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1.
Cell Microbiol ; 17(6): 843-59, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25486861

RESUMO

Salmonella enterica Typhimurium induces intestinal inflammation through the activity of type III secreted effector (T3SE) proteins. Our prior results indicate that the secretion of the T3SE SipA and the ability of SipA to induce epithelial cell responses that lead to induction of polymorphonuclear transepithelial migration are not coupled to its direct delivery into epithelial cells from Salmonella. We therefore tested the hypothesis that SipA interacts with a membrane protein located at the apical surface of intestinal epithelial cells. Employing a split ubiquitin yeast-two-hybrid screen, we identified the tetraspanning membrane protein, p53 effector related to PMP-22 (PERP), as a SipA binding partner. SipA and PERP appear to have intersecting activities as we found PERP to be involved in proinflammatory pathways shown to be regulated by SipA. In sum, our studies reveal a critical role for PERP in the pathogenesis of S. Typhimurium, and for the first time demonstrate that SipA, a T3SE protein, can engage a host protein at the epithelial surface.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Inflamação/microbiologia , Inflamação/patologia , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Salmonella typhimurium/imunologia , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Genes Supressores de Tumor , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas , Migração Transendotelial e Transepitelial , Técnicas do Sistema de Duplo-Híbrido
2.
Blood ; 119(5): 1228-39, 2012 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-22086418

RESUMO

Survival signaling by the erythropoietin (Epo) receptor (EpoR) is essential for erythropoiesis and for its acceleration in hypoxic stress. Several apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here we used mouse models of acute and chronic stress, including a hypoxic environment and ß-thalassemia, to identify two markedly different response dynamics for two erythroblast survival pathways in vivo. Induction of the antiapoptotic protein Bcl-x(L) is rapid but transient, while suppression of the proapoptotic protein Bim is slower but persistent. Similar to sensory adaptation, however, the Bcl-x(L) pathway "resets," allowing it to respond afresh to acute stress superimposed on a chronic stress stimulus. Using "knock-in" mouse models expressing mutant EpoRs, we found that adaptation in the Bcl-x(L) response occurs because of adaptation of its upstream regulator Stat5, both requiring the EpoR distal cytoplasmic domain. We conclude that survival pathways show previously unsuspected functional specialization for the acute and chronic phases of the stress response. Bcl-x(L) induction provides a "stop-gap" in acute stress, until slower but permanent pathways are activated. Furthermore, pathologic elevation of Bcl-x(L) may be the result of impaired adaptation, with implications for myeloproliferative disease mechanisms.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Células Precursoras Eritroides/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteína bcl-X/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Sobrevivência Celular/genética , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Células Precursoras Eritroides/metabolismo , Eritropoese/genética , Eritropoese/fisiologia , Fígado/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT5/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
3.
bioRxiv ; 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38659745

RESUMO

The recA gene, encoding Recombinase A (RecA) is one of three Mycobacterium tuberculosis (Mtb) genes encoding an in-frame intervening protein sequence (intein) that must splice out of precursor host protein to produce functional protein. Ongoing debate about whether inteins function solely as selfish genetic elements or benefit their host cells requires understanding of interplay between inteins and their hosts. We measured environmental effects on native RecA intein splicing within Mtb using a combination of western blots and promoter reporter assays. RecA splicing was stimulated in bacteria exposed to DNA damaging agents or by treatment with copper in hypoxic, but not normoxic, conditions. Spliced RecA was processed by the Mtb proteasome, while free intein was degraded efficiently by other unknown mechanisms. Unspliced precursor protein was not observed within Mtb despite its accumulation during ectopic expression of Mtb recA within E. coli. Surprisingly, Mtb produced free N-extein in some conditions, and ectopic expression of Mtb N-extein activated LexA in E. coli. These results demonstrate that the bacterial environment greatly impacts RecA splicing in Mtb, underscoring the importance of studying intein splicing in native host environments and raising the exciting possibility of intein splicing as a novel regulatory mechanism in Mtb.

4.
PLoS Biol ; 8(9)2010 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-20877475

RESUMO

Hematopoietic progenitors undergo differentiation while navigating several cell division cycles, but it is unknown whether these two processes are coupled. We addressed this question by studying erythropoiesis in mouse fetal liver in vivo. We found that the initial upregulation of cell surface CD71 identifies developmentally matched erythroblasts that are tightly synchronized in S-phase. We show that DNA replication within this but not subsequent cycles is required for a differentiation switch comprising rapid and simultaneous committal transitions whose precise timing was previously unknown. These include the onset of erythropoietin dependence, activation of the erythroid master transcriptional regulator GATA-1, and a switch to an active chromatin conformation at the ß-globin locus. Specifically, S-phase progression is required for the formation of DNase I hypersensitive sites and for DNA demethylation at this locus. Mechanistically, we show that S-phase progression during this key committal step is dependent on downregulation of the cyclin-dependent kinase p57(KIP2) and in turn causes the downregulation of PU.1, an antagonist of GATA-1 function. These findings therefore highlight a novel role for a cyclin-dependent kinase inhibitor in differentiation, distinct to their known function in cell cycle exit. Furthermore, we show that a novel, mutual inhibition between PU.1 expression and S-phase progression provides a "synchromesh" mechanism that "locks" the erythroid differentiation program to the cell cycle clock, ensuring precise coordination of critical differentiation events.


Assuntos
Ciclo Celular , Eritropoese , Proteínas Proto-Oncogênicas/metabolismo , Fase S , Transativadores/metabolismo , Animais , Antígenos CD/metabolismo , Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Metilação de DNA , Replicação do DNA , Regulação para Baixo , Camundongos , Receptores da Transferrina/metabolismo
5.
J Microbiol Biol Educ ; 24(1)2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37089221

RESUMO

This learning activity teaches the difficult concept of V(D)J recombination as it occurs in B cells. Following the traditional lecture, this hands-on activity uses pipe cleaners of various colors representing variable, joining, and diversity gene segments and recombination signal sequences. Students are provided with instructions for using the pipe cleaners to assemble specific light and heavy immunoglobulin chains. Students each assemble their own light and heavy chains and compare the products made by classmates. This activity uses materials that are easy and affordable to acquire and provides a tactile approach to reinforcing concepts that students often struggle to visualize and master from lecture and textbook material alone.

6.
Cell Mol Life Sci ; 68(22): 3687-97, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21984608

RESUMO

Acute gastroenteritis caused by Salmonella enterica serovar typhimurium is a significant public health problem. This pathogen has very sophisticated molecular machinery encoded by the two pathogenicity islands, namely Salmonella Pathogenicity Island 1 and 2 (SPI-1 and SPI-2). Remarkably, both SPI-1 and SPI-2 are very tightly regulated in terms of timing of expression and spatial localization of the encoded effectors during the infection process within the host cell. This regulation is governed at several levels, including transcription and translation, and by post-translational modifications. In the context of a finely tuned regulatory system, we will highlight how these effector proteins co-opt host signaling pathways that control the ability of the organism to infect and survive within the host, as well as elicit host pro-inflammatory responses.


Assuntos
Proteínas de Bactérias/imunologia , Interações Hospedeiro-Patógeno/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas , Humanos , Imunidade Inata/imunologia , Processamento de Proteína Pós-Traducional , Infecções por Salmonella/fisiopatologia , Salmonella typhimurium/genética , Transdução de Sinais/fisiologia , Vacúolos/metabolismo , Vacúolos/microbiologia
7.
Gut Microbes ; 7(2): 136-45, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27078059

RESUMO

Salmonella enterica Typhimurium employs type III secreted effectors to induce cellular invasion and pathogenesis. We previously reported the secreted effector SipA is in part responsible for inducing the apical accumulation of the host membrane protein PERP, a host factor we have shown is key to the inflammatory response induced by Salmonella. We now report that the S. Typhimurium type III secreted effector SipC significantly contributes to PERP redistribution to the apical membrane surface. To our knowledge, this is the first report demonstrating a role for SipC in directing the trafficking of a host membrane protein to the cell surface. In sum, facilitation of PERP trafficking appears to be a result of type III secreted effector-mediated recruitment of vesicles to the apical surface. Our study therefore reveals a new role for SipC, and builds upon previous reports suggesting recruitment of vesicles to the cell surface is important for Salmonella invasion.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhimurium/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Genes Supressores de Tumor , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Membrana/genética , Transporte Proteico , Infecções por Salmonella/genética , Salmonella typhimurium/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo
8.
Front Microbiol ; 2: 88, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21747800

RESUMO

Salmonella enterica serotypes are invasive enteric pathogens spread through fecal contamination of food and water sources, and represent a constant public health threat around the world. The symptoms associated with salmonellosis and typhoid disease are largely due to the host response to invading Salmonella, and to the mechanisms these bacteria employ to survive in the presence of, and invade through the intestinal mucosal epithelia. Surmounting this barrier is required for survival within the host, as well as for further dissemination throughout the body, and subsequent systemic disease. In this review, we highlight some of the major hurdles Salmonella must overcome upon encountering the intestinal mucosal epithelial barrier, and examine how these bacteria surmount and exploit host defense mechanisms.

9.
PLoS One ; 6(7): e21192, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21760888

RESUMO

Erythropoiesis maintains a stable hematocrit and tissue oxygenation in the basal state, while mounting a stress response that accelerates red cell production in anemia, blood loss or high altitude. Thus, tissue hypoxia increases secretion of the hormone erythropoietin (Epo), stimulating an increase in erythroid progenitors and erythropoietic rate. Several cell divisions must elapse, however, before Epo-responsive progenitors mature into red cells. This inherent delay is expected to reduce the stability of erythropoiesis and to slow its response to stress. Here we identify a mechanism that helps to offset these effects. We recently showed that splenic early erythroblasts, 'EryA', negatively regulate their own survival by co-expressing the death receptor Fas, and its ligand, FasL. Here we studied mice mutant for either Fas or FasL, bred onto an immune-deficient background, in order to avoid an autoimmune syndrome associated with Fas deficiency. Mutant mice had a higher hematocrit, lower serum Epo, and an increased number of splenic erythroid progenitors, suggesting that Fas negatively regulates erythropoiesis at the level of the whole animal. In addition, Fas-mediated autoregulation stabilizes the size of the splenic early erythroblast pool, since mutant mice had a significantly more variable EryA pool than matched control mice. Unexpectedly, in spite of the loss of a negative regulator, the expansion of EryA and ProE progenitors in response to high Epo in vivo, as well as the increase in erythropoietic rate in mice injected with Epo or placed in a hypoxic environment, lagged significantly in the mutant mice. This suggests that Fas-mediated autoregulation accelerates the erythropoietic response to stress. Therefore, Fas-mediated negative autoregulation within splenic erythropoietic tissue optimizes key dynamic features in the operation of the erythropoietic network as a whole, helping to maintain erythroid homeostasis in the basal state, while accelerating the stress response.


Assuntos
Envelhecimento/metabolismo , Eritropoese , Homeostase , Estresse Fisiológico , Receptor fas/metabolismo , Envelhecimento/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Atmosfera , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Eritroblastos/citologia , Eritroblastos/efeitos dos fármacos , Eritroblastos/metabolismo , Eritropoese/efeitos dos fármacos , Eritropoetina/farmacologia , Proteína Ligante Fas/deficiência , Proteínas de Homeodomínio/metabolismo , Homeostase/efeitos dos fármacos , Camundongos , Modelos Biológicos , Oxigênio/farmacologia , Receptores Fc/metabolismo , Transdução de Sinais/efeitos dos fármacos , Baço/citologia , Estresse Fisiológico/efeitos dos fármacos , Receptor fas/deficiência
10.
Mol Biol Evol ; 24(8): 1731-43, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17513882

RESUMO

We investigated the origin and diversification of the high-affinity nitrate transporter NRT2 in fungi and other eukaryotes using Bayesian and maximum parsimony methods. To assess the higher-level relationships and origins of NRT2 in eukaryotes, we analyzed 200 amino acid sequences from the Nitrate/Nitrite Porter (NNP) Family (to which NRT2 belongs), including 55 fungal, 41 viridiplantae (green plants), 11 heterokonts (stramenopiles), and 87 bacterial sequences. To assess evolution of NRT2 within fungi and other eukaryotes, we analyzed 116 amino acid sequences of NRT2 from 58 fungi, 40 viridiplantae (green plants), 1 rhodophyte, and 5 heterokonts, rooted with 12 bacterial sequences. Our results support a single origin of eukaryotic NRT2 from 1 of several clades of mostly proteobacterial NNP transporters. The phylogeny of bacterial NNP transporters does not directly correspond with bacterial taxonomy, apparently due to ancient duplications and/or horizontal gene transfer events. The distribution of NRT2 in the eukaryotes is patchy, but the NRT2 phylogeny nonetheless supports the monophyly of major groups such as viridiplantae, flowering plants, monocots, and eudicots, as well as fungi, ascomycetes, basidiomycetes, and agaric mushrooms. At least 1 secondary origin of eukaryotic NRT2 via horizontal transfer to the fungi is suggested, possibly from a heterokont donor. Our analyses also suggest that there has been a horizontal transfer of nrt2 from a basidiomycete fungus to an ascomycete fungus and reveal a duplication of nrt2 in the ectomycorrhizal mushroom genus, Hebeloma.


Assuntos
Proteínas de Transporte de Ânions/genética , Células Eucarióticas/fisiologia , Fungos/genética , Proteínas de Transporte de Ânions/metabolismo , Eucariotos/classificação , Eucariotos/genética , Evolução Molecular , Fungos/metabolismo , Regulação da Expressão Gênica de Plantas , Transportadores de Nitrato , Nitratos/metabolismo , Filogenia , Alinhamento de Sequência
11.
J Org Chem ; 71(3): 1080-4, 2006 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-16438524

RESUMO

Heteroaromatic thiols may be oxidized to the sulfonyl chloride at low temperature (-25 degrees C) by using 3.3 equiv of aqueous sodium hypochlorite. The reaction is rapid, avoids the use of chlorine gas, and succeeds with substrates that have previously been found to afford little or none of the sulfonamide product with other procedures. The method allows the preparation of the sulfonyl fluorides, which are stable enough to be purified and stored, making them potentially useful monomers in parallel chemistry efforts.


Assuntos
Compostos de Sulfidrila/química , Ácidos Sulfínicos/química , Sulfonamidas/química , Estrutura Molecular , Oxirredução
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