Research Article: Ablation of Arginylation in the Mouse N-End Rule Pathway: Loss of Fat, Higher Metabolic Rate, Damaged Spermatogenesis, and Neurological Perturbations

Date Published: November 13, 2009

Publisher: Public Library of Science

Author(s): Christopher S. Brower, Alexander Varshavsky, Immo A. Hansen.

Abstract: In the N-end rule pathway of protein degradation, the destabilizing activity of N-terminal Asp, Glu or (oxidized) Cys residues requires their conjugation to Arg, which is recognized directly by pathway’s ubiquitin ligases. N-terminal arginylation is mediated by the Ate1 arginyltransferase, whose physiological substrates include the Rgs4, Rgs5 and Rgs16 regulators of G proteins. Here, we employed the Cre-lox technique to uncover new physiological functions of N-terminal arginylation in adult mice. We show that postnatal deletion of mouse Ate1 (its unconditional deletion is embryonic lethal) causes a rapid decrease of body weight and results in early death of ∼15% of Ate1-deficient mice. Despite being hyperphagic, the surviving Ate1-deficient mice contain little visceral fat. They also exhibit an increased metabolic rate, ectopic induction of the Ucp1 uncoupling protein in white fat, and are resistant to diet-induced obesity. In addition, Ate1-deficient mice have enlarged brains, an enhanced startle response, are strikingly hyperkinetic, and are prone to seizures and kyphosis. Ate1-deficient males are also infertile, owing to defects in Ate1−/− spermatocytes. The remarkably broad range of specific biological processes that are shown here to be perturbed by the loss of N-terminal arginylation will make possible the dissection of regulatory circuits that involve Ate1 and either its known substrates, such as Rgs4, Rgs5 and Rgs16, or those currently unknown.

Partial Text: N-terminal arginylation of intracellular proteins by Arg-tRNA-protein transferase (R-transferase) is a part of the N-end rule pathway of protein degradation (Fig. 1A). In eukaryotes, this pathway is a part of the ubiquitin (Ub)-proteasome system. The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue (reviewed in [1], [2], [3], [4]). Degradation signals (degrons) that can be targeted by the N-end rule pathway are of two distinct kinds: N-terminal degrons, called N-degrons, and internal (non-N-terminal) degrons [1], [5]. The main determinant of an N-degron is a destabilizing N-terminal residue of a substrate protein (Fig. 1A). The other determinants of N-degron are a substrate’s internal Lys residue (the site of formation of a poly-Ub chain) and a nearby unstructured region [6], [7]. An N-degron is produced from a precursor, called a pre-N-degron, through a protease-mediated cleavage of a substrate that exposes a destabilizing N-terminal residue.

A cell is alive owing to a cell-wide dynamic network of structurally or functionally interacting biopolymers. Some parts of this network can be sufficiently insulated, through their design, to be considered, in the first approximation, as distinct circuits. The N-end rule pathway is one such circuit. Its enzymes receive as their input specific degron-bearing proteins and convert them, through deamidation, arginylation, polyubiquitylation and processive degradation, into an output of proteolysis-derived short peptides (Fig. 1A). The rate and selectivity of the proteasome-mediated protein degradation by the N-end rule pathway are modulated by physiological effectors, including specific phosphokinases, short peptides, redox, heme and nitric oxide (see Introduction). Some of N-end rule substrates are produced by proteases that include MetAPs, separases, caspases and calpains. These and other nonprocessive proteases, which function as upstream components of the N-end rule pathway, have in common their ability to convert, through a cleavage, a pro-N-degron into an N-degron.



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