Project description:Population adaptation to strong selection can occur through the sequential or parallel accumulation of competing beneficial mutations. The dynamics, diversity and rate of fixation of beneficial mutations within and between populations are still poorly understood. To study the changes in the mutational landscape across populations during adaptation, we performed experimental evolutions on seven parallel populations of Saccharomyces cerevisiae continuously cultured in limiting sulfate medium. By combining qPCR, array CGH, restriction, digestion and CHEF gels, and whole genome sequencing, we followed the trajectory of evolution to determine the identity and fate of beneficial mutations. Over a period of 200 generations, the yeast populations displayed parallel evolutionary dynamics that are driven by the coexistence of independent beneficial mutations. Segmental amplifications are rapidly gained under this selective pressure, including, common inverted amplifications containing the sulfate transporter gene SUL1. Detailed analysis of the populations uncovers a deep complexity where by multiple subpopulations arise and compete with each another. The most common trajectories to adaptation in these populations are incomplete soft sweeps, with adaptive variants replacing one another. These are CGH arrays. Each experiment compares the DNA content of an experimentally evolved strain with its ancestor.
Project description:The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides was lacking. To this end, Akhilesh Pandey's lab reported a draft map of the human proteome based on high resolution Fourier transform mass spectrometry-based proteomics technology, which included an in-depth proteomic profiling of 30 histologically normal human samples including 17 adult tissues, 7 fetal tissues and 6 purified primary hematopoietic cells ( http://dx.doi.org/10.1038/nature13302 ). The profiling resulted in identification of proteins encoded by greater than 17,000 genes accounting for ~84% of the total annotated protein-coding genes in humans. This large human proteome catalog (available as an interactive web-based resource at http://www.humanproteomemap.org) complements available human genome and transcriptome data to accelerate biomedical research in health and disease. Pandey's lab and collaborators request that those considering use of this primary dataset for commercial purposes contact pandey@jhmi.edu. The full details of this study can be found in the PRIDE database: www.ebi.ac.uk/pride/archive/projects/PXD000561/. This ArrayExpress entry represents a top level summary of the metadata only which formed the basis of the reanalysis performed by Joyti Choudhary's team ( jc4@sanger.ac.uk ), results of which are presented in the Expression Atlas at EMBL-EBI : http://www.ebi.ac.uk/gxa/experiments/E-PROT-1.
Project description:Human phosphoglycerate kinase 1 is a key glycolytic enzyme that regulates the balance between ADP and ATP concentrations inside the cell. Phosphorylation of hPGK1 at S203 and S256 has been associated with enzyme import from the cytosol to the mitochondria and the nucleus, respectively. These changes in subcellular location drive tumorigenesis and are likely associated with site-specific changes in protein stability. In this work, we investigate the effects of site-specific phosphorylation on thermal and kinetic stability and protein structural dynamics by hydrogen-deuterium exchange (HDX). We also investigate the binding of 3-phosphoglycerate and Mg-ADP using these approaches. We show that the phosphomimetic mutation S256D reduces hPGK1 kinetic stability by 50-fold, with no effect of the mutation S203D. Calorimetric studies of ligand binding show a large decrease in affinity for Mg-ADP in the S256D variant whereas Mg-ADP binding to the WT and S203D can be accurately investigated using protein kinetic stability and binding thermodynamic models. HDX studies structurally confirmed the destabilization caused by the mutation S256D (with some long-range effects on stability) and its reduced affinity for Mg-ADP due to the strong destabilization of its binding site (particularly in the apo-state). Our work thus support that alterations in protein stability might facilitate import of hPGK1 to the nucleus in cancer, whereas the structural and energetic basis of its mitochondrial import remain unknown. The general implications of our work for protein import to certain subcellular locations are discussed in a wider context.
Project description:Primary hyperoxaluria type I (PH1) is a genetic disease caused by a deficiency in the peroxisomal alanine:glyoxylate aminotransferase (AGT) activity. Mutations in AGT mostly cause protein mistargeting and enhanced aggregation, although the molecular and structural basis of these mechanisms are unknown. In this work, we use hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) to provide novel insight into these pathogenic mechanisms. We characterize the wild-type (WT) protein, the LM variant (containing the mutations P11L and I340M, a haplotype more frequent in PH1 patients) and the LM G170R (the most common genotype in PH1, introducing the G170R mutation on the LM background). Our study provides the first experimental analysis of the local stability and dynamics of AGT, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants destabilize locally the structure. Enzymatic transamination of the PLP bound to AGT hardly affects stability. Our study thus supports that AGT misfolding is not caused by dramatic effects on the stability and dynamics of the holo-protein.
Project description:We Investigate of the mice hearts from embryonic day 10.5 to postnatal week 8 and reveal developmental changes in phosphoproteome, proteome encompassing cardiogenesis and cardiac maturation.
Project description:Five replicates of GV, GVBD, and MII oocytes were subjected to the 15-plex TMT labeling, HP-RP fractionation, and LC-MS/MS analysis. For each replicate, 2,000 oocytes were collected from each of the GV, GVBD, and MII stages. Ti4+-IMAC was used to enrich phosphopeptides.
Project description:We employed CapitalBio Corporation to investigate the global transcriptional profiling of Saccharomyces cerevisiae treated with thymol. Keywords: gene expression array-based, count S. cerevisiae strain L1190 was incubated in a rotary shaker. Subsequently, thymol was added to the culture and DMSO to the control. Then, the yeast cells were further incubated for a certain time and total RNA was extracted by a hot acidic phenol method. The experimental sample and control sample were incorporated and dissolved in hybridization solution after labelled. Arrays hybridization was preformed in a CapitalBio BioMixerTM II Hybridization Station overnight and washed with two consecutive solutions. Arrays were scanned with a confocal LuxScanTM scanner and the images obtained were then analyzed using LuxScanTM 3.0 software. A space- and intensity-dependent normalization based on a LOWESS program was employed.
Project description:We employed CapitalBio Corporation to investigate the global transcriptional profiling of Saccharomyces cerevisiae treated with dictamnine. S. cerevisiae strain L1190 was incubated in a rotary shaker. Subsequently, dictamnine was added to the culture and DMSO to the control. Then, the yeast cells were further incubated for a certain time and total RNA was extracted by a hot acidic phenol method. The experimental sample and control sample were incorporated and dissolved in hybridization solution after labelled. Arrays hybridization was preformed in a CapitalBio BioMixerTM II Hybridization Station overnight and washed with two consecutive solutions. Arrays were scanned with a confocal LuxScanTM scanner and the images obtained were then analyzed using LuxScanTM 3.0 software. A space- and intensity-dependent normalization based on a LOWESS program was employed.
Project description:Response of Saccharomyces cerevisiae to Ammonium, L-alanine, or L-glutamine Limitation. The protrotophic laboratory strain CEN.PK113-7D (MAT a) was grown in laboratory fermentors with a working volume of 1 litre at dilution rate (D) of 0.20 per hour (in triplicate for each nitrogen limited condition). At steady state, samples from each of the 12 continuous cultures were taken and cooled below 2 degree C within ten seconds by mixing 40% sample and 60% crushed ice.