Regulation of Cullin RING E3 Ubiquitin Ligases by CAND1 In Vivo

Abstract

Cullin RING ligases are multi-subunit complexes consisting of a cullin protein which forms a scaffold onto which the RING protein Rbx1/2 and substrate receptor subunits assemble. CAND1, which binds to cullins that are not conjugated with Nedd8 and not associated with substrate receptors, has been shown to function as a positive regulator of Cullin ligases in vivo. Two models have been proposed to explain this requirement: (i) CAND1 sequesters cullin proteins and thus prevents autoubiquitination of substrate receptors, and (ii) CAND1 is required to promote the exchange of bound substrate receptors. Using mammalian cells, we show that CAND1 is predominantly cytoplasmically localized and that cullins are the major CAND1 interacting proteins. However, only small amounts of CAND1 bind to Cul1 in cells, despite low basal levels of Cul1 neddylation and approximately equal cytoplasmic endogenous protein concentrations of CAND1 and Cul1. Compared to F-box protein substrate receptors, binding of CAND1 to Cul1 in vivo is weak. Furthermore, preventing binding of F-box substrate receptors to Cul1 does not increase CAND1 binding. In conclusion, our study suggests that CAND1 does not function by sequestering cullins in vivo to prevent substrate receptor autoubiquitination and is likely to regulate cullin RING ligase activity via alternative mechanisms.

COMMD1 (Copper Metabolism MURR1 Domain-containing Protein 1) Regulates Cullin RING Ligases by Preventing CAND1 (Cullin-associated Nedd8-dissociated Protein 1) Binding

“…Although the SRC can dissociate the CRL1-CAND1 complex in vitro, various in vivo experiments indicate that other factors regulate this interaction. Disrupting Cul1-SRC complex formation in vivo has limited effects on CRL1-CAND1 interactions (39). Similarly, inhibiting CRL neddylation in cells did not promote CRL1-CAND1 interactions, nor did it destabilize CRL1-SRC complexes (40).…”

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

“…The proteins were Cullin‐associated NEDD8‐dissociated protein 1 (Cand1) as well as protein SON. Cand1 is involved in the regulation of SCF ubiquitin ligases (Chua et al, 2011; Olma and Dikic, 2013). Ubiquitination plays a crucial role in the nuclear factor‐κB (NFκB) pathway, endocytic trafficking, DNA repair, and protein degradation (Grabbe et al, 2011).…”

Intracellular Internalization Mechanism of Protein Transfection Reagents

“…Nevertheless, those studies focused primarily on the functions of the delivered proteins and demanded high transfection efficiency and no cytotoxicity for protein transfection reagents, and less attention has been paid to the intracellular trafficking of the proteins. 12,13) In this study, we evaluated three protein transfection reagents, Pro-DeliverIN, Xfect, and TurboFect, that deliver the proteins into HeLa cells, and especially the intracellular internalization routes of their complexes with BSA.…”