LysoSENS Progress and Prospects

Posted by Jacques Mathieu on August 02, 2010 | Clearing cells of age-related wastes

The 7KC-degrading bacterium I’ve been studying, Rhodococcus jostii RHA1, has two large gene clusters that are up-regulated by 7KC, but not cholesterol. In these two gene clusters lie a number of enzymes we believe are involved in 7KC degradation, including an enzyme that could reduce the 7-keto group to a hydroxyl. What makes this interesting to us is that while 7KC is highly cytotoxic, 7α-hydroxycholesterol (7αOH) is relatively harmless. So I am now methodically going through suspected candidates, searching for reductase activity against 7KC. Currently I am looking at nine different enzymes, and am in various stages of cloning the genes into expression vectors and assaying their products for activity.

While I’ve uncovered some interesting findings, so far I haven’t found the reductase I’m looking for. This could be for several reasons, the first being that I haven’t assayed all the enzymes I need to. As I still have the majority remaining, this is a likely scenario. However, one possibility is that the normal substrate for the enzyme is not 7KC, but some downstream metabolite. If this is the case then I have several approaches I could take.

The first would be to test downstream metabolites, though this would be very time consuming as most of these compounds are not commercially available. The second approach would be to use mutagenesis techniques on the enzymes I have to see if I can alter their substrate specificity to accept 7KC. I could also systematically delete candidate genes and monitor 7KC degradation, though that could be time consuming as well. Another alternative is to obtain hamster 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD). 11β-HSD has been found to interconvert 7KC, 7βOH, and 7αOH. Unfortunately 7βOH is more cytotoxic than 7KC, so this activity isn’t beneficial, though this enzyme could potentially be engineered for altered stereoisomerism. The ideal path may be to attempt all of these approaches in parallel, but I’d like to finish subcloning and expressing the genes I have before making that decision.