Analysis of proteome bound to D-loop region of mitochondrial DNA by DNA-linked affinity chromatography and reverse-phase liquid chromatography/tandem mass spectrometry.
Mitochondrial dysfunction has been suggested as a causal factor for insulin resistance and diabetes. Previously we have shown a decrease of mitochondrial DNA (mtDNA) content in tissues of diabetic patients. The mitochondrial proteins, which regulate the mitochondrial biogensis, including transcription and replication of mtDNA, are encoded by nuclear DNA. Despite the potential function of the proteins bound to the D-loop region of mtDNA in regulating mtDNA transcription/replication, only a few proteins are known to bind the D-loop region of mtDNA. The functional association of these known proteins with insulin resistance is weak. In this study, we applied proteomic analysis to identify a group of proteins (proteome) that physically bind to D-loop DNA of mtDNA. We amplified D-loop DNA (1.1 kb) by PCR and conjugated the PCR fragments to CNBr-activated sepharose. Mitochondria fractions were isolated by both differential centrifugation and Optiprep-gradient ultracentrifugation. The D-loop DNA binding proteome fractions were enriched via this affinity chromatography and analyzed by SDS-PAGE. The proteins on the gel were transferred onto PVDF membrane and the peptide sequences of each band were subsequently analyzed by capillary reverse-phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS). We identified many D-loop DNA binding proteins, including mitochondrial transcription factor A (mtTFA, Tfam) and mitochondrial single-stranded DNA binding protein (mtSSBP) which were known to bind to mtDNA. We also report the possibility of novel D-loop binding proteins such as histone family proteins and high-mobility group proteins.
Choi
Yon-Sik
YS
Asan Institute for Life Sciences, College of Medicine, University of Ulsan, Songpa-Ku Pungnap-Dong 388-1, Seoul, 138-736, Korea.
Ryu
Bo-Kyung
BK
Min
Hye-Ki
HK
Lee
Sang-Won
SW
Pak
Youngmi Kim
YK
D000595:Amino Acid Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D004272:DNA, Mitochondrial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D020543:Proteome