Fig 2: DIRAS family GTPase 2 (DIRAS2) interacts with 26S proteasome non-ATPase regulatory subunit 2 (PSMD2). (A) Flow chart of liquid chromatography-tandem mass spectrometry (LC-MS). (B) oe-DIRAS2 RKO cells and their controls were harvested and immunoprecipitated. Coomassie blue staining of the image before LC-MS detection is shown. Mass spectrometric results show the top 10 proteins according to protein score. (C) The interaction between DIRAS2 and PSMD2 proteins in RKO and HEK293 cells. (D) Immunofluorescent staining of DIRAS2 and PSMD2 in RKO and HEK293 cells.

Fig 3: The tumor-suppressing activity of DIRAS family GTPase 2 (DIRAS2) can be overridden by 26S proteasome non-ATPase regulatory subunit 2 (PSMD2). (A) DIRAS2 and PSMD2 were identified by western blot in RKO cells respectively transfected with control-vector, Flag-tagged DIRAS2, Ha-PSMD2, or both. (B) The effect of PSMD2 and DIRAS2 on cellular proliferation in RKO cells. (C) The impact of PSMD2 and DIRAS2 on cellular growth in RKO cells. (D) The correlation between DIRAS2 and PSMD2 expression. (E) A schematic diagram of DIRAS2 inhibiting cell growth through nuclear factor kappa light-chain enhancer of activated B-cells signaling pathways and being degraded by PSMD2 in a proteasome-mediated way (all *P<.05, ***P<.001).

Fig 4: The structure of PSMD2 reveals a new ligand binding site.a, Structure of PSMD2 in complex with two Fabs and macrocycle MC1. Macrocycle MC1 (orange) binds between C-terminal and solenoid domains of PSMD2 (teal). PSMD2-interacting Fab chaperones are shown in gray. b, Ribbon diagram of PSMD2 (teal) with MC1 shown as orange sticks; CTD, C-terminal domain; NTD, N-terminal domain. c, Close-up of the binding site shows residues HRYxGW of macrocycle MC1 interact with K773, L873, E336, H868, E878, V846, H770 and N737 on PSMD2.

Fig 5: Validation by PCR and immunoblotting of a clone with a knock-in biotin tag at the hRpn1 C terminus.A, the binding site of each primer pairing in the PSMD2-edited (left) and PSMD2 parental (right) region is displayed including (1) Biotin-FP1 and 3′-UTR (purple) (2), FP101 and Biotin-RP1 (orange), and (3) FP101 and 3′-UTR (pink) with anticipated amplicon size. The measured amplicon size for each primer set is listed for parental, C1, and C4 in a table to the right. ‘X’ indicates not detected. B–D, representative agarose gel image showing the PCR amplicon generated from (B) primer set 1, (C) primer set 2, and (D) primer set 3, with inclusion of a 1-kilobase-pair (kbp) and 100 base-pair (bp) DNA ladder, as indicated. E, immunoblotting of the whole cell extract from C1 to C6 along with parental WT cells by anti-hRpn1 antibody (left) and HRP-conjugated streptavidin antibody (right). β-actin was also probed as a loading control. The bands corresponding to hRpn1 and hRpn1-biotin are labeled. HRP, horseradish peroxide, UTR, untranslated region.