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Systematic detection of amino acid substitutions in proteome reveals a mechanistic basis of ribosome errors and selection for translation fidelity

ABSTRACT: The protein translation machinery and the genes it decodes co-evolved to achieve production throughput and accuracy. Nonetheless translation errors are frequent and they affect physiology, fitness and protein evolution. Mapping translation errors in proteomes and understanding their causes was hindered by lack of a proteome-wide experimental methodology. Here, we present the first methodology for systematic detection and quantification of errors in entire proteomes. Following proteome mass-spectrometry, we identify in E. coli and S. cerevisiae peptides whose mass indicates specific amino acid substitutions. Most substitutions occur due to codon-anticodon mispairing within the ribosome. we performed a deep RNA sequencing of the bacterial tRNA pool at the same time points in which we measured the proteome during E. coli culture growth. We compared ratio of abundances between congate and near-cognate tRNAs and found the changes corespond with changes in translation errors.

ORGANISM(S): Escherichia coli BW25113 Escherichia coli

PROVIDER: GSE128812 | GEO | 2019/04/18

REPOSITORIES: GEO

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Project description:Mutant KRAS (mut-KRAS) is present in 30% of all human cancers and plays a critical role in cancer cell growth and resistance to therapy. There is evidence from colon cancer that mut-KRAS is a poor prognostic factor and negative predictor of patient response to molecularly targeted therapy. However, evidence for such a relationship in non small cell lung cancer (NSCLC) is conflicting. KRAS mutations are primarily found at codons 12 and 13, where different base changes lead to alternate amino acid substitutions that lock the protein in an active state. The patterns of mut-KRas amino acid substitutions in colon cancer and NSCLC are quite different, with aspartate (D) predominating in colon cancer (50%) and cysteine (C) in NSCLC (47%). Through an analysis of a recently completed biopsy biomarker-driven, molecularly targeted multi-arm trial of 215 evaluable patients with refractory NSCLC we show that mut-KRas-G12C/V but not total mut-KRAS predicts progression free survival for the overall group, and for the sorafenib and vandetanib treatment arms. Transcriptome microarray data shows differential expression of cell cycle genes between mut-KRas-G12C/V and G12D patient tumors. A panel of NSCLC cell lines with known mut-KRas amino acid substitutions was used to identify pathways activated by the different mut-KRas, showing that mut-KRas-G12D activates both PI-3-K and MEK signaling, while mut-KRas G12C does not, and alternatively activates RAL signaling. This finding was confirmed using immortalized human bronchial epithelial cells stably transfected with wt-KRAS and different forms of mut-KRAS. Molecular modeling studies show that the different conformation imposed by mut-KRas-G12C could lead to altered association with downstream signaling transducers compared to wild type and mut-KRas-G12D. The significance of the findings for developing mut-KRAS therapies is profound, since it suggests that not all mut-KRas amino acid substitutions signal to effectors in a similar way, and may require different therapeutic interventions. Gene expression profiles were measured in 22 core biopsies from patients with refractory non-small cell lung cancer included in the Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE). All tumors were KRAS mutants, but with different patterns of amino acid substitutions. Supervised analysis of transcriptome profiling was performed to compare cysteine or valine KRAS mutants with other KRAS mutants.

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Systematic detection of amino acid substitutions in proteome reveals a mechanistic basis of ribosome errors and selection for translation fidelity

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