Yeast techniques for modeling drugs targeting Bcl-2 and caspase family members

Abstract

Development of drugs targeting Bcl-2 relatives and caspases, for treating diseases including cancer and inflammatory disorders, often involves measuring interactions with recombinant target molecules, and/or monitoring cancer cell killing in vitro. Here, we present yeast-based methods for evaluating drug-mediated inhibition of Bcl-2 relatives or caspases. Active Bax and caspases kill Saccharomyces cerevisiae, and pro-survival Bcl-2 proteins can inhibit Bax-induced yeast death. By measuring the growth or adenosine triphosphate content of transformants co-expressing Bax with pro-survival Bcl-2 relatives, we found that the Bcl-2 antagonist drugs ABT-737 or ABT-263 abolished Bcl-2 or Bcl-xL function and reduced Bcl-w activity, but failed to inhibit Mcl-1, A1 or the poxvirus orthologs DPV022 and SPPV14. Using this technique, we also demonstrated that adenoviral E1B19K was resistant to these agents. The caspase inhibitor Q-VD-OPh suppressed yeast death induced by caspases 1 and 3. Yeast engineered to express human apoptotic regulators enable simple, automatable assessment of the activity and specificity of candidate drugs targeting Bcl-2 relatives or caspases.

NK4 inhibits the proliferation and induces apoptosis of human rheumatoid arthritis synovial cells

“…9 In addition, flow cytometry analysis showed that NK4 could induce apoptosis and cell cycle arrest at G0/G1 in RA-FLS and MH7A cells. 15 In the present study, we found that NK4 decreased Bcl-2 protein level and increased Bax and cleaved caspase 3 protein levels, indicating an essential role of NK4 in the induction of the apoptosis of RA-FLS and MH7A cells.…”

“…The activation of caspase 3 is the key to cause apoptosis . Antiapoptotic Bcl‐2 and proapoptotic Bax belong to Bcl‐2 family and are the main regulators of caspase activation pathway during apoptosis . In the present study, we found that NK4 decreased Bcl‐2 protein level and increased Bax and cleaved caspase 3 protein levels, indicating an essential role of NK4 in the induction of the apoptosis of RA‐FLS and MH7A cells.…”

Sequence and partial functional analysis of canine Bcl-2 family proteins

“…As previously described for the human and/or murine orthologs (Ink et al, 1997;Tao et al, 1997;Ligr et al, 1998), expression of canine Bak and Bax showed a lethal effect in yeast. Moreover, this effect was abrogated on co-expression of the anti-apoptotic members Bcl-xL, Mcl-1 and Bcl-w as previously demonstrated for large part of their human orthologs in yeast (Tao et al, 1997;Beaumont et al, 2013). To our knowledge, abrogation of the effect of Bak by Bcl-w has not been previously reported in this model.…”

“…This particular phenomenon -a hallmark of mammalian Bax action -has been demonstrated following Bax expression in yeast (Manon et al, 1997). To date, several studies of mammalian Bcl-2 family proteins in S. cerevisiae have focused on genetic analyses of Bax, Bcl-xL and Bcl-2, while this model has rarely been used, if at all, to characterize further members such as Bak, Bcl-w, and Mcl-1 for instance (Bodrug et al, 1995;Tao et al, 1997;Beaumont et al, 2013).…”

Reconstituting the Mammalian Apoptotic Switch in Yeast

“…As the survival of different cancer cells depends on different specific antiapoptotic proteins, an easy method for testing the effect of these compounds in vivo, in system with individual antiapoptotic Bcl-2 family members, appears to be very useful. The effect of several BH3-mimetic compounds was tested in yeast cells coexpressing Bax together with different individual antiapoptotic proteins of the Bcl-2 family including Bcl-XL, Bcl-2, Bcl-w, Mcl-1 [81]. Two of tested drugs (ABT-737 and ABT-263) were shown to inhibit cell growth both on solid and liquid media in Bcl-2 family protein-dependent manner.…”