RelA
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The alarmone nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p)ppGpp-mediated signaling a promising target for development of antibacterials. Although ppGpp itself is an activator of the ribosome-associated ppGpp synthetase RelA, several ppGpp mimics have been developed as RelA inhibitors. However promising, the currently available ppGpp mimics are relatively inefficient, with IC50 in the sub-mM range. In an attempt to identify a potent and specific inhibitor of RelA capable of abrogating (p)ppGpp production in live bacterial cells, we have tested a targeted nucleotide library using a biochemical test system comprised of purified Escherichia coli components. While none of the compounds fulfilled this aim, the screen has yielded several potentially useful molecular tools for biochemical and structural work.
RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.
- MeSH
- bakteriální toxiny genetika metabolismus farmakologie MeSH
- fosforylace účinky léků MeSH
- grampozitivní nesporulující tyčinky chemie metabolismus MeSH
- guanosinpentafosfát chemie metabolismus MeSH
- inhibitory syntézy proteinů farmakologie MeSH
- ligasy chemie genetika metabolismus MeSH
- proteosyntéza účinky léků fyziologie MeSH
- pyrofosfatasy MeSH
- ribozomy metabolismus MeSH
- RNA transferová metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p)ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p)ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p)ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p)ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to β-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p)ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.
- MeSH
- antibakteriální látky farmakologie MeSH
- beta-laktamy farmakologie MeSH
- chloramfenikol farmakologie MeSH
- dihydrofolátreduktasa genetika metabolismus MeSH
- Escherichia coli chemie genetika metabolismus MeSH
- guanosintetrafosfát analogy a deriváty metabolismus MeSH
- isoleucin-tRNA-ligasa genetika MeSH
- lékové interakce MeSH
- ligasy antagonisté a inhibitory genetika metabolismus MeSH
- mupirocin farmakologie MeSH
- proteosyntéza účinky léků MeSH
- ribozomy účinky léků metabolismus MeSH
- RNA transferová genetika metabolismus MeSH
- subcelulární frakce chemie účinky léků metabolismus MeSH
- tetracyklin farmakologie MeSH
- thiostrepton farmakologie MeSH
- tolerance léku * MeSH
- trimethoprim farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
NF-κB pathway is involved in inflammation; however, recent data shows its role also in cancer development and progression, including metastasis. To understand the role of NF-κB interactome dynamics in cancer, we study the complexity of breast cancer interactome in luminal A breast cancer model and its rearrangement associated with NF-κB modulation. Liquid chromatography-mass spectrometry measurement of 160 size-exclusion chromatography fractions identifies 5460 protein groups. Seven thousand five hundred sixty eight interactions among these proteins have been reconstructed by PrInCE algorithm, of which 2564 have been validated in independent datasets. NF-κB modulation leads to rearrangement of protein complexes involved in NF-κB signaling and immune response, cell cycle regulation, and DNA replication. Central NF-κB transcription regulator RELA co-elutes with interactors of NF-κB activator PRMT5, and these complexes are confirmed by AlphaPulldown prediction. A complementary immunoprecipitation experiment recapitulates RELA interactions with other NF-κB factors, associating NF-κB inhibition with lower binding of NF-κB activators to RELA. This study describes a network of pro-tumorigenic protein interactions and their rearrangement upon NF-κB inhibition with potential therapeutic implications in tumors with high NF-κB activity.
- MeSH
- karcinogeneze metabolismus MeSH
- lidé MeSH
- mapování interakce mezi proteiny MeSH
- mapy interakcí proteinů * MeSH
- nádorové buněčné linie MeSH
- nádory prsu * metabolismus patologie MeSH
- NF-kappa B * metabolismus MeSH
- proteinarginin-N-methyltransferasy metabolismus MeSH
- signální transdukce MeSH
- transkripční faktor RelA * metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
2. vydání 78 stran : ilustrace ; 25 cm
Objectives Nuclear factor-kappaB (NF-kappaB), especially p65 subunit, seems to be associated with origin and progression of cancer. The aim of the study was to determine expression of NF-kappaB/p65 in rectal cancer patients before and after radiotherapy as well as to assess the relationship between NF-kappaB/p65 expression, other tumor characteristics, and disease progression. Further aim was to evaluate whether expression of NF-kappaB/p65 in tumor tissue may serve as a predictive marker of patient outcome. Patients and methods Twenty-five patients with rectal cancer undergoing pre-operative radiotherapy were included in the study. Unirradiated rectal cancer specimens were obtained from diagnostic colonoscopy. Irradiated rectal cancer specimens were obtained from surgically removed part of the rectum with the tumor. NF-kappaB/p65 expression was determined by immunohistochemistry. Results Cytoplasmic positivity in cancer cells and nuclear positivity in lymphocytes were detected. In post-radiotherapy specimens single tumor cells or small clones of them deeply infiltrating the wall of the rectum, that were characterized by high NF-kappaB/p65 expression, were found. Patients with presence of these cells in post-radiotherapy specimens have worse clinical outcome in terms of overall survival and disease-free interval. Conclusion While the NF-kappaB/p65 positive staining of the epithelial cells did not have any clinical implications in this study, it may be of clinical significance in the future. Residual invasively growing cancer cells with high NF-kappaB/p65 positivity found in specimens after radiotherapy and surgery may be used to find what patients have a worse outcome. Thus, patients being at risk of cancer progression and requiring more aggressive anti-cancer therapy may be identified.
- MeSH
- dospělí MeSH
- financování organizované MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádorové biomarkery analýza MeSH
- nádory rekta chemie patologie radioterapie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- transkripční faktor RelA analýza MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
Hepatocellular carcinomas (HCC) contain a subpopulation of cancer stem cells (CSCs), which exhibit stem cell-like features and are responsible for tumor relapse, metastasis, and chemoresistance. The development of effective treatments for HCC will depend on a molecular-level understanding of the specific pathways driving CSC emergence and stemness. MacroH2A1 is a variant of the histone H2A and an epigenetic regulator of stem-cell function, where it promotes differentiation and, conversely, acts as a barrier to somatic-cell reprogramming. Here, we focused on the role played by the histone variant macroH2A1 as a potential epigenetic factor promoting CSC differentiation. In human HCC sections we uncovered a significant correlation between low frequencies of macroH2A1 staining and advanced, aggressive HCC subtypes with poorly differentiated tumor phenotypes. Using HCC cell lines, we found that short hairpin RNA-mediated macroH2A1 knockdown induces acquisition of CSC-like features, including the growth of significantly larger and less differentiated tumors when injected into nude mice. MacroH2A1-depleted HCC cells also exhibited reduced proliferation, resistance to chemotherapeutic agents, and stem-like metabolic changes consistent with enhanced hypoxic responses and increased glycolysis. The loss of macroH2A1 increased expression of a panel of stemness-associated genes and drove hyperactivation of the nuclear factor kappa B p65 pathway. Blocking phosphorylation of nuclear factor kappa B p65 on Ser536 inhibited the emergence of CSC-like features in HCC cells knocked down for macroH2A1. Conclusion: The absence of histone variant macroH2A1 confers a CSC-like phenotype to HCC cells in vitro and in vivo that depends on Ser536 phosphorylation of nuclear factor kappa B p65; this pathway may hold valuable targets for the development of CSC-focused treatments for HCC. (Hepatology 2018;67:636-650).
- MeSH
- buňky Hep G2 MeSH
- fosforylace MeSH
- hepatocelulární karcinom patologie MeSH
- histony fyziologie MeSH
- lidé MeSH
- nádorové kmenové buňky patologie MeSH
- nádory jater patologie MeSH
- proliferace buněk MeSH
- stanovení celkové genové exprese MeSH
- transkripční faktor RelA metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
