An Essential Role of N-Terminal Arginylation in Cardiovascular Development

Abstract

Addendum to the main text (i) ATE1-/-embryos from ATE1 +/-intercrosses were present at the expected (~25%) frequency up to ~E13.5, but virtually no ATE1 -/-embryos were recovered alive by E17. Specifically, no ATE1 -/-mice were recovered amongst either 954 F 2 -generation pups of the C57BL/6J-129SvEv (mixed) background or 267 F 2 -generation pups of the 129SvEv (inbred) background. Timed intercrosses of ATE1 +/-mice were used to determine that ATE1 -/-embryos were present at approximately the expected (25%) frequency up to ~E13.5, but no ATE1 -/-embryos were recovered alive by E17.Until E12.5, ATE1 -/-embryos appeared to be morphologically normal; however, their growth stopped during E13.5-E15.5. By E14.5-E15.5, ~50% of ATE1 -/-embryos were still alive, but growth-retarded. Live E14.5-E15.5 embryos were capable of opening their mouths and flexing their bodies, suggesting the absence of gross neuromuscular defects. Sections through E13.5 ATE1-/-embryos indicated the presence and apparently normal appearance of major organs, except for the phenotypes described in the main text and below.(ii) We examined the expression of ATE1 mRNA during embryogenesis using Northern hybridization with total RNA from +/+ embryos ranging in age from E4.5 to E18.5. ATE1 mRNA was present at least as early as E4.5, and a strong spike of ATE1 expression was observed during E7.5-9.5 (Fig. S1E). The ~2 kb ATE1 transcript detected in adult mouse testis (1) was also clearly present during the spike of ATE1 expression in E7.5-E9.5 embryos (Fig. S1E). The ATE1 -allele was marked with NLS-β-galactosidase (hereafter βgal), expressed from the ATE1 promoter

Cell biology of embryonic migration

“…Analysis of these phenotypes provides insights into the underlying molecular reasons for birth defects, and may allow us to predict major functional roles for proteins, whose functions have not been well established. An example of such a protein is arginyltransferase, Ate1, whose knockout in mice results in embryonic lethality with specific defects in heart development and angiogenesis that suggest that this gene and the corresponding posttranslational modification plays a critical role in cell migration in situ (Kwon et al, 2002). In agreement with that, Ate1 has been recently shown to regulate actin cytoskeleton, lamella formation, and lamellipodial protrusion (Karakozova et al, 2006), however the exact role of Ate1 in embryogenesis and cell motility remains to be uncovered.…”

“…(Carmeliet et al, 1999) L1-CAM Cell-cell adhesion Total Malformations of the nervous system, decreased nerve sensitivity, weak and uncoordinated hindlegs. (Dahme et al, 1997) Ate1 Arginyltransferase Total Embryonic lethality at E12-17 with defects in cardiovascular development and angiogenesis (Kwon et al, 2002) Calpain IV (Capn4) Protease Total Embryonic lethality at midgestation with defects in the cardiovascular system, (Arthur et al, 2000) Birth Defects Res C Embryo Today. Author manuscript; available in PMC 2009 June 1.…”

The N-end rule and regulation of apoptosis

“…The N-end rule relates the in vivo halflife of a protein to the identity of its N-terminal residue [9][10][11][12] ; the underlying proteolytic pathway is called the N-end rule pathway (Fig. 1).…”

“…With N-terminal Asn and Gln, their conjugation to Arg is preceded by their enzymatic deamidation 14 . In metazoans, but not in fungi such as S. cerevisiae, N-terminal Cys is yet another tertiary destabilizing residue, in that the arginylation of Cys is preceded by its (apparently) enzymatic oxidation 12 (Fig. 1).…”

Global cellular regulation including cardiac function by post-translational protein arginylation

“…This intuition turned out to be correct, and arginylation is now well accepted to be just as important as the phosphorylation [26,27]. The very first work, which clearly established the essential nature of ATE1 in mammals, is the experiment where the ATE1 gene was knocked out in mice, causing lethality due to heart defects in cardiovascular development and angiogenic remodeling during embryogenesis [28]. Their studies revealed that in the knockout mice, abnormal cardiac morphogenesis occurred.…”