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Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and plays multifunctional roles in the immune system. In the present study, a homolog of TRAIL from the Mongolian horse (named ecTRAIL) was identified and characterized. The 870-bp open reading frame encodes a polypeptide of 289 amino acid residues with a predicted molecular weight of 33.47 kDa and p I of 8.47. The genomic structure of ecTRAIL shares a five-exon/six-intron arrangement similar to its orthologs. Multiple alignments show that ecTRAIL is a type II transmembrane protein with a typical transmembrane region, three conserved cysteine residues (Cys56, Cys77, Cys238) and a TNF family signature sequence ([LV]-x-[LIVM]-x(3)-G-[LIVMF]-Y-[LIVMFY](2)-x(2)-[QEKHL]-[LIVMGT]-x-[LIVMFY]). Three-dimensional structure prediction based on the same template revealed that the positional arrangement of the key amino acid residues, Cys238 and Cys230 in ecTRAIL and human TRAIL, respectively, is significantly conserved. Evolutionary analysis suggests that ecTRAIL is most closely related to its ortholog from pigs, with an identity of 83.99%. The solubilizing small ubiquitin-related modifier (SUMO) tag fused recombinant protein SUMO-ecsTRAIL was successfully expressed in E. coli and exhibited binding activity and cytotoxicity to HeLa cells in a cross-species manner in vitro . These results provide a better understanding of TRAILs in mammals and indicate that ecTRAIL may play an important role in the immune response in horses. [ABSTRACT FROM AUTHOR]

Abstract: TWEAK is a member of the tumor necrosis factor superfamily. The interaction of TWEAK with its receptor Fn14 regulates multiple cellular responses, including stimulation of proliferation, migration, apoptosis, angiogenesis, and induction of proinflammatory cytokines. This paper reports for the first time the molecular cloning of dog TWEAK and Fn14. The open reading frame (ORF) of dog TWEAK is 750bp, its genomic DNA consists of seven exons and six introns and is approximately 10kb in size by computer-assisted analysis. Sequence similarity at the amino acid level between dog TWEAK and human or mouse was 95 and 92%, respectively. The ORF of dog Fn14 contains 390bp. Sequence similarity at the amino acid level between dog Fn14 and human, or mouse, or frog was 95, 93 and 64%, respectively. Real-time quantitative PCR analysis revealed that both TWEAK and Fn14 are constitutively expressed in various tissues in dog. Furthermore, we verified dTWEAK interacted with dFn14 by yeast two-hybrid assay. Our results suggest that the TWEAK–Fn14 pathway is evolutionarily highly conserved. It will be helpful for investigation on the biological role of the TWEAK/Fn14 system in this important animal model. Furthermore, it provides insight into the molecular evolution of the emerging TWEAK and Fn14 families. [ABSTRACT FROM AUTHOR]