Identification of 2′-Phosphodiesterase, Which Plays a Role in the 2-5A System Regulated by Interferon

Abstract

The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5-triphosphorylated, 2,5-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2,5-oligoadenylate synthetases (2,5-OASs), inactivated by 5-phosphatase and completely degraded by 2-phosphodiesterase (2-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2,5-OASs and RNase L have been molecularly cloned and studied well, the identification of 2-PDE has remained elusive. Here, we describe the first identification of 2-PDE, the third key enzyme of the 2-5A system. We found a putative 2-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2-PDE by mass spectrometry. Based on the full-length sequence of the human 2-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2-PDE expressed in mammalian cells certainly cleaved the 2,5-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2-PDE were found, the human 2-PDE was considered to be a unique enzyme without isoform. Suppression of 2-PDE by a small interfering RNA and a 2-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments.

A scientific journey through the 2-5A/RNase L system

“…The principal species of 2-5A found in such cells is the trimeric form, p 3 (A2′p5′) 2 A [21]. 2-5A is a transient signaling molecule that is degraded within minutes by the combined action of 2′-phosphodiesterase and 5′-phosphatase(s) [24,25]. The only well-established function of 2-5A is activation of the latent endoribonuclease, RNase L [26].…”

Diverse functions of RNase L and implications in pathology

“…It is dephosphorylated by general phosphatases at its 5′ end, leaving the so-called core oligoadenylate that is unable to efficiently activate RNase L [14]. 2-5A can also be degraded from the 2′,3′-termini by a 2′-phosphodiesterase [15].…”

Role of 2-5A-dependent RNase-L in senescence and longevity

“…2-5A induces the dimerization and activation of a latent endoribonuclease, RNase-L, which cleaves single-stranded viral, messenger and ribosomal RNAs. RNase-L activity is attenuated by proteasomal degradation (Chase et al, 2003), 2-5A inactivation by cellular phosphatases and a 2 0 phosphodiesterase (Kubota et al, 2004), and by an RNase-L inhibitor, RLI (Bisbal et al, 1995). Experimental modulation of RNase-L expression and activity has confirmed its role in the antiviral, antiproliferative and proapoptotic activities of IFN (Hassel et al, 1993;Castelli et al, 1997;Zhou et al, 1997).…”