UBR box N-recognin-4 (UBR4), an N-recognin of the N-end rule pathway, and its role in yolk sac vascular development and autophagy

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

KCMF1 (potassium channel modulatory factor 1) Links RAD6 to UBR4 (ubiquitin N-recognin domain-containing E3 ligase 4) and Lysosome-Mediated Degradation*

“…Interestingly, we identify two new RAD6-associated E3 proteins, KCMF1 and UBR4, along with several additional interactors previously linked (via genetic and/or biochemical evidence) to intracellular vesicle trafficking and mitochondrial function. Using a combination of in vivo and in vitro binding assays combined with NMR spectroscopy, our data suggest that the KCMF1 protein recruits RAD6 to UBR4, a noncanonical N-recognin recently implicated in bulk lysosomal degradation and autophagy (11,12). Consistent with this observation, knockdown of KCMF1 or RAD6A expression alters late endosome-lysosome vesicle trafficking.…”

“…UBR4 interacts with the Dengue virus protein NS5, and both KCMF1 and UBR4 interact with the Human Papilloma Virus (HPV) E7 proteins across a wide range of HPV isotypes (32)(33)(34). Notably, like the other N-recognins, UBR4 can bind to N-end rule substrates, and was recently linked to lysosomemediated degradation and autophagy (11,12). Consistent with these findings, UBR4 is also a candidate gene for episodic ataxia (EA), a group of rare human neurological channelopathies characterized by a lack of balance and coordination (35), and D. melanogaster strains mutant for the UBR4 ortholog pushover display sluggishness, a lack of coordina- (36).…”

A Ca2+-dependent Mechanism of Neuronal Survival Mediated by the Microtubule-associated Protein p600

“…1B). The embryonic lethality of two different p600 null mouse strains (24,25), however, precluded an investigation of the mechanistic roles of p600 in adult brain neurons but hints at the requirement of p600 for cell survival in vivo. Because of this lethal phenotype and expression of p600 in the adult hippocampus (15) (Fig.…”

“…Because we were unable to detect signs of the former two mechanisms, we are bound to suspect the latter. This suspicion is well grounded in the published role of p600 in the N-end rule pathway (50) and especially in new work (24) showing that p600 knock-out mice die during embryogenesis, with yolk sac development defects characterized by aberrant autophagy (elaborated in Ref. 51).…”

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

“…31 More recent studies have suggested that the Arg/N-end rule pathway is also implicated in autophagy and mitophagy. 32,33 A set of endoplasmic reticulum (ER)-residing chaperone proteins, including HSPA5/BiP/ GRP78 (heat shock protein family A [HSP70] member 5), P4HB/PDI (prolyl 4-hydroxylase subunit b), and DNAJC10/ ERdj5 (DnaJ heat shock protein family [Hsp40] member C10), were identified to be N-terminally arginylated, retrotranslocated to the cytosol, bound to misfolded proteins, and recognized by the autophagic receptor SQSTM1/p62 (sequestosome 1). 34 Although these proteins are likely to be cotargeted to the autophagosome, the biological functions of arginylated ER proteins, their catabolic regulation (for example, via UPS, autophagy, or both), and the implication of these proteins in autophagic flux remain essentially uncharacterized.…”