A New Efficient Method for Production of Recombinant Antitumor Cytokine TRAIL and Its Receptor-Selective Variant DR5-B.

The cytokine TRAIL induces apoptosis in tumor cells of various origin without affecting normal cells. Clinical trials of TRAIL-receptor (DR4 and DR5) agonists (recombinant TRAIL or death receptors antibodies) have largely failed because most human tumors were resistant to them. Currently, a second generation of agents targeted at TRAIL-R with increased efficiency has been developed. To this end, we have developed DR5-B, a variant of TRAIL selectively interacting with DR5. We have developed a new efficient method for production of TRAIL and DR5-B using expression of these proteins in Escherichia coli strain SHuffle B. The proteins were isolated from the cytoplasmic fraction of cells and purified to a high degree of homogeneity using metal-affinity and ion-exchange chromatography. The protein yield was 211 and 173 mg from one liter of cell culture for DR5-B and TRAIL, respectively, which significantly exceeded the results obtained by other methods. DR5-B killed tumor cells of different origin more efficiently and rapidly compared with TRAIL. The resulting preparations can be used for the study of TRAIL signaling pathways and in preclinical and clinical trials as antitumor agents.

Isolation, characterisation and reconstitution of cell death signalling complexes.

Apoptosis and necroptosis are dependent on the formation/activation of distinct multi-protein complexes; these include the Death-Inducing Signalling Complex (DISC), apoptosome, piddosome, necrosome and ripoptosome. Despite intense research, the mechanisms that regulate assembly/function of several of these cell death signalling platforms remain to be elucidated. It is now increasingly evident that the composition and stoichiometry of components within these key signalling platforms not only determines the final signalling outcome but also the mode of cell death. Characterising these complexes can therefore provide new insights into how cell death is regulated and also how these cell death signalling platforms could potentially be targeted in the context of disease. Large multi-protein complexes can initially be separated according to their size by gel filtration or sucrose density gradient centrifugation followed by subsequent affinity-purification or immunoprecipitation. The advantage of combining these techniques is that you can assess the assembly of individual components into a complex and then assess the size and stoichiometric composition of the native functional signalling complex within a particular cell type. This, alongside reconstitution of a complex from its individual core components can therefore provide new insight into the mechanisms that regulate assembly/function of key multi-protein signalling complexes. Here, we describe the successful application of a range of methodologies that can be used to characterise the assembly of large multi-protein complexes such as the apoptosome, DISC and ripoptosome. Together with their subsequent purification and/or reconstitution, these approaches can provide novel insights into how cell death signalling platforms are regulated in both normal cell physiology and disease.

Isolation of a TRAIL antagonist from the serum of HIV-infected patients.

Virus-host interactions are characterized by the selection of adaptive mechanisms by which to evade pathogenic and defense mechanisms, respectively. In primary T cells infected with HIV, HIV infection up-regulates TNF-related apoptosis inducing ligand (TRAIL) and death-inducing TRAIL receptors, but blockade of TRAIL:TRAIL receptor interaction does not alter HIV-induced cell death. Instead, HIV infection results in a novel splice variant that we call TRAIL-short (TRAIL-s), which antagonizes TRAIL-R2. In HIV patients, plasma TRAIL-s concentration increases with increasing viral load and renders cells resistant to TRAIL-induced death. Knockdown of TRAIL-s abrogates this resistance. We propose that TRAIL-s is a novel adaptive mechanism of apoptosis resistance acquired by HIV-infected cells to avoid their elimination by TRAIL-dependent effector mechanism.

Characterization of a novel anti-DR5 monoclonal antibody WD1 with the potential to induce tumor cell apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL) is a TNF family member capable of inducing apoptosis. Death receptor 5 (DR 5) is a key receptor of TRAIL and plays an important role in TRAIL-induced apoptosis. To prepare monoclonal antibodies (mAbs) against DR5, cDNA encoding soluble DR5 (sDR5) was firstly amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with specific primers, and then inserted into a prokaryotic expression vector pET-30a. The recombinant plasmid was expressed in Escherichia coli strain BL21 (DE3), and sDR5 was purified by nickel affinity chromatography. As an antigen, sDR5 was used to immunize mice. Hybridomas secreting antibodies against sDR5 were identified. One positive clone was selected to produce antibody, WD1. ELISA and immunofluorescence demonstrated that WD1 could bind recombinant sDR5 and membrane-bound DR5 (mDR5) on Jurkat and Molt-4 cells. ATPLite assays showed that Jurkat and Molt-4 cells were sensitive to the antibody in a dose dependent manner. The Annexin V/PI assays and Giemsa's staining both showed that WD1 could induce Jurkat cell apoptosis efficiently. Transient transfection of 293T cells and indirect immunofluorescence assay demonstrated that mAb (WD1) couldn't cross-react with DR4. Our findings indicated that the novel antibody, WD1 could act as a direct agonist, bind DR5 characteristically, and initiate efficient apoptotic signaling and tumor regression. Thus, WD1 would be a leading candidate for potential cancer therapeutics.

The structure of the death receptor 4-TNF-related apoptosis-inducing ligand (DR4-TRAIL) complex.

The structure of death receptor 4 (DR4) in complex with TNF-related apoptosis-inducing ligand (TRAIL) has been determined at 3 Šresolution and compared with those of previously determined DR5-TRAIL complexes. Consistent with the high sequence similarity between DR4 and DR5, the overall arrangement of the DR4-TRAIL complex does not differ substantially from that of the DR5-TRAIL complex. However, subtle differences are apparent. In addition, solution interaction studies were carried out that show differences in the thermodynamics of binding DR4 or DR5 with TRAIL.

[Gene expression and primary functional characterization of human DR5 extracellular fragment].

AIM: To acquire human DR5 extracellular fragment with bioactivity. METHODS: Total RNA was prepared from Jurkat cells by Trizol. Human DR5 extracellular fragment gene was amplified by RT-PCR, cloned into pGEM-T Easy vector, and confirmed by sequence analysis. Then the gene was subcloned into expression vector pET30a with a His-tag at the amino terminus and expressed in E.coli BL21 (DE3). The products was purified by Ni-NTA chromatography column and identified by SDS-PAGE and Western blot. ELISA method was used to detect its binding activity to anti-DR5 monoclonal antibody (mAb) mDRA-6. RESULTS: Human DR5 extracellular fragment gene was successfully amplified and high level expression was obtained in E.coli BL21 (DE3) induced by 0.1 mmol/L IPTG. The DR5 extracellular fragment protein was identified by SDS-PAGE and Western blot analysis. ELISA results showed that the purified DR5 could be recognized by mDRA-6. CONCLUSION: The extracellular region of DR5 with bioactivity has been successfully expressed and purified, which lay the foundation for further study.