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1.
Nat Commun ; 12(1): 56, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397922

RESUMO

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2-/- Vav3-/- Tiam1-/-), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Intestinos/patologia , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Carcinogênese/genética , Homeostase , Intestinos/ultraestrutura , Camundongos Knockout , Mutação/genética , Especificidade de Órgãos , Fenótipo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismo , Regulação para Cima , Via de Sinalização Wnt
2.
Proc Natl Acad Sci U S A ; 110(17): 7032-7, 2013 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-23572593

RESUMO

Rearrangement hotspot (Rhs) and related YD-peptide repeat proteins are widely distributed in bacteria and eukaryotes, but their functions are poorly understood. Here, we show that Gram-negative Rhs proteins and the distantly related wall-associated protein A (WapA) from Gram-positive bacteria mediate intercellular competition. Rhs and WapA carry polymorphic C-terminal toxin domains (Rhs-CT/WapA-CT), which are deployed to inhibit the growth of neighboring cells. These systems also encode sequence-diverse immunity proteins (RhsI/WapI) that specifically neutralize cognate toxins to protect rhs(+)/wapA(+) cells from autoinhibition. RhsA and RhsB from Dickeya dadantii 3937 carry nuclease domains that degrade target cell DNA. D. dadantii 3937 rhs genes do not encode secretion signal sequences but are linked to hemolysin-coregulated protein and valine-glycine repeat protein G genes from type VI secretion systems. Valine-glycine repeat protein G is required for inhibitor cell function, suggesting that Rhs may be exported from D. dadantii 3937 through a type VI secretion mechanism. In contrast, WapA proteins from Bacillus subtilis strains appear to be exported through the general secretory pathway and deliver a variety of tRNase toxins into neighboring target cells. These findings demonstrate that YD-repeat proteins from phylogenetically diverse bacteria share a common function in contact-dependent growth inhibition.


Assuntos
Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Toxinas Bacterianas/genética , Bactérias Gram-Positivas/genética , Interações Microbianas/genética , Northern Blotting , Bactérias Gram-Positivas/fisiologia , Indóis , Microscopia de Fluorescência , Oligonucleotídeos/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , Especificidade da Espécie
3.
PLoS One ; 8(2): e57609, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23469034

RESUMO

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CDI systems deploy a variety of distinct toxins, which are contained within the polymorphic C-terminal region (CdiA-CT) of CdiA proteins. Several CdiA-CTs are nucleases, suggesting that the toxins are transported into the target cell cytoplasm to interact with their substrates. To analyze CdiA transfer to target bacteria, we used the CDI system of uropathogenic Escherichia coli 536 (UPEC536) as a model. Antibodies recognizing the amino- and carboxyl-termini of CdiA(UPEC536) were used to visualize transfer of CdiA from CDI(UPEC536+) inhibitor cells to target cells using fluorescence microscopy. The results indicate that the entire CdiA(UPEC536) protein is deposited onto the surface of target bacteria. CdiA(UPEC536) transfer to bamA101 mutants is reduced, consistent with low expression of the CDI receptor BamA on these cells. Notably, our results indicate that the C-terminal CdiA-CT toxin region of CdiA(UPEC536) is translocated into target cells, but the N-terminal region remains at the cell surface based on protease sensitivity. These results suggest that the CdiA-CT toxin domain is cleaved from CdiA(UPEC536) prior to translocation. Delivery of a heterologous Dickeya dadantii CdiA-CT toxin, which has DNase activity, was also visualized. Following incubation with CDI(+) inhibitor cells targets became anucleate, showing that the D.dadantii CdiA-CT was delivered intracellularly. Together, these results demonstrate that diverse CDI toxins are efficiently translocated across target cell envelopes.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Membrana/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Técnicas de Cocultura , Citoplasma/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Imagem Molecular , Transporte Proteico
4.
Proc Natl Acad Sci U S A ; 109(52): 21480-5, 2012 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-23236156

RESUMO

Contact-dependent growth inhibition (CDI) systems encode polymorphic toxin/immunity proteins that mediate competition between neighboring bacterial cells. We present crystal structures of CDI toxin/immunity complexes from Escherichia coli EC869 and Burkholderia pseudomallei 1026b. Despite sharing little sequence identity, the toxin domains are structurally similar and have homology to endonucleases. The EC869 toxin is a Zn(2+)-dependent DNase capable of completely degrading the genomes of target cells, whereas the Bp1026b toxin cleaves the aminoacyl acceptor stems of tRNA molecules. Each immunity protein binds and inactivates its cognate toxin in a unique manner. The EC869 toxin/immunity complex is stabilized through an unusual ß-augmentation interaction. In contrast, the Bp1026b immunity protein exploits shape and charge complementarity to occlude the toxin active site. These structures represent the initial glimpse into the CDI toxin/immunity network, illustrating how sequence-diverse toxins adopt convergent folds yet retain distinct binding interactions with cognate immunity proteins. Moreover, we present visual demonstration of CDI toxin delivery into a target cell.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Inibição de Contato/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/imunologia , Burkholderia pseudomallei/metabolismo , Cristalografia por Raios X , DNA/metabolismo , Endonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismo , Modelos Moleculares , Família Multigênica/genética , Estrutura Secundária de Proteína
5.
Proc Natl Acad Sci U S A ; 101(25): 9223-8, 2004 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-15197281

RESUMO

Association of two proteins can be described as a two-step process, with the formation of an encounter complex followed by desolvation and establishment of a tight complex. Here, by using the computer algorithm PARE, we designed a set of mutants of the Ras effector protein Ral guanine nucleotide dissociation stimulator (RalGDS) with optimized electrostatic steering. The fastest binding RalGDS mutant, M26K,D47K,E54K, binds Ras 14-fold faster and 25-fold tighter compared with WT. A linear correlation was found between the calculated and experimental data, with a correlation coefficient of 0.97 and a slope of 0.65 for the 24 mutants produced. The data suggest that increased electrostatic steering specifically stabilizes the encounter complex and transition state. This conclusion is backed up by Phi analysis of the encounter complex and transition state of the RalGDS(M26K,D47K,E54K)/Ras complex, with both values being close to 1. Upon further formation of the final complex, the increased Coulombic interactions are probably counterbalanced by the cost of desolvation of charges, keeping the dissociation rate constant almost unchanged. This mechanism is also reflected by the mutual compensation of enthalpy and entropy changes quantified by isothermal titration calorimetry. The binding constants of the faster binding RalGDS mutants toward Ras are similar to those of Raf, the most prominent Ras effector, suggesting that the design methodology may be used to switch between signal transduction pathways.


Assuntos
Proteínas ral de Ligação ao GTP/química , Proteínas ras/metabolismo , Sítios de Ligação , Clonagem Molecular , Escherichia coli , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade Estática , Termodinâmica , Proteínas ral de Ligação ao GTP/metabolismo
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