A novel mouse model for inhibition of DOHH mediated hypusine modification reveals crucial function for embryonic development, proliferation and oncogenic transformation

Abstract

The central importance of translational control by post-translational modification has spurred major interest in regulatory pathways that control translation. One such pathway uniquely adds hypusine to eukaryotic initiation factor 5A (eIF5A), and thereby affects protein synthesis and, subsequently, cellular proliferation through an unknown mechanism. Using a novel conditional knockout mouse model and a Caenorhabditis elegans knockout model, we found an evolutionarily conserved role for the DOHH-mediated second step of hypusine synthesis in early embryonic development. At the cellular level, we observed reduced proliferation and induction of senescence in 3T3 Dohh−/− cells as well as reduced capability for malignant transformation. Furthermore, mass spectrometry showed that deletion of DOHH results in an unexpected complete loss of hypusine modification. Our results provide new biological insight into the physiological roles of the second step of the hypusination of eIF5A. Moreover, the conditional mouse model presented here provides a powerful tool for manipulating hypusine modification in a temporal and spatial manner, to analyse both how this unique modification normally functions in vivo as well as how it contributes to different pathological conditions.

Biological Relevance and Therapeutic Potential of the Hypusine Modification System

“…3G). According to previous data (33,45), these spots represent the unhypusinated (Fig. 3G, red arrow) and the more acidic unhypusinated and acetylated form of eIF-5A1 (Fig.…”

“…However, we have shown recently that deletion of Dohh and subsequent loss of hypusine modification induced an embryonic lethal phenotype in mice (33).…”

“…(33). The cells were cultured in DMEM (all cell culture media and additives were from Invitrogen) supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 g/ml streptomycin, 25 M ␤-mercaptoethanol, and 4 mM L-glutamine (37°C, 5% CO 2 , humidified atmosphere).…”

“…Morphological and Histopathological Analysis of Mouse Tissue-Mice were transcardially perfused with a mixture of 4% paraformaldehyde (and 1% glutaraldehyde) in 0.1 M PB buffer at pH 7.4 for tissue preparation, and bone marrow was morphologically analyzed as described before (33). Briefly, after postfixation overnight at 4°C, tibiae were decalcified for 4 days in 10% (w/v) EDTA in PBS and cut in 150-m sagittal sections with a vibratome.…”

“…Mice-To study the biological relevance and functional requirement of the hypusine modification in embryonic development (43,44) and for viability, we used a recently described conditional knock-out strain for Dohh (33) and established a new conditional knock-out mouse model for Dhs (B6.Dhps tm2a(EUCOMM)Wtsi ). For the generation of the latter strain, embryonic stem cell clones were obtained from the International Knock-out Mouse Consortium (30).…”