title: Identification and characterization of the direct interaction between methotrexate (MTX) and high-mobility group box 1 (HMGB1) protein.
abstract: BACKGROUND
  Methotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood. METHODOLOGY/RESULT: Here, we performed a screening of MTX-binding proteins using T7 phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 phage display screen, we identified T7 phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD) of 0.50±0.03 µM for Al and 0.24 ± 0.01 µM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macrophage-like cells (RAW 264.7), TNF-α release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited in the presence of MTX.
  
  
  CONCLUSIONS
  These data demonstrate that HMGB1 is a direct binding protein of MTX. Moreover, binding of MTX to RAGE-binding region in HMGB1 inhibited the HMGB1/RAGE interaction at the molecular and cellular levels. These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.
authors: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Yamazaki, Noda, Chiba, Japan.
authors: Yuki
authors: Y
authors: Kuroiwa
authors: Yoichi
authors: Y
authors: Takakusagi
authors: Tomoe
authors: T
authors: Kusayanagi
authors: Kouji
authors: K
authors: Kuramochi
authors: Takahiko
authors: T
authors: Imai
authors: Tomoko
authors: T
authors: Hirayama
authors: Ichiaki
authors: I
authors: Ito
authors: Michiteru
authors: M
authors: Yoshida
authors: Kengo
authors: K
authors: Sakaguchi
authors: Fumio
authors: F
authors: Sugawara
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D017123:Bacteriophage T7 / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D024243:HMGB1 Protein / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008727:Methotrexate / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D000964:Antimetabolites, Antineoplastic; D024243:HMGB1 Protein; D019151:Peptide Library; D010455:Peptides; D014409:Tumor Necrosis Factor-alpha; D008727:Methotrexate
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