Evidence for msiCAT-tailing on mitochondrial proteins in GBM cells

(A) RQC gene expression levels in GBM tumor tissues (n=153) compared to normal brain tissues (n=206) (unpaired Student’s t-test; *, logFC (fold change)>1; adj.P.Val<0.001). (B) Western blot analysis of msiCAT-tailed mitochondrial proteins and RQC factors in patient-derived GSC and control NSC cells, using ACTIN as the loading control. Red arrowheads indicate short CAT-tailed mitochondrial proteins; “short” and “long” refer to exposure time; the red numbers represent fold changes compared to controls (NSC). (C) Western blot of 5×FLAG-tagged β-globin reporter proteins in GBM and control cells, showing more CAT-tailed proteins in GBM cells, using ACTIN as the loading control. The red numbers represent fold changes compared to controls (NHA without any treatment); the purple numbers represent the ratio of red (CAT-tailed) to green (non-CAT-tailed) sections. (D) Western blot of overexpressed ATP5α-AT3 and ATP5α-AT20 in GBM and control cells, using GAPDH as the loading control; arrowheads indicate endogenous ATP5α, ATP5α-AT3, ATP5α-AT20, and oligomers/aggregates of msiCAT-tailed ATP5α proteins. The purple numbers represent the ratio of red (exogenous) to green (endogenous) sections. (E) Immunofluorescence staining shows endogenous ATP5α protein aggregates in GBM cells, with TOM20 (red) as a mitochondrial marker. White arrows indicate ATP5α protein aggregates. (F) Quantification of E (n=3; chi-squared test; ***, P < 0.001; ****, P < 0.0001); the total number of cells counted is indicated in the columns.

Impact of msiCAT-tailed ATP5IZ proteins on mitochondrial functions in GBM cells

(A) TMRM staining shows a high mitochondrial membrane potential in patient-derived GSC cells (n=3; unpaired Student’s t-test; ***, P < 0.001; ****, P < 0.0001). (B) ATP measurement shows a low mitochondrial ATP production in patient-derived GSC cells (n=3; unpaired Student’s t-test; **, P < 0.01; ***, P < 0.001). (C, D) JC-10 staining reveals a reduced mitochondrial membrane potential in GBM cells, but not in NHA control cells, upon both genetic (C) and pharmacological (D) inhibition of the msiCAT-tailing pathway (n=3; unpaired Student’s t-test; ***, P < 0.001; ****, P < 0.0001; ns, not significant). (E) JC-10 staining reveals an increased mitochondrial membrane potential in GBM cells, but not in control cells, upon overexpression of ATP511-AT3 and ATP511-AT20 (n=3; unpaired Student’s t-test; ****, P < 0.0001; ns, not significant). (F) Western blot of FLAG-tagged ATP511, NEMF, and ANKZF1 in GBM cells and control cells, using ACTIN as the loading control. (G) JC-10 staining reveals an increased mitochondrial membrane potential in GBM cells, but not in NHA control cells, upon overexpression of ATP511-AT3 and ATP511-AT20 with concurrent genetic inhibition of the endogenous msiCAT-tailing pathway (n=3; unpaired Student’s t-test; *, P < 0.05; **, P < 0.01). (H) BN-PAGE western blot of ATP511 and Flag shows that ATP511-AT3 is incorporated into the mitochondrial Complex-V (ATP synthase), while ATP511-AT20 forms high molecular weight protein aggregates in GBM cells. SC: respiratory supercomplex; C-V: Complex-V/ATP synthase. (I, K) Oxygen consumption rate (OCR) data indicate a reduction in mitochondrial oxygen consumption in SF268 cells expressing ATP511-AT3 and ATP511-AT20. Oligomycin (1.5 µM), FCCP (1.0 µM), and rotenone/antimycin A (R/A, 0.5 µM) were sequentially added. (J, L) Statistics of mitochondrial respiration parameters in (I, K), including non-mitochondrial respiration, basal respiration, maximum respiration, spare respiration, proton leaks, and ATP production (n=3; unpaired Student’s t-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).

msiCAT-tailing product regulates MPTP status in GBM cells

(A, C) MPTP activity assay shows reduced mitochondrial permeability transition pore opening in GSC cells compared to NHA (control) cells. Pharmacological (A, anisomycin 200 nM) and genetic (sgNEMF) inhibition of CAT-tailing reverse it. (B, D) Quantification of (A, C) (n=3; unpaired Student’s t-test; ****, P < 0.0001; ns, not significant). (E, G) Immunofluorescence staining shows that anisomycin treatment (E) and sgNEMF (G) inhibit endogenous ATP511 protein aggregation in GBM cells, using TOM20 (red) as a mitochondrial marker. (F, H) Quantification of (E, G) (n=3; chi-squared test; *, P < 0.05; ***, P < 0.001); the total number of cells counted is indicated in the columns. (I) Calcium retention capacity (CRC) assay of isolated mitochondria, measured with Calcium Green-5N dye, shows high CRC in GBM cells compared to control NHA cells. CsA (Cyclosporin A, MPTP inhibitor) serves as a positive control. (J) Statistic of (I) shows attenuated CRC in mitochondria pretreated with anisomycin or with sgNEMF (n=3; unpaired Student’s t-test; ***, P < 0.001; ****, P < 0.0001). (K) MPTP activity assay shows that ectopic expression of ATP511-AT3 and ATP511-AT20 inhibits MPTP opening in GBM cells. (L) Quantification of (K) (n=3; unpaired Student’s t-test; ****, P < 0.0001). (M) BN-PAGE western blot shows that ATP511-AT3 and ATP511-AT20 expression alters ANT1/2 protein patterns in GBM cells, resulting in a missing band (circled in yellow dashed line), and formation of high molecular weight aggregates. SC: respiratory supercomplex; C-V: Complex V/ATP synthase.\

msiCAT-tailed ATP5IZ protein promotes GBM progression

(A) MTT assay indicates increased proliferation caused by ATP511-AT3 and ATP511-AT20 expression in GBM cells (n=3; unpaired Student’s t-test; **, P < 0.01; ***, P < 0.001). (B) MTT assay indicates no change in proliferation caused by ATP511-AT3 and ATP511-AT20 expression in NHA cells (n=3; unpaired Student’s t-test; ns, not significant). (C) Transwell assay reveals enhanced migration induced by ATP511-AT3 and ATP511-AT20 expression in GBM (SF) cells but not in control (NHA) cells. (D) Quantification of (C) shows the number of migrated cells (n=3; unpaired Student’s t-test; ****, P < 0.0001; ns, not significant). (E) MTT assay indicates an increased proliferation in GBM cells, upon overexpression of ATP511-AT3 and ATP5-AT20 with concurrent genetic inhibition of the endogenous msiCAT-tailing pathway (n=3; unpaired Student’s t-test; *, P < 0.05; **, P < 0.01). (F) Transwell assay reveals enhanced migration upon overexpression of ATP511-AT3 and ATP511-AT20 with concurrent genetic inhibition of the endogenous msiCAT-tailing pathway. (G) Quantification of (F) shows the number of migrated cells (n=3; unpaired Student’s t-test; ***, P < 0.001; ****, P < 0.0001). (H) TUNEL staining shows that staurosporine (STS, 1 µM)-induced apoptosis is attenuated by ATP511-AT3 and ATP511-AT20 expression in GBM cells, using TUNEL-Cy3 as an apoptotic cell indicator and DAPI as a nucleus indicator. (I) Quantification of (H) shows the percentage of TUNEL-positive cells in the population (n=3; unpaired student’s t-test; ***, P < 0.001), using DMSO as the vehicle control. (J) MTT assay indicates an enhanced resistance to temozolomide (TMZ, 150 µM) induced by ATP511-AT3 and ATP511-AT20 expression. The TMZ-treated/SF-Ctrl group is used as the control (n=3; unpaired Student’s t-test; ***, P < 0.001).

Inhibition of msiCAT-tailing impedes GBM progression

(A) Cell viability assay shows greater sensitivity to anisomycin treatment in patient-derived GSC cells than control NSC cells at 48 hours (n=3; unpaired Student’s t-test; ***, P < 0.001; ****, P <0.0001; compared to controls at the corresponding dose). (B) MTT assay indicates reduced GBM cell proliferation by genetic inhibition of the msiCAT-tailing pathway (n=3; unpaired Student’s t-test; **, P < 0.01; ***, P < 0.001; ****, P <0.0001, compared to controls at the corresponding time). (C) MTT assay indicates reduced NHA cell proliferation by genetic inhibition of the msiCAT-tailing pathway (n=3; unpaired Student’s t-test; **, P < 0.01; ****, P <0.0001, compared to controls at the corresponding time). (D, F) Transwell assay reveals that both genetic (D) and pharmacological (F) inhibition of the msiCAT-tailing pathway hampers the migration of GBM cells but not control cells. (E, G) Quantification of (D, F) showing the number of migrated cells (n=3; unpaired Student’s t-test; ***, P < 0.001; ****, P <0.0001; ns, not significant). (H, J) TUNEL staining shows that both genetic (H) and pharmacological (J) inhibition of the msiCAT-tailing pathway enhances STS-induced apoptosis in GBM cells, using TUNEL-Cy3 as an apoptotic cell indicator and DAPI as a nucleus indicator. (I, K) Quantification of (H, J) showing the percentage of TUNEL-positive cells in the population (n=3; unpaired Student’s t-test; **** P < 0.0001), using DMSO as the vehicle control. (L) MTT assay shows that pharmacological inhibition of the msiCAT-tailing pathway decrease the resistance of GBM cells to temozolomide (TMZ, 150 µM) treatment (n=3; unpaired Student’s t-test; * P < 0.05; **, P < 0.01). (M) Neurosphere formation assay shows that reduced spheroid formation caused by pharmacological inhibition of the msiCAT-tailing pathway can synergize with TMZ in GBM cells. (N) Quantification of (M) (n=3; unpaired Student’s t-test; **, P < 0.01).

Impact of msiCAT-tail modified ATP5α protein on mitochondrial function in GBM cells

In healthy cells, ATP5α protein, encoded by the nuclear genome, is imported into the mitochondrial matrix via the TOM/TIM complex through co-translational import and incorporated into ATP synthase (Left). Conversely, in GBM cells, the CAT-tailed ATP5α protein can either form aggregates near the mitochondrial outer membrane or be imported into the mitochondria. Within the mitochondrial matrix, proteins with shorter CAT-tails readily integrate into ATP synthase, disrupting its functionality. This dysfunction is characterized by a reduced ATP synthesis rate and proton (H+) accumulation, leading to an elevated mitochondrial membrane potential (ΔΨm). These alterations in ATP synthase ultimately trigger malfunction of the MPTP, consequently affecting cell proliferation, migration, and resistance to drug-induced apoptosis (Right).