Environmental Factor, October 2010, National Institute of Environmental Health Sciences
JBC selects NIEHS study as paper of the week
A recent report from researchers in the NIEHS Mitochondrial DNA Replication Group (http://www.niehs.nih.gov/research/atniehs/labs/lmg/mdnar/index.cfm) was chosen as The Journal of Biological Chemistry (JBC) Paper of the Week - an honor awarded to the top one percent of reviewed manuscripts for their significance and overall importance.
Published Sept. 24, the study(http://www.ncbi.nlm.nih.gov/pubmed/20659899) coauthored by Matthew Longley, Ph.D., Margaret Humble, Farida Sharief, and William Copeland, Ph.D., describes novel purification methods for the mitochondrial helicase encoded by the human C10orf2 gene. The research team also characterized partial biochemical defects in twenty mutant variants identified in patients with mitochondrial disease.
The mitochondrial helicase is involved in mitochondrial genome (mtDNA) maintenance. Mutations in C10orf2 co-segregate with people afflicted with heritable fatal mitochondrial diseases, such as the adult-onset Progressive External Ophthalmoplegia, hepatocerebral mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. These mitochondrial diseases are characterized by mtDNA depletion and deletions leading to decreases in energy production in tissues.
Biochemical defects discovered in helicase disease variants
Helicases are enzymes responsible for unwinding the two DNA strands at the DNA replication fork, which is necessary for mtDNA stability. However, the extent that mtDNA instability causes mitochondrial diseases is unknown, partly because the mechanism for replicating mtDNA remains unclear.
Longley said he believes that a biochemical approach begins to address these uncertainties. "The accurate determination of biochemical dysfunction is critical to identifying molecular mechanisms underlying disease."
The study describes four basic measurements of the mitochondrial helicase required for its function:
- Binding to single-stranded and double-stranded DNA as measured by fluorescence anisotropy, a method that detects the change in the speed of rotation of DNA when a large molecule is bound
- ATPase activity, which is required for helicase function
- Protein stability as estimated by the rate of inactivation of ATPase activity at temperatures that induce unfolding of less stable proteins
- The ability to unwind double-stranded DNA
All of the mutant variants exhibited variable helicase activity, suggesting, as Copeland explains, that changes in helicase function contribute to mtDNA instability. "We believe these subtle changes are consistent with the late presentation of disease in most of these patients."
Determination of optimal purification conditions
Protein instability and aggregation, which have hampered previous analyses of this enzyme by other labs, complicated purification of the mitochondrial helicase. Longley and colleagues described purification schemes for the His-tagged and untagged mitochondrial helicase that had been overproduced in E. coli cells, utilizing combinations of immobilized metal affinity, anion exchange, and heparin affinity chromatography. The protein precipitated without careful consideration of the buffer components. Precipitation was minimized by increasing the concentrations of both salt and glycerol, as well as by the addition of detergents and magnesium ions.
The survey of optimal purification conditions helped to determine appropriate conditions for enzymatic assays in vitro. Others have reported a lack of helicase activity for certain purified mitochondrial helicase disease variants. However, Longley explains that these discrepancies demonstrate the differences in optimal and suboptimal experimental conditions. "This work emphasizes the importance of maintaining enzyme stability and assessing enzymatic activities under carefully optimized conditions."
Future biochemical work could help to determine why a single mutant copy of the C10orf2 gene causes disease. The native helicase is a complex of six identical subunits. Mixtures of wild type and mutant proteins in affected patients could form so-called "heterohexameric" helicases with enzymatic functions altered in complex ways. Alternatively, reduced abundance of the wildtype helicase may be sufficient to cause mtDNA instability. The optimal biochemical conditions worked out in this study will be instrumental in defining the dominant-negative biochemical effect expected to underlie the dominant nature of these diseases.
Citation: Longley MJ, Humble MM, Sharief FS, Copeland WC. 2010.(http://www.ncbi.nlm.nih.gov/pubmed/20659899) Disease variants of the human mitochondrial DNA helicase encoded by C10orf2 differentially alter protein stability, nucleotide hydrolysis and helicase activity. J Biol Chem. DOI: 10.1074/jbc.M110.151795.
(Jeffrey Stumpf, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Mitochondrial DNA Replication Group.)