csn5 protein  (Human Protein Atlas)

Journal: Acta Pharmaceutica Sinica. B

Article Title: Berberine diminishes cancer cell PD-L1 expression and facilitates antitumor immunity via inhibiting the deubiquitination activity of CSN5

doi: 10.1016/j.apsb.2020.06.014

Figure Lengend Snippet: BBR directly binds to and inhibits CSN5 activity. (A) The 293T or 293T expressing Flag-CSN5 cell lysates were incubated with BBR-biotin at 4 °C overnight, the lysates were used for streptavidin–agarose pull-down assays, and the precipitates were resolved by IB for CSN5. (B) The recombinant Flag–CSN5 proteins were incubated with BBR–biotin for 1 h at 37 °C, followed by IB with biotin (upper band) or Flag (lower band). (C) The recombinant Flag–CSN5 protein was incubated with BBR–biotin in the absence or presence of indicated concentration of unlabelled BBR for 1.5 h at 37 °C, and the mixtures were IB for biotin or flag. (D) SPR analysis of the binding between BBR and CSN5. Recombinant human CSN5 protein was immobilized on an activated CM5 sensor chip, BBR was then flowed across the chip. (E) CSN5 activity in an in vitro deubiquitination assay. The activity was measured by 7-amino-4-methylcoumarin (AMC) released from the fluorogenic substrate, ubiquitin–AMC ( n = 3). ∗ ∗ P < 0.01 and ∗ ∗ ∗ P < 0.001 compared with DMSO group. (F) IB analysis of the P53 and P27 levels in H460 cells treated with indicated concentration of BBR for 24 h. (G and H) H460 cells were transfected with siRNA control, siRNA targeting CSN5 for 24 h (G), or transfected with 2 μg empty vector, 2 μg Flag–CSN5 for 24 h (H), followed by BBR (10 μmol/L) treatment for 24 h, the PD-L1 expression level was determined by IB. Data shown are mean value of three independent experiments±standard error of mean (SEM).

Article Snippet: Recombinant human CSN5 (TP300979; Origene, Rockville, MD, USA) was immobilized in parallel-flow channels of CM5 chip by using amine-coupling kit (BR-1000-50; GE Healthcare).

Techniques: Activity Assay, Expressing, Incubation, Recombinant, Concentration Assay, Binding Assay, In Vitro, Ubiquitin Proteomics, Transfection, Control, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Berberine diminishes cancer cell PD-L1 expression and facilitates antitumor immunity via inhibiting the deubiquitination activity of CSN5

doi: 10.1016/j.apsb.2020.06.014

Figure Lengend Snippet: Glu76 of CSN5 is critical for binding to BBR. (A) Molecular docking model carried out by Discovery Studio 4.5 revealed that BBR binds to the JAMA domain of CSN5. (B) BBR forms an ionic bond with the backbone of Glu76 and interacts favorably with several residues including Met78, His138, Tyr143, and Asp151. (C) 293T-cells were transfected with WT-CSN5 and CSN5-mutant plasmids for 48 h, cell lysates were incubated with BBR–biotin at 37 °C for 2 h, followed by pull-down with streptavidin–agarose; the precipitates were then immunoblotted by flag antibody. (D) 293T-cells were co-transfected with Myc–PD-L1, HA–Ub and Flag–CSN5 WT or Flag–CSN5 E76A, followed by BBR treatment for 8 h. The ubiquitination of PD-L1 were analyzed by IB. (E) Proposed model of BBR-mediated PD-L1 degradation. BBR specific binds to and subsequently inactivates CSN5, which led to degradation of PD-L1 and activation of tumor-infiltrating T-cells.

Article Snippet: Recombinant human CSN5 (TP300979; Origene, Rockville, MD, USA) was immobilized in parallel-flow channels of CM5 chip by using amine-coupling kit (BR-1000-50; GE Healthcare).

Techniques: Binding Assay, Transfection, Mutagenesis, Incubation, Ubiquitin Proteomics, Activation Assay