LC-MS/MS analyses of MMP cleavage fragments of tyrosyl-tRNA synthetase
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ABSTRACT: Tyrosyl-tRNA synthetase was digested with MMPs after which cleavage fragments were resolved using SDS-PAGE. Major bands were digested with trypsin and analyzed by LC-MS/MS.
Project description:Tryptophanyl-tRNA synthetase was incubated by MMPs, cleavage products were resolved on SDS-PAGE. Cleavage products were trypsin digested and analyzed by LC-MS/MS.
Project description:Recombinant human tyrosyl-tRNA synthetase (YRS) was cleaved using recombinant MMPs. Terminal amine oriented mass spectrometry of substrates (ATOMS) was used to identify MMP generated cleavage sites from these mixtures with LC-MS/MS analysis.
Project description:Human tryptophanyl-tRNA synthetase (WRS) was digested by human MMPs, and digestion mixtures were subjected to terminal amine oriented mass spectrometry of substrates (ATOMS) protocol and LC-MS/MS analysis to identify MMP cleavage sites in WRS.
Project description:During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a transcriptional GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. The GCN4 response was confirmed using SILAC proteomics, using heavy isotope labelling to identify changes in protein expression during glutamine tRNA synthetase depletion.
Project description:Airway epithelium is the initial point of host-pathogen interaction in Pseudomonas aeruginosa infection, an important pathogen in cystic fibrosis and nosocomial pneumonia. We used global gene expression analysis to determine airway epithelial transcriptional responses dependent on matrilysin (MMP-7) and stromelysin-2 (MMP-10), two matrix metalloproteinases induced by acute P. aeruginosa pulmonary infection. Extraction of Differential Gene Expression (EDGE) analysis of gene expression changes in P. aeruginosa infected organotypic tracheal epithelial cell cultures from wildtype, Mmp7-/-, and Mmp10-/- mice identified 2,089 matrilysin-dependent and 1,628 stromelysin-2-dependent genes that were differentially expressed. Key node network analysis showed that these MMPs controlled distinct gene expression programs involved in proliferation, cell death, immune responses, and signal transduction, among other host defense processes. Our results demonstrate discrete roles for these MMPs in regulating epithelial responses to pseudomonas infection and show that a global genomics strategy can be used to assess MMP function. Experiment Overall Design: C57Bl6, Mmp7-/- and Mmp10-/- mouse epithelium at an organotypic air liquid interface culture was exposed to Pseudomonas aeruginosa for 1 and 24 h. RNA was collected from these samples as well as uninfected 0 h and assessed for expression changes using Affymetrix Mouse 430 2.0 Arrays. Triplicate samples were processed for each genotype at each time point.
Project description:Airway epithelium is the initial point of host-pathogen interaction in Pseudomonas aeruginosa infection, an important pathogen in cystic fibrosis and nosocomial pneumonia. We used global gene expression analysis to determine airway epithelial transcriptional responses dependent on matrilysin (MMP-7) and stromelysin-2 (MMP-10), two matrix metalloproteinases induced by acute P. aeruginosa pulmonary infection. Extraction of Differential Gene Expression (EDGE) analysis of gene expression changes in P. aeruginosa infected organotypic tracheal epithelial cell cultures from wildtype, Mmp7-/-, and Mmp10-/- mice identified 2,089 matrilysin-dependent and 1,628 stromelysin-2-dependent genes that were differentially expressed. Key node network analysis showed that these MMPs controlled distinct gene expression programs involved in proliferation, cell death, immune responses, and signal transduction, among other host defense processes. Our results demonstrate discrete roles for these MMPs in regulating epithelial responses to pseudomonas infection and show that a global genomics strategy can be used to assess MMP function. Keywords: time course
Project description:As a multifunctional RBP, CELF1 is known to preferentially binds to GU-rich elements (GREs) predominantly located in 3’ untranslated regions(UTRs) of target mRNA to regulate various post-transcriptional process. However, the targeted genes that regulated by CELF1 during cataractogenesis remains unknown. In present study,the function of CELF1 in SRA01/04 cells was investigated with CELF1 overexpression, the expression of MMPs was regulated by CELF1.
Project description:Substantial data indicate that microRNA-21 (miR-21) is significantly elevated in glioblastoma (GBM) and in many other tumors of various origins. This miRNA has been implicated in various aspects of carcinogenesis, including cellular proliferation, apoptosis and migration. We demonstrate that miR-21 regulates multiple genes associated with glioma cell apoptosis, migration, and invasiveness, including RECK and TIMP3, suppressors of malignancy and inhibitors of matrix metalloproteinases (MMPs). Specific inhibition of miR-21 with antisense olgonucleotides leads to elevated levels of RECK and TIMP3 and therefore reduces MMP activities in vitro and in a human model of glioma in nude mice. Moreover, down-regulation of miR-21 in glioma cells leads to decrease of their migratory and invasion abilities. Our data suggest that miR-21 contributes to the glioma malignancy by down-regulation of MMP inhibitors that leads to activation of MMPs thus promoting invasiveness of cancer cells. Our results also indicate that inhibition of a single oncomir like miR-21 with specific antisense molecules can provide a novel therapeutic approach for “physiological” modulation of multiple proteins whose expression is de-regulated in cancer.
Project description:Heterozygous mutations in six tRNA synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT-mutant glycyl- or tyrosyl-tRNA synthetases inhibit global protein synthesis by an unknown mechanism, independent of aminoacylation activity. We report that tRNAGly overexpression rescues protein synthesis and peripheral neuropathy phenotypes in Drosophila and mouse models of CMT caused by glycyl-tRNA synthetase (GlyRS) mutations (CMT2D). Kinetic experiments revealed that CMT-mutant GlyRS bind tRNAGly, but display markedly slow release rates. This tRNAGly sequestration may deplete the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly supply to the ribosome and translation deficit.
Project description:Purpose: Primary cutaneous squamous cell carcinoma (SCC) can be an invasive cancer in skin and has the potential to metastasize. We aimed to define the cancer related molecular changes that distinguish non-invasive from invasive SCC. Experimental design: We used laser capture microdissection technique in combination with cDNA microarray analysis in order to determine molecular changes that associate with SCC progression. Results: We defined invasion-associated genes as those udifferentially regulated only in SCC invasive nests, but not in actinic keratosis-like dysplasia or SCC in situ regions, compared to normal epidermis. We designated these genes as “invasion signature gene set of cutaneous SCC”. Overall we found 383 up- and 354 down-regulated probe-sets that constitute the invasion signature gene set. As part of this profile, SCC invasion is associated with aberrant gene expression changes of numerous MMPs including MMP7 (FCH=5.43, FDR<0.01) and MMP13 (FCH=12.53, FDR<0.01). IL-24 is also up-regulated in the leading invasive edge of SCC (FCH=6.74, FDR<0.01). IL-24 enhanced mRNA expression of both MMP7 and MMP13 in a human SCC cell line. Laminin332, which is one of the target molecules of MMP7, had altered expression at the leading edge of SCC invasion nests at both the genomic and protein level. Conclusions: We defined the distribution of MMPs within human cutaneous SCC tissue showing distinct expression with progression from normal skin to actinic keratosis to SCC in situ to invasive carcinoma. We further suggest a potential role for IL-24 in progression to invasion via MMP7 and MMP13. Laser capture microdissection was performed on 5 cases of actinic keratosis, 5 cases of in situ SCC, and 5 cases of invasive SCC.