Influence of DPH1 and DPH5 Protein Variants on the Synthesis of Diphthamide, the Target of ADPRibosylating Toxins

Abstract

The diphthamide on eukaryotic translation elongation factor 2 (eEF2) is the target of ADP-ribosylating toxins and -derivatives that serve as payloads in targeted tumor therapy. Diphthamide is generated by seven DPH proteins; cells deficient in these (DPHko) lack diphthamide and are toxin-resistant. We have established assays to address the functionality of DPH1 (OVCA1) and DPH5 variants listed in dbSNP and cosmic databases: plasmids encoding wildtype and mutant DPHs were transfected into DPHko cells. Supplementation of DPH1 and DPH5 restores diphthamide synthesis and toxin sensitivity in DPH1ko and DPH5ko cells, respectively. Consequently, the determination of the diphthamide status of cells expressing DPH variants differentiates active and compromised proteins. The DPH1 frameshift variant L96fs* (with N-terminal 96 amino acids, truncated thereafter) and two splice isoforms lacking 80 or 140 amino acids at their N-termini failed to restore DPH1ko deficiency. The DPH1 frameshift variant R312fs* retained some residual activity even though it lacks a large C-terminal portion. DPH1 missense variants R27W and S56F retained activity while S221P had reduced activity, indicated by a decreased capability to restore diphthamide synthesis. The DPH5 nonsense or frameshift variants E60*, W136fs* and R207* (containing intact N-termini with truncations after 60, 136 or 207 amino acids, respectively) were inactive: none compensated the deficiency of DPH5ko cells. In contrast, missense variants D57G, G87R, S123C and Q170H as well as the frequently occurring DPH5 isoform delA212 retained activity. Sensitivity to ADP-ribosylating toxins and tumor-targeted immunotoxins depends on diphthamide which, in turn, requires DPH functionality. Because of that, DPH variants (in particular those that are functionally compromised) may serve as a biomarker and correlate with the efficacy of immunotoxin-based therapies.

Diphthamide-deficiency syndrome: a novel human developmental disorder and ribosomopathy

“…Therefore, toxin-ADPR assays can be applied to probe the presence or absence of diphthamide on human eEF2. To investigate the effect of the huDPH2 c.922C > T (p.(Gln308*)) and c.601C > T (p.(Arg201Cys)) variants on diphthamide synthesis in human cells using ADPR assays, protein extracts were prepared from DPH2-deficient MCF7 cells recombinantly supplemented with wild-type or mutated DPH2 genes as previously described [ 8 , 24 ]. The ADPR reaction transfers ADP to the diphthamide moiety of eEF2, using cell extracts as a source of diphthamide-eEF2 and NAD to donate ADP.…”

“…This places a biotin-label onto eEF2. Presence of bioADP-diphthamide-eEF2 in cell extracts was subsequently monitored by a Western blot procedure, detecting the modified eEF2 via labeled streptavidin [ 24 ]. Because the presence of diphthamide is essential for toxin-mediated ADPR, eEF2 that lacks diphthamide does not become labeled via ADPR.…”

“…One explanation for the developmental phenotypes of diphthamide deficiency may be that it modulates stress- and development-related pathways and transcriptional patterns in mammalian cells. These include phosphorylation events involved in regulation of translation [ 47 ], responses to oxidative stress [ 24 ], NF-κB responses, and pathways including tumor necrosis factor-mediated pathways [ 8 ]. The latter processes, in particular NF-κB-associated transcriptional responses, are relevant for general as well as neuronal development [ 48 ].…”

“…ADPR of human diphthamide-modified eEF2 was evaluated via toxin-mediated transfer of biotinylated ADP to eEF2 present in total cell extracts of transfected or non-transfected MCF7 DPH2 ko cells. The assays were established based on the principle previously described for the analyses of DPH1 and DPH5 variants [ 24 ] except that DPH2 -deficient cells and DPH2 expression plasmids were applied instead of DPH1 ko or DPH5 ko cells and corresponding DPH1/5 plasmids. Radioimmunoprecipitation assay buffer (RIPA) extracts of DPH2 ko cells [ 8 ] were exposed to DT and biotinylated NAD 1 h at 25 °C, subsequently subjected to reducing SDS-PAGE and blotted onto membranes.…”

Diphthamide affects selenoprotein expression: Diphthamide deficiency reduces selenocysteine incorporation, decreases selenite sensitivity and pre-disposes to oxidative stress

“…The post-translational diphthamide modification of eukaryotic translation elongation factor eEF2 is highly conserved in eukaryotes as well as in the archaeal eEF2 counterpart [1] , [2] , [3] , [4] , [5] . It consists of a histidine in elongation factor 2 (His 715 in human eEF2), modified by the concert action of diphthamide synthesis enzymes encoded by DPH genes DPH1–7 in humans, [6] , [7] , [8] , [9] , [10] , [11] , [12] . High conservation of diphthamide and -synthesis genes would suggest that this modification may be rather important for eEF2 functionality and hence for protein synthesis.…”