Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor.
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.
Roche Pharma Research & Early Development, Large Molecule Research, Roche Innovation Center Penzberg, 82377 Penzberg, Germany;
Sebastian
S
Stahl
Roche Pharma Research & Early Development, Discovery Oncology, Roche Innovation Center Penzberg, 82377 Penzberg, Germany;
Ana Rita
AR
da Silva Mateus Seidl
Roche Pharma Research & Early Development, Pharmaceutical Sciences, Translational Technology and Bioinformatics, Roche Innovation Center Basel, 4070 Basel, Switzerland;
Axel
A
Ducret
Roche Pharma Research & Early Development, Pharmaceutical Sciences, Translational Technology and Bioinformatics, Roche Innovation Center Basel, 4070 Basel, Switzerland;
Sabine
S
Kux van Geijtenbeek
Roche Pharma Research & Early Development, Pharmaceutical Sciences, Translational Technology and Bioinformatics, Roche Innovation Center Basel, 4070 Basel, Switzerland;
Sven
S
Michel
Roche Pharma Research & Early Development, Pharmaceutical Sciences, Translational Technology and Bioinformatics, Roche Innovation Center Penzberg, 82377 Penzberg, Germany;
Tomas
T
Racek
Roche Pharma Research & Early Development, Pharmaceutical Sciences, Translational Technology and Bioinformatics, Roche Innovation Center Penzberg, 82377 Penzberg, Germany;
Fabian
F
Birzele
Roche Pharma Research & Early Development, Large Molecule Research, Roche Innovation Center Penzberg, 82377 Penzberg, Germany;
Alexander K
AK
Haas
Roche Pharma Research & Early Development, Pharmaceutical Sciences, Translational Technology and Bioinformatics, Roche Innovation Center Penzberg, 82377 Penzberg, Germany;
Ruediger
R
Rueger
Roche Diagnostics, Roche Professional Diagnostics, Biological Rare Reagents, 82377 Penzberg, Germany;
Michael
M
Gerg
Roche Pharma Research & Early Development, Discovery Oncology, Roche Innovation Center Penzberg, 82377 Penzberg, Germany;
Gerhard
G
Niederfellner
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda MD 20892-4263 pastani@mail.nih.gov ulrich.brinkmann@roche.com.
Ira
I
Pastan
Roche Pharma Research & Early Development, Large Molecule Research, Roche Innovation Center Penzberg, 82377 Penzberg, Germany; pastani@mail.nih.gov ulrich.brinkmann@roche.com.
Ulrich
U
Brinkmann
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D001426:Bacterial Proteins / Q000494:pharmacology; D001943:Breast Neoplasms / Q000473:pathology; D019731:Carbon-Nitrogen Ligases / Q000172:deficiency / Q000502:physiology; D045744:Cell Line, Tumor; D004167:Diphtheria Toxin / Q000494:pharmacology; D005260:Female; D018628:Gene Dosage; D055786:Gene Knockout Techniques; D006639:Histidine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000172:deficiency; D006801:Humans; D016328:NF-kappa B / Q000502:physiology*; D009363:Neoplasm Proteins / Q000502:physiology; D020652:Peptide Elongation Factor 2 / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D053218:Receptors, Death Domain / Q000502:physiology*
D001426:Bacterial Proteins; D004167:Diphtheria Toxin; D016328:NF-kappa B; D009363:Neoplasm Proteins; D020652:Peptide Elongation Factor 2; D053218:Receptors, Death Domain; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa; D006639:Histidine; C027527:diphthamide; D019731:Carbon-Nitrogen Ligases
ADP-ribosylation of eEF2; DPH gene knockout; Pseudomonas exotoxin; diphtheria toxin; translation