RESUMO
The growing prevalence of antibiotic resistance in numerous pathogenic bacteria is a major public health concern and urgently requires the development of new therapeutic approaches. Multidrug resistant species that remain sensitive to chloramphenicol (CAM) treatment have engendered renewed interest in using this drug as a modern day antimicrobial agent. High-level resistance to CAM commonly is mediated by chloramphenicol acetyltransferase (CAT) which catalyzes the acetylation of CAM and renders the drug inactive. Of the three main types (CATI, CATII and CATIII), CATI is of broad clinical significance. Despite this importance, understanding of the catalytic mechanism of CATI largely is extrapolated from studies of CATIII. Here, pentapeptide scanning mutagenesis was used to generate a library of random insertions in CATI to gain a better understanding of structure-function relationships in the enzyme. Pentapeptide insertions in secondary structure elements which contain residues that form part of the CATI active site abolished CAM resistance in Escherichia coli. Insertions in secondary structures that have key roles in protein folding and CAM binding led to a reduction in resistance. In contrast, insertions in loop regions between the major secondary structure features exerted modest, if any, effects on CAM resistance. The analysis pinpoints regions of CATI that may serve as targets for the design of novel inhibitors that prevent the spread of CAM-resistant pathogens thereby enabling the drug to be re-deployed as a broad range antimicrobial agent. Moreover, regions of CATI that are tolerant of insertions may be suitable for the construction of bifunctional enzymes in which peptides, mini-proteins or amino acid tags are introduced at the permissive sites.
Assuntos
Cloranfenicol , Escherichia coli , Sequência de Bases , Cloranfenicol/farmacologia , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Peptídeos/genéticaRESUMO
Mosaic variants in genes GNAQ or GNA11 lead to a spectrum of vascular and pigmentary diseases including Sturge-Weber syndrome, in which progressive postnatal neurological deterioration led us to seek biologically targeted therapeutics. Using two cellular models, we find that disease-causing GNAQ/11 variants hyperactivate constitutive and G-protein coupled receptor ligand-induced intracellular calcium signaling in endothelial cells. We go on to show that the aberrant ligand-activated intracellular calcium signal is fueled by extracellular calcium influx through calcium-release-activated channels. Treatment with targeted small interfering RNAs designed to silence the variant allele preferentially corrects both the constitutive and ligand-activated calcium signaling, whereas treatment with a calcium-release-activated channel inhibitor rescues the ligand-activated signal. This work identifies hyperactivated calcium signaling as the primary biological abnormality in GNAQ/11 mosaicism and paves the way for clinical trials with genetic or small molecule therapies.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Subunidades alfa de Proteínas de Ligação ao GTP , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Mutação , Cálcio , Células Endoteliais/metabolismo , Mosaicismo , Sinalização do Cálcio/genética , LigantesRESUMO
Among children with multiple congenital melanocytic nevi, 25% have no established genetic cause, of whom many develop a hyperproliferative and severely pruritic phenotype resistant to treatment. Gene fusions have been reported in individual cases of congenital melanocytic nevi. We studied 169 patients with congenital melanocytic nevi in this study, 38 of whom were double wild type for pathogenic NRAS/BRAF variants. Nineteen of these 38 patients had sufficient tissue to undergo RNA sequencing, which revealed mosaic BRAF fusions in 11 of 19 patients and mosaic RAF1 fusions in 1 of 19. Recurrently, fusions involved the loss of the 5´ regulatory domain of BRAF or RAF1 but preserved the kinase domain. We validated all cases and detected the fusions in two separate nevi in 5 of 12 patients, confirming clonality. The absence of the fusion in blood in 8 of 12 patients indicated mosaicism. Primary culture of BRAF-fusion nevus cells from 3 of 12 patients demonstrated highly increased MAPK activation, despite only mildly increased BRAF expression, suggesting additional mechanisms of kinase activation. Trametinib quenched MAPK hyperactivation in vitro, and treatment of two patients caused rapid improvement in bulk tissue, improving bodily movement and reducing inflammation and severe pruritus. These findings offer a genetic diagnosis to an additional group of patients and trametinib as a treatment option for the severe associated phenotypes.
Assuntos
Nevo de Células Epitelioides e Fusiformes , Nevo Pigmentado , Neoplasias Cutâneas , Criança , Humanos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Mutação , Nevo Pigmentado/tratamento farmacológico , Nevo Pigmentado/genética , Nevo Pigmentado/congênitoRESUMO
RAS proteins regulate cell division, differentiation, and apoptosis through multiple downstream effector pathways. Oncogenic RAS variants are the commonest drivers in cancers; however, they also drive many benign lesions predisposing to malignancy, such as melanocytic nevi, thyroid nodules, and colonic polyps. Reversal of these benign lesions could reduce cancer incidence; however, the effects of oncogenic RAS have been notoriously difficult to target with downstream pathway inhibitors. In this study, we show effective suppression of oncogenic and currently undruggable NRASQ61K in primary cells from melanocytic nevi using small interfering RNA targeted to the recurrent causal variant. This results in striking reduction in expression of ARL6IP1, a known inhibitor of endoplasmic reticulum stress-induced apoptosis not previously linked to NRAS. We go on to show that a single dose of small interfering RNA in primary cells triggers an apoptotic cascade, in contrast to treatment with a MAPK/extracellular signal-regulated kinase kinase inhibitor. Protective packaging of the targeted small interfering RNA into lipid nanoparticles permits successful delivery into a humanized mouse model of melanocytic nevi and results in variant NRAS knockdown in vivo. These data show that RAS-induced protection from apoptosis is involved in persistence of NRAS-driven melanocytic nevi and anticipate that targeted small interfering RNA could form the basis of clinical trials for RAS-driven benign tumors.
RESUMO
Triple-negative breast cancer (TNBC) has poorer prognosis compared to other types of breast cancers due to the lack of effective therapies and markers for patient stratification. Loss of PTEN tumor suppressor gene expression is a frequent event in TNBC, resulting in over-activation of the PI 3-kinase (PI3K) pathway and sensitivity to its inhibition. However, PI3K pathway inhibitors show limited efficacy as monotherapies on these tumors. We report a whole-genome screen to identify targets whose inhibition enhanced the effects of different PI3K pathway inhibitors on PTEN-null TNBC. This identified a signaling network that relies on both the G protein-coupled receptor for thrombin (PAR1/F2R) and downstream G protein ßγ subunits and also epidermal growth factor receptor (EGFR) for the activation of the PI3K isoform p110ß and AKT. Compensation mechanisms involving these two branches of the pathway could bypass PI3K blockade, but combination targeting of both EGFR and PI3Kß suppressed ribosomal protein S6 phosphorylation and exerted anti-tumor activity both in vitro and in vivo, suggesting a new potential therapeutic strategy for PTEN-null TNBC.