Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer drug that selectively induces apoptosis in a variety of cancer cells by interacting with death receptors DR4 and DR5. TRAIL can also bind to decoy receptors (DcR1, DcR2, and osteoprotegerin receptor) that cannot induce apoptosis. The occurrence of DR5-responsive tumor cells indicates that a DR5 receptor-specific TRAIL variant will permit tumor-selective therapies. By using the automatic design algorithm FOLD-X, we successfully generated DR5-selective TRAIL variants. These variants do not induce apoptosis in DR4-responsive cell lines but show a large increase in biological activity in DR5-responsive cancer cell lines. Even wild-type TRAIL-insensitive ovarian cancer cell lines could be brought into apoptosis. In addition, our results demonstrate that there is no requirement for antibody-mediated cross-linking or membrane-bound TRAIL to induce apoptosis through DR5.computational protein design ͉ receptor selectivity ͉ biopharmaceutical ͉ death receptor ͉ apoptosis-inducing ligand 2 T umor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is currently attracting great interest as a potential anticancer therapeutic. TRAIL, in its soluble form, selectively induces apoptosis in tumor cells in vitro and in vivo by a death receptor-mediated process. Unlike other apoptosis-inducing TNF family members, soluble TRAIL appears to be inactive against normal healthy tissue (1). Reports in which TRAIL induces apoptosis in normal cells could be attributed to the specific preparations of TRAIL used (2). TRAIL shows a high degree of promiscuity as it binds to five cognate receptors: DR4 (TRAIL-R1) and DR5 (TRAIL-R2) and the decoy receptors DcR1 (TRAIL-R3), DcR2 (TRAIL-R4), and osteoprotegerin (OPG) (3). Upon binding to TRAIL, DR4 and DR5 receptors recruit Fas-associated death domain, which binds and activates the initiator caspase 8, leading to apoptosis (4-6). DcR1 or DcR2 do not contain a death domain or a truncated death domain, respectively, and therefore could prevent apoptosis by sequestering available TRAIL or by interfering in the formation of a TRAIL-DR4 or -DR5 signaling complex (7).Use of TRAIL receptor-selective variants could permit better tumor-specific therapies through escape from the decoy receptor-mediated antagonism, resulting in a lower administrated dose with possibly fewer side effects and as alternatives to existing agonistic receptor antibodies (8-10). In experimental anticancer treatments, the receptors DR4 and͞or DR5 were shown to be up-regulated after treatment with DNA-damaging chemotherapeutic drugs, and the response to TRAIL-induced apoptosis was significantly increased (3, 11). In addition, irradiation appears to specifically up-regulate DR5 receptor expression, and the combination of irradiation and TRAIL treatment has been demonstrated to have an additive or synergistic effect (12). Thus, we chose to develop DR5 receptor-selective TRAIL variants by using a computational design strategy. Computatio...

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The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.

The RET proto-oncogene encodes a receptor tyrosine kinase whose dysfunction plays a crucial role in the development of several neural crest disorders. Distinct activating RET mutations cause multiple endocrine neoplasia type 2A (MEN2A), type 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC). Despite clear correlations between the mutations found in these cancer syndromes and their phenotypes, the molecular mechanisms connecting the mutated receptor to the different disease phenotypes are far from completely understood. Luciferase reporter assays in combination with immunoprecipitations, and Western and immunohistochemistry analyses were done in order to characterize the signaling properties of two FMTC-associated RET mutations, Y791F and S891A, respectively, both affecting the tyrosine kinase domain of the receptor. We show that these RET-FMTC mutants are monomeric receptors which are autophosphorylated and activated independently of glial cell line-derived neurotrophic factor. Moreover, we show that the dysfunctional signaling properties of these mutants, when compared with wild-type RET, involve constitutive activation of signal transducers and activators of transcription 3 (STAT3). Furthermore, we show that STAT3 activation is mediated by a signaling pathway involving Src, JAK1, and JAK2, differing from STAT3 activation promoted by RET(C634R) which was previously found to be independent of Src and JAKs. Three-dimensional modeling of the RET catalytic domain suggested that the structural changes promoted by the respective amino acids substitutions lead to a more accessible substrate and ATP-binding monomeric conformation. Finally, immunohistochemical analysis of FMTC tumor samples support the in vitro data, because nuclear localized, Y705-phosphorylated STAT3, as well as a high degree of RET expression at the plasma membrane was observed.

Fluorescence resonance energy transfer (FRET)-based detection of protein interactions is limited by the very narrow range of FRET-permitting distances. We show two different strategies for the rational design of weak helper interactions that co-recruit donor and acceptor fluorophores for a more robust detection of bimolecular FRET: (i) in silico design of electrostatically driven encounter complexes and (ii) fusion of tunable domain-peptide interaction modules based on WW or SH3 domains. We tested each strategy for optimization of FRET between (m)Citrine and mCherry, which do not natively interact. Both approaches yielded comparable and large increases in FRET efficiencies with little or no background. Helper-interaction modules can be fused to any pair of fluorescent proteins and could, we found, enhance FRET between mTFP1 and mCherry as well as between mTurquoise2 and mCitrine. We applied enhanced helper-interaction FRET (hiFRET) probes to study the binding between full-length H-Ras and Raf1 as well as the drug-induced interaction between Raf1 and B-Raf.