Helicobacter pylori is classified as the definitive carcinogen for stomach cancer in humans: It induces inflammation in cancer microenvironment of the stomach associated with induction of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), an endogenous tumor promoter. The finding of tumor promoting activity by Helicobacter pylori membrane protein 1 (HP-MP1) made it possible to identify HP0596 gene from the complete genome sequence of H. pylori strain 26695. The HP0596 protein, consisting of 172 amino acids with 19 kDa, was named TNF-α-inducing protein (Tipα), a new carcinogenic factor of H. pylori. To study the mechanism of Tipα, we prepared His-tagged recombinant Tipα protein (rTipα), the His-tag attached to N-terminal methionine, and deletion mutant of Tipα (rdel-Tipα) lacking the N-terminal 6 amino acids including 2 cysteines. The rTipα protein formed an active homodimer, and rdel-Tipα was an inactive monomer. Crystal structure of rdel-Tipα, however, showed a heart-shaped homodimer without covalent bonds, indicating the importance of N-terminal region. Nucleolin was identified as a receptor of Tipα using the pull-down assay with anti-FLAG antibody in a mixture of mouse gastric cancer cell lysates and rTipα-FLAG. Specifically, the homodimer of rTipα directly binds to two-thirds of C-terminal nucleolin on cell surface. Binding inhibitors of nucleolin, such as AS1411 and lactoferrin, are discussed as tools of gastric cancer treatment in humans.
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