No abstract

The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/ E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases. cardiovascular system | ubiquitin ligase

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial electron transport chain, is regulated by isozyme expression, allosteric effectors such as the ATP/ADP ratio, and reversible phosphorylation. Of particular interest is the ‘allosteric ATP-inhibition’, which has been hypothesized to keep the mitochondrial membrane potential at low healthy values (< 140 mV), thus preventing the formation of superoxide radical anions, which have been implicated in multiple degenerative diseases. It has been proposed that the ‘allosteric ATPinhibition’ is switched on by the protein kinase A-dependent phosphorylation of COX. The goal of this study was to identify the phosphorylation site(s) involved in the ‘allosteric ATPinhibition’ of COX. We report the mass spectrometric identification of four new phosphorylation sites in bovine heart COX. The identified phosphorylation sites include Tyr-218 in subunit II, Ser-1 in subunit Va, Ser-2 in subunit Vb, and Ser-1 in subunit VIIc. With the exeption of for Ser- 2 in subunit Vb, the identified phosphorylation sites were found in enzyme samples with and without ‘allosteric ATP inhibition’, making Ser-2 of subunit Vb a candidate site enabling allosteric regulation. We therefore hypothesize that additional phosphorylation(s) may be required for the ‘allosteric ATP-inhibition’, and that these sites may be easily dephosphorylated or difficult to identify by mass spectrometry.

Background: ATE1 transfers Arg to protein N termini, generating the degron for the N-end rule pathway. Results: ATE1-deficient cardiomyocytes are impaired in the PLC/PKC-MEK1-ERK axis of G␣ q -mediated cardiac signaling. Conclusion:The arginine branch of the N-end rule pathway controls G-protein signaling in cardiomyocytes in part through hypoxia-sensitive degradation of GAP proteins. Significance: This study provides a cellular mechanism underlying cardiovascular defects observed in ATE1-deficient mice.