Chapter Three: A 37-kDa RNase L: A Novel Form of RNase L Associated with Chronic Fatigue Syndrome by Robert J. Suhadolnik, Susan E. Shetzline, Camille Martinand-Mari, and Nancy L. Reichenbach
(This chapter broadens the discussion of the 37-kDa fragment. For the first time RNase L fragmentation is correlated with symptoms in CFS. The activity of the fragment and RLI is described. )
Several viruses, including HHV6 and Epstein-Barr, appear to be associated with CFS. Changed cytokine levels, depressed natural killer (NK) function, and increased numbers of activated cytotoxic T-cells may result in persistent illness and fatigue.
These indicators of immune activation suggested to researchers that defects in the ‘dsRNA-dependent, interferon inducible 2-5A RNase L antiviral defense pathways’ could occur.
The first examination of the 2-5A/RNase L pathway in connection with CFS in 1994 revealed that CFS patients had significantly lower levels of inactive or latent 2-5A synthetase (2-5OAS) and significantly higher levels of bioactive 2-5A and RNase L..
They found that concentrations of 2-5A were up to 220x’s higher in severely disabled CFS patients. Further testing indicated that in some of these individuals, RNase L activity was up to 1500x’s higher than that of the controls, and that certain strands (28S, 18S) ribosomal RNA (rRNA) were being completely hydrolyzed. Complete hydrolysis of ribosomal RNA was observed in almost all the severely disabled CFS patients tested (n=15).
The authors noted that Ampligen therapy resulted in down regulation of the 2-5A/RNase L pathway, a significant decrease in HHV6 activity, and in decreased symptoms in 15 severely disabled CFS patients. 2-5A levels, in fact, declined to normal after 25 weeks of Ampligen therapy. (Ampligen (poly (I)-poly (C12U)) is a reconfigured dsRNA that has antiviral and immunomodulatory activities. It has the capability, at times, in returning the 2-5OAS system to proper functioning.)
A subsequent larger study confirmed not only that bioactive 2-5A, RNase L activity, and 37-kDa RNase L were all upregulated in CFS patients (p<.001, .002, .001) but that clinical measures of fatigue were positively correlated with RNase L upregulation. Studies of cleavage efficiency indicated that the 37-kDa fragment hydrolyses RNA 3x’s faster than the native RNase L.
A positive correlation between the 37-kDa fragment and reduced maximum oxygen consumption (VO2 max) and lower exercise duration suggested that low exercise tolerance in CFS may be a product of abnormal oxidative metabolism. Another study indicated that high RNase L activity is a good predictor of fatigue, muscle pain and reduced mood in CFS.
Another study confirmed the 37-kDa fragment was much more abundant in CFS patients than healthy controls or fibromyalgia patients. Only the 37-kDa enzyme was found in a subgroup of the most severely disabled CFS patients. A very significant correlation between the native 80-kDa RNase L and the 37-kDa RNase L (p<.0001) suggested that the 37-kDa fragment was derived from the breakup of the native 80-kDa RNase L. (80 and 83- kDa are used interchangeably through the book for the native RNase L).
The upregulation of the IFN inducible RNA degrading pathway in CFS has been verified by four independent laboratories in North America, Europe, and Australia.
RLI inhibits 2-5A binding with both the native RNase L and the 37-kDa fragment. RLI expression, however, is decreased in CFS patients. (Both the activity of the native RNase L and the 37-kDa fragment are increased in CFS. Decreased RLI expression in CFS patients appears to provide a mechanism for increased RNase L activity even in the absence of chemical or viral activators.).
In contrast to the native RNase L, the 37-kDa fragment is active without homodimerizing. (This essentially means that, except for when it is bound by RLI, it is active all the time.)
Chapter Four – Ribonuclease L Inhibitor: A Member of the ATP-Binding Cassette Superfamily By Patrick Englebienne, C. Vincent Hearst, Anne D’Haese, Kenny De MeirLeir, Lionel Bastide, Edith Demettre, and Bernard Lebleu
A protein called ribonuclease inhibitor (RLI) regulates RNase L activity probably by preventing 2-5A from binding with RNase L and dimerizing and thus activating it. While all the other proteins in the RNase L pathway are activated by the IFN’s, RLI interestingly enough, is not.
Plasma membranes protect the integrity of every cell in our body. The cells, however, need chemicals and ions to fuel the factories and create the products the body needs. Channels are used to take in these substances and to excrete metabolic wastes derived from them.
ABC transporters bind onto the chemicals the body needs and transport them across the protective membrane. (ABC transporters contain ‘ATP binding cassettes’ which bind ATP and cleave off phosphate groups. ATP fragmentation provides energy and the phosphate groups change the protein conformation at the membrane causing channels in the membrane to open. ABC transporters ferry ions, amino acids, sugars, vitamens, and peptides across membrane boundaries.)
RLI is the sole member of one of the eight families that make up the ABC superfamily. Although RLI’s primary role is RNase L inhibition, its membership in the ABC superfamily suggests it may have other biological functions.
The RNase L inhibitor and chronic fatigue syndrome
Besides fatigue, CFS is characterized by a series of symptoms that are reminiscent of‘voltage-gated channelopathies (hypakaliemic periodic paralysis, skeletal muscle pain, ventricular hypercontractility, drenching night sweats, and cognitive defects). Improper ion channel function may account for many of these symptoms.
After classifying, once again, blood samples by their degree of RNase L fragmentation (37-kDa/83-kDa), an analysis of RLI in CFS patients found that not only did the native 68-kDa RLI progressively disappear as RNase L fragmentation increased, but that fragments of RLI begin to appear. This suggests, of course, that the same agent may be fragmenting both RNase L and RLI.
Ankyrins are proteins that can link cell membranes with the cells skeleton. By controlling the shape of the cell membrane, its elasticity, and the proteins it is composed of, ankyrins play integral roles in many biological activities.
One end of the ankyrin domain found on the native whole RNase L enzyme is blocked from interacting with membranes because it is sequestered inside the protein. Cleavage of the native RNase L, however, frees this formerly sequestered end of the ankyrin domain thus potentially restoring its ability to interact with components of the cells membranes.
This would not necessarily be a problem because RLI would normally bind with that end of the ankyrin fragment and render it inactive. Because RLI is down regulated in CFS, however, the ankyrin domains found on the 37-kDa fragment appear to be active.
The motif that is released (AIIK) is very similar to the motif (ALLK) that other ABC transporters interact with. (I is isoleucine, L is leucine; isoleucine is a close enough analogue to leucine that it probably interacts just as leucine does).
The striking similarity between the two motifs suggests that the 37-kDa fragment may be capable of interacting with ABC transporters to alter ion channel functioning. Interestingly, the disruption of these ion channels could account for many of the symptoms and abnormalities found in CFS.
Many of these ABC transporters are members of the multi-drug resistance (MDR) group that binds cytotoxic compounds and pumps them out of the cell. The hypersensitivity to chemicals often reported in CFS could result of dysfunctional transporters.
(The MDR group was discovered when oncologists noticed that some cells were impervious to chemotherapy. The protein responsible for this is not, despite its name, primarily involved in clearing ‘drugs’ from the cell. Rather it transports natural and metabolic toxins into the bile, intestines or urine. Its high abundance in liver cells limits the effectiveness of chemotherapeutic drugs in those cells.)
Besides the multi-drug resistant channels or transporters (the transporters form channels when activated), RLI’s amino acid sequence bears strong similarity to 10 other ABC transporters and Na+K+ATPase.
Some of the processes and symptoms these transporters are associated with include: exocrine functions of epithelial tissues (night sweats, irritable bowel syndrome, increased pain sensitivity, insulin excretion (transient hypoglycemia), Na+ channels (increased pain sensitivity), excretion of cytotoxic compounds (chemical hypersensitivity), macrophage cholesterol (immuno-deficiency), antigen processing (Th1/Th2 imbalance), heme transport (anemia, CNS abnormalities), etc.
All these symptoms are found in CFS patients. The disregulation of one ABC transporter involved uptake of tryptophan, a precursor of serotonin, could lead to CNS abnormalities.
(The Na+K+ATPase channels are involved in some of the most fundamental processes that occur in animal cells. Among them are nerve cell activity and amino acid uptake (the nervous system and energy generation). The process of simply maintaining the proper Na+, K+, and Ca+ gradients between the cell and its environment uses up enormous amounts of energy.)
*Update – (2003, Clin Sci, Apr. 23) – Putative amino acid modulators of serotonin and dopamine function were measured in CFS and controls in an investigation of the central neural system (CNS) in CFS. Levels of free tryptophan, the rate-limiting serotonin precursor were significantly higher and levels of tyrosine, the dopamine precursor and branch chain and large neutral amino acids were significantly lower in CFS patients. The authors state that these finding implicate the CNS in CFS pathology. Here we have confirmation of the authors supposition that tryptophan levels may be altered in CFS patients.
*Update – (2003, JCFS vol. 11, #1, -from abstract) – Preliminary observations were made of whole body potassium, serum electrolytes (sodium, calcium, potassium), immune cells, blood cells counts and ESR. More than 50% of CFS patients had either abnormally increased or depleted whole body potassium. CFS patients could be differentiated from controls through higher B-cell and reduced NK cell levels. Reduced NK cell counts were very strongly associated with high RNase L fragmentation rates and reduced calcium. These findings suggest an RNase L induced channelopathy is present in a subset of CFS patients.
*Update – (2003, Medical Hypotheses, Jan 60 (1) 65-8) – Several of the authors of this chapter further explore the implications of the channelopathies believed to occur in CFS. They state that ‘pathophysiological observations suggest that besides the channelopathies present, increased demands for calcium may occur as a result of two factors often found in CFS. By stimulating prostaglandins Mycoplasma fermentans may stimulate calcium uptake from bones. The low levels of insulin-like growth factor (IGF) often found in CFS may inhibit the proliferation of the main bone producing cells, the osteoblasts. These factors may put CFS patients at risk from osteopenia.
*Update – (2003 Neruromuscular Disorders, Aug 13 (6): 479-84) Disregulations in both the Na+K+ATPase and Ca2+ATPase pumps in the sacroplasmic reticulum in muscles supports the hypothesis that reactive oxygen species contribute to fatigue in CFS. Pump disregulation may result from increased membrane fluidity.