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Proteome-wide effects of single gene perturbations

ABSTRACT: Protein expression levels are controlled at the transcriptional, translational and post-translational level and their regulatory principles are starting to emerge. While transcriptional outcomes, which are commonly also used as a proxy for protein abundance, have been investigated on a larger scale, the study of translational output requires large-scale proteomics data. However, data for proteome alterations by systematic assessment of knockouts genome-wide is not available yet. We here determined the individual proteome changes for 3,308 non-essential genes in the yeast S. pombe. We observed that genes with high proteome remodeling are predominantly involved in gene expression regulation, in particular acting as translational regulators. Focusing on those knockout strains with a large number of altered proteins, we performed paired transcriptome/proteome measurements to uncover translational regulators and features of translational regulation. Furthermore, by similarity clustering of these proteome changes, we infer gene functionality that can be extended to other species such as human or baker’s yeast.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Schizosaccharomyces Pombe 927

TISSUE(S): Bile

SUBMITTER: F Butter

LAB HEAD: Falk Butter

PROVIDER: PXD024383 | Pride | 2022-09-21

REPOSITORIES: Pride

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Proteome-wide effects of single gene perturbations

Project description:Protein expression levels are controlled at the transcriptional, translational and post-translational level and their regulatory principles are starting to emerge. While transcriptional outcomes, which are commonly also used as a proxy for protein abundance, have been investigated on a larger scale, the study of translational output requires large-scale proteomics data. However, data for proteome alterations by systematic assessment of knockouts genome-wide is not available yet. We here determined the individual proteome changes for 3,308 non-essential genes in the yeast S. pombe. We observed that genes with high proteome remodeling are predominantly involved in gene expression regulation, in particular acting as translational regulators. Focusing on those knockout strains with a large number of altered proteins, we performed paired transcriptome/proteome measurements to uncover translational regulators and features of translational regulation. Furthermore, by similarity clustering of these proteome changes, we infer gene functionality that can be extended to other species such as human or baker’s yeast.

2022-09-21 | PXD024332 | Pride

Rubisco acetylation stoichiometry in Arabidopsis Thaliana

Project description:Multiple studies have shown Rubisco to be subject to Lys-acetylation at various residues; however, conflicting reports exist about the biological significance of these post-translational modifications. One aspect of the Lys-acetylation that has not been addressed in plants generally, or with Rubisco specifically, is the stoichiometry at which these Lys-acetylation events occur. As a method to ascertain which Lys-acetylation sites on Arabidopsis Rubisco might be of regulatory importance, we purified Rubisco from leaves in both the day and night-time and performed independent mass-spectrometry based methods to determine the stoichiometry of Rubisco Lys-acetylation events.

2020-09-28 | PXD019268 | Pride

Validation of the expression of clathrin heavy chain muscle isoform including exon 31 at the protein level

Project description:A single exon alternative splicing in the CLTC gene determines clathrin coat organization. This event controls plasticity from clathrin-coated pits to plaques, structures that are essential for muscle fiber function and maintenance

2020-06-19 | PXD018333 | Pride

Dynamic profiling of the protein life cycle in response to pathogens (protein)

Project description:Protein expression is regulated by production and degradation of mRNAs and proteins, but their specific relationships remain unknown. We combine measurements of protein production and degradation and mRNA dynamics to build a quantitative genomic model of the differential regulation of gene expression in LPS stimulated mouse dendritic cells. Changes in mRNA abundance play a dominant role in determining most dynamic fold changes in protein levels. Conversely, the preexisting proteome of proteins performing basic cellular functions is remodeled primarily through changes in protein production or degradation, accounting for over half of the absolute change in protein molecules in the cell. Thus, the proteome is regulated by transcriptional induction of novel cellular functions and remodeling of preexisting functions through the protein life cycle. Mouse primary dendritic cells were treated with LPS or mock stimulus and profiled over a 12-hour time course. Cells were grown in M-labeled SILAC media, which was replaced with H-labeled SILAC media at time 0. Aliquots were taken at 0, 0.5, 1, 2, 3, 4, 5, 6, 9, and 12 hours post-stimulation and added to equal volumes of a master mix of unlabeled (L) cells for the purpose of normalization. RNA-Seq was performed at 0, 1, 2, 4, 6, 9, and 12 hours post-stimulation.

2015-03-06 | E-GEOD-59792 | biostudies-arrayexpress

Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides

Project description:Chronic low dose exposure to organophosphorus pesticides is associated with risk of neurodegenerative disease. The mechanism of neurotoxicity is unknown, though it is independent of acetylcholinesterase inhibition. Adducts on tyrosine, lysine, threonine, and serine can occur after exposure to organophosphorus pesticides, the most stable being adducts on tyrosine. Rabbit monoclonal 1C6 to diethoxyphosphate modified tyrosine (depY) was created by single B cell cloning. The amino acid sequence and binding constant (Kd 3.2 x 10-‐8 M) were determined. Cultured human neuroblastoma SH-‐SY5Y and mouse neuroblastoma N2a cells incubated with a sub-‐cytotoxic dose of 10 µM chlorpyrifos oxon contained depY-‐modified proteins detected by monoclonal 1C6 on Western blots. DepY-labeled peptides from tryptic digests of cell lysates were imunopurified by binding to immobilized 1C6. Peptides released with 50% acetonitrile, 1% formic acid were analyzed by LC-‐MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Protein Prospector database searches identified 51 peptides modified on tyrosine by diethoxyphosphate in human SH-‐SY5Y cell lysate and 73 diethoxyphosphate-‐modified peptides in mouse N2a cell lysate. Adducts appeared most frequently on the cytoskeleton proteins tubulin, actin, and vimentin. It was concluded that rabbit monoclonal 1C6 can be useful for studies that aim to understand the mechanism of neurotoxicity resulting from low dose exposure t organophosphorus pesticides.

2022-02-17 | PXD027203 | Pride

Proteome-wide effects of single gene perturbations in a eukaryotic cell

Project description:Eukaryotic gene expression is controlled at the transcriptional, translational and protein degradation level and its principles are starting to emerge. While transcriptional outcomes, which are commonly also used as a proxy for protein abundance, have been investigated on a larger scale, the study of translational output requires large-scale proteomics data. However, data for proteome alterations by systematic assessment of knockouts genome-wide is not available yet. We here determined the individual proteome changes for 3,308 non-essential genes in the yeast S. pombe (www.butterlab.org/SpProtQuant). We observed that genes with high proteome remodeling are predominantly involved in gene expression regulation, in particular acting as translational regulators. Focusing on those knockout strains with a large number of altered proteins, we performed paired transcriptome/proteome measurements to uncover translational regulators and features of translational regulation. Furthermore, by similarity clustering of these proteome changes, we infer gene functionality that can be extended to other species such as human or baker’s yeast.

2022-09-20 | GSE167543 | GEO

Expression data of Soybean (Glycine max) roots from different iron treatments.

Project description:Two Near Isogenic soybean (Glycine max) lines were grown in hydroponic conditions with either 50uM ferric nitrate or 100uM ferric nitrate. After 10 days, half the plants were harvested (total root tissue). At 12 days after planting, iron was added to plants grown in low iron conditions bringing them up to sufficient iron growth conditions. Root tissue was harvested for the remaining plants at 14 days after planting. Gene expression analysis from root tissue of two Near Isogenic Lines (NILs), Clark (PI548553) and IsoClark (PI547430), grown in iron stress or iron stress recovered conditions. A total of 24 samples from four growth conditions, three biological replicates per treatment

2010-09-06 | E-GEOD-22227 | biostudies-arrayexpress

Human connective tissue mast cell proteome

Project description:Mast cells are hematopoietic cells that reside preferentially in tissues exposed to internal and external environments. Mast cells sense immunological, inflammatory and environmental stimuli, and can be activated to release granules and generate inflammatory mediators. Mast cell-derived products confer protection against snake venoms and some parasite infections. Aberrant activation of mast cells is a major contributor to human pathology, including allergy, asthma and adverse drug reactions. Their strict tissue location has largely impeded the isolation of large numbers of primary mast cell for further analysis. To better understand the biology of mast cells, we analyzed the proteome of primary human and mouse mast cells by quantitative mass spectrometry. We identified a mast cell-specific protein signature that was conserved from mouse to man. Compared to a comprehensive set of other immune cell lineages, proteome analysis identified a unique and distant mast cell cluster. The mast cell signature included proteins governing granule biosynthesis and secretion, as well as proteoglycan- and neurotransmitter metabolism. Proteome conservation across species suggests evolutionary maintenance of mast cell functions.

2020-02-12 | PXD014978 | Pride

Expression profile of melanoma cells following p300 HAT inhibition

Project description:Epigenetic events, including covalent post-translational modification of histones, have frequently been demonstrated to play critical roles in tumor development and progression. The transcriptional coactivator, p300/CBP, possesses both histone acetyltransferase (HAT) activity as well as scaffolding properties that directly influence transcriptional activation of targeted genes. We have used a recently reported small molecule inhibitor of p300 HAT activity, C646, to explore the specific contribution of p300/CBP HAT activity to tumor development and progression. We find that C646 inhibits the growth of lineage-specific tumor cell lines including human melanomas through direct transcriptional regulation of cell cycle regulatory proteins. Further evaluation of the p300 HAT transcriptome in human melanoma cells using comprehensive gene expression profiling reveals that p300 HAT activity globally promotes cell cycle progression, nucleosome assembly, and the DNA damage checkpoint through direct transcriptional regulatory mechanisms. Additionally, C646 promotes sensitivity to DNA damaging agents leading to enhanced apoptosis of melanoma cells following combination treatment with cisplatin. Together our data suggest that p300 HAT activity regulates critical growth regulatory pathways in tumors and may serve as a novel therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents. Keywords: p300, small molecule inhibitor, melanoma WM35 cells grown under normal culture conditions were treated for 6 and 24 hours with compound C646 to block p300 HAT activity. A vehicle control (DMSO) was included at each time point. RNA was extracted from all 4 samples using the Qiagen RNeasy kit. RNA quality check, labeling, hybridization, initial data processing and analysis was performed by the JHMI Microarray Core Facility. Affymetrix GeneChip Human Exon 1.0 ST Array was used for this study.

2013-08-01 | E-GEOD-45981 | biostudies-arrayexpress

An evolutionarily distinct chaperone is required for 20S proteasome α-ring assembly in Arabidopsis

Project description:26S proteasomes direct the degradation of many short-lived regulatory proteins and dysfunctional polypeptides, and thus are essential effectors of eukaryotic proteostasis. Proteolysis by this multi-subunit complex occurs inside a barrel-shaped 20S core protease (CP) whose quaternary structure is conserved across all domains of life, and comprises four co-axially stacked heptameric rings formed by structurally related α- and β-subunits in an αββα configuration. CP biogenesis typically begins with the assembly of the α-rings, which then serve as templates for β-subunit integration, three of which present a peptidase active site within the central β-ring chamber. In eukaryotes, α-ring assembly is partially mediated by two hetero-dimeric chaperones, termed Pba1-Pba2 and Pba3-Pba4 in yeast (or PAC1-PAC2 and PAC3-PAC4 in mammals). Pba1-Pba2 initially promotes orderly recruitment of the α-subunits through interactions between their C-terminal HbYX/HbF motifs and shallow pockets at the α5-α6 and α6-α7 interfaces. Here, we identify PBAC5 as a fifth α-ring assembly chaperone in Arabidopsis that directly associates with the Pba1 homolog PBAC1 to form a trimeric PBAC5-PBAC1-PBAC2 complex that can functionally replace the yeast Pba1-Pba2 pair. PBAC5 contains a HbYX motif that likely docks with the pocket formed between the α4 and α5 subunits during α-ring formation. Arabidopsis missing PBAC5, PBAC1, and/or PBAC2 are hypersensitive to proteotoxic, salt and osmotic stress, and display proteasome assembly defects, consistent with a role in CP assembly. Remarkably, while PBAC5 is evolutionarily conserved in plants, it is also present in other kingdoms, with homologs evident in a limited array of fungal, metazoan, and oomycete species.

2020-05-18 | PXD018717 | Pride

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