Temperature sensitive Mutant Proteome Profiling: a novel tool for the characterization of the global impacts of missense mutants on the proteome
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ABSTRACT:
PROVIDER: PRJNA599371 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10842942_1.fastq.gz | Fastqsanger.gz | |||
| SRR10842942_2.fastq.gz | Fastqsanger.gz | |||
| SRR10842943_1.fastq.gz | Fastqsanger.gz | |||
| SRR10842943_2.fastq.gz | Fastqsanger.gz | |||
| SRR10842944_1.fastq.gz | Fastqsanger.gz |
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Project description:RNA-seq experiments measuring global RNA abundance Temperature sensitive (TS) mutants are a tool that have been foundational for the study of many essential life processes. Despite the long-term use of TS mutants, the mechanisms that lead to temperature sensitivity are not fully understood. Furthermore, a high-throughput workflow to characterize biophysical changes occurring in TS mutants is lacking. We developed Temperature sensitive Mutant Proteome Profiling (TeMPP), a novel application of mass spectrometry based thermal proteome profiling (TPP) as a way to measure the effects of missense mutations on protein stability and protein-protein interactions. This study characterized the global changes in mRNA abundance, protein abundance, and protein thermal stability as a result of missense mutants within two subunits of the yeast ubiquitin-proteasome system. Global protein abundance measurements and RNA sequencing data resulted in a large number of possible candidates that could be causing the phenotypic changes observed in the mutant strains. The additional information gained from TeMPP along with complementary proteomic and transcriptomic experiments allows for multiomic intersection analysis that may reveal interesting regulatory categories to pursue in follow-up mechanistic experiments.
2020-01-08 | GSE143236 | GEO
Project description:Temperature sensitive (TS) missense mutants have been foundational in the characterization essential gene function. However, an unbiased approach for the analysis of the biochemical and biophysical changes in TS missense mutants within the context of their functional proteomes is lacking. We applied mass spectrometry (MS) based thermal proteome profiling (TPP) to investigate the proteome-wide effects of missense mutations in a specific application that we refer to as mutant Thermal Proteome Profiling (mTPP). This study characterized the global changes in mRNA abundance, protein abundance, and protein thermal stability as a result of missense mutants within two subunits of the yeast ubiquitin-proteasome system. Global protein abundance measurements and RNA sequencing data resulted in a large number of possible candidates that could be causing the phenotypic changes observed in the mutant strains. The additional information gained from mTPP along with complementary proteomic and transcriptomic experiments allows for multiomic intersection analysis that may reveal interesting regulatory categories to pursue in follow-up mechanistic experiments.
2020-11-03 | PXD017222 | Pride
Project description:RNase E has an important role in mRNA turnover and stable RNA processing, although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium, an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (TS) for growth. Four different TS mutations were identified, all in the N-terminal domain of RNase E: Gly66→Cys, Ile207→Ser, Ile207→Asn, and Ala327→Pro. We also selected second-site mutations in RNase E that reversed temperature sensitivity. The complete set of RNase E mutations (53 primary mutations including the TS mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E by using the available three-dimensional (3-D) structures. Most single mutations were predicted to destabilize the structure, while second-site mutations that reversed the TS phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (TS, and TS plus second-site mutation) were tested for their effects on the degradation, accumulation, and processing of mRNA, rRNA, and tRNA. The greatest defect was observed on rne mRNA autoregulation, and this correlated with the ability to rescue the tufA499-associated slow-growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.
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Project description:Streptococcus agalactiae is one of the most important pathogens associated with outbreaks of streptococcis in Nile tilapia farms around the world. High water temperature (above 27°C) have been described as factor predisposing for disease in fish. On the other hand, at low temperature (below 25°C) fish mortalities are no usually observed in farms. The temperature variation can modulate the expression of genes and proteins involved with metabolism, adaptation and bacterial pathogenicity, increasing or decreasing the host susceptibility to infection. The aim of this study was to evaluate the transcriptome and proteome of fish-pathogenic S. agalactiae strain (SA53) submitted to in vitro growth under different temperatures using microarray and label-free shotgun LC-HDMSE approach, and to compare the expression trends of proteins shared among GBS strains from different hosts (SA53 and NEM316). Biological triplicates of isolates were cultured in BHIT broth at 22°C or 32°C for RNA and protein isolation and submitted to transcriptomic and proteomic analysis. Total of 1730 transcripts were identified in SA53, being 107 genes differentially expressed among the temperature evaluated. A higher number of genes related with metabolism were detected as up-regulated proteins at 32°C, mainly PTS system and ABC transport system. In proteome analysis, 1046 proteins were identified in SA53 strain, being 81 proteins differentially regulated at 22 and 32ºC. Proteins involved in Defense mechanisms (V), Lipid transport and metabolism (I), and Nucleotide transport and metabolism (F) were up-regulated at 32ºC. A higher number of interactions was observed in the category F. The induction of genes/proteins involved in virulence were detected in both temperatures evaluated. A low correlation between transcriptome and proteome datasets was observed. And there is a distinct adaptation between fish and human GBS strains at the proteome level. Our study showed that the transcriptome and proteome of fish-adapted GBS strain are modulated by temperature, especially regulating the differential expression of genes/proteins involved with metabolism, adaptation and virulence, and revealing a host specificity at proteome regulation for human and fish hosts
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