2V ,5V -Oligoadenylate size is critical to protect RNase L against proteolytic cleavage in chronic fatigue syndrome Marc Fre ´mont a , Karim El Bakkouri a , Freya Vaeyens a , C. Vincent Herst a , Kenny De Meirleir b , Patrick Englebienne a,c, T a RED Laboratories, Pontbeek 61, B-1731 Zellik, Belgium b Vrije Universiteit Brussel, Department of Human Physiology and Medicine, Pleinlaan 2, B-1051 Brussels, Belgium c Universite ´ Libre de Bruxelles, Department of Nuclear Medicine, Brugmann Hospital, Place Van Gehuchten 4, B-1020 Brussels, Belgium Received 23 December 2004 Available online 2 March 2005 Abstract A dysregulation in the 2V ,5V -oligoadenylate (2-5A)-dependent RNase L antiviral pathway has been detected in peripheral blood mononuclear cells (PBMC) of chronic fatigue syndrome (CFS) patients, which is characterized by upregulated 2-5A synthetase and RNase L activities, as well as by the presence of a low molecular weight (LMW) 2-5A-binding protein of 37-kDa related to RNase L. This truncated protein has been shown to originate from proteolytic cleavage of the native 83-kDa RNase L by m-calpain and human leukocyte elastase (HLE). We investigated the possible role of 2-5A oligomers in the proteolytic action toward the endonuclease and show that incubation of CFS PBMC extracts with 2-5A trimer and tetramer, but not with the dimer, results in a significant protection of the native 83-kDa RNase L against cleavage by endogenous and purified proteases. Similar results are obtained with a purified recombinant RNase L. An analysis of the size of 2-5A oligomers produced by the catalytic activity of the 2-5A synthetase present in PBMC extracts further shows that samples containing the 37-kDa RNase L preferentially produce 2-5A dimers instead of higher oligomers. Taken together, our results indicate that homodimerization of RNase L by 2-5A oligomers higher than the dimer prevents its cleavage by proteolytic enzymes. The presence of the truncated 37-kDa RNase L in PBMC extracts is therefore likely to result, not only from the abnormal activation of inflammatory proteases, but also from a dysregulation in 2-5A synthetase induction or activation towards the preferential production of 2-5A dimers. D 2005 Elsevier Inc. All rights reserved. Keywords: Ribonuclease L; 2V ,5V -oligoadenylate synthetase; Chronic fatigue syndrome; 2V ,5V -oligoadenylates; Protein cleavage; M-calpain; Human leukocyte elastase Introduction The interferon (IFN)-inducible 2V ,5V -oligoadenylate (2- 5A) synthetase/ribonuclease L (RNase L) pathway (2-5A pathway) is involved in eukaryotic cell protection against viruses. IFN triggers the production of nearly micromolar concentrations of 2-5A oligomers by virus-infected cells (Williams et al., 1979). The 2-5A are generated from ATP by any of several isozymes of an 2-5A synthetase (2-5OAS) specifically activated by double-stranded RNA (ds-RNA) of viral origin (Maitra et al., 1995). The regulation by IFN of the 2-5A anti-viral mechanisms involves also the enhanced expression of an 2-5A-dependent ribonuclease, termed RNase L. This 83-kDa monomeric latent protein is activated by binding 2-5A oligomers made of at least a trimer and homodimerizes during this activation process (Dong and Silverman, 1995). The 2-5A dimer binds to, but does not trigger, the homodimerization and catalytic activation of the human enzyme (Dong and Silverman, 1995; Naik et al., 1998). The activated homodimeric enzyme cleaves single- stranded RNA of viral and cellular origins, primarily after UpNp sequences (Wreschner et al., 1981), which results in the inhibition of protein synthesis. 0014-4800/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2005.01.003 T Corresponding author. RED Laboratories, Pontbeek 61, B-1731 Zellik, Belgium. Fax: +32 2 481 5311. E-mail address: penglebi@ulb.ac.be (P. Englebienne). Experimental and Molecular Pathology 78 (2005) 239 – 246 www.elsevier.com/locate/yexmp