Effect of TOPBEffect of TOPBP1 on BLM phosphosites
Ontology highlight
ABSTRACT: Identification of BLM phosphosites. The experiment is composed by 2 parts: i) identification of BLM phosphosites with DDA method ii) quantification of phosphosites with a targeted method.
Project description:We used microarrays to identify differentially expressed genes after DNA-damaging agent bleomycin (BLM) and/or immune inducer 2, 6-dichloroisonicotinic acid (INA) treatment. We focused on those genes that were synergistically induced by co-treatment (BLM+INA). Arabidopsis seedlings were treated with 4 μg/ml BLM and/or low INA (10 μM). There are 4 treatments: control (CK), INA, BLM and BLM+INA. Each treatments have three biological replicates. There are 12 samples in total.
Project description:Background: Pulmonary Fibrosis (PF) is an interstitial lung disease of unknown etiology, characterized by excessive accumulation of extracellular matrix in the lungs, which disrupts the structure and gas exchange of the alveoli. There are only two approved therapies for PF, nintedanib (Nib) and pirfenidone. Therefore, the use of Chinese medicine for PF is attracting attention. Tianlongkechuanling (TL) is an effective Chinese formula that has been applied clinically to alleviate PF, which can enhance lung function and quality of life. Purpose: The potential effects and specific mechanisms of TL have not been fully explored, yet. In the present study, proteomics was performed to explore the therapeutic protein targets of TL on Bleomycin (BLM)-induced Pulmonary Fibrosis. Method: BLM-induced PF mice models were established. Hematoxylineosin staining and Masson staining were used to analyze histopathological changes and collagen deposition. To screen the differential proteins expression between the Control, BLM, BLM+TL and BLM+ Nib (BLM+ Nintedanib) groups, quantitative proteomics was performed using tandem mass tag (TMT) labeling with nanoLC-MS/MS [nano liquid chromatographymass spectrometry]). Changes in the profiles of the expressed proteins were analyzed using the bioinformatics tools Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The protein–protein interactions (PPI) were established by STRING. Expressions of α-smooth muscle actin (α-SMA), Collagen I (Col1a1), Fibronectin (Fn1) and enzymes in arginase-ornithine pathway were detected by Western blot or RT-PCR.
Project description:The Bloom syndrome DNA helicase BLM contributes to chromosome stability through its roles in double-strand break repair by homologous recombination and DNA replication fork restart during the replication stress response. Loss of BLM activity leads to Bloom syndrome, which is characterized by extraordinary cancer risk and small stature. Here, we have analyzed the composition of the BLM complex in unperturbed cells and identified a direct physical interaction with the Mcm6 subunit of the minichromosome maintenance (MCM) complex by co-immunopecipitations using endogenous and recombinant proteins as well as two-hybrid analysis.
Project description:We report for the first time that secretoglobin (SCGB) 3A2, a novel cytokine-like molecule, predominantly expressed in pulmonary airways epithelium, drastically reduced the severity of lung fibrosis using bleomycin-induced mouse model. DNA microarrays were performed using RNAs isolated from mouse lungs in two experiments. In the first experiments, mice were adminitered intravenously SCGB3A2 or PBS once daily for a week, starting on day 14 after BLM administration, and in the second experiments, SCGB3A2 or PBS was administered intravenously for 12 hrs.
Project description:BLM protein has been shown to play an important role in homologous recombination (HR) repair. We report that BLM interacts with RAD54 (another HR repair protein) to enhance its chromatin remodeling function. This experiment identifies that BLM gets recruited to different genetic loci even in the absence of damage, possibly along with RAD54 chromatin remodeler.
Project description:Idiopathic pulmonary fibrosis is a chronic devastating disease of unknown etiology. No therapy is currently available. A growing body of evidence supports the role of TGFβ1 as the major player in the pathogenesis of the disease. This study designed novel human- and mouse-specific siRNAs and siRNA/DNA chimeras targeting both human and mouse common sequences and evaluated their inhibitory activity in pulmonary fibrosis induced by bleomycin and lung-specific transgenic expression of human TGFβ1. Selective novel sequences of siRNA and siRNA/DNA chimeras efficiently inhibited pulmonary fibrosis, indicating their applicability as tools for treating fibrotic disease in humans. Total RNA was extracted from lung tissue from mice with bleomycin (BLM)-induced lung fibrosis treated with mouse TGFβ1 siRNAs or vehicle on different days after BLM infusion.
Project description:The discovery of the IDH1 R132H (IDH1 mut) mutation in low-grade glioma and the associated change in function of the IDH1 enzyme has increased the interest in glioma metabolism. In an earlier study, we found that changes in expression of genes involved in the aerobic glycolysis and the TCA-cycle are associated with IDH1 mut. Here we apply proteomics to FFPE samples of diffuse gliomas with or without IDH1 mutations, in order to map changes in protein levels associated with this mutation. We observed significant changes in the enzyme abundance associated with aerobic glycolysis, glutamate metabolism and the TCA-cycle in IDH1 mut gliomas. Specifically, the enzymes involved in the metabolism of glutamate, lactate and enzymes involved in the conversion of α-ketoglutarate were increased in IDH1 mut gliomas. In addition, the bicarbonate transporter (SLC4A4) was increased in IDH1 mut gliomas, supporting the idea that a mechanism preventing intracellular acidification is active. We also found that enzymes that convert proline, valine, leucine and isoleucine into glutamate were increased in IDH1 mut glioma. We conclude that in IDH1 mut glioma metabolism is rewired (increased input of lactate and glutamate) to preserve TCA cycle activity in IDH1 mut gliomas.
Project description:Owing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs rapidly associated with translating ribosomes in the cytoplasm and that this incorporation is independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (following oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing neuronal synapses a rapid means to regulate local protein synthesis.