tpc2 a1 n  (MedChemExpress)

Journal: Acta pharmacologica Sinica

Article Title: Tetrandrine augments melanoma cell immunogenicity via dual inhibition of autophagic flux and proteasomal activity enhancing MHC-I presentation.

doi: 10.1038/s41401-025-01507-9

Figure Lengend Snippet: Fig. 8 Mechanistic illustration of tetrandrine-mediated enhance- ment of melanoma cell recognition and killing by CD8+ T cells through the inhibition of autophagy and proteasomal activity. The diagram illustrates how MHC-I molecules in melanoma cells can be degraded through both autophagy and proteasomal pathways, leading to a reduction in surface MHC-I molecules and facilitating immune escape of the melanoma cells. Tetrandrine disrupts this degradation process by concurrently inhibiting late- stage autophagic flux, through lysosomal acidification disrup- tion, and suppressing proteasomal activity. This dual inhibition prevents MHC-I degradation, thereby increasing MHC-I-mediated antigen presentation on the surface of melanoma cells. The elevated antigen presentation enhances CD8+ T cell recognition and cytotoxicity against melanoma cells. Further mechanistic exploration revealed that tetrandrine exerts its effects by blocking the lysosomal calcium efflux channel TPC2, leading to elevated lysosomal calcium levels and reduced cytosolic calcium concentrations. This calcium imbalance inhibits lysosomal acidification and suppresses cytoplasmic proteasomal activity, collectively contributing to reduced MHC-I degradation. The schematic emphasizes tetrandrine’s pivotal role in modulating both autophagic and proteasomal pathways, ultimately enhan- cing the immunogenicity of melanoma cells and increasing their susceptibility to CD8+ T cell-mediated cytotoxicity. Tet tetrandrine.

Article Snippet: The TPC2 agonist TPC2-A1-N (Cat. No. HY-131614) was obtained from MedChemExpress (Monmouth, NJ, USA).

Techniques: Inhibition, Activity Assay, Immunopeptidomics, Blocking Assay

Journal: The Journal of Clinical Investigation

Article Title: OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca 2+ release, exocytosis, and pancreatic tissue damage

doi: 10.1172/JCI169428

Figure Lengend Snippet: ( A ) Analysis of spontaneous amylase exocytosis from acinar cells. Acini from C57BL/6N ( n = 9, black) and OCaR1 –/– ( n = 8, red) mice were incubated for the indicated time periods and the fraction of released amylase measured. ( B ) The total amylase content was unaltered in acinar cells of OCaR1 –/– compared with littermate OCaR1 +/+ mice ( n = 14 preparations per genotype). ( C ) Plasma cholecystokinin levels are unchanged in OCaR1 –/– mice ( OCaR1 +/+ , n = 7; OCaR1 –/– , n = 8). ( D and E ) Representative traces (25 cells per genotype, normalized to the value at 295 seconds) of Fura-2 fluorescence in C57BL/6N ( D ) and OCaR1 –/– ( E ) acinar cells in Ca 2+ -free physiological solutions. Carbachol (CCh; 10 μM) was applied at the end of the experiment as a positive control. ( F – I ) Spontaneous Ca 2+ transients in acinar cells of OCaR1 –/– mice can be inhibited by 90-minute preincubation with bafilomycin A1 (Baf A1) (C57BL/6N, n = 5; OCaR1 –/– , n = 5) ( F ) or by additional deletion of both TPC1 and TPC2 ( OCaR1 –/– TPC2 –/– TPC1 –/– triple knockout) ( G – I ). ( J ) Comparison of frequency of spontaneous Ca 2+ oscillations between genotypes of the experiments in D – I (C57BL/6N, n = 7; OCaR1 –/– , n = 6; C57BL/6N + Baf A1, n = 5; OCaR1 –/– + Baf A1, n = 5; OCaR1 –/– TPC1 –/– , n = 5; OCaR1 –/– TPC2 –/– , n = 5; OCaR1 –/– TPC1 –/– TPC2 –/– , n = 7). Statistical analysis was done by 2-tailed Student’s t test ( A – C ) or 1-way ANOVA with Bonferroni’s post hoc test ( J ) (** P < 0.01, *** P < 0.001).

Article Snippet: TPC2-A1-N and TPC2-A1-P were purchased from MedChemExpress.

Techniques: Incubation, Fluorescence, Positive Control, Triple Knockout, Comparison

Journal: The Journal of Clinical Investigation

Article Title: OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca 2+ release, exocytosis, and pancreatic tissue damage

doi: 10.1172/JCI169428

Figure Lengend Snippet: ( A ) Model indicating localization of OCaR1 to secretory granules and lysosomes in acinar cells. NAADP-mediated Ca 2+ release via TPC2 channels (blue) is monitored by the activity of adjacent Ca 2+ -activated chloride channels (brown). ( B ) CCK-8–induced (2 pM) Ca 2+ -activated Cl – currents in C57BL/6N and OCaR1 –/– acinar cells in the absence of extracellular Ca 2+ . ( C ) AUC of inward currents over 20 minutes and average amplitude ( n = 16 cells, 5 mice per genotype). ( D ) Model of C-terminal fusion construct of TPC2 and GCaMP6m. ( E ) Representative (17 images, 3 mice) confocal microscopy images of TPC2-GCaMP6m–expressing acinar cells costained with anti-GFP and anti-Rab27B and merged image. Scale bars: 5 μm. ( F ) CCK-8 (2 pM)–induced oscillations in GCaMP6m fluorescence in acinar cells from mice expressing TPC2-GCaMP6m. Acute application of Ned-19 (50 μM) abolishes CCK-8–induced responses ( n = 99). ( G ) Representative traces ( n = 20, from 100 cells per mouse, 5 mice) from CCK-8–evoked global Ca 2+ oscillations in WT acinar cells. ( H ) Representative traces ( n = 20) of CCK-8 (2 pM)–evoked oscillations in GCaMP6m fluorescence in TPC2-GCaMP6m–expressing acinar cells. ( I ) Frequency and duration of Ca 2+ oscillations detected by TPC2-GCaMP6m during stimulation with 2 pM CCK-8 of acinar cells from C57BL/6N ( n = 77 cells, 3 mice) and OCaR1 –/– mice ( n = 135 cells, 4 mice). ( J ) Current densities elicited by coapplication of PI(3,5)P 2 and TPC2-A1-N in vacuolin-enlarged endo-lysosomal vesicles isolated from HEK293 cells overexpressing mTPC2-RFP with or without OCaR1-YFP . Activated currents were blocked with 1 mM ATP (overlapping the basal current) ( n = 4–6). Right: Average current densities at –100 mV. Comparison was done by 2-sample t test ( C and I ) or 1-way ANOVA and Tukey’s post hoc test ( J ) (* P < 0.05, *** P < 0.001). IM, 10 μM ionomycin.

Article Snippet: TPC2-A1-N and TPC2-A1-P were purchased from MedChemExpress.

Techniques: Activity Assay, CCK-8 Assay, Construct, Confocal Microscopy, Expressing, Fluorescence, Isolation, Comparison