Overexpression of various HK proteins in HEK293 cells. (A) Plasmid constructs used in our studies. All protein products contain GFP at their C termini. Since only the C-terminal half of HKI (C-HKI) is catalytically active, a mutation (D657A) was introduced in that half to make the enzyme inactive. Both halves of HKII have catalytic activity; thus, mutations in both halves (D209A and D657A) were made. The truncated proteins lack the N-terminal 21-amino-acid hydrophobic region and are unable to bind to the mitochondria. Each individual half of HKI and HKII was also expressed as a GFP fusion protein. M, mitochondrial binding domain; N, N-terminal half; Mito binding, mitochondrial binding; Glc phos, glucose phosphorylation; Trunc, truncated; Mut, mutant. (B) Western blots of extracts from transfected cells. Endogenous protein is at 100 kDa, while exogenously expressed GFP fusion proteins are at 120 kDa. The top panel was probed with HKI, and the bottom panel was probed with HKII antibody. M-HKI and M-HKII, mutant HKI and HKII, respectively; Tr-HKI and Tr-HKII, truncated HKI and HKII, respectively. (C) Confocal images of cells transfected with HK constructs and stained with TMRE. Full-length and mutant HKs localize mostly to mitochondria, while truncated proteins are cytoplasmic. Control cells were sham-transfected cells.

HKI and HKII inhibit cyto c release, Bax translocation into the mitochondria, and the generation of ROS. (A) cyto c release and Bax translocation into the mitochondria in response to H₂O₂ in cells transfected with various HK constructs. Mitochondrial extracts from cells were isolated and membranes were probed with cyto c and Bax antibodies. The levels of mitochondrial cyto c were significantly reduced in GFP-transfected cells with H₂O₂ treatment, while mitochondrial Bax was increased under those conditions. However, the overexpression of full-length HKI and HKII reversed those effects. Truncated and mutant HKI and HKII had partial effects. An antibody against ATP synthase was used as an internal control. (B) Immunofluorescence of control cells and cells overexpressing GFP or various HK constructs stained with cyto c antibody in the presence and absence of H₂O₂. While treatment of control and GFP-transfected cells resulted in cyto c redistribution within the cells, the overexpression of HKs (at least partially) reversed this process. (C) Confocal images of cells treated with various HK constructs and treated with an ROS marker, mitoSOX. While GFP-transfected cells showed an increase in the levels of intracellular ROS in the presence of H₂O₂, this was significantly reduced in cells transfected with HK constructs. The bar graph represents a summary of flow cytometry experiments from mitoSOX studies. Data are represented as mean ± SEM. n = 3 in each group. Abbreviations: Cont., control; FL, full-length; Tr, truncated; M, mutant.

Full-length HKI and HKII protect against H₂O₂-induced cell death in NRCM. (A) Confocal images of NRCM transduced with full-length and truncated HKI and HKII adenoviruses. More than 90% of the cells displayed green fluorescence when the adenovirus was added. (B) Western blots of cells transduced with GFP and various HK adenoviruses. The HK adenoviruses contain GFP and the HK cDNAs under different promoters; thus, the overexpressed HKs are the same size as the endogenous proteins. There are a significantly higher levels of HK in cells transduced with HK adenoviruses compared to GFP-only adenovirus. The top and bottom panels were probed with HKI and HKII antibodies, respectively. (C) Summary data of flow cytometry of NRCM treated with various HK constructs and loaded with TMRE. Data are represented as mean ± SEM. n = 3 in each group. Abbreviations: FL, full-length; Tr, truncated.

Full-length HKI and HKII increase VDAC phosphorylation in a PKCɛ-dependent pathway. (A) Western blots of extracts from HEK293 cells transfected with various HK constructs. The extracts were loaded on a column with VDAC antibody, and the eluate was probed with a phosphothreonine antibody, thus representing phosphorylated VDAC (top). The relative intensity of phosphorylated VDAC to total VDAC in each lane is represented in the bar graph next to the blots. Full-length HKI and HKII resulted in a significant increase in VDAC phosphorylation, while the truncated and mutant forms led to a smaller response. (B) Western blot of cell extracts treated with full-length HKI or HKII in the presence or absence of a PKCɛ inhibitor peptide. The addition of the PKCɛ inhibitor peptide resulted in a significant decrease in VDAC phosphorylation both in the presence and in the absence of HK overexpression, suggesting that VDAC phosphorylation is mediated through PKCɛ. The membranes in each panel were probed for phospho-VDAC (top) or total VDAC (bottom). The bar graphs next to each membrane represent the relative intensity of phosphorylated to total VDAC. Abbreviations: FL, full-length; M, mutant; Tr, truncated; In, inhibitor.

Full-length HKI and HKII protect against H₂O₂-induced cell death, while the truncated and mutant proteins exert partial protection. (A) Confocal images of cells transfected with various HK constructs and loaded with TMRE at baseline and 15 min after the addition of H₂O₂. The first image in each series is a merged image of green and red fluorescence (i.e., TMRE and GFP). While the full-length proteins maintained TMRE in the mitochondria after H₂O₂ treatment, truncated and mutant proteins led to only partial effects. (B) Flow cytometry of transfected cells treated with TMRE showed that H₂O₂ treatment leads to about 35% cell death in GFP-transfected cells. Full-length proteins led to significant protection, while the mutant and truncated proteins exerted only partial protection. Truncated and mutant proteins (i.e., TM-HKI and TM-HKII) did not protect against cell death. (C) Summary of trypan blue exclusion studies of cells transfected with various HK constructs. In accordance with TMRE results, full-length HK proteins protected against cell death, while the mutant and truncated proteins exerted partial protection. (D and E) Time course of cell death in the presence of various HKI and HKII constructs in response to H₂O₂ as assessed by trypan blue staining. Data are represented as mean ± standard error of the mean (SEM). n ≥ 6 in each group, except for TM-HKI and -HKII (n = 3). Abbreviations: Tr, truncated; TM, truncated and mutant; M, mutant F, full length.

Overexpression of individual HK halves results in partial protection. (A) Western blot of extracts from cells overexpressing the N- and C-terminal halves of HKI (N-HKI and C-HKI, respectively) and HKII. The GFP fusion protein of each half yields a protein of about 68 kDa. The left panels represent extracts from cells treated with N- and C-terminal halves of HKI, and the right panels show extracts from cells transfected with N- and C-terminal halves of HKII. (B) Flow cytometry of cells transfected with HKI and HKII halves and loaded with TMRE. The overexpression of each half led to partial protection against H₂O₂-induced cell death. (C) Similar results were obtained when trypan blue staining was used to measure cell death. Data are represented as mean ± SEM. n ≥ 6 in the HK groups.

Full-length HKs possibly protect against MPT in response to H₂O₂. Confocal images of HEK293 cells transfected with HK constructs and loaded with CellTrace calcein blue AM and TMRE. Transfected cells were treated with H₂O₂, and confocal images were obtained at baseline and at 15- and 30-min intervals after the addition of H₂O₂. Results suggest that full-length HKI and HKII may prevent MPT, while the truncated proteins exert a less significant effect. Abbreviations: FL, full-length; Tr, truncated.