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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 human pathogenic NDUFV2 deletion mutant lost most of its mitochondrial targeting ability. (a) Schematic representation of the genomic structure of NDUFV2 in the first three exons. The 4-bp deletion of human pathogenic IVS2+5_+8delGTAA mutation is indicated by underlined red letters (GTAA). Dotted line represents the wild-type splicing form, and continuous line indicates the abnormal splicing form. Schematic structures of c-myc fusion proteins corresponding to the wild-type splicing form and the abnormal splicing form are also shown here. E1, E2 and E3 represent exon 1, exon 2 and exon 3, respectively. (b) The protein distribution patterns of wild-type and NDUFV2 IVS2+5_+8delGTAA mutant. The expressed proteins were labeled by an anti-c-myc-FITC antibody in transfected cells (green color), mitochondria were labeled by Mito Tracker Red (red color), and colocalization of expressed protein and mitochondria is shown as a merged image and indicated by yellow signals. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization. The (++++) symbol indicates all of the FITC fluorescence signals in transfected cells are fully colocalized with mitochondria. Scale bars = 10 μm. (c) Subcellular localization of the wild-type and NDUFV2 pathogenic mutant. Western blot analyses were conducted using cytosolic (Cy) and mitochondrial (Mi) extracts from T-REx-293 cells transiently transfected with the wild-type or NDUFV2 pathogenic mutant construct. (c) The expression levels of the wild-type and the △19-40 NDUFV2 mutant proteins in the whole cell lysates. Western blot analyses were conducted using the whole cell lysates from T-REx-293 cells transiently transfected with the wild-type or the △19-40 NDUFV2 mutant construct. The expressed proteins were detected by an anti-c-myc antibody. β-tubulin was used as a cytosolic marker and ATP synthase subunit α (ATP α) was used as a mitochondrial marker.
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Figure 8: The human pathogenic NDUFV2 deletion mutant lost most of its mitochondrial targeting ability. (a) Schematic representation of the genomic structure of NDUFV2 in the first three exons. The 4-bp deletion of human pathogenic IVS2+5_+8delGTAA mutation is indicated by underlined red letters (GTAA). Dotted line represents the wild-type splicing form, and continuous line indicates the abnormal splicing form. Schematic structures of c-myc fusion proteins corresponding to the wild-type splicing form and the abnormal splicing form are also shown here. E1, E2 and E3 represent exon 1, exon 2 and exon 3, respectively. (b) The protein distribution patterns of wild-type and NDUFV2 IVS2+5_+8delGTAA mutant. The expressed proteins were labeled by an anti-c-myc-FITC antibody in transfected cells (green color), mitochondria were labeled by Mito Tracker Red (red color), and colocalization of expressed protein and mitochondria is shown as a merged image and indicated by yellow signals. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization. The (++++) symbol indicates all of the FITC fluorescence signals in transfected cells are fully colocalized with mitochondria. Scale bars = 10 μm. (c) Subcellular localization of the wild-type and NDUFV2 pathogenic mutant. Western blot analyses were conducted using cytosolic (Cy) and mitochondrial (Mi) extracts from T-REx-293 cells transiently transfected with the wild-type or NDUFV2 pathogenic mutant construct. (c) The expression levels of the wild-type and the △19-40 NDUFV2 mutant proteins in the whole cell lysates. Western blot analyses were conducted using the whole cell lysates from T-REx-293 cells transiently transfected with the wild-type or the △19-40 NDUFV2 mutant construct. The expressed proteins were detected by an anti-c-myc antibody. β-tubulin was used as a cytosolic marker and ATP synthase subunit α (ATP α) was used as a mitochondrial marker.

Mentions: The patients of early-onset hypertrophic cardiomyopathy and encephalopathy were shown to have a homozygous mutation, a 4-bp deletion in intron 2 (IVS2+5_+8delGTAA), in NDUFV2 gene [33]. This mutated gene finally produced a shortened NDUFV2 that lacks 19-40 residues due to a splicing donor site is affected (Figure 8a). The affected patients had a significant complex I deficiency and NDUFV2 missing. In a study using yeast Y. lipolytica as the model, the corresponding amino acids 17-32 from the orthologous NUHM protein have been deleted to mimic the disease condition. However, it was found that the resulting mutant produced a normal amount of NUHM, and this protein was fully assembled into complex I with a normal function [19]. This finding contradicted the situation described for the patients with early-onset hypertrophic cardiomyopathy and encephalopathy and thus prompted us to test the same mutation using the human cell model. The DNA fragment encoded residues 19-40 of NDUFV2 was removed from the wild-type NDUFV2 construct and the resulting plasmid was introduced into T-REx-293 cells for analysis. When confocal microscopy was used for tracking the expressed human disease associated NDUFV2 mutant protein (△19-40 NDUFV2), diffuse fluorescence was present throughout the cytoplasm and only a very limited mitochondrial localization was observed (Figure 8b). To confirm the immunofluorescent results, subcellular fractions prepared from T-Rex-293 cells transiently transfected with the wild-type and human disease mutant NDUFV2 constructs were applied for Western blotting analyses. As controls for proper cytosolic and mitochondrial separation, tubulin and ATP synthase α-subunit was used as a marker for the cytosol and mitochondria, respectively. In accordance with the immunofluorescent results, the wild-type NDUFV2 was found to be localized only in mitochondria whereas the △19-40 NDUFV2 mutant protein was detected mainly in the cytosol (Note: Equal amounts of total protein were loaded in each lane of gel and the △19-40 NDUFV2 mutant was expected to be less concentrated in the cytosol than in mitochondria) (Figure 8c). In addition, the size of △19-40 NDUFV2 (227 amino acids) observed in the Western blotting was slightly larger than that of the mature wild-type NDUFV2 (217 amino acids), implying that the △19-40 NDUFV2 mutant protein was not processed.


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 human pathogenic NDUFV2 deletion mutant lost most of its mitochondrial targeting ability. (a) Schematic representation of the genomic structure of NDUFV2 in the first three exons. The 4-bp deletion of human pathogenic IVS2+5_+8delGTAA mutation is indicated by underlined red letters (GTAA). Dotted line represents the wild-type splicing form, and continuous line indicates the abnormal splicing form. Schematic structures of c-myc fusion proteins corresponding to the wild-type splicing form and the abnormal splicing form are also shown here. E1, E2 and E3 represent exon 1, exon 2 and exon 3, respectively. (b) The protein distribution patterns of wild-type and NDUFV2 IVS2+5_+8delGTAA mutant. The expressed proteins were labeled by an anti-c-myc-FITC antibody in transfected cells (green color), mitochondria were labeled by Mito Tracker Red (red color), and colocalization of expressed protein and mitochondria is shown as a merged image and indicated by yellow signals. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization. The (++++) symbol indicates all of the FITC fluorescence signals in transfected cells are fully colocalized with mitochondria. Scale bars = 10 μm. (c) Subcellular localization of the wild-type and NDUFV2 pathogenic mutant. Western blot analyses were conducted using cytosolic (Cy) and mitochondrial (Mi) extracts from T-REx-293 cells transiently transfected with the wild-type or NDUFV2 pathogenic mutant construct. (c) The expression levels of the wild-type and the △19-40 NDUFV2 mutant proteins in the whole cell lysates. Western blot analyses were conducted using the whole cell lysates from T-REx-293 cells transiently transfected with the wild-type or the △19-40 NDUFV2 mutant construct. The expressed proteins were detected by an anti-c-myc antibody. β-tubulin was used as a cytosolic marker and ATP synthase subunit α (ATP α) was used as a mitochondrial marker.
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Figure 8: The human pathogenic NDUFV2 deletion mutant lost most of its mitochondrial targeting ability. (a) Schematic representation of the genomic structure of NDUFV2 in the first three exons. The 4-bp deletion of human pathogenic IVS2+5_+8delGTAA mutation is indicated by underlined red letters (GTAA). Dotted line represents the wild-type splicing form, and continuous line indicates the abnormal splicing form. Schematic structures of c-myc fusion proteins corresponding to the wild-type splicing form and the abnormal splicing form are also shown here. E1, E2 and E3 represent exon 1, exon 2 and exon 3, respectively. (b) The protein distribution patterns of wild-type and NDUFV2 IVS2+5_+8delGTAA mutant. The expressed proteins were labeled by an anti-c-myc-FITC antibody in transfected cells (green color), mitochondria were labeled by Mito Tracker Red (red color), and colocalization of expressed protein and mitochondria is shown as a merged image and indicated by yellow signals. The number of (+) symbols indicates that the proportion of cells exhibiting FITC fluorescence have a typical punctuated staining pattern and mitochondrial colocalization. The (++++) symbol indicates all of the FITC fluorescence signals in transfected cells are fully colocalized with mitochondria. Scale bars = 10 μm. (c) Subcellular localization of the wild-type and NDUFV2 pathogenic mutant. Western blot analyses were conducted using cytosolic (Cy) and mitochondrial (Mi) extracts from T-REx-293 cells transiently transfected with the wild-type or NDUFV2 pathogenic mutant construct. (c) The expression levels of the wild-type and the △19-40 NDUFV2 mutant proteins in the whole cell lysates. Western blot analyses were conducted using the whole cell lysates from T-REx-293 cells transiently transfected with the wild-type or the △19-40 NDUFV2 mutant construct. The expressed proteins were detected by an anti-c-myc antibody. β-tubulin was used as a cytosolic marker and ATP synthase subunit α (ATP α) was used as a mitochondrial marker.
Mentions: The patients of early-onset hypertrophic cardiomyopathy and encephalopathy were shown to have a homozygous mutation, a 4-bp deletion in intron 2 (IVS2+5_+8delGTAA), in NDUFV2 gene [33]. This mutated gene finally produced a shortened NDUFV2 that lacks 19-40 residues due to a splicing donor site is affected (Figure 8a). The affected patients had a significant complex I deficiency and NDUFV2 missing. In a study using yeast Y. lipolytica as the model, the corresponding amino acids 17-32 from the orthologous NUHM protein have been deleted to mimic the disease condition. However, it was found that the resulting mutant produced a normal amount of NUHM, and this protein was fully assembled into complex I with a normal function [19]. This finding contradicted the situation described for the patients with early-onset hypertrophic cardiomyopathy and encephalopathy and thus prompted us to test the same mutation using the human cell model. The DNA fragment encoded residues 19-40 of NDUFV2 was removed from the wild-type NDUFV2 construct and the resulting plasmid was introduced into T-REx-293 cells for analysis. When confocal microscopy was used for tracking the expressed human disease associated NDUFV2 mutant protein (△19-40 NDUFV2), diffuse fluorescence was present throughout the cytoplasm and only a very limited mitochondrial localization was observed (Figure 8b). To confirm the immunofluorescent results, subcellular fractions prepared from T-Rex-293 cells transiently transfected with the wild-type and human disease mutant NDUFV2 constructs were applied for Western blotting analyses. As controls for proper cytosolic and mitochondrial separation, tubulin and ATP synthase α-subunit was used as a marker for the cytosol and mitochondria, respectively. In accordance with the immunofluorescent results, the wild-type NDUFV2 was found to be localized only in mitochondria whereas the △19-40 NDUFV2 mutant protein was detected mainly in the cytosol (Note: Equal amounts of total protein were loaded in each lane of gel and the △19-40 NDUFV2 mutant was expected to be less concentrated in the cytosol than in mitochondria) (Figure 8c). In addition, the size of △19-40 NDUFV2 (227 amino acids) observed in the Western blotting was slightly larger than that of the mature wild-type NDUFV2 (217 amino acids), implying that the △19-40 NDUFV2 mutant protein was not processed.

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