Project description:Targeting proximity labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes with minimal perturbance to normal physiology. Here, we generate transgenic mice expressing a mitochondrial matrix-targeted ascorbate peroxidase (MTS-APEX2) to enable analysis of tissue-specific matrix proteomes. Desthiobiotin-phenol labeling of the mitochondrial proteome revealed 263 mitochondrial-localized proteins in mouse muscle tissues. Comparison with the MTS-APEX2-labeled proteome in HEK293T cells revealed enrichment in mitochondrial proteins related to energy production in the muscle tissues of transgenic mice. We found Reticulon 4 Interacting Protein 1(RTN4IP1) or Optic Atrophhy-10 (OPA10) was highly enriched in cardiac and soleus muscle and contained a strong mitochondrial matrix-targeting sequence. Structural analysis shows RTN4IP1 is an NADPH oxidoreductase with protein structure resembling quinone oxidoreductase (QOR) in bacterial species. Enzymatic activity assays, interactome analysis, and metabolite profiling confirmed that RTN4IP1 functions in coenzyme Q biosynthesis in the mitochondria. Due to reduced CoQ9 levels, Rtn4ip1-knockout C2C12 cells were vulnerable to oxidative stress and had decreased oxygen consumption rates and ATP production.
Project description:This metabolite dataset was collected from bacterial channel rhodopsin expressing transgenic mouse models subjected to optic nerve crush (ONC) with subsequent stimulation with light (promoting regeneration) or non-stimulation (lacking axon regeneration). ONC retains retinal ganglion cells within the retina, while degenerating axons. In transgenic bacterial channel rhodopsin expressing cells, light stimulation promotes regeneration. Genetically matched wild-type uninjured optic nerves, or control transgenic mice, were also analyzed. Metabolites were carefully extracted from finely minced optic nerve tissue using a solvent system (initial separation using 1:1 Methanol and H2O and second extraction using 8:1:1 of Acetonitrile:Acetone:Methanol). Untargeted liquid chromatography-mass spectrometry (LC-MS/MS) profiling was performed using fractionation on a Vanquish Horizon Binary UHPLC. Subsequent analyses were performed on an inline coupled Q-Exactive Orbitrap instrument. Metabolites were identified using Compound DiscovererTM software. Statistical analysis was performed using MetaboAnalyst 5.0.
Project description:The A238L gene has been selectively expressed in mouse T lymphocytes using tissue specific promoter, enhancer and locus control region sequences for CD2. The resulting two independently derived transgenic mice expressed the transgene and developed a metastatic, angiogenic and transplantable CD4+CD8+ CD69- lymphoma. The absence of CD69 from the tumour cells suggests that they were derived from T-cells at a stage prior to positive selection. In contrast, transgenic mice similarly expressing a mutant A238L, mutA238L, solely inhibiting transcription mediated by NF-B, were indistinguishable from wild type mice. Expression of Rag 1, Rag2, TCRbeta V8.2, CD25, FoxP3, Bcl3, Bcl2 l14, Myc, IL-2, NFAT1, Itk, by purified CD4+CD8+ CD69- thymocytes from A238L transgenic mice was consistent with the phenotype. Similarly evaluated expression profiles of CD4+CD8+ CD69- thymocytes from the mutant A238L transgenic mice were comparable to those of wild type mice. Purified CD4+CD8+CD69- thymocytes were prepared for wild-type control FVB/N mice, as well as from the FVB/N transgenic mice expressing the A238L and the mutated A238L, mutA238L. Comparisons were performed between the two transgenic mice lines and the control mice.
Project description:We employed isobaric labeled peptides to do bottom-up proteomics to quantify both non-enriched total peptides and enriched phospho-peptides obtained from hearts of control mice expressing wild-type cardiac troponin I and transgenic mice expressing the truncated N-terminal cardiac troponin I.
Project description:Thymocytes from HEBAlt-transgenic mice expressing HA-tagged HEBAlt were subjected to IP using anti-HA, trypsin digested, and subjected to mass spectrometry