- 作者: Hsin-Yu Liu; Pin-Chao Liao; Kai-Tun Chuang; Mou-Chieh Kao
- 作者服務機構: Institute of Molecular Medicine & Department of Life Science, National Tsing Hua University, 101, Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan, R.O.C.
- 中文摘要: --
- 英文摘要:
Background: NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2), containing one
iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), is one of the core nuclearencoded
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. - 中文關鍵字: --
- 英文關鍵字: --