RESUMEN
The diphthamide on eukaryotic translation elongation factor 2 (eEF2) is the target of ADPribosylating 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 Nterminal 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.
Asunto(s)
Histidina/análogos & derivados , Antígenos de Histocompatibilidad Menor/genética , Proteínas Supresoras de Tumor/genética , ADP Ribosa Transferasas/toxicidad , Adenosina Difosfato Ribosa/metabolismo , Toxinas Bacterianas/toxicidad , Toxina Diftérica/toxicidad , Exotoxinas/toxicidad , Histidina/biosíntesis , Humanos , Inmunotoxinas/toxicidad , Células MCF-7 , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Factores de Virulencia/toxicidad , Exotoxina A de Pseudomonas aeruginosaRESUMEN
The ubiquitin-proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer.