Journal: bioRxiv

Article Title: Tryptoline Stereoprobe Elaboration Identifies Inhibitors of the GRPEL1-HSPA9 Chaperone Complex

doi: 10.1101/2025.10.20.683548

Figure Lengend Snippet: a , Structures of parent and alkynylated tryptoline acrylamide stereoprobes used previously . b, Heatmap showing DFT calculations for elaborated tryptoline acrylamide stereoprobes compared to unelaborated counterparts (* parent stereoprobes, ** other analogs included for analysis), suggesting minimal impact of C6-and C7-substitution on tryptoline acrylamide core geometry. c, Workflow for cysteine-directed ABPP experiments where stereoprobe reactivity with cysteines is determined by blockade of iodoacetamide-desthiobiotin (IA-DTB) labeling, streptavidin enrichment, and identification and quantification by multiplexed (tandem mass tagging, TMT 10p lex ) MS-based proteomics, as described previously . d, Bar graph showing the number of experiments in which stereoprobe-liganded cysteines were quantified (shown for all cysteines liganded by elaborated, parent, and/or alkyne stereoprobes). e, Bar graphs showing representative liganding profiles for cysteines preferentially engaged by elaborated (left) or parent stereoprobes (right) or showing no preference (middle). Data represent average values ± SD of four independent experiments. f, Pie chart showing number of cysteines preferentially engaged (> 1.5-fold) by elaborated stereoprobes or alkyne stereoprobes. Cysteines not quantified in either elaborated or alkyne stereoprobe datasets were excluded from the analysis (12 total cysteines). g, Bar graph comparing the number of liganded cysteines for each elaborated tryptoline acrylamide stereoprobe where blue and grey designate cysteines that were engaged solely by the indicated stereoprobe vs multiple stereoprobes, respectively.

Article Snippet: Raw files were uploaded to the Integrated Proteomics Pipeline (IP2, version 6.0.2) available at http://ip2.scripps.edu/ ip2/mainMenu.html, and MS2 and MS3 files were extracted from the raw files using RAW Converter (version 1.1.0.22, available at http://fields.scripps.edu/rawconv/ ) and searched using the ProLuCID algorithm using a reverse concatenated, non-redundant variant of the Human UniProt database (release 2016-07).

Techniques: Labeling

Journal: bioRxiv

Article Title: Tryptoline Stereoprobe Elaboration Identifies Inhibitors of the GRPEL1-HSPA9 Chaperone Complex

doi: 10.1101/2025.10.20.683548

Figure Lengend Snippet: a , Colocalization of MTS-EGFP and Mitotracker Deep Red FM for 10 images from a single independent experiment performed in MTS-EGFP-inducible parental HCT-116 cells treated with doxycycline (0.5 µg/mL) and DMSO or WX-71b or WX-71d (20 µM, 8 h) as described in . Data represent average values ± SD of ten technical replicates. b, Generation of sgGRPEL1 cells. HCT-116 cells stably expressed Flag epitope-tagged WT or C124A-GRPEL1 were subject to CRISPR/Cas9 disruption of endogenous GRPEL1 and analyzed at the population level. c, Workflow for pulsed-SILAC labeling with tandem-mass tag (TMT 16p lex )-based multiplexing. sgGRPEL1 HCT-116 cells expressing Flag-epitope tagged WT-or C124A-GRPEL1 were pretreated in light media with DMSO or WX-71b or WX-71d (20 µM, 4 h) followed by shifting the cells to heavy amino acid media in the continued presence of stereoprobes (5 µM, 8 h). Mitochondria were biochemically enriched and analyzed by quantitative proteomics. d, Violin plot showing heavy-labeled protein abundance for the indicated treatment groups in pulse-SILAC experiments. Protein signals were corrected to light-labeled protein signals and normalized to heavy-labeled DMSO-treated WT-GRPEL1 or C124A-GRPEL1 signals. Statistical significance evaluated with parametric, two-tailed, paired t-test. e, Violin plot showing relative heavy-labeled protein abundance for WT-GRPEL1 cells across the indicated submitochondrial localizations as annotated from MitoCarta3.0 in combination with information retrieved from Uniprot and Human Protein Atlas. OMM, outer mitochondrial membrane; IMS, mitochondrial intermembrane space; IMM, inner mitochondrial membrane. f, Histograms (cell count (y axis)) versus mt-mKeima excitation (x axis)) showing mitophagy induction in (left) parental HCT-116 cells or sgGRPEL1 cells expressing WT-GRPEL1 (middle) or C124A-GRPEL1 (right) and also expressing mt-mKeima and Parkin treated with DMSO or WX-71b or WX-71d (20 µM, 8 h). Data show a single experiment representative of three independent experiments ( , present quantification).

Article Snippet: Raw files were uploaded to the Integrated Proteomics Pipeline (IP2, version 6.0.2) available at http://ip2.scripps.edu/ ip2/mainMenu.html, and MS2 and MS3 files were extracted from the raw files using RAW Converter (version 1.1.0.22, available at http://fields.scripps.edu/rawconv/ ) and searched using the ProLuCID algorithm using a reverse concatenated, non-redundant variant of the Human UniProt database (release 2016-07).

Techniques: Stable Transfection, FLAG-tag, CRISPR, Disruption, Multiplex sample analysis, Labeling, Multiplexing, Expressing, Quantitative Proteomics, Two Tailed Test, Membrane, Cell Counting