Novel Mutants of Human Tumor Necrosis Factor with Dominant-Negative Properties

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L. N. Shingarova^1*, E. F. Boldyreva¹, S. A. Yakimov¹, S. V. Guryanova¹, D. A. Dolgikh^1,2, S. A. Nedospasov^2,3, and M. P. Kirpichnikov^1,2

¹Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (495) 330-6983; E-mail: lshing@mx.ibch.ru

²Biological Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 330-6983; E-mail: dolgikh@nmr.ru

³Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, 19991 Moscow, Russia; fax: (499) 135-1405; E-mail: sergei.nedospasov@gmail.com

^* To whom correspondence should be addressed.

Received June 30, 2010; Revision received July 14, 2010

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Tumor necrosis factor (TNF) is a polyfunctional cytokine, one of the key mediators of inflammation and innate immunity. On the other hand, systemic or local TNF overexpression is typical of such pathological states as rheumatoid arthritis, psoriasis, Crohn’s disease, septic shock, and multiple sclerosis. Neutralization of TNF activity has a marked curative effect for some diseases; therefore, the search for various TNF blockers is a promising field of protein engineering and biotechnology. According to the previously developed concept concerning the possibility of designing dominant-negative mutants, the following TNF variants have been studied: TNFY87H + A145R, TNFY87H + A96S + A145R, and TNFV91N + A145R. All of these form inactive TNF heterotrimers with the native protein. The ability of mutants to neutralize the effect of TNF was investigated. The addition of mutants to the native protein was shown to provide a concentration-dependent suppression of TNF cytotoxicity against the mouse fibroblast cell line L929. Thus, novel inhibitors of human TNF can be engineered on the basis of these muteins.

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KEY WORDS: tumor necrosis factor (TNF), mutants, TNF inhibitors

DOI: 10.1134/S0006297910120060

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