ORIGINAL PAPER Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to death receptor 5 Marine E. Gasparian Æ Boris V. Chernyak Æ Dmitry A. Dolgikh Æ Anne V. Yagolovich Æ Ekaterina N. Popova Æ Anna M. Sycheva Æ Sergey A. Moshkovskii Æ Mikhail P. Kirpichnikov Published online: 2 May 2009 Ó Springer Science+Business Media, LLC 2009 Abstract TRAIL (tumor necrosis factor (TNF) related apoptosis-inducing ligand) has been introduced as an extrinsic pathway inducer of apoptosis that does not have the toxicities of Fas and TNF. However, the therapeutic potential of TRAIL is limited because of many primary tumor cells are resistant to TRAIL. Despite intensive investigations, little is known in regards to the mechanisms underlying TRAIL selectivity and efficiency. A major reason likely lies in the complexity of the interaction of TRAIL with its five receptors, of which only two DR4 and DR5 are death receptors. Binding of TRAIL with decoy receptors DcR1 and DcR2 or soluble receptor osteopro- tegerin (OPG) fail to induce apoptosis. Here we describe design and expression in Escherichia coli of DR5-selective TRAIL variants DR5-A and DR5-B. The measurements of dissociation constants of these mutants with all five receptors show that they practically do not interact with DR4 and DcR1 and have highly reduced affinity to DcR2 and OPG receptors. These mutants are more effective than wild type TRAIL in induction of apoptosis in different cancer cell lines. In combination with the drugs targeted to cytoskeleton (taxol, cytochalasin D) the mutants of TRAIL induced apoptosis in resistant Hela cells overexpressing Bcl-2. The novel highly selective and effective DR5-A and DR5-B TRAIL variants will be useful in studies on the role of different receptors in TRAIL-induced apoptosis in sen- sitive and resistant cell lines. Keywords TRAIL Á Apoptosis Á Death receptors Á Decoy receptors Introduction Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also called apoptosis-inducing ligand 2 (Apo2L), triggers programmed cell death in various types of cancer cells but not in most normal cells [1]. TRAIL is a homo- trimeric protein that interacts with five receptors. Binding with DR4 and DR5 receptors induces death signals to the intracellular apoptotic machinery [2]. DcR1 and DcR2 are designated as decoy receptors that compete with DR4 and DR5 for TRAIL binding and antagonize TRAIL-induced apoptosis [3]. Osteoprotegerin (OPG) is a soluble TRAIL- interacting molecule which may have a more prominent role in bone and myeloid cell development than in regu- lating TRAIL-induced apoptosis [4]. Many cancer cell lines express both DR4 and DR5, and each of these receptors can initiate apoptosis independently of the other. Recent evidence indicates that both tumor and normal cells can acquire resistance to TRAIL-induced kill- ing by upregulating either of the two antagonistic receptors, DcR1 and DcR2 so the four TRAIL receptors are involved in regulation of TRAIL-induced apoptosis [5–9]. DcR1 and Electronic supplementary material The online version of this article (doi:10.1007/s10495-009-0349-3) contains supplementary material, which is available to authorized users. M. E. Gasparian (&) Á D. A. Dolgikh Á A. V. Yagolovich Á M. P. Kirpichnikov Laboratory of Protein Engineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 16/10 Miklukho-Maklaya, 117997 Moscow, Russia e-mail: marine_gasparian@yahoo.com B. V. Chernyak Á E. N. Popova A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia A. M. Sycheva Á S. A. Moshkovskii V. N. Orekhovich Institute of Biomedical Chemistry, RAMS, 119121 Moscow, Russia 123 Apoptosis (2009) 14:778–787 DOI 10.1007/s10495-009-0349-3