Figure 7. Overexpression of HKIII increases expression of markers associated with mitochondrial biogenesis and mito- chondrial DNA content. (A) HKIII overexpression lead to significant increases in the expression of NRF-1, NRF-2a, and PGC-1a, which regulate mitochondrial biogenesis. (B) Genes encoding regulatory elements of the TCA cycle, fatty acid transport, and b-oxidation were also upregulated after HKIII overexpression. Data represent transcript levels first normalized to an internal housekeeping control and presented relative to GFP expression. (C) HEK293 cells were transfected with HKIII-GFP or GFP; 48 hours later, changes in mitochondrial DNA content were determined by measuring the levels of the mitochondrial encoded gene CO1 and the nuclear encoded 18S gene. Overexpression of HKIII significantly increased the ratio of mitochondrial encoded to nuclear-encoded DNA. *P,0.05. Data are presented as mean6SEM, N = 3. CO1: cytochrome c oxidase subunit 1; Cpt1-b: Carnitine O- palmitoyltransferase I, muscle isoform; CS: citrate synthase; MCAD: Medium-chain acyl-CoA dehydrogenase; NRF: Nuclear Regulatory Factor 1,2a; PDK4: pyruvate dehydrogenase kinase 4; PGC-1a: Peroxisome proliferator-activated receptor gamma coactivator-1 alpha. doi:10.1371/journal.pone.0013823.g007 HK3 Regulation and Protection The N-terminal 32-amino-acid domain of HKIII is required for proper protein expression Previous studies have shown that the binding of HKI and HKII to mitochondria protects against cell death [27]. Thus, we investigated whether the protective effects of HKIII, which is a cytoplasmic protein, could be enhanced by replacing its N-terminal 32 amino acids (encoded by exon 1b of HKIII) with the 21-amino- acid mitochondrial-targeting (Mt) sequence of HKII (encoded by exon 1 of HKII) (Figure 8A). Overexpression of the chimeric Mt- HKIII protein (containing the HKII mitochondrial-targeting sequence in place of the N-terminal 32 amino acids of HKIII) did not lead to co-localization with the mitochondria, which was observed with overexpression of HKII (Figure 8B) [27]. In addition, overexpression of the mitochondrial targeted chimera (Mt-HKIII) led to the formation of protein aggregates (Figure 8B). To determine whether aggregation was caused by the removal of the native HKIII 32amino acid sequence, or by the addition of the mitochondrial- targeting sequence of HKII, we deleted the native sequence only (Figure 8A). Overexpression of this truncated construct (Tr-HKIII) also caused protein aggregation (Figure 8B), suggesting that the N- terminal 32 amino acids of HKIII are essential for proper folding of the protein. Consistent with these findings, overexpression of the C- terminal domain of HKIII formed aggregates, whereas the N- terminal domain was soluble, and its cytosolic expression was similar to that of the full-length protein (Figure S5A and B). In an attempt to identify specific N-terminal HKIII residues required to maintain solubility, we progressively deleted 8-amino- acid sequences in this region, and expressed these mutants in HEK293 cells (Figure S6A). The overexpressed proteins were soluble; indicating that partial truncation of this N-terminal region does not result in aggregate formation (Figure S6B). Western blot demonstrating protein expression in HEK293 cell lysates is shown for all constructs in Figure S7A. Overall, these results suggest that the N-terminal 32-amino-acid sequence of HKIII is necessary to maintain solubility and proper expression of the protein. Discussion Three 100-kDa HK isozymes are expressed in mammalian tissue [7,8,27,38,39]. The regulation, localization, activity, and cytoprotective capacity of HKI and HKII have been extensively studied. [10–17,21,22,27]. However, the regulation of HKIII and its potential cytoprotective role are not well characterized. Here, we show that: 1) HKIII is regulated by hypoxia through a HIF- dependent pathway, 2) glucose and G6P binding to the N-terminal domain regulates the catalytic activity of the C-terminal domain, 3) overexpression of HKIII protects cells from oxidant-induced cell death, and 4) HKIII overexpression increases cellular ATP levels, reduces ROS production, preserves mitochondria membrane potential, and promotes the expression of genes that regulate mitochondrial biogenesis. The latter results suggest that the protective effects of HKIII may evolve through a decline in ROS levels and increases in ATP and mitochondrial biogenesis. Our results also demonstrate that the 32 amino acids encoded by exon 1b of HKIII and located at the protein’s N-terminus appear to be essential for proper folding, because deletion of these residues led to protein aggregation. Previous studies have identified distinct differences between the regulation of HKI and HKII [7,8]. Analysis of the HKI promoter, as well as the ubiquitous tissue expression of this isoform, indicate that HKI is constitutively expressed, while HKII expression is tissue specific and regulated by stimuli such as insulin and hypoxia [7,30,31,32,39,40]. HKIII is primarily expressed in the lung, kidney, and liver [7,9,41], and an Oct-1 binding site is necessary PLoS ONE | www.plosone.org 9 November 2010 | Volume 5 | Issue 11 | e13823