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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PROVIDER: PRJNA599371 | ENA |
REPOSITORIES: ENA
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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: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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Project description:1 Novel Missense Variants in Patients with Primary Immunodeficiency and Immune dysregulation
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Project description:Missense mutations account for nearly 50% of pathogenic mutations in human genetic diseases, most lack effective treatments. Gene therapies, CRISPR-based gene editing, and RNA therapies including transfer RNA (tRNA) modalities are common strategies for potential treatments of genetic diseases. However, reported tRNA therapies are for nonsense mutations, how tRNAs can be engineered to correct missense mutations have not been explored. Here, we describe missense correcting tRNAs (mc-tRNAs) as a potential therapeutic modality for correcting pathogenic missense mutations. Mc-tRNAs are engineered tRNAs that are charged with one amino acid and read codons of another amino acid in translation in human cells. We first developed a series of fluorescence protein (FP)-based reporters that indicate successful correction of missense mutations via restoration of fluorescence signals. We engineered mc-tRNAs that effectively corrected Serine and Arginine missense mutations in the reporters and confirmed the amino acid substitution by protein mass spectrometry and mc-tRNA expression by tRNA sequencing. We examined the transcriptome response to the expression of mc-tRNAs and found some mc-tRNAs induced minimum transcriptomic changes. Furthermore, we applied an Arg-tRNAGln(CUG) mc-tRNA to rescue the autolytic activity of a pathogenic CAPN3 Arg-to-Gln mutant involved in limb-girdle muscular dystrophy type 2A. These results establish a versatile pipeline for mc-tRNA engineering and demonstrate the potential of mc-tRNA as an alternative therapeutic platform for the treatment of genetic disorders.
2024-01-04 | GSE236623 | GEO
Project description:Missense mutations account for nearly 50% of pathogenic mutations in human genetic diseases, most lack effective treatments. Gene therapies, CRISPR-based gene editing, and RNA therapies including transfer RNA (tRNA) modalities are common strategies for potential treatments of genetic diseases. However, reported tRNA therapies are for nonsense mutations, how tRNAs can be engineered to correct missense mutations have not been explored. Here, we describe missense correcting tRNAs (mc-tRNAs) as a potential therapeutic modality for correcting pathogenic missense mutations. Mc-tRNAs are engineered tRNAs that are charged with one amino acid and read codons of another amino acid in translation in human cells. We first developed a series of fluorescence protein (FP)-based reporters that indicate successful correction of missense mutations via restoration of fluorescence signals. We engineered mc-tRNAs that effectively corrected Serine and Arginine missense mutations in the reporters and confirmed the amino acid substitution by protein mass spectrometry and mc-tRNA expression by tRNA sequencing. We examined the transcriptome response to the expression of mc-tRNAs and found some mc-tRNAs induced minimum transcriptomic changes. Furthermore, we applied an Arg-tRNAGln(CUG) mc-tRNA to rescue the autolytic activity of a pathogenic CAPN3 Arg-to-Gln mutant involved in limb-girdle muscular dystrophy type 2A. These results establish a versatile pipeline for mc-tRNA engineering and demonstrate the potential of mc-tRNA as an alternative therapeutic platform for the treatment of genetic disorders.
2024-01-04 | GSE236622 | GEO
Project description:Determining the pathogenicity of human genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes will likely require generalizable, scalable assays. We previously developed Variant Abundance by Massively Parallel Sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance in a single experiment. Here, we reapplied VAMP-seq to quantify the abundances of additional PTEN missense variants which were missing from the original experiment.
2021-09-13 | GSE159469 | GEO
Project description:To identify conditional alleles which disrupt the splicing pathway, we screened a collection of ~2000 chemically mutagenized temperature-sensitive fission yeast isolates for intron accumulation in three introns, including a naturally occurring U2-dependent AT-AC intron. We identified 54 strains which accumulate unspliced message at the non-permissive temperature. A combination of mutation effect prediction and genetic mapping techniques revealed missense mutations in core splicing genes which function at various steps of spliceosome assembly and activation including: prp10, prp28, sap61, ntr1, spp42, sap114, cwf22, and prp22. Interrogation of structural data and genome-wide analysis of the splicing defects in these mutations revealed intron-specific defects which suggest the biochemical basis for these mutations’ effects on splicing.
2020-11-13 | GSE161333 | GEO
Project description:TP53, encoding for the tumor suppressor p53, is the most frequently mutated gene in human cancer. The selective pressures shaping its mutational spectrum, dominated by missense mutations, have remained enigmatic, and neomorphic gain-of-function (GOF) activities have been implicated. We generated isogenic human leukemia cell lines of the most common TP53 missense mutations using CRISPR/Cas9. Functional, DNA binding, and transcriptional analyses revealed loss-of-function (LOF) without GOF effects of missense mutations. Comprehensive mutational scanning of p53 single amino acid variants demonstrated that DNA-binding domain missense variants exert dominant-negative effects (DNE). In mice, DNE of p53 missense variants confer a selective advantage on hematopoietic cells upon DNA damage in vivo. Clinical outcomes in acute myeloid leukemia patients showed no evidence of GOF for TP53 missense mutations. These findings establish dominant-negativity as the primary unit of selection for TP53 missense mutations in myeloid malignancies.
2019-08-30 | GSE131592 | GEO