Project description:To better understand proteostasis in health and disease, determination of protein half-lives is essential. We improved the precision and accuracy of peptide-ion intensity based quantification in order to enable accurate determination of protein turnover in non-dividing cells using dynamic-SILAC. This enabled precise and accurate protein half-life determination ranging from 10 to more than 1000 hours. We achieve good proteomic coverage ranging from four to six thousand proteins in several types of non-dividing cells, corresponding to a total of 9699 unique proteins over the entire dataset. Good agreement was observed in half-lives between B-cells, natural killer cells and monocytes, while hepatocytes and mouse embryonic neurons showed substantial differences. Our comprehensive dataset enabled extension and statistical validation of the previous observation that subunits of protein complexes tend to have coherent turnover. Furthermore, we observed complex architecture dependent turnover within complexes of the proteasome and the nuclear pore complex. Our method is broadly applicable and might be used to investigate protein turnover in various cell types.
Project description:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. MicroRNAs are small nucleatides that function as regulators of gene expression in almost any biological process. However, few microRNAs are reported to have a role in the pathological process of OPLL. Therefore, we performed high-throughput microRNA sequencing and transcriptome sequencing of primary OPLL and PLL cells in order to decipher the interacting network of microRNAs in OPLL. MRNA and microRNA profiles were done using primary culture cells of human ossification of the posterior longitudinal ligament (OPLL) tissue and normal posterior longitudinal ligament (PLL) tissue.
Project description:Purpose: The aim of this study is to investigate the translational regulation of skeletal muscle during acute endurance exercise. Methods: We used mRNA-Seq and ribosome profiling to examine transcriptional and translational regulation, respectively. Result: There were clear distinctions between the profiles of transcription and translation even at a basal condition. TOP-motif genes were translationally suppressed immediately after the exercise. Other genes, such as Slc25a25 was significantly translationally up-regulated presumably in a mTOR-independent manner. Conclusion: There were diverse regulation between transcription and translation. Although many focused on overall protein synthesis to understand the effect of exercise, translational regulation of individual genes are required. Transcriptional and translational profiles of mouse gastrocnemius with or without acute endurance exercise were generated using Ion PGM sequencer.