Journal: Nature Communications

Article Title: OSGEP regulates islet β-cell function by modulating proinsulin translation and maintaining ER stress homeostasis in mice

doi: 10.1038/s41467-024-54905-8

Figure Lengend Snippet: ITT assay detected the activity of insulin extracted from WT and Osgep- Ins2 - Cre mice pancreas at CD ( a ) or HFD ( b )-fed, and insulin standard as positive control ( n = 4). c AUC of ITT curve ( n = 4). d Schematic of the universal t 6 A 37 biosynthesis pathway in mammalian cell. Kinase, Endopeptidase, and Other Proteins of Small size (KEOPS) compound, with OSGEP as the active catalytic enzyme, utilizes threonylcarbamoyl adenylate (TC-AMP) to catalyze the formation of t 6 A 37 modification in ANN-decoding tRNA substrates. e HPLC-MS/MS chromatograms of t 6 A 37 in islet tRNA and relative t 6 A 37 levels were showed in WT and Osgep- Ins2 - Cre mice islet ( n = 4). f Circular dichroism spectra of protein extracted from WT and Osgep- Ins2 - Cre islet at CD. Proinsulin protein was analyzed by reducing western blots, and misfolded Proinsulin protein was analyzed by non-reducing Western blots in islets of mice under CD ( g ) and HFD ( h )-fed condition. The ratios of Monomer to total Proinsulin were calculated. The ratios of Dimers, Trimers plus High-Molecular-Weight (HMW) to total Proinsulin were calculated ( n = 3 biological replicates). Results are repeated three times independently with similar results. i Experimental procedure of in vitro transcription and translation for proinsulin protein. j Western blots analyzed the proinsulin product in addition of tRNAs from islets of HFD-fed WT and Osgep- Ins2 - Cre mice in vitro reticulocytes translation system ( n = 3). a – c , e , g , h , j Data are presented as the mean ± SD. a , b One-way ANOVA with Tukey’s post-test. c , e , g , h , j Unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data are provided as a Source Data file. CD Chow diet, HFD High fat diet, KEOPS Kinase, Endopeptidase, and Other Proteins of Small size, TC-AMP threonylcarbamoyl adenylate, RRL Rabbit Reticulocyte Lysate.

Article Snippet: After blocking with 5% skim milk, the membrane was incubated overnight at 4 °C with one of the following primary antibodies: Osgep (SANTA CRUZ, sc-393199, 1:500 dilution in blocking buffer), β-Tubulin (Proteintech, 10094-1-AP, 1:20000 in blocking buffer), Insulin (Proteintech, 66198-1-Ig, 1:2000 in blocking buffer), Proinsulin, (Novus, NB100-73013, 1:1000 in blocking buffer), IRE1α (Cell Signaling Technology, 3294, 1:1000 in blocking buffer), Bip (Proteintech, 66574-1-Ig, 1:1000 in blocking buffer), PERK (Sigma, P0074, 1:2000 in blocking buffer), Phospho-PERK (Sigma, SAB5700521, 1:1000 in blocking buffer), eIF2α (Cell Signaling Technology, 5324, 1:2000 in blocking buffer), Phospho-eIF2α (Cell Signaling Technology, 3398, 1:1000 in blocking buffer), ATF4 (Cell Signaling Technology, 11815, 1:1000 in blocking buffer), CHOP (Cell Signaling Technology, 2895, 1:1000 in blocking buffer), ATF6 (Proteintech, 66563-1-Ig, 1:1000 in blocking buffer), AKT (Proteintech, 60203-2-Ig, 1:2000 in blocking buffer), p-Akt1/2/3 (SANTA CRUZ, SC-514032, 1:500 in blocking buffer) or Gapdh (Proteintech, 60004-1-Ig, 1:50000 in blocking buffer).

Techniques: Activity Assay, Positive Control, Modification, Tandem Mass Spectroscopy, Circular Dichroism, Western Blot, High Molecular Weight, In Vitro, Two Tailed Test