Project description:During autophagy, the contents enclosed within autophagosomes are degraded, while the outer membrane components are recycled from autolysosomes by the recycler complex through the recently discovered autophagosomal components recycling (ACR) process. This recycling is essential for maintaining autophagic activity. However, the molecular machinery and upstream regulatory mechanisms driving this recycling process remain poorly understood. Here, we identify SNX16 as a key component of the recycler complex, which localizes to autolysosomes and is required for ACR. SNX16 functions in ACR by regulating recycler complex formation, facilitating cargo recognition, and mediating the connection between STX17-SNX4-SNX5 and dynein-dynactin complexes. In addition, SNX16-cargo interactions are regulated by two ACR-related small GTPases, Rab32 and Rab38. Importantly, mTORC1 phosphorylates SNX16 to regulate ACR by inhibiting its interactions with STX17 and other recycler components, thus preventing recycler complex formation. Taken together, our findings identify SNX16 as a novel recycler component and establish a link between mTORC1 and ACR.
Project description:Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr(193) of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr(215) of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.
| S-EPMC3459221 | biostudies-literature
Project description:Multi-omics of colorectal peritoneal malignancy
Project description:Mechanistic target of rapamycin complex 1 (MTORC1) is a critical negative regulator of general autophagy. We hypothesized that MTORC1 may specifically regulate autophagic clearance of damaged mitochondria. To test this, we used cells lacking tuberous sclerosis complex 2 (TSC2-/- cells), which show constitutive MTORC1 activation. TSC2-/- cells show MTORC1-dependent impaired autophagic flux after chemical uncoupling of mitochondria, increased mitochondrial-protein aging, and accumulation of p62/SQSTM1-positive mitochondria. Mitochondrial autophagy (mitophagy) was also deficient in cells lacking TSC2, associated with altered expression of PTEN-induced putative kinase 1 (PINK1) and PARK2 translocation to uncoupled mitochondria, all of which were recovered by MTORC1 inhibition or expression of constitutively active forkhead box protein O1 (FoxO1). These data prove the necessity of intact MTORC1 signaling to regulate two synergistic processes required for clearance of damaged mitochondria: (i) general autophagy initiation and (ii) PINK1/PARK2-mediated selective targeting of uncoupled mitochondria to the autophagic machinery.
Project description:We used genome-scale modeling and multi-omics (transcriptomics, proteomics, and metabolomics) analysis to assess metabolic features that are critical for macrophage activation. We constructed a genome-scale metabolic network for the RAW 264.7 cell line to determine metabolic modulators of activation. Metabolites well-known to be associated with immunoactivation (glucose and arginine) and immunosuppression (tryptophan and vitamin D3) were among the most critical effectors. Intracellular metabolic mechanisms were assessed, identifying a suppressive role for de-novo nucleotide synthesis. Finally, underlying metabolic mechanisms of macrophage activation are identified by analyzing multi-omic data obtained from LPS-stimulated RAW cells in the context of our flux-based predictions. Two condition (flagellin and LPS) time course exposure of RAW 264.7 cell line at 1, 2, 4, and 24 hours. Two replicates for each condition and time point. All conditions compared to a pool of untreated cells at a 0 hour time point.
Project description:LARP1 is a key repressor of TOP mRNA translation. It binds the m7Gppp cap moiety and the adjacent 5'TOP motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 controls TOP mRNA translation via LARP1, but the details of the mechanism are unclear. Herein we elucidate the mechanism by which mTORC1 controls LARP1's translation repression activity. We demonstrate that mTORC1 phosphorylates LARP1 in vitro and in vivo, activities that are efficiently inhibited by rapamycin and torin1. We uncover 26 rapamycin-sensitive phospho-serine and -threonine residues on LARP1 that are distributed in 7 clusters. Our data show that phosphorylation of a cluster of residues located proximally to the m7Gppp cap-binding DM15 region is particularly sensitive to rapamycin and regulates both the RNA-binding and the translation inhibitory activities of LARP1. Our results unravel a new model of translation control in which the La module (LaMod) and DM15 region of LARP1, both of which can directly interact with TOP mRNA, are differentially regulated: the LaMod remains constitutively bound to PABP (irrespective of the activation status of mTORC1), while the C-terminal DM15 'pendular hook' engages the TOP mRNA 5'-end to repress translation, but only in conditions of mTORC1 inhibition.
Project description:Serotonin is important material in our body. 5-HT is synthesized by TPH1 starting with tryptophan and this compound exists on colorectal cancer. And serotonin combines with various receptors to conduct the metabolic pathway in colorectal cancer. Each receptor relates with various metabolism, such as lipid metabolism, growth, immunity, etc. In this way, medicine which inhibits these receptors can expect for therapy of colorectal cancer. Especially HTR 2B has correlation with growth factor and using the inhibitor of HTR2B, SB204741, can have effect on metabolic pathway in colorectal cancer. So, we set the hypothesis that HTR2B antagonist can have negative effect on growth of colorectal cancer.
Project description:The homeodomain transcription factor Prep1 was previously shown to regulate insulin sensitivity. Our aim was to study the specific role of Prep1 for the regulation of energy metabolism in skeletal muscle. Muscle-specific ablation of Prep1 resulted in increased expression of respiratory chain subunits. This finding was consistent with an increase in mitochondrial enzyme activity without affecting mitochondrial volume fraction as assessed by electron microscopy. Metabolic phenotyping revealed no differences in daily energy expenditure or body composition. However, during treadmill exercise challenge, Prep1 ablation resulted in a higher maximal oxidative capacity and better endurance. Elevated PGC-1? expression was identified as a cause for increased mitochondrial capacity in Prep1 ablated mice. Prep1 stabilizes p160 Mybbp1a, a known inhibitor of PGC-1? activity. Thereby, p160 protein levels were significantly lower in the muscle of Prep1 ablated mice. By a chromatin immunoprecipitation-sequencing (ChIP-seq) approach, PREP1 binding sites in genes encoding mitochondrial components (e.g., Ndufs2) were identified that might be responsible for elevated proteins involved in oxidative phosphorylation (OXPHOS) in the muscle of Prep1 null mutants. These results suggest that Prep1 exhibits additional direct effects on regulation of mitochondrial proteins. We therefore conclude that Prep1 is a regulator of oxidative phosphorylation components via direct and indirect mechanisms.
Project description:DNA methylation array data generated from epidermal samples (suction blister roofs) of healthy female subjects between 21 and 76 years. Aim of the project was the investigation of non-linearities in the human aging progression using an integrative multi-omics analysis. DNA was extracted from suction blisters taken from the volar forearms of each subject, bisulfite converted, and profiled using Illumina Infinium HumanMethylation450 BeadChip arrrays.