The
diphthamide on human eukaryotic translation
elongation factor 2 (eEF2) is the target of
ADP ribosylating
diphtheria toxin (DT) and Pseudomonas
exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis
proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7
breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on
diphthamide synthesis and toxin sensitivity, and to address the
biological consequence of
diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly
diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall
diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without
diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (
diphthine-precursor) modified eEF2. Loss of
diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other
protein synthesis inhibitors, such as
saporin or
cycloheximide. Surprisingly, cells without
diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light
polypeptide gene enhancer in B cells (NF-κB) and
death-receptor pathways without crossing lethal thresholds. In consequence, loss of
diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of
diphthamide in modulating NF-κB,
death receptor, or apoptosis pathways.