Characterization of a 2′,5′-Oligoadenylate (2–5A)-dependent 37-kDa RNase L

Abstract

Upregulation of key components of the 2,5-oligoadenylate (2-5A) synthetase/RNase L pathway have been identified in extracts of peripheral blood mononuclear cells from individuals with chronic syndrome, including the presence of a low molecular weight form of RNase L. In this study, analysis of 2,5-Oligoadenylate (2-5A) binding and activation of the 80-and 37-kDa forms of RNase L has been completed utilizing photolabeling/immunoprecipitation and affinity assays, respectively. Saturation of photolabeling of the 80-and the 37-kDa RNase L with the 2-5A azido photoprobe, [ Previous studies from this laboratory (1) described structural similarities between two forms of 2Ј,5Ј-oligoadenylate (2-5A) 1 -dependent RNase L in extracts of peripheral blood mononuclear cells (PBMC). The 2-5A-dependent 80-kDa RNase L is the terminal enzyme in the 2-5A synthetase/RNase L antiviral defense pathway (2). RNase L is distinguished from other RNases in that it requires 2-5A for allosteric activation (reviewed in Refs. 1 and 3-6). The 80-kDa RNase L plays an essential role in the inhibition of viral mRNA and thus the inhibition of viral protein synthesis (3, 4). In addition to the 80-kDa form of RNase L, a 40/42-kDa form has been identified and is thought to be a proteolytic degradation product of the 80-kDa RNase L (7, 8).As part of ongoing studies on the up-regulation of the 2-5OAS/RNase L antiviral pathway, a previously unknown 37-kDa form of RNase L has recently been identified in extracts of PBMC from individuals who met the diagnostic criteria for chronic fatigue syndrome (CFS) (9 -12). We had previously demonstrated that the 2-5OAS/RNase L pathway is constitutively active in PBMC extracts from individuals with CFS; 2-5OAS is predominantly in its activated form and levels of bioactive 2-5A and RNase L enzyme activity are elevated in CFS PBMC extracts compared with healthy control PBMC extracts (13,14). Continued analyses of CFS PBMC extracts have revealed the presence of a novel low molecular mass RNase L that binds 2-5A, exhibits 2-5A-dependent RNase L activity, and has a molecular mass of 37-kDa under denaturing conditions (1, 10). The 37-kDa RNase L has not been observed in PBMC extracts from individuals diagnosed with fibromyalgia or depression (15).Two subsets of individuals with CFS have been identified in PBMC extracts using azido photoaffinity labeling with [ 32 P]pApAp(8-azidoA) and immunoprecipitation with a polyclonal antibody specific to the human 80-kDa RNase L (1, 10). One subset contains only the 37-kDa RNase L and the second subset contains both the 80-and 37-kDa RNase L. The subset of CFS PBMC containing only the 37-kDa RNase L had the higher level of RNase L activity compared with the subset containing both the 80-and 37-kDa forms of RNase L (2). In a two-site randomized-coded study, the presence of the 37-kDa RNase L also correlated with the level of clinical disability experienced by the study subjects (10).The elucidation of the molecular structure of the 37-kDa RNase L has been a major effort in this laborat...

Ribonuclease L Proteolysis in Peripheral Blood Mononuclear Cells of Chronic Fatigue Syndrome Patients

“…This assay confirms that the biological activity of proteolyzed RNase L is not dramatically different from native RNase L, in agreement with Shetzline and Suhadolnik (13). A 2-5A-dependent nuclease activity remains after the proteolysis of recombinant RNase L in its 30-and 37-kDa major cleavage fragments.…”

“…Moreover, an increased nuclease activity has been reported in a subset of CFS patients whose PBMC extracts did not contain any full-size 83-kDa RNase L (13). This observation has remained puzzling since the P-loop region (which is responsible for 2-5A binding) and the C-terminal end (which contains the catalytic site of RNase L) are far apart and cannot be fitted within a single 37-kDa polypeptide.…”

Pathologic effects of RNase-L dysregulation in immunity and proliferative control

“…This proteolysis results in the production of two fragments, RNL-37 and a 30 kDa protein that encodes the C-terminal nuclease domain of RNase-L. Based on the current model of RNase-L activation (figure 2B), RNL-37 may bind and inhibit the nuclease activity of the 30 kDa fragment. Once 2-5A binds RNL-37, it disassociates from the nuclease domain, allowing RNA hydrolysis at a rate three times that of the wild type enzyme (151). In addition to this increased rate of RNA decay, this truncated endonuclease may not be regulated in the same way as the full length protein, possibly exhibiting altered target specificity that may contribute to CFS pathology.…”

The Roles of RNase-L in Antimicrobial Immunity and the Cytoskeleton-Associated Innate Response

“…Tumor suppressive activities, including antiproliferative and proapoptotic functions, were quickly ascribed and, in 2002, a genetic association was made when RNASEL was identified as the hereditary prostate cancer 1 (HPC1) locus on chromosome 1q25 [ 11 , 12 , 13 , 14 , 15 ]. Novel roles and mechanisms have continued to emerge, such as the correlation between a 37 kDa truncated form of RNase-L and chronic fatigue syndrome [ 16 , 17 , 18 ]. Other non-canonical functions for RNase-L include the induction of senescence and shortened lifespan, cellular differentiation, colitis susceptibility, the demyelination of axons, lipid storage, and the development of diabetes [ 19 , 20 , 21 , 22 , 23 , 24 ].…”