Abstract 1361: Characterization of TpeL, a RAS specific clostridial toxin

Abstract

Abstract Clostridium perfringes lethal toxin (TpeL) belongs to the family of large clostridial glycosylating cytotoxins. Clostridial toxins are glycosyltransferases that modify and deactivate small GTPases of the RHO and RAS subfamily. TpeL mono-glycosylates in the switch I domain (Thr35) of RAC and RAS small GTPases. Glycosylation of RAS at Thr35 prevents binding to the primary effector, RAF kinase and results in a blockade RAS signal transduction. RAS family proteins function as key regulators of cell proliferation, differentiation, survival and gene expression. Moreover, mutations in RAS proteins are highly prevalent in human cancers. Considering the specificity of TpeL for RAS, we decided to investigate the biochemical interaction between TpeL and RAS, and potentially develop tools to disrupt the RAS signaling pathway. In order to assess the effect of TpeL in vivo, we use RAS-dependent MEFs expressing different KRAS mutations and BRAF V600E. We expect that proliferation should be inhibited in MEF cells expressing RAS isoforms, but MEF cells expressing BRAF V600E should not be affected by any toxin. We found that TpeL treatment did not affect the viability of the BRAF V600E cells and only induced toxicity in RAS expressing cells. Consistent with this result, TpeL treatment inhibited MAPK signal transduction in the RAS expressing cells, but not in the BRAF V600E cells. Surprisingly, both cell viability as well as pERK levels remained unaffected in the KRAS Q61R MEF cells treated with TpeL, suggesting that KRAS Q61R is resistant to glycosylation. This may be due in part to the relatively slow intrinsic rate of GTP hydrolysis and very high levels of KRAS-GTP in KRAS Q61R cells. We also developed and adapted biochemical assays to study TpeL-KRAS interaction in vitro. Based on Alpha Assay and UDP Glow, TpeL activity does not appear to be nucleotide specific, in contrast to previously reported findings. We are currently testing if TpeL RAS glycosylation is competitive with or is inhibited by other molecules that bind to switch I, such as the RBD domain of RAF1 or GEF/GAP proteins. These data may indicate that the lack of activity in KRAS Q61R expressing MEF cells is due not to the high levels of RAS-GTP, but to the constitutive binding to effector molecules, which prevent TpeL from accessing KRAS. We also hope to identify the critical residues required for TpeL binding and catalysis. By elucidating the mechanism of substrate binding and substrate specificity we may gain insight into novel binding pockets that could be exploited therapeutically. Citation Format: Maria T. Abreu-Blanco. Characterization of TpeL, a RAS specific clostridial toxin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1361. doi:10.1158/1538-7445.AM2017-1361