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Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy.

Liu HY, Liao PC, Chuang KT, Kao MC - J. Biomed. Sci. (2011)

Bottom Line: A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein.The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability.The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan, People's Republic of China.

ABSTRACT

Background: NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2), containing one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), is one of the core nuclear-encoded subunits existing in human mitochondrial complex I. Defects in this subunit have been associated with Parkinson's disease, Alzheimer's disease, Bipolar disorder, and Schizophrenia. The aim of this study is to examine the mitochondrial targeting of NDUFV2 and dissect the pathogenetic mechanism of one human deletion mutation present in patients with early-onset hypertrophic cardiomyopathy and encephalopathy.

Methods: A series of deletion and point-mutated constructs with the c-myc epitope tag were generated to identify the location and sequence features of mitochondrial targeting sequence for NDUFV2 in human cells using the confocal microscopy. In addition, various lengths of the NDUFV2 N-terminal and C-terminal fragments were fused with enhanced green fluorescent protein to investigate the minimal region required for correct mitochondrial import. Finally, a deletion construct that mimicked the IVS2+5_+8delGTAA mutation in NDUFV2 gene and would eventually produce a shortened NDUFV2 lacking 19-40 residues was generated to explore the connection between human gene mutation and disease.

Results: We identified that the cleavage site of NDUFV2 was located around amino acid 32 of the precursor protein, and the first 22 residues of NDUFV2 were enough to function as an efficient mitochondrial targeting sequence to carry the passenger protein into mitochondria. A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein. The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability.

Conclusions: The mitochondrial targeting sequence of NDUFV2 is located at the N-terminus of the precursor protein. Maintaining a net positive charge and an amphiphilic structure with the overall balance and distribution of basic and hydrophobic amino acids in the N-terminus of NDUFV2 is important for mitochondrial targeting. The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.

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The α-helical wheel diagram of the first 32 amino acids of NDUFV2. The α-helical wheel model for the first 32 residues of NDUFV2 was constructed using Helical wheel projections [39]. The output presents the hydroxylated residues as yellow circles, hydrophobic residues as green diamonds, potentially basic (or positively charged) residues as blue pentagons, and the remaining residues as grey circles.
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Figure 5: The α-helical wheel diagram of the first 32 amino acids of NDUFV2. The α-helical wheel model for the first 32 residues of NDUFV2 was constructed using Helical wheel projections [39]. The output presents the hydroxylated residues as yellow circles, hydrophobic residues as green diamonds, potentially basic (or positively charged) residues as blue pentagons, and the remaining residues as grey circles.

Mentions: As shown in Figure 1a, the first 1-32 amino acids which we just demonstrated to function as the MTS have a net positive charge (contributing by 4 arginines, 1 lysine, 3 histidines, and the N-terminal methionine) but no acidic amino acids. Based on the Eisenberg method of hydrophobic moment calculation with Hmoment server [37], the MTS of NDUFV2 had a hydrophobic region roughly in the middle of the presequence. The secondary structure prediction using PSIPRED server [38] indicated that the first 1-32 residues of NDUFV2 contain two α-helical structures (one in residues 4-16, the other in residues 22-30) with one short coil structure in between (Figure 1b). When Helical Wheel Projections program [39] was applied to construct the α-helical wheel model for the N-terminus of NDUFV2, it was clear that the N-terminal region of NDUFV2 contains a typical amphiphilic structure with hydrophobic residues on one side and polar residues on the other side of the α-helix (Figure 5).


Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy.

Liu HY, Liao PC, Chuang KT, Kao MC - J. Biomed. Sci. (2011)

The α-helical wheel diagram of the first 32 amino acids of NDUFV2. The α-helical wheel model for the first 32 residues of NDUFV2 was constructed using Helical wheel projections [39]. The output presents the hydroxylated residues as yellow circles, hydrophobic residues as green diamonds, potentially basic (or positively charged) residues as blue pentagons, and the remaining residues as grey circles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117770&req=5

Figure 5: The α-helical wheel diagram of the first 32 amino acids of NDUFV2. The α-helical wheel model for the first 32 residues of NDUFV2 was constructed using Helical wheel projections [39]. The output presents the hydroxylated residues as yellow circles, hydrophobic residues as green diamonds, potentially basic (or positively charged) residues as blue pentagons, and the remaining residues as grey circles.
Mentions: As shown in Figure 1a, the first 1-32 amino acids which we just demonstrated to function as the MTS have a net positive charge (contributing by 4 arginines, 1 lysine, 3 histidines, and the N-terminal methionine) but no acidic amino acids. Based on the Eisenberg method of hydrophobic moment calculation with Hmoment server [37], the MTS of NDUFV2 had a hydrophobic region roughly in the middle of the presequence. The secondary structure prediction using PSIPRED server [38] indicated that the first 1-32 residues of NDUFV2 contain two α-helical structures (one in residues 4-16, the other in residues 22-30) with one short coil structure in between (Figure 1b). When Helical Wheel Projections program [39] was applied to construct the α-helical wheel model for the N-terminus of NDUFV2, it was clear that the N-terminal region of NDUFV2 contains a typical amphiphilic structure with hydrophobic residues on one side and polar residues on the other side of the α-helix (Figure 5).

Bottom Line: A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein.The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability.The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan, People's Republic of China.

ABSTRACT

Background: NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2), containing one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), is one of the core nuclear-encoded subunits existing in human mitochondrial complex I. Defects in this subunit have been associated with Parkinson's disease, Alzheimer's disease, Bipolar disorder, and Schizophrenia. The aim of this study is to examine the mitochondrial targeting of NDUFV2 and dissect the pathogenetic mechanism of one human deletion mutation present in patients with early-onset hypertrophic cardiomyopathy and encephalopathy.

Methods: A series of deletion and point-mutated constructs with the c-myc epitope tag were generated to identify the location and sequence features of mitochondrial targeting sequence for NDUFV2 in human cells using the confocal microscopy. In addition, various lengths of the NDUFV2 N-terminal and C-terminal fragments were fused with enhanced green fluorescent protein to investigate the minimal region required for correct mitochondrial import. Finally, a deletion construct that mimicked the IVS2+5_+8delGTAA mutation in NDUFV2 gene and would eventually produce a shortened NDUFV2 lacking 19-40 residues was generated to explore the connection between human gene mutation and disease.

Results: We identified that the cleavage site of NDUFV2 was located around amino acid 32 of the precursor protein, and the first 22 residues of NDUFV2 were enough to function as an efficient mitochondrial targeting sequence to carry the passenger protein into mitochondria. A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein. The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability.

Conclusions: The mitochondrial targeting sequence of NDUFV2 is located at the N-terminus of the precursor protein. Maintaining a net positive charge and an amphiphilic structure with the overall balance and distribution of basic and hydrophobic amino acids in the N-terminus of NDUFV2 is important for mitochondrial targeting. The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.

Show MeSH
Related in: MedlinePlus