Project description:Using a newly-developed workflow for quantitative newly synthesized proteome analysis (QuaNPA), featuring automated sample processing and multiplexed DIA (plexDIA) analysis, changes in the newly synthesized proteome of IFN-gamma treated Hela cells were monitored over time.
Project description:To analyse the genome-wide impact of inactivation of the ATAC or SAGA coactivator complexes on RNA polymerase II (Pol II) transcription, we purified newly synthesized RNA from mutant mouse embryonic stem (ES) cell lines in which subunits of ATAC (Yeats2, Zzz3) or SAGA (Supt7l) are inactivated or depleted. We also performed this analysis in mutant cell lines in which a subunit (Tada3) of the shared histone acetyltransferase activity of these two complexes is depleted. Newly synthesized RNA was purified following the 4sU labelling method (more details in extract protocol). For two wildtype samples, the total RNA input was also analysed.
Project description:Innate stimulation with TLR ligands leads to the activation of various genes in macrophages and various populations of these cells may exhibit different responses. Here wanted to delineate and characterize these transcriptional responses in newly established, self-renewing, in vitro grown non-transformed lines (MPI cells) and bone marrow derived macrophages We used microarrays to detail the global programme of gene expression Newly synthesized RNA from independently established MPI lines and bone marrow derived macrophages was extracted and hybridized to Affymetrix microarrays
Project description:THP-1 macrophages were infected with four strains of Mycobacterium tuberculosis to study the temporal dynamics of newly synthesized proteins in the secretome. Temporal snapshots of secretome reflect the macrophage response to pathogenicity which in combination with intracellular events, completes the disease picture. However, such studies are compromised by limitations of quantitative proteomics. Metabolic labeling by SILAC allows a 3-plex experiment while isobaric chemical labeling by iTRAQ/TMT allows up to 8 to 10-plex respectively. This makes studying temporal proteome dynamics an intangible and elusive proposition. We have developed a new variant of hyperplexing method, combining triplex SILAC with 6-plex iTRAQ to achieve 18-plex quantitation in a single MS run. THP-1 macrophages were infected with H37Ra, H37Rv, BND433 and JAL2287 and the newly synthesized secreted host proteins were studied over six temporal frame still 30 hours post infection, at a difference of 4 hours each. For quantitation, the strains were encoded with two sets of triple SILAC- H37Ra & H37Rv in one and BND433 & JAL2287 in another with a control in each. These sets were then iTRAQ labeled to encode for temporal profiles across six time points in 6-plex iTRAQ. Effectively a 36-plex design with 4 replicates of each set, these experiments were completed within few days on the mass spectrometer. Using MaxQuant and in house developed tools and pipelines, we have analysed the data to map the temporal and strain specific dynamics of newly synthesized proteins in host. Hyperplexing enables large scale spatio-temporal systems biology studies where large number of samples can be processed simultaneously and in quantitative manner.
Project description:Energy metabolism and extracellular matrix function are closely connected to orchestrate and maintain tissue organization, but the crosstalk is poorly understood. Here, we used scRNA-seq analysis to uncover the importance of respiration for extracellular matrix homeostasis in mature cartilage. A combined approach of high-resolution single cell RNA sequencing, mass spectrometry/matrisome analysis and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. Genetic inhibition of respiration in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage showing disorganized chondrocytes and increased deposition of extracellular matrix material. scRNA-seq analysis identified a cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of extracellular matrix-related genes in nonarticular chondrocyte clusters. These changes were associated with alterations in extracellular matrix composition, a shift in the collagen/non-collagen protein content and an increase of collagen crosslinking and ECM stiffness. The results demonstrate, based on findings of the scRNA-seq analysis, that respiration is a key factor contributing to ECM integrity and mechanostability in cartilage and presumably also in many other tissues.
Project description:Growth plate chondrocytes are regulated by numerous factors and hormones as they mature during endochondral bone formation. Chondrocytes in the growth plate’s growth zone (GC cells) produce and export matrix vesicles (MVs) under the regulation of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. 1α,25(OH)2D3 secreted by the cells acts on the MV membrane, releasing its contents. This study examined the regulatory role 1α,25(OH)2D3 has over the production and packaging of microRNA into MVs by GC cells and the ability to release microRNA from MVs once produced. We treated GC cells with 1α,25(OH)2D3 and then sequenced the microRNA in the cells and MVs. We also treated MVs with 1α,25(OH)2D3 and determined if the microRNA was released. To assess whether MVs can act directly with chondrocytes and if this is regulated by 1α,25(OH)2D3, we stained MVs with a membrane dye and treated GC cells with them. 1α,25(OH)2D3 regulated the production and packaging of microRNA into matrix vesicles. MVs did not release microRNA when treated with 1α,25(OH)2D3, indicating a heterogeneous MV population or a protective factor. Stained MVs were endocytosed by GC cells and this was increased with 1α,25(OH)2D3 treatment. This study adds new regulatory roles for 1α,25(OH)2D3 with respect to packaging and transport of MV microRNAs.
Project description:Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively-fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1alpha, ERO1beta and PRDX4, compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly-modest delay in disulfide bond formation in secreted proteins but a profound, five-fold lower procollagen 4 hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content, in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H2O2 generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation and a non-canonical form of scurvy. double and triple mutants and wild type
Project description:The precise regulation of transcription requires the coordinated activity of numerous proteins and protein complexes. Although significant progress has been made in understanding the mechanisms that regulate transcription, many questions remain unresolved. Accurately defining the direct effects of transcriptional regulators is critical to addressing these questions. An effective approach for identifying the direct targets of transcriptional regulators is combining rapid protein depletion and quantification of newly synthesized RNA. The auxin-inducible degron (AID) system and thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) are powerful methods to rapidly degrade a target protein and directly quantify newly synthesized RNA, respectively. Both methods have been widely applied to study transcriptional regulation. To address unresolved questions in transcription, we assembled an end-to-end workflow to deplete proteins of interest using the AID system and measure newly synthesized RNA using SLAM-seq in the model eukaryote, Saccharomyces cerevisiae. We provide an open-source, step-by-step protocol to support rapid implementation of this workflow. We include methods for targeted protein degradation, 4-thiouracil (4tU) incorporation, rapid methanol fixation, RNA purification, RNA alkylation, mRNA-seq library construction, and data analysis. Additionally, we demonstrate that this workflow can help define the direct effects of transcriptional regulators using the bromodomain and extra-terminal domain (BET) proteins, Bdf1 and Bdf2, as an example. We provide evidence that data generated using this workflow is robust to normalization using whole-cell spike-in or total read counts, correlates well with 4tU-seq data, and identifies extensive differential expression due to the depletion of Bdf1 and Bdf2. Taken together, this workflow will help address outstanding questions underlying the molecular basis of transcriptional regulation and other processes in S. cerevisiae and other eukaryotes.