Kits to Quantitate and Analyze Mitochondrial Proteins

Mitosciences, Inc, Eugene OR

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): Mitochondria play a key role in cellular energy metabolism: they are the seat of oxidative phosphorylation (OXPHOS), which produces 98 percent of all the ATP utilized by mammalian cells. OXPHOS is catalyzed by 5 large, membrane-bound, protein complexes together made up of 90 polypeptides. Substrate entry into the OXPHOS system is controlled by a sixth, large enzyme-complex, the pyruvate dehydrogenase complex. Genetic defects in these six complexes cause a heterogeneous set of human diseases collectively classified as mitochondrial disorders, which affect around 1 in 6000 live births. Mitochondrial diseases remain hard to diagnose and the genotype-phenotype relationships of these disorders is still obscure. There are several late-onset diseases where mitochondrial dysfunction is thought to be causal and/or predictive, including Parkinson's disease, Alzheimer's, early- and late-onset Diabetes, schizophrenia and some forms of heart disease. In common to all of these conditions is the idea that they result from accumulation of oxidative damage leading to cell death caused by and involving the mitochondrion and the OXPHOS components in particular. As with mitochondrial genetic diseases, the etiology of the late-onset diseases listed above remains obscure and their diagnosis is still difficult because of a lack of adequate, simple high-throughput methods for analyzing the composition, enzyme activities and post-translational (oxidative) modifications of the OXPHOS components. This grant is to establish immunologically-based methods for such analyses. Monoclonal antibodies (mAbs) have been generated from small amounts of human and other mammalian tissue, which capture functionally active intact forms of 4 of the 50XPHOS complexes and pyruvate dehydrogenase. Screening is in progress for a mAb to capture Complex III. These will be fabricated into several 96-well based microassays. One will compare the levels of each complex in an experimental/ diagnostic sample versus a control sample by a twodye fluorometric approach. A second will compare the enzymatic activities of each complex in an experimental sample with a control using fluorometric and colorimetric activity assays. A third set will immunocapture the various complexes in amount sufficient for proteomic analysis of post-translational modifications. These kits will be invaluable, not only for researchers studying early-onset and late-onset mitochondrial disorders, but also for studies of mitochondria physiology, and for high-throughput screening for environmental toxins that cause, and drugs that ameliorate, these conditions.