Demettre, E, Bastide, E, D’Haese, A., De Smet, K., DeMeirLeir, K., Tiev, P., Englebienne, P. and B. Lebleu. 2002. Ribonuclease L proteolysis in peripheral blood mononuclear cells of Chronic Fatigue Syndrome patients. Journal of Biological Chemistry 277 (38) 35746-34571.
If you are an RNase L junkie, as I have become, this is a fascinating paper. We find here that contrary to our understanding from CFS ABA RNase L fragmentation produces not one but two major fragments.
Some review may be necessary. In Section 2.8 of “Chronic Fatigue Syndrome: A Biological Approach” we learned a 37-kDa fragment produced by the cleavage of the native 83-kDa RNase L enzyme appeared to contain both the catalytic and binding sites of the native enzyme.
(2-5A latches onto RNase L’s binding sites and prompts it to ‘homodimerize’ or ‘mate’ with another RNase L. When it does this the enzyme changes shape and its catalytic site is exposed and it begins searching for RNA to destroy. It appeared that the 37-kDa fragment contained both the binding and catalytic sites.)
Because the 37-kDa fragment was so small and the binding and catalytic sites were so far apart on the native enzyme a di-sulphide bridge was believed to join the two sites together.
The disintegration of the native enzyme was studied in further detail by another group in Chapter 3 of “Chronic Fatigue Syndrome: A Biological Approach”. Here it appeared that the enzyme was destroyed bit by bit leaving behind one large 30-kDa (not a 37-kDa) fragment. The authors accounted for the disparity in weight by noting that measurements on this scale often varied slightly; indeed, the native 83-kDa enzyme appeared to weigh 80-kDa in one study.
This study revealed, however, that the kits the two groups used each revealed different fragments; one that weighed 37-kDa and one that weighed 30-kDa. It turns out that the 37-kDa fragment does not contain RNase L’s catalytic site; it contains its binding site and the 30-kDa fragment contains the catalytic site.
If the binding and catalytic sites are found on separate fragments how could RNase L activity still be a function of 2-5A binding? How could 2-5A prompt RNase L to expose its catalytic site if the fragment it binds to does not contain the catalytic site?
‘Pull down’ experiments indicated that the 30 and 37-kDa fragments remain associated with each other after RNase L breaks up. This suggests they are linked together by a disulphide bridge or by non-covalent bonds. If I am reading this correctly this means we are essentially back to where we started, a little smarter to be sure, but still back to page one: a disulphide bridge (or bond) still links the binding and catalytic sites but now they are found on two fragments instead of one.