Overview
Cellular processes are carefully controlled to maintain homeostasis, in part by transcriptional regulation of protein expression. Recent studies underscore the complexity of this regulation by showing the extent to which cellular stress fine-tunes protein levels through multiple different transcriptional factors along with their co-activators, to which we can now add regulators of these co-activators. Screening for drug induced transcriptional changes by RNA expression using gene chip analyses is now commonplace. However such analyses alone do not provide unambiguous information about the critical parameter in understanding cellular responses to compounds, i.e. the alterations in the protein levels of the components of the many different metabolic pathways. This is because a determination of message levels as a surrogate for final protein levels does not take account of either regulation at the level of transcript stability or protein half-life.

The MT-PE1 is the first assay to measure the levels of proteins key to mitochondrial metabolism directly. It is a focused expression screen involving a set of key enzymes of metabolism that represent multiple transcriptional programs. Important for its utility, the assay does not require complicated, time and labor consuming informatics analyses to obtain an overview of the changes in metabolic pathways caused by a drug effect.

Assay Details
The MT-PE assays are available as 96-well sandwich ELISA's which use a monoclonal antibody specific to each target to capture, and then a second monoclonal antibody against a different epitope to detect, the protein of interest. The use of 2 monoclonals per target makes the assay highly specific for each protein. Samples for analysis can be whole cell (or tissue) extract, which is suspended in the detergent lauryl maltoside, conditions which leave proteins as functional complexes. The advantage is that the expression assay can be multiplexed with measurement of oxidative modifications or other post-translational modifications. Moreover, multisubunit complexes and functional associations of proteins are kept intact through the procedure so that the level of complex I, for example, is determined as the amount of the assembled 45 subunit complex, not as the RNA levels of each of the subunits individually.

The proteins available for the expression array are involved in several mitochondrial functions, and the list is constantly growing. Each plate is arrayed to assay 8 target proteins in triplicate.

Target proteins are:
1. Complex I of the respiratory chain
2. Complex II of the respiratory chain
3. Complex III of the respiratory chain
4. Complex IV or cytochrome c oxidase of the respiratory chain
5. Cytochrome c, involved in electron transfer and in apoptosis
6. ATP synthase or F1F0.
7. Frataxin, involved in iron metabolism
8. AIF, involved in apoptosis
9. Trifunctional protein, involved in fatty acid oxidation of long chain fatty acids
10. SCHAD, involved in FAO of short chain fatty acids
11. MCAD, involved in FAO of medium chain fatty acids
12. NNT, involved in oxidative stress and energy metabolism
13. pyruvate dehydrogenase, involved in glucose metabolism
14. mitofilin, involved in mitochondrial morphology maintainance and apoptosis.

After broad screening to identify those proteins most altered by a compound, the MitoTox Expression assay can be focused to this set for subsequent evaluation of other compounds of the same class examined in cell culture, and importantly in animal studies using many different tissues. As very little sample is needed for the screen, it is possible to monitor time dependent effects of compounds in animals and in human trials with this assay using needle biopsy of tissue, blood, cheek swabs or urine samples.

Figure 1. Percent change in expression of 8 proteins in HepG2 cells after dosing with 20µM rosiglitazone (green) and 20µM troglitazone (red) for 24 hours.

Frequently Asked Questions

Q1. Just how finely controlled is regulation of protein levels?
A1. Based on studies in yeast, multiple transcription factors can cooperate at individual (gene) promoters to very precisely determine the levels of mRNA that are made. Also the stability of this mRNA can be regulated by binding of proteins, which are often enzymes in pathways that support or compete with the metabolic process in which the protein of interest is involved. In toto, it may prove that these regulatory processes add up to provide a "molecular counter" of stress events and allow a precise response.