrap and embed bacterial cells, thereby largely increasing the adherence of Clostridia. The study describes a novel type of microtubule structure caused by less efficient microtubule capture and offers a new perspective for the pathogenetic role of CDT and other binary actin-ADP-ribosylating toxins in host-pathogen interactions.