Identification of 2'-phosphodiesterase, which plays a role in the 2-5A system regulated by interferon.
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.
Kubota
Kazuishi
K
Biomedical Research Laboratories, Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan. kkubot@sankyo.co.jp
Nakahara
Kaori
K
Ohtsuka
Toshiaki
T
Yoshida
Shuku
S
Kawaguchi
Junko
J
Fujita
Yoko
Y
Ozeki
Yohei
Y
Hara
Ayako
A
Yoshimura
Chigusa
C
Furukawa
Hidehiko
H
Haruyama
Hideyuki
H
Ichikawa
Kimihisa
K
Yamashita
Makoto
M
Matsuoka
Tatsuji
T
Iijima
Yasuteru
Y
D000227:Adenine Nucleotides / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers; D005095:Exoribonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D014779:Virus Replication / Q000187:drug effects
D000227:Adenine Nucleotides; D017931:DNA Primers; D009843:Oligoribonucleotides; D010726:Phosphodiesterase Inhibitors; C023505:2',5'-oligoadenylate; D005095:Exoribonucleases; C029364:2'-phosphodiesterase