EMBO/soda-nel huggingface dataset:
EZH2 cooperates with gain-of-function p53 mutants to promote cancer growth and metastasis (2019)
Figure 2

Fig 2. EZH2 upregulates p53 expression via binding to the IRES motif in 5'UTR

(A) Biotin pull down assay by incubating biotin-labelled different fragments of p53 mRNA and HOTAIR (positive control) with C4-2 cell lysate followed by western blot with EZH2 antibody.

(B) Biotin pull down assay as in (A) using unmutated and various internally deleted mutants of 5'UTR of p53 mRNA. Top, diagram of different p53 5'UTR deletion mutants.

(C) Upper, the linear map of the pRF bicistronic report plasmid. The SV40 promoter (purple box) was used to drive firefly luciferase (Fluc) and Renilla luciferase (Rluc) gene transcription. Different p53 5'UTR fragments were inserted between the Fluc and Rluc genes. Lower, at 24 h after transfection, cells were lysed and luciferase activities were measured using a dual-luciferase kit and the ratio of Fluc/Rluc was calculated. Data shown as means±SD (n=3). Statistical significance was determined by two-tailed Student's t-test. * P<0.01.

(D) EZH2 knockdown C4-2 cells were transfected with the bicistronic reporter vector in combination with empty vector, Myc-tagged EZH2 WT or deletion mutants followed by western blot analysis with indicated antibodies (bottom) and luciferase assay (top). Data shown as means±SD (n=3). * P<0.01.

(E) C4-2 and U2OS cell lines were transfected with non-specific control (siC) or two independent EZH2-specific siRNAs and harvested for western blot analysis with indicated antibodies. ERK2 and β-TUBULIN, loading controls.

(F) U2OS cells were transfected with control (siC) or EZH2-specific siRNAs and treated with 200 nM of CPT followed by western blot analysis for indicated proteins.

(G) C4-2 cells were transfected with control (siC) or EZH2-specific siRNAs and plasmids for empty vector, EZH2 WT or deletion mutants followed by western blot analysis for indicated proteins.
EZH2 cooperates with gain-of-function p53 mutants to promote cancer growth and metastasis (2019)
Figure 3

Fig 3. EZH2 binds with cap-independent translation complex and increases p53 protein translation

(A) List of top hits of EZH2-bound proteins identified by TAP-MS. The number of peptides and coverage of each protein are indicated.

(B) Reciprocal co-IP of endogenous EZH2 and eIF4G2 or PABP1 in C4-2 cells.

(C) Co-IP of endogenous EZH2 with eIF4G2 or PABP1 from C4-2 cell lysate pre-treated with RNaseA in 37oC for 30 min. The effectiveness of RNaseA treatment was monitored by RT-PCR analysis of the presence of GAPDH mRNA.

(D) C4-2 cells were transfected control (siC) or EZH2-specific siRNA and lysed for polysome fractionation. RNA was extracted from even-number fractions followed by RT-qPCR analysis for p53 and GAPDH mRNA. The β-ACTIN mRNA was used as an internal control. Data shown as means±SD (n=3). Statistical significance was determined by two-tailed Student's t-test. * P<0.01; NS, no significance.

(E) C4-2 cells were transfected with control (siC) or EZH2-specific siRNAs in combination with empty vector, EZH2 WT or different deletion mutants followed by polysome fractionation and RT-qPCR. β-ACTIN mRNA was used as an internal control. Data shown as means±SD (n=3). * P<0.01.
EZH2 cooperates with gain-of-function p53 mutants to promote cancer growth and metastasis (2019)
Figure 4

Fig 4. Ezh2 knockout decreases p53 expression in the Pten-null prostate cancer mouse model

(A) IHC analysis of Pten, Ezh2 and p53 proteins in prostatic tissues of 4-month-old mice with different genotypes: Ptenpc-/- (n=11); Ezh2pc-/- (n=8); Ptenpc-/-;Ezh2pc-/- (n=10) and 'wild-type' littermate controls (n=10). Scale bars, 50 µm.

(B) Western blot analysis of expression of indicated proteins in four different groups of mice as described in (A) (n=2 mice/group).

(C) Analysis of correlation of Ezh2 and p53 protein levels determined by IHC in Pten knockout (Ptenpc-/-) mice (n=11). See IHC scoring details in Methods section.

(D) RT-qPCR analysis of p53 mRNA expression in four different genotypes of mice as described in (A). Statistical significance was determined by two-tailed Student's t-test. * P<0.01.

(E) RT-qPCR analysis of mRNA expression of p53 downstream target gene Bax in four different genotypes of mice as indicated. Statistical significance was determined by two-tailed Student's t-test. * P< 0.01.

(F) H&E analysis of ventral prostate (VP) of 4-month-old mice with indicated genotypes. The inset shows a high magnification image of the representative (framed) area in each panel. Scale bars, 50 µm. Scale bars in insets, 10 µm.

(G) Quantification of nonmalignant, low-grade PIN (LGPIN) and high-grade PIN (HGPIN) or cancerous acini in the prostates (including anterior prostate (AP), ventral prostate (VP) and dorsolateral prostate (DLP)) of mice with the indicated genotypes and number as in (A).
EZH2 cooperates with gain-of-function p53 mutants to promote cancer growth and metastasis (2019)
Figure 5

Fig 5. EZH2 correlates with p53 expression in various types of human cancer

(A) Correlation analysis of EZH2 and p53 mRNA expression measured by RT-qPCR in primary and metastatic prostate cancer specimens of a cohort of patients from University of Washington.

(B) IHC analysis of EZH2 and p53 protein expression in human prostate cancer tissues of a cohort of patients from Mayo Clinic. Case 1 and 2 show low and high expression in primary tissues, respectively; case 3 shows high expression in lymph node metastasis. Scale bar, 50 µm. Scale bars in insets, 10 µm.

(C) Correlation analysis of EZH2 and p53 protein expression in primary and metastatic prostate cancer specimens of the Mayo Clinic cohort.

(D) IHC analysis of EZH2 and p53 protein expression in commercially purchased TMA with patient specimens of brain, colorectal, pancreatic cancer and sarcoma. Scale bars in low magnification images, 200 µm; Scale bars in high magnification images, 50 µm. Arrows, indicated the zoom out-box.

(E) IHC data as shown in (D) were quantified and used for correlation analysis of EZH2 and p53 protein expression in patient specimens of brain, colorectal, pancreatic cancer and sarcoma. Pearson correlation r and P values in each cancer type are indicated.
EZH2 cooperates with gain-of-function p53 mutants to promote cancer growth and metastasis (2019)
Figure 6

Fig 6. EZH2 enhances p53 GOF mutant-mediated cancer growth and metastasis independently of its methyltransferase activity

(A, B) VCaP cells stably expressing control (sh-control) or p53-specific shRNA were infected with lentivirus for empty vector (EV) or deletion mutants of EZH2. Cell growth in 2D (A) and 3D (B) conditions were determined by MTS assay and measurement of clone size, respectively. Statistical significance was determined by two-tailed Student's t-test. * P < 0.05, *** P < 0.001.

(C, D) VCaP cells (1x107) infected with lentivirus as in (A) were injected subcutaneously into NSG mice (n = 8/group). Tumors were measured by caliper twice a week. Tumors at the end point of measurement were isolated and photographed (C), and data are shown as means ± SD (D). Statistical significance was determined by two-tailed Student's t-test for tumors at day 56. *** P < 0.001.

(E-G) Luciferase-expressing MIA PaCa-2 cells (2×106) infected with lentivirus as in (A) were injected via tail vein into NSG mice (n = 6/group). At 12 weeks after injection, mice were subjected to bioluminescent imaging, and images were recorded (E) and bioluminescent signals were quantified (F). Bioluminescent flux (photons s-1 sr-1 cm-2) was determined for lesions in lung, the ends of the box are the upper and lower quartiles and the box spans the interquartile range; the median is marked by a vertical line inside the box; the whiskers are the two lines outside the box that extend to the highest and lowest observations. Lungs were isolated from mice, stained with Bouin's solution, and photographed (G). The white spots on lungs (stained in yellow) are metastatic tumors. * P < 0.05; NS, no significance.
EZH2 cooperates with gain-of-function p53 mutants to promote cancer growth and metastasis (2019)
Figure 7

Fig 7. Depletion of EZH2 expression inhibits growth of p53 GOF-mutated cancer cells

(A) Schematic diagram depicting the distinctive impacts of the functional interplay between EZH2 protein and p53 mRNA on progression of cancers expressing WT p53, no p53 or GOF mutated p53. MTase, methyltransferase.

(B) Kaplan-Meier plots showing the association of EZH2 overexpression with biochemical recurrence of prostate cancer in patients from the TCGA cohort. Tumors were separated into two groups according to EZH2 expression levels (high or low) and three groups based on the mutation status of p53 (WT, loss or mutated).

(C, D) VCaP and C4-2 cells were treated with GSK-126, DZNep (5 µM for VCaP and 10 µM for C4-2), and EZH2 ASO, followed by western blots with indicated antibodies (C) and MTT assay (D). Data shown as means±SD (n=6). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.

(E, F) Effect of Ezh2 ASOs on growth of murine prostate cancer allografts. Mice (n=10) bearing the Ptenpc-/-;Trp53pc-/- (E) and Ptenpc-/-;Trp53pc-/R172H allografts (F) were treated according the scheme shown in (Appendix Fig S2G) with control ASOs (50 mg kg-1) or Ezh2 ASOs at different doses (25 mg kg-1 and 50 mg kg-1). Tumor growth was measured twice a week for 3 weeks and the data are shown in the bottom panels. Each allograft was isolated by the end of ASO treatment and photographed (top panels). Data shown as means±SD (n=10). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.

Expanded View Figure Legends
PEBP1 suppresses HIV transcription and induces latency by inactivating MAPK/NF-κB signaling (2020)
Figure 2

Figure 2

Validation of the candidate genes screened from the lentiCRISPR v2.0 library in HIV-1 latently infected cell lines.

A. Validation of the top candidate genes in the C11 cell line. C11 cells were infected by lentiCRISPR v2.0 packaged lentiviruses with sgRNA following by screening for 14 days with 2 µg/ml puromycin. The percentage of GFP-positive cells was measured by flow cytometry to determine the level of HIV-1 reactivation.

B-C. The effect of candidate genes on HIV latency was further verified in J-Lat 10.6 (B) and ACH2 (C) models of HIV latency. GFP expression in J-Lat 10.6 cells and p24 in ACH2 cells was analyzed by flow cytometry and ELISA, respectively.

D. PEBP1 was deleted after CRISPR/Cas9 knockout. PCR products related to PEBP1 from control or PEBP1 knockout cells were cloned, and then sequenced. PEBP1-sg1 target sites are shown in red letters. Dashes indicate the deleted bases relative to the wild-type PEBP1 gene sequence.

E. PEBP1 protein levels were measured by Western blot after knock out in C11 cells by LvPEBP1-sg1. Mock C11 cells served as control.

Data information: Data represent the mean ± SD of three independent experiments (n=3), and were analyzed with T-test. ***p < 0.001.
PEBP1 suppresses HIV transcription and induces latency by inactivating MAPK/NF-κB signaling (2020)
Figure 3

Figure 3

PEBP1 inhibits HIV-1 reactivation by inactivating Raf1/IKK/NF-κB signaling pathways.

A. PEBP1 expression was measured by qPCR (left panel) and Western blot (right panel) in the latently infected C11 cells and the parental Jurkat cells.

B. The immunoprecipitation assay was performed in C11 cell lysates with anti-PEBP1 antibody followed by Western blot with anti-Raf1 or anti-IKK antibodies.

C-D. Effect of PEBP1 on the Raf1/ERK/IκB and IKK/IκB/NF-κB signaling pathways. The levels of indicated proteins in total protein lysates (C) or the levels of NF-κB/p65 protein in nucleus (D) were analysed by Western blot in C11-PEBP1-KO cells and mock C11 cells.

E. NF-κB/p65 protein recruitment into HIV LTR was analyzed by ChIP-qPCR with specific primers targeting the HIV LTR after normalization to the input.

F. The impact of PEBP1 on the activation of the HIV LTR was explored by luciferase reporter assay in 293T cells. 293T cells were co-transfected with PEBP1-sg1 alone or PEBP1-sg1 with HIV-1 LTR-empty plasmids, HIV-1 wild type LTR-luciferase plasmids, HIV-1 LTR-Δsp1-luciferase, HIV-1 LTR-ΔNF-κB-luciferase, HIV-1 LTR-ΔAp1-luciferase or HIV-1 LTR-ΔYY1-luciferase. Transcription of HIV-1 was determined by luciferase reporter assay.

Data information: Data are normalized to the HIV-LTR-empty plasmid transfection group. Data represent the mean ± SD of three independent experiments (n=3) and were analyzed with T-test. *p < 0.05, ***p < 0.001.
PEBP1 suppresses HIV transcription and induces latency by inactivating MAPK/NF-κB signaling (2020)
Figure 4

Figure 4

PEBP1 restricts HIV-1 transcription to induce HIV latency.

A. The expression of PEBP1 was analysed by Western blot in TZM-bl cells infected with mock, Lv-PCDH-empty or Lv-PCDH-PEBP1 plasmids.

B-C. Overexpression of PEBP1 suppressed HIV-1 replication. TZM-bl cells were transfected with Lv-PCDH-empty or Lv-PCDH-PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies/ml. The transcription of HIV-1 was evaluated 72 h post-infection by luciferase activity (B) and levels of p24 in the supernatants (C).

D. The expression level of PEBP1 was detected by Western blot using the whole cell lysate of C11 cells treated with 10 μM EGCG or DHA for 24 h.

E. Induction of PEBP1 by EGCG or DHA in HIV-1 infected primary CD4+ T cells. Primary CD4+ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV-1 infection. Similar to Panel B, HIV-1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. Seventy-two hours post-treatment with 10 µM EGCG or DHA, the expression of PEBP1 was detected by qPCR.

F. The induction of PEBP1 by EGCG or DHA suppressed HIV replication in the primary CD4+ T cells. The primary CD4+ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV-1 infection. Similar to Panel B and E, HIV-1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. The supernatants from HIV-1-infected CD4+ T cell were collected 1, 2, 3 or 4 days post-infection. Replication of HIV-1 was analyzed by p24 ELISA.

Data information: Data represent the mean ± SD of three independent experiments (n=3) and were analyzed with T-test and compared with TZMBl cells infected with Lv-PCDH-empty vector or mock. **p < 0.01; ***p<0.001.
PEBP1 suppresses HIV transcription and induces latency by inactivating MAPK/NF-κB signaling (2020)
Figure 5

Figure 5

PEBP1 induction by EGCG inhibits the reactivation of latent HIV in primary CD4+ T cells isolated from patients receiving ART.

A. EGCG inhibited α-CD3/CD28 reactivation of latent HIV-1. Primary CD4+ T cells were isolated from HIV-1 positive patients on antiretroviral therapy (n=5, P1-P5). Primary CD4+ T cells were treated with α-CD3/CD28 alone or α-CD3/CD28 and 10 µM EGCG. Cell-associated RNA was extracted 7 days post-treatment. The transcription of HIV-1 was determined by real-time qPCR.

B. PEBP1 mRNA was induced by EGCG in patient primary CD4+ T cells. Primary CD4+ T cells were treated with 10 μM EGCG. The expression of PEBP1 in the cells was measured by qPCR.

C. PEBP1 protein was induced by EGCG in patient primary CD4+ T cells, which was determined by Western blot of whole cell lysate from primary CD4+ T cells.

D. EGCG-induced PEBP1 suppressed nuclear entry of NF-κB/p65. This was measured by Western blot of nuclear protein of primary CD4+ T cells treated with α-CD3/CD28 or α-CD3/CD28 plus 10 μM EGCG.

Data information: Data represent the mean ± SD of three independent experiments (n=3) and were analyzed with T-test. *p < 0.05; **p<0.01, compared with mock treatment.
PEBP1 suppresses HIV transcription and induces latency by inactivating MAPK/NF-κB signaling (2020)
Figure 6

Figure 6

PEBP1 reactivates latent HIV-1 in the primary CD4+ T model of latency.

A. Outline of latency establishment in the primary CD4+ T cells. Human primary CD4+ T cells were activated and expanded with α-CD3/CD28 beads at day 1. The α-CD3/CD28 beads were removed at day 3. Cells were then infected with HIV-1 NL4.3-nanoluc at 3rd day after expansion and maintained over 7 days with a decreasing concentration of IL-2 to establish latency until day 12. At day 12, cells were infected with CRISPR/Cas9 and PEBP1-sgRNA lentivirus.

B. Transcription of HIV-1 in the primary CD4+ T cells was determined by nanoluc luciferase assays during HIV-1 infection.

C. After infection with VSV-G pseudotyped HIV-1 NL4.3-nanoluc, the mRNA expression of PEBP1 was measured by qPCR. D-E. Jurkat CD4+ T cells were treated with IFN-α, IFN-β and IFN-γ for 24 h. The mRNA (A) or protein (B) expression levels of PEBP1 were measured by qPCR or Western blot. F. PEBP1 knockout enhanced HIV-1 transcription in the primary CD4+ T cell model of latency. The expression of HIV-1 was measured by nanoluc after gene knockout where α-CD3/CD28 stimulation served as a positive control.

G. PEBP1 gene deletion after Lv-PEBP1-sg1 knockout in the primary CD4+ T cells. The PCR products of PEBP1 were cloned and then sequenced. PEBP1-sg1 target gene sequences are shown in red letters. Dashes indicate deleted bases relative to the wild-type sequence.

Data information: Data represent the mean ± SD of three independent experiments (n=3) and were analyzed with T-test. *p < 0.05; ***p < 0.001.
The human GID complex engages two independent modules for substrate recruitment (2021)
Figure 1

Figure 1: The hGID complex uses distinct substrate modules to target different substrates. A. Immunoblot of cell extracts following depletion of WDR26, RanBP9, ARMC8, and GID4 using pools of siRNAs for 72 hr in HeLa Kyoto cells. Endogenous levels of the indicated proteins were monitored by western blotting (n=3). B., C. Western blotting of samples after ectopic overexpression of HBP1 (B) or ZMYND19 (C) alone, or together with WDR26 or GID4 in HEK-293T cells. HBP1 and ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3). D. Schematic representation visualizing the hGID E3 ligase complex using two distinct modules for substrate recruitment.
The human GID complex engages two independent modules for substrate recruitment (2021)
Figure 2

Figure 2: Distinct substrate recruitment modules are required to ubiquitinate HBP1 and ZMYND19 in vitro. A. Coomassie-stained SDS-PAGE showing purified hGID sub-complexes used for in vitro ubiquitination assays. The catalytic core composed of MAEA, RMND5a and TWA1 is colored in blue, WDR26 in dark cyan, RanBP9 in light magenta, ARMC8 in dark red and GID4 in orange. B. Coomassie-stained SDS-PAGE showing the purified hGID substrates, HBP1 and ZMYND19. C., D. Western blot analysis of in vitro ubiquitinated HBP1, which was performed by mixing purified HBP1 with ubiquitin E1, UBCH5a, and ubiquitin in the presence of the indicated hGID sub-complexes (n=3). E. Immunoblots of in vitro ubiquitinated ZMYND19, which was performed by mixing purified ZMYND19 with ubiquitin E1, UBE2H, ubiquitin and the 6-subunit hGID complex (ARCM8, RanBP9, WDR26, MAEA, RMND5a and TWA1) in the presence or absence of GID4 and a 10-fold excess of the PGLV GID4-specific peptide (n=2) F. Comparison of the N-terminal sequences of the first five amino acids of the Pro-/N-degron consensus motif (Dong et al. 2020) and human ZMYND19 (Q96E35).
The human GID complex engages two independent modules for substrate recruitment (2021)
Figure 3

Figure 3: WDR26/RanBP9-containing hGID complexes assemble ring-shaped tetramers A. Chromatogram of the SEC-MALS analysis at a flow rate of 0.5 ml/min, showing the UV curve and the Rayleigh ratio (1/cm) at a scattering angle of 90 degrees (left y-axis), together with the molar mass (MDa) of the peaks determined by MALS (right y-axis). The peak fraction with a homogenous size distribution at around 1.1 MDa is labeled with gray dotted lines. B. XL-MS analysis of the 5-subunit hGID complex (RanBP9, WDR26, RMND5a, MAEA and TWA1). Cross-links within different complex subunits are indicated by green lines, and cross-links within the same subunit with purple lines. The predicted domain boundaries of the different subunits are colored as follows: LisH domain in light orange, CTLH domain in dark orange, RING domains in blue, TWA1's CRA domain in light blue and RanBP9's CRA domain in light grey, WD40 in dark cyan, and SPRY in light magenta. C. Rotational views of the cryo-EM map of the 5-subunit hGID complex (RanBP9, WDR26, RMND5a, MAEA and TWA1) at 11.2 Å resolution. The higher resolution cryo-EM map at 9 Å produced by particle symmetry expansion is shown in blue. The dotted rectangle highlights the positions of the fitted WD40 domains from two tetrameric building blocks. D. Western blotting of samples following ectopic overexpression of HBP1 either alone, or with full-length (FL) or WD40-truncated WDR26 (ΔWD40) in HEK-293T cells. HBP1 levels were monitored in cells treated with MG132 or DMSO for 12-14 h (n=3). E. Western blot analysis of in vitro ubiquitinated HBP1 in the presence of wild type WDR26 (WT) or the WDR26 (ΔWD40) mutant (n=2).
The human GID complex engages two independent modules for substrate recruitment (2021)
Figure 4

Figure 4: ARMC8α but not ARMC8β recruits GID4 to the core complex in an assembly that does not prevent binding of the WDR26/RanBP9 module. A. Schematic representation of ARMC8α (Q8IUR7-1), ARMC8β (Q8IUR7-6) and GID4 (Q8IVV7-1) proteins. The binding site of GID4 and the C-terminus of ARMC8α is indicated. B. Western blot analysis showing the levels of ARMC8α and ARMC8β in HeLa Kyoto cells treated for 72 hr with control siRNA or siRNA pools against ARMC8 (n=3). C. Transiently expressed FLAG-GID4 and HSS-tagged ARMC8 isoforms (α or β) in HEK293T cells. The presence of GID4 in isoform-specific ARMC8 immunoprecipitates was visualized by immunoblotting (left panels). The right panel shows a western blot of transiently expressed and immunoprecipitated HSS-WDR26 from HEK-293T cells and the presence of endogenous ARMC8 isoforms (α or β) was probed by immunoblotting (n=2). D. Baculoviral co-expression in Sf9 cells of the 5-subunit hGID complex (5mer; Strep-RanBP9, His-WDR26, FLAG-MAEA, His-RMND5a and His-TWA1) along with His-GID4 in the presence of ARMC8α or ARMC8β. Strep- or His-pulldowns revealed the presence of GID4 in ARMC8α, but not in ARMC8β, complexes (n=3). E. Immunoblot analysis of in vitro ubiquitinated GID4-hGID complexes containing either ARMC8α or ARMC8β (6mer; ARMC8, RanBP9, WDR26, MAEA, RMND5a and TWA1) . Where indicated, the reaction was carried out in the presence of 20-fold molar excess of the PGLV GID4-binding peptide (n=3). F. Western blot analysis of in vitro ubiquitinated HBP1 by the hGID 5-subunit complex in the presence of the different ARMC8 isoforms (α or β) (n=2). G. Schematic representation illustrating that in contrast to ARMC8α, incorporation of ARMC8β prevents hGID activity towards GID4 substrates. H. XL-MS analysis of the 6-subunit hGID complex (RanBP9, WDR26, RMND5a, MAEA, TWA1 and ARMC8β). Cross-links within the different complex subunits are indicated by green lines, and cross-links within the same subunit by purple lines. The predicted domain boundaries within the different subunits are colored as follows: LisH domain in light orange, CTLH domain in dark orange, RING domains in blue, TWA1's CRA domain in light blue and RanBP9's CRA domain in light grey, WD40 in dark cyan, ARMC8β in dark red, and the SPRY domain in light magenta. I. Comparison of the cryo-EM maps of the 5-subunit hGID complex (RanBP9, WDR26, RMND5a, MAEA and TWA1) and the 6-subunit hGID complex (RanBP9, WDR26, RMND5a, MAEA, TWA1 and ARMCβ). J. A difference map (red) shows the extra density in the 6-subunit hGID complex corresponding to ARMC8β.