A. PKC - key enzymes in many diseases.
Our research focuses on protein kinase C (PKC) and its role in normal signal transduction and in disease. PKC is a family of highly homologous enzymes whose functions include regulation of gene expression, response to ischemic insult, regulation of hormone responses and ion channels, to name a few. The challenge was to determine what is the role of each of the isozymes in these responses.
Most of the effort in developing PKC-specific drugs focused so far on the generation of PKC antagonists. However, many of the antagonists resulting from these studies were found not to be isozyme selective. Similarly, agonists of PKC were also not isozyme-selective. A possible reason for this outcome is the focus that has been put on mimicking cofactors or substrates of the enzymes. Because most of the isozymes do not differ substantially in their substrate specificity or sensitivity to activators and cofactors it is possible that selectivity of such agents could not be readily achieved. My laboratory has capitalized on the findings that the activated isozymes are differentially localized within cells and originally developed peptides that selectively block or induce this localization and the resulting function of the isozymes.
Binding of activated PKC isozymes to their RACKs determines the functional specificity of PKC
PKC isozymes translocate from one cell compartment to another when activated by the appropriate signal with each isozyme translocating to a unique subcellular site. We suggested that this unique localization is mediated by binding of each of the activated isozymes to their corresponding isozyme-specific anchoring proteins, termed RACKs (for Receptors for Activated C-Kinase).
Based on that idea, we have rationally identified isozyme-selective inhibitory peptides for all the PKC isozymes. The translocation inhibitors are 6-8 amino acids long and they selectively inhibit translocation and function of their corresponding isozymes at intracellular concentrations of 3-10 nM.
Translocation activators were also developed. These are short peptides that induce translocation of PKC by mimicking the action of the RACK on the enzyme. These peptides are called pseudo-RACK peptides [Figure 3 (4,8,9)]
How are the peptides introduced into cells?
We used Antennapedia, Tat, or poly-arginine to deliver the peptides into cardiac cells, with all methods yielding effective delivery.
What did we learn on the role of PKC isozymes?
There are over 200 published studies using these regulators that were carried out by our group and independently by other laboratories. Our studies focused mainly on the role of PKC in heart function. Using these peptide regulators of PKC we found:
- Beta II PKC mediates heart failure.
- Epsilon PKC activation regulates the activity of the L-type calcium channel and sodium channel and are therefore potential therapeutic target for cardiac arrhythmia (16-18).
- Epsilon PKC activation reduces damage induced by ischemia both in isolated myocytes and in intact heart.
- Delta PKC inhibition reduces damage induced by heart attack, stroke and damage to the brain induced by chronic hypertension. The delta PKC inhibitor was also used in human patient with acute myocardial infarction.
- Epsilon and gamma PKC peptide inhibitors that inhibit pain sensation are currently in human clinical trials.
- We are also interested in the role of PKC isozymes in breast cancer growth and in the angiogenic response associated with it.
Finally, these peptides have been made available to many other laboratories and studies by others have elucidated the role of individual PKC isozymes in other organs and disease states including chronic pain, diabetes, early embryo development, bone regeneration and others.
Note: If you wish to use the above figures in your presentations, click on the image for 2 sec and a menu allowing coping of the image will appear.
B. Aldehyde dehydrogenases
Aldehyde dehydrogenases (ALDHs) are important enzymes that eliminate oxidative stress-induced reactive aldehydes. Unfortunately, aldehydes directly inactivate the enzymes that detoxify them, thus removing a critical cell protective mechanism. We recently identified a set of small molecules that increase the activity of these protective detoxifying ALDHs, and protect them from inactivation by their substrates. Our goal is to apply this technology, which restores the cell’s healing ability, for the treatment of acute and chronic oxidative stress diseases for the benefit of patients.
The human ALDHs vary in their tissue localizations and substrate specificities. Several family members have received significant attention due to their widespread tissue distributions and links to disease progression. ALDH1, located in the cytosol, has been associated with the generation of retinoic acid and the detoxification of chemotherapeutic agents. ALDH3 is important in eye physiology, preventing cataract. ALDH2, located in the mitochondria, detoxifies oxidative stress-induced reactive aldehydes (Fig. 4).
A naturally occurring mutation in ALDH2 (ALDH2*2) is found in East Asians and significantly reduces enzyme function. Patients bearing this mutation are at higher risk for alcohol intolerance, cancers associated with exposure to aldehyde-producing toxins, and a number of diseases of aging, including late-onset Alzheimer’s disease, etc. The existence of this mutation enables correlative studies linking ALDH2 function with disease progression and prognosis.
Using Stanford’s high throughput facility, our lab has identified molecules that activate as well as protect select aldehyde dehydrogenases from substrate-induced inactivation. These compounds, called Aldas for aldehyde dehydrgeanse activators, preserve enzyme function in the presence of 4-hydroxynonenal (4HNE), a common toxic aldehyde byproduct of lipid peroxidation. For example, Alda-1 increases the catalytic activity of the wildtype ALDH2 and completely blocks 4HNE-induced inactivation of the enzyme. Importantly, Alda-1 increases the catalytic activity of the almost inactive ALDH2*2 by over 10 fold.
In addition to biochemical characterization of the signaling role of these enzymes, multiple in vivo studies currently test the therapeutic hypothesis that activating and protecting ALDHs will reduce the tissue damage caused by the toxic aldehydes produced by reactive oxygen species.
These diseases include the following:
- Ischemic damage: We showed that injection of Alda-1 reduces infarct size by 60% in a rat acute myocardial infarction model (Chen et al., Science, 321, 1493; Sept 12, 2008).
- Nitroglycerin response: ALDH2 is also a critical enzyme in converting nitroglycerin to the bioactive, NO, used in patients with heart attack.
- CNS diseases: The role of ALDHs in chronic CNS diseases including Parkinson’s disease and Alzheimer’s disease triggered our current efforts in using animal models to test the benefits of treatment with Aldas.