Pig Mitochondria Research Articles

Mitochondrial DNA enrichment reduced NUMT contamination in porcine NGS analyses.

Genetic associations between mitochondrial DNA (mtDNA) and economic traits have been widely reported for pigs, which indicate the importance of mtDNA. However, studies on mtDNA heteroplasmy in pigs are rare. Next generation sequencing (NGS) methodologies have emerged as a promising genomic approach for detection of mitochondrial heteroplasmy. Due to the short reads, flexible bioinformatic analyses and the contamination of nuclear mitochondrial sequences (NUMTs), NGS was expected to increase false-positive detection of heteroplasmy. In this study, Sanger sequencing was performed as a gold standard to detect heteroplasmy with a detection sensitivity of 5% in pigs and then one whole-genome sequencing method (WGS) and two mtDNA enrichment sequencing methods (Capture and LongPCR) were carried out. The aim of this study was to determine whether mitochondrial heteroplasmy identification from NGS data was affected by NUMTs. We find that WGS generated more false intra-individual polymorphisms and less mapping specificity than the two enrichment sequencing methods, suggesting NUMTs indeed led to false-positive mitochondrial heteroplasmies from NGS data. In addition, to accurately detect mitochondrial diversity, three commonly used tools-SAMtools, VarScan and GATK-with different parameter values were compared. VarScan achieved the best specificity and sensitivity when considering the base alignment quality re-computation and the minimum variant frequency of 0.25. It also suggested bioinformatic workflow interfere in the identification of mtDNA SNPs. In conclusion, intra-individual polymorphism in pig mitochondria from NGS data was confused with NUMTs, and mtDNA-specific enrichment is essential before high-throughput sequencing in the detection of mitochondrial genome sequences.

The occurrence of l-lactate dehydrogenase in the inner mitochondrial compartment of pig liver

Although pig represents a model species in biomedical research including studies dealing with liver patho-physiology, some aspects of liver metabolism need to be addressed. In particular, whether and how pig mitochondria can metabolize l-lactate remains to be established. We show here that pig liver mitochondria (PLM) possess their own l-lactate dehydrogenase (mL-LDH). This was shown both via immunological analysis and by assaying photometrically the L-LDH reaction in solubilised PLM. The mL-LDH reaction shows hyperbolic dependence on the substrate concentration, it is inhibited by oxamate and proves to differ from the cytosolic activity (cL-LDH), as revealed by the difference found in both pH profiles and temperature dependence of m- and cL-LDH. Titration experiments with digitonin show that mL-LDH is restricted in mitochondrial inner compartment. In agreement with the above findings, three genes in Sus scrofa genome encoded for L-LDH subunits which are predicted to have mitochondrial localization, as investigated by Target P 1.1 and PredSL analysis.

EST analysis on pig mitochondria reveal novel expression differences between developmental and adult tissues

BackgroundThe mitochondria are involved in many basic functions in cells of vertebrates, and can be considered the power generator of the cell. Though the mitochondria have been extensively studied there appear to be only few expression studies of mitochondrial genes involving a large number of tissues and developmental stages. Here, we conduct an analysis using the PigEST resource [1] which contains expression information from 35 tissues distributed on one normalized and 97 non-normalized cDNA libraries of which 24 are from developmental stages. The mitochondrial PigEST resource contains 41,499 mitochondrial sequences.ResultsThe mitochondrial EST (Expressed Sequence Tag) sequences were assembled into contigs which covers more than 94 percent of the porcine mitochondrial genome, with an average of 976 EST sequences per nucleotide. This data was converted into expression values for the individual genes in each cDNA library revealing differential expression between genes expressed in cDNA libraries from developmental and adult stages. For the 13 protein coding genes (and several RNA genes), we find one set of six genes, containing all cytochrome oxidases, that are upregulated in developmental tissues, whereas the remaining set of seven genes, containing all ATPases, that are upregulated in adult muscle and brain tissues. Further, the COX I (Cytochrome oxidase subunit one) expression profile differs from that of the remaining genes, which could be explained by a tissue specific cleavage event or degradation pattern, and is especially pronounced in developmental tissues. Finally, as expected cDNA libraries from muscle tissues contain by far the largest amount (up to 20%) of expressed mitochondrial genes.ConclusionOur results present novel insight into differences in mitochondrial gene expression, emphasizing differences between adult and developmental tissues. Our work indicates that there are presently unknown mechanisms which work to customize mitochondrial processes to the specific needs of the cell, illustrated by the different patterns between adult and developmental tissues. Furthermore, our results also provide novel insight into how in-depth sequencing can provide significant information about expression patterns.

Mitochondrial respiration and membrane potential after low-flow ischemia are not affected by ischemic preconditioning

Mitochondrial function following prolonged ischemia and subsequent reperfusion is better preserved by ischemic preconditioning (IP). In the present study, we analyzed whether or not IP has an impact on mitochondrial function at the end of a sustained ischemic period. Göttinger minipigs were subjected to 90-min low-flow ischemia without ( n = 5) and with ( n = 5) a preconditioning cycle of 10-min ischemia and 15-min reperfusion. Mitochondria were isolated from the ischemic or preconditioned anterior wall (AW) and the control posterior wall (PW) at the end of ischemia. Basal mitochondrial respiration was not different between AW and PW. The ADP-stimulated (state 3) respiration in AW mitochondria compared to PW mitochondria was equally decreased in non-preconditioned and preconditioned pigs. The uncoupled respiration as well as the membrane potential (rhodamine 123 fluorescence) were not significantly different between groups. However, the recovery of the membrane potential (Δ rhodamine 123 fluorescence/s) after the addition of ADP was delayed in mitochondria obtained from AW compared to PW, both in non-preconditioned and in preconditioned pig hearts. Neither the amount of marker proteins for complexes of the electron transport chain nor the level of reactive oxygen species were affected by ischemia without or with IP. State 3 respiration and recovery of membrane potential were impaired in pig mitochondria after 90 min of low-flow ischemia. IP did not improve mitochondrial function during ischemia. Therefore, the preservation of mitochondrial function by IP may occur during reperfusion rather than during the sustained ischemic period.

Identification of Mn2+-binding Aspartates from α, β, and γ Subunits of Human NAD-dependent Isocitrate Dehydrogenase

The human NAD-dependent isocitrate dehydrogenase (IDH), with three types of subunits present in the ratio of 2alpha:1beta:1gamma, requires a divalent metal ion to catalyze the oxidative decarboxylation of isocitrate. With the aim of identifying ligands of the enzyme-bound Mn(2+), we mutated aspartates on the alpha, beta, or gamma subunits. Mutagenesis target sites were based on crystal structures of metal-isocitrate complexes of Escherichia coli and pig mitochondrial NADP-IDH and sequence alignments. Aspartates replaced by asparagine or cysteine were 206, 230, and 234 of the alpha subunit and those corresponding to alpha-Asp-206: 217 of the beta subunit and 215 of the gamma subunit. Each expressed, purified mutant enzyme has two wild-type subunits and one subunit with a single mutation. Specific activities of WT, alpha-D206N, alpha-D230C, alpha-D234C, beta-D217N, and gamma-D215N enzymes are 22, 29, 1.4, 0.2, 7.3 and 3.7 micromol of NADH/min/mg, respectively, whereas alpha-D230N and alpha-D234N enzymes showed no activity. The K(m,Mn(2+)) for alpha-D230C and gamma-D215N are increased 32- and 100-fold, respectively, along with elevations in K(m,isocitrate). The K(m,NAD) of alpha-D230C is increased 16-fold, whereas that of beta-D217N is elevated 10-fold. For all the mutants K(m,isocitrate) is decreased by ADP, indicating that these aspartates are not needed for normal ADP activation. This study demonstrates that alpha-Asp-230 and alpha-Asp-234 are critical for catalytic activity, but alpha-Asp-206 is not needed; alpha-Asp-230 and gamma-Asp-215 may interact directly with the Mn(2+); and alpha-Asp-230 and beta-Asp-217 contribute to the affinity of the enzyme for NAD. These results suggest that the active sites of the human NAD-IDH are shared between alpha and gamma subunits and between alpha and beta subunits.

Crystal structures of medium-chain acyl-CoA dehydrogenase from pig liver mitochondria with and without substrate.

The three-dimensional structure of medium-chain acyl-CoA dehydrogenase from pig mitochondria in the native form and that of a complex of the enzyme and a substrate (product) have been solved and refined by x-ray crystallographic methods at 2.4-A resolution to R factors of 0.172 and 0.173, respectively. The overall polypeptide folding and the quaternary structure of the tetramer are essentially unchanged upon binding of the ligand, octanoyl (octenoyl)-CoA. The ligand binds to the enzyme at the rectus (re) face of the FAD in the crevice between the two alpha-helix domains and the beta-sheet domain of the enzyme. The fatty acyl chain of the thioester substrate is buried inside of the polypeptide and the 3'-AMP moiety is close to the surface of the tetrameric enzyme molecule. The alkyl chain displaces the tightly bound water molecules found in the native enzyme and the carbonyl oxygen of the thioester interacts with the ribityl 2'-hydroxyl group of the FAD and the main-chain carbonyl oxygen of Glu-376. The C alpha--C beta of the fatty acyl moiety lies between the flavin and the gamma-carboxylate of Glu-376, supporting the role of Glu-376 as the base that abstracts the alpha proton in the alpha--beta dehydrogenation reaction catalyzed by the enzyme. Trp-166 and Met-165 are located at the sinister (si) side of the flavin ring at the surface of the enzyme, suggesting that they might be involved in the interactions with electron transferring flavoprotein. Lys-304, the prevalent mutation site found in patients with medium-chain acyl-CoA dehydrogenase deficiency, is located approximately 20 A away from the active site of the enzyme.

Lambda-crystallin, a major rabbit lens protein, is related to hydroxyacyl-coenzyme A dehydrogenases.

It has recently been discovered that several lens proteins in birds and lower vertebrates are active enzymes or enzyme-related proteins (Wistow, G., Mulders, J. W. M., and de Jong, W. W. (1987) Nature 326, 622-624; Wistow, G., and Piatigorsky, J. (1987) Science 236, 1554-1556). We report here a novel lens protein, designated as lambda-crystallin, that occurs in rabbit and hare. It constitutes 7-8% of the total lens protein and has a subunit molecular mass of 35 kDa. Sequencing of cDNA clones encoding rabbit lambda-crystallin revealed 30% homology (at the amino acid sequence level) with L-3-hydroxyacyl-CoA dehydrogenase from pig mitochondria and 26% homology with enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase from rat peroxisomes. Also, the presence of a putative beta-alpha-beta nucleotide-binding fold and low levels of non-lens expression are indicative of some enzymatic function for lambda-crystallin (or highly related sequences) in non-lens tissues. lambda-Crystallin thus represents the first example of an enzyme-related crystallin in lenses from mammalian species. The recruitment of enzymes as lens structural proteins apparently is an evolutionary strategy which has been applied independently in different lineages.