Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to death receptor 5.
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 osteoprotegerin (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 sensitive and resistant cell lines.
Laboratory of Protein Engineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 16/10 Miklukho-Maklaya, 117997, Moscow, Russia. marine_gasparian@yahoo.com
Marine E
ME
Gasparian
Boris V
BV
Chernyak
Dmitry A
DA
Dolgikh
Anne V
AV
Yagolovich
Ekaterina N
EN
Popova
Anna M
AM
Sycheva
Sergey A
SA
Moshkovskii
Mikhail P
MP
Kirpichnikov
D019943:Amino Acid Substitution / Q000187:drug effects; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D015638:Cytochalasin D / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D017239:Paclitaxel / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*; D014461:Ultracentrifugation
D050505:Mutant Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand; D015638:Cytochalasin D; D017239:Paclitaxel