Impaired neurogenesis and cardiovascular development in mice lacking the E3 ubiquitin ligases UBR1 and UBR2 of the N-end rule pathway

Abstract

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. A subset of degradation signals recognized by the N-end rule pathway comprises the signals, called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified a family of at least four mammalian E3 ubiquitin ligases, including UBR1 and UBR2, that share the UBR box and recognize N-degrons. These E3 enzymes mediate the multifunctional N-end rule pathway, but their individual roles are just beginning to emerge. Mutations of UBR1 in humans are the cause of Johanson-Blizzard syndrome. UBR1 and UBR2 are 46% identical and appear to be indistinguishable in their recognition of N-degrons. UBR1 ؊/؊ mice are viable but have defects that include pancreatic insufficiency, similarly to UBR1 ؊/؊ human patients with Johanson-Blizzard syndrome. UBR2 ؊/؊ mice are inviable in some strain backgrounds and are defective in male meiosis. To examine functional relationships between UBR1 and UBR2, we constructed mouse strains lacking both of these E3s. We report here that UBR1 ؊/؊ UBR2 ؊/؊ embryos die at midgestation, with defects in neurogenesis and cardiovascular development. These defects included reduced proliferation as well as precocious migration and differentiation of neural progenitor cells. The expression of regulators such as D-type cyclins and Notch1 was also altered in UBR1 ؊/؊ UBR2 ؊/؊ embryos. We conclude that the functions of UBR1 and UBR2 are significantly divergent, in part because of differences in their expression patterns and possibly also because of differences in their recognition of protein substrates that contain degradation signals other than N-degrons. ubiquitylation ͉ proteolysis ͉ N-recognin ͉ UBR box ͉ arginylation

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

“…Constitutional nullification of these genes resulted in embryonic lethality. 18,44 WT MEFs showed similar responses to PCA that were observed in HeLa cells-stabilization of LC3-II by PCA but no additive effects with BafA1 ( Fig. 2A)-thereby also revealing the inhibitory effect of PCA on autophagosome-lysosome fusion.…”

Profiling human protein degradome delineates cellular responses to proteasomal inhibition and reveals a feedback mechanism in regulating proteasome homeostasis

“…In UBR1 −/− UBR2 −/− mouse embryonic fibroblast (MEF) cells, both UBR1 and UBR2, the two major E3 ubiquitin ligases, were genetically ablated to impair the functionality of the N-end rule pathway, thus stabilizing RGS4 [35,36]. Indeed, protein RGS4-mEG-FP fu was unstable in HEK293FT cells, but significantly accumulated in UBR1 −/− UBR2 −/− MEF cells ( Figure 1E).…”

Inability to fully suppress sterol synthesis rates with exogenous sterol in embryonic and extraembyronic fetal tissues

“…SREBP-2 is degraded via the ubiquitin-proteosome pathway after its phosphorylation and ubiquination [50][51][52]. Though the ubiquitin-proteosome system for degradation is active during development [53,54], it seems that the SREBPs would not be readily modified and/or degraded during this time of rapid growth since sterol synthesis rates remain relatively active in the presence of cholesterol. Lack of SREBP-2 degradation may be the result of a change in proteosome activity [55] or rate of de-ubiquitination [41].…”