Tag: Suhadolnik

Structural and functional features of the 37-kDa 2-5A-dependent RNase L in chronic fatigue syndrome

Abstract:

A 2′,5′-oligoadenylate (2-5A)-dependent 37-kDa form of RNase L has been reported in extracts of peripheral blood mononuclear cells (PBMC) from individuals with chronic fatigue syndrome (CFS). In the current study, analytic gel permeation FPLC, azido photoaffinity labeling, two-dimensional (2-D) gel electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been used to examine the biochemical relationship between the 80-kDa RNase L in healthy control PBMC and the 37-kDa RNase L in PBMC from individuals with CFS.

Like the 80-kDa RNase L, the 37-kDa RNase L is present as a catalytically inactive heterodimer complex with the RNase L inhibitor (RLI). Formation of a 37-kDa RNase L-RLI complex indicates that the 37-kDa RNase L is structurally similar to the 80-kDa RNase L at the N-terminus, which contains the 2-5A binding domain. The enzymatically active monomer form of 37-kDa RNase L resolved by 2-D gel electrophoresis has a pI of 6.1. RT-PCR and Southern blot analyses demonstrated that the 37-kDa RNase L is not formed by alternative splicing. In-gel tryptic digestion of the 37-kDa RNase L that was excised from 2-D gels and subsequent MALDI-MS analysis identified three peptide masses that are identical to three predicted peptide masses in the 80-kDa RNase L. The electrophoretic mobility of 2-5A azido photolabeled/immunoprecipitated 37-kDa RNase L was the same under reducing and nonreducing conditions. The results presented show that the 37-kDa form of RNase L in PBMC shares structural and functional features with the native 80-kDa RNase L, in particular in the 2-5A binding and catalytic domains.

 

Source: Shetzline SE, Martinand-Mari C, Reichenbach NL, Buletic Z, Lebleu B, Pfleiderer W, Charubala R, De Meirleir K, De Becker P, Peterson DL, Herst CV,Englebienne P, Suhadolnik RJ. Structural and functional features of the 37-kDa 2-5A-dependent RNase L in chronic fatigue syndrome.  J Interferon Cytokine Res. 2002 Apr;22(4):443-56. http://www.ncbi.nlm.nih.gov/pubmed/12034027

 

Antiviral pathway activation in chronic fatigue syndrome and acute infection

Comment on: Antiviral pathway activation in patients with chronic fatigue syndrome and acute infection. [Clin Infect Dis. 2001]

 

SIR—We read the very engaging report by Gow et al. [1] with the utmost interest. However, we feel that this article raises more questions than clear-cut answers regarding the hypothesis that motivated the study—that is, that the previously reported activation of the antiviral pathway in chronic fatigue syndrome (CFS) might be linked to infection rather than to CFS specifically. To verify their hypothesis, Gow and colleagues used PCR to measure the genetic expression of 3 IFN-regulated genes—namely, the latent ribonuclease (RNase L), RNA-regulated protein kinase (PKR), 2,5 synthetase, and the RNase L inhibitor (RLI)—in patients with acute infection (in their study, severe gastroenteritis; group 1), patients with CFS (group 2), and healthy control subjects (group 3).

First, surprisingly enough, although they recognized that acute infection is supposed to induce the expression of the genes selected for their study (see figure 1 of [1]), Gow and colleagues failed to find any significant increase in the expression of 2 major genes (RNase L and 2,5 synthetase) in group 1, as compared with groups 2 and 3; they observed only increased mRNA for PKR and RLI. Although it is recognized that genetic expression of PKR, RNase L, and 2,5 synthetase is under the control of interferon, RLI is definitely not [2]. Upregulation of RLI genetic expression with a normal genetic expression of both 2,5 synthetase and RNase L (although PKR is overexpressed!) during acute infection, as was observed in the study of Gow et al. [1], would indicate not only that RNase L is not activated (normal expression of RNase L and, more importantly, of 2,5 synthetase), but that it is further inhibited by an overexpressed RLI [2]. Such a scenario, if verified, would be in complete disagreement with the current understanding of the IFN pathway [3]. Therefore, we cannot help but wonder how Gow and colleagues reconcile their observations with the acute infection status of study group 1. In our view, this inconsistency severely undermines their conclusions.

You can read the rest of this comment here:  http://cid.oxfordjournals.org/content/34/10/1420.long

 

Source: De Meirleir K, Suhadolnik RJ, Lebleu B, Englebienne P. Antiviral pathway activation in chronic fatigue syndrome and acute infection. Clin Infect Dis. 2002 May 15;34(10):1420-1; author reply 1421-2. http://cid.oxfordjournals.org/content/34/10/1420.long (Full article)

 

Characterization of a 2′,5′-oligoadenylate (2-5A)-dependent 37-kDa RNase L: azido photoaffinity labeling and 2-5A-dependent activation

Erratum in: J Biol Chem 2001 Aug 24;276(34):32392.

Abstract:

Upregulation of key components of the 2′,5′-oligoadenylate (2-5A) synthetase/RNase L pathway has been identified in extracts of peripheral blood mononuclear cells from individuals with chronic fatigue [corrected] 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, [(32)P]pApAp(8-azidoA), was achieved. Half-maximal photoinsertion of [(32)P]pApAp(8-azidoA) occurred at 3.7 x 10(-8) m for the 80-kDa RNase L and at 6.3 x 10(-8) m for the 37-kDa RNase L. Competition experiments using 100-fold excess unlabeled 2-5A photoaffinity probe, pApAp(8-azidoA), and authentic 2-5A (p(3)A(3)) resulted in complete protection against photolabeling, demonstrating that [(32)P]pApAp(8-azidoA) binds specifically to the 2-5A-binding site of the 80- and 37-kDa RNase L. The rate of RNA hydrolysis by the 37-kDa RNase L was three times faster than the 80-kDa RNase L. The data obtained from these 2-5A binding and 2-5A-dependent activation studies demonstrate the utility of [(32)P]pApAp(8-azidoA) for the detection of the 37-kDa RNase L in peripheral blood mononuclear cell extracts.

 

Source: Shetzline SE, Suhadolnik RJ. Characterization of a 2′,5′-oligoadenylate (2-5A)-dependent 37-kDa RNase L: azido photoaffinity labeling and 2-5A-dependent activation. J Biol Chem. 2001 Jun 29;276(26):23707-11. Epub 2001 Apr 25. http://www.jbc.org/content/276/26/23707.long (Full article)

 

Biochemical evidence for a novel low molecular weight 2-5A-dependent RNase L in chronic fatigue syndrome

Abstract:

Previous studies from this laboratory have demonstrated a statistically significant dysregulation in several key components of the 2′,5′-oligoadenylate (2-5A) synthetase/RNase L and PKR antiviral pathways in chronic fatigue syndrome (CFS) (Suhadolnik et al. Clin Infect Dis 18, S96-104, 1994; Suhadolnik et al. In Vivo 8, 599-604, 1994). Two methodologies have been developed to further examine the upregulated RNase L activity in CFS.

First, photoaffinity labeling of extracts of peripheral blood mononuclear cells (PBMC) with the azido 2-5A photoaffinity probe, [32P]pApAp(8-azidoA), followed by immunoprecipitation with a polyclonal antibody against recombinant, human 80-kDa RNase L and analysis under denaturing conditions. A subset of individuals with CFS was identified with only one 2-5A binding protein at 37 kDa, whereas in extracts of PBMC from a second subset of CFS PBMC and from healthy controls, photolabeled/immunoreactive 2-5A binding proteins were detected at 80, 42, and 37 kDa.

Second, analytic gel permeation HPLC was completed under native conditions. Extracts of healthy control PBMC revealed 2-5A binding and 2-5A-dependent RNase L enzyme activity at 80 and 42 kDa as determined by hydrolysis of poly(U)-3′-[32P]pCp. A subset of CFS PBMC contained 2-5A binding proteins with 2-5A-dependent RNase L enzyme activity at 80, 42, and 30 kDa. However, a second subset of CFS PBMC contained 2-5A binding and 2-5A-dependent RNase L enzyme activity only at 30 kDa. Evidence is provided indicating that the RNase L enzyme dysfunction in CFS is more complex than previously reported.

 

Source: Suhadolnik RJ, Peterson DL, O’Brien K, Cheney PR, Herst CV, Reichenbach NL, Kon N, Horvath SE, Iacono KT, Adelson ME, De Meirleir K, De Becker P,Charubala R, Pfleiderer W. Biochemical evidence for a novel low molecular weight 2-5A-dependent RNase L in chronic fatigue syndrome. J Interferon Cytokine Res. 1997 Jul;17(7):377-85. http://www.ncbi.nlm.nih.gov/pubmed/9243369

 

Changes in the 2-5A synthetase/RNase L antiviral pathway in a controlled clinical trial with poly(I)-poly(C12U) in chronic fatigue syndrome

Abstract:

Latent 2′, 5′-oligoadenylate (2-5A) synthetase activity, bioactive 2-5A and RNase L activity were measured in extracts of peripheral blood mononuclear cells (PMBC) before and during a randomized, multicenter, placebo-controlled, double-blind study of poly(I)-poly(C12U) in individuals with chronic fatigue syndrome (CFS) as defined by the Centers for Disease Control and Prevention. The mean values for bioactive 2-5A and RNase L activity were significantly elevated at baseline compared to controls (p < .0001 and p = .001, respectively). In individuals that presented with elevated RNase L activity at baseline, therapy with poly(I)-poly(C12U) resulted in a significant decrease in both bioactive 2-5A and RNase L activity (p = .09 and p = .005, respectively). Decrease in RNase L activity in individuals treated with poly(I)-poly(C12U) correlated with cognitive improvement (p = .007). Poly(I)-poly(C12U) therapy resulted in a significant decrease in bioactive 2-5A and RNase L activity in agreement with clinical and neuropsychological improvements (Strayer DR, et al., Clin. Infectious Dis. 18:588-595, 1994). The results described show that poly(I)-poly(C12U) is a biologically active drug in CFS.

 

Source: Suhadolnik RJ, Reichenbach NL, Hitzges P, Adelson ME, Peterson DL, Cheney P, Salvato P, Thompson C, Loveless M, Müller WE, et al. Changes in the 2-5A synthetase/RNase L antiviral pathway in a controlled clinical trial with poly(I)-poly(C12U) in chronic fatigue syndrome. In Vivo. 1994 Jul-Aug;8(4):599-604. http://www.ncbi.nlm.nih.gov/pubmed/7893988

 

Upregulation of the 2-5A synthetase/RNase L antiviral pathway associated with chronic fatigue syndrome

Abstract:

Levels of 2′,5′-oligoadenylate (2-5A) synthetase, bioactive 2-5A, and RNase L were measured in extracts of peripheral blood mononuclear cells (PBMCs) from 15 individuals with chronic fatigue syndrome (CFS) before and during therapy with the biological response modifier poly(I).poly(C12U) and were compared with levels in healthy controls.

Patients differed significantly from controls in having a lower mean basal level of latent 2-5A synthetase (P < .0001), a higher pretreatment level of bioactive 2-5A (P = .002), and a higher level of pretherapy RNase L activity (P < .0001). PBMC extracts from 10 persons with CFS had a mean basal level of activated 2-5A synthetase higher than the corresponding control value (P = .009). All seven pretherapy PBMC extracts tested were positive for the replication of human herpesvirus 6 (HHV-6).

Therapy with poly(I).poly(C12U) resulted in a significant decrease in HHV-6 activity (P < .01) and in downregulation of the 2-5A synthetase/RNase L pathway in temporal association with clinical and neuropsychological improvement. The upregulated 2-5A pathway in CFS before therapy is consistent with an activated immune state and a role for persistent viral infection in the pathogenesis of CFS. The response to therapy suggests direct or indirect antiviral activity of poly(I).poly(C12U) in this situation.

 

Source: Suhadolnik RJ, Reichenbach NL, Hitzges P, Sobol RW, Peterson DL, Henry B, Ablashi DV, Müller WE, Schröder HC, Carter WA, et al. Upregulation of the 2-5A synthetase/RNase L antiviral pathway associated with chronic fatigue syndrome. Clin Infect Dis. 1994 Jan;18 Suppl 1:S96-104. http://www.ncbi.nlm.nih.gov/pubmed/8148461