Inositol 1,4,5-Trisphosphate Receptor Ubiquitination Is Mediated by Mammalian Ubc7, a Component of the Endoplasmic Reticulum-associated Degradation Pathway, and Is Inhibited by Chelation of Intracellular Zn2+

Abstract

In response to activation of certain cell surface receptors, inositol 1,4,5-trisphosphate receptors (InsP 3 Rs), which are located in the endoplasmic reticulum, can be rapidly ubiquitinated and then degraded by the proteasome. Ubiquitination is mediated by the concerted action of ubiquitin-conjugating enzymes (Ubcs or E2s) and ubiquitin-protein ligases (E3s). In the present study we have examined the enzymology of ubiquitination of endogenous InsP 3 Rs in muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells, focusing our attention on two mammalian E2s, MmUbc6 and MmUbc7, that have been implicated in endoplasmic reticulum-associated degradation (ERAD) and are homologous to the yeast ERAD E2s, Ubc6p and Ubc7p. Analysis of SH-SY5Y cells stably expressing these enzymes and their dominant-negative mutants revealed that MmUbc7 mediates InsP 3 R ubiquitination and down-regulation, but that MmUbc6 does not. These data indicate that InsP 3 Rs are processed by a component of the ERAD pathway and suggest that MmUbc7 may be employed selectively to ubiquitinate proteins, like InsP 3 Rs, that are subject to regulated ERAD. Additional studies showed that the Zn 2؉ chelator N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine blocked InsP 3 R ubiquitination, suggesting that a RING finger domain-containing E3 is also involved in this process. Finally, muscarinic agonist-induced InsP 3 R ubiquitination was seen in rat brain slices, indicating that the results obtained from SH-SY5Y cells reflect a physiological process.

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

“…Transfection efficiencies and expression levels of the first exon of htt (httex)1Q97-GFP were monitored by flow cytometry on a Moflo FACS machine (Cytomation). Cell lysis, preparation of Triton X-100-soluble lysates, and immunoblots were carried out as described (24). Triton X-100-insoluble fractions were prepared by resuspending the Triton X-100-insoluble pellet in water, followed by sonication and centrifugation at 16,000 ϫ g for 10 min; the final pellet was resuspended in SDS gel-loading buffer before processing in immunoblots with a monoclonal anti-htt recognizing the first 17 aa of the htt protein (m445).…”

A Point Mutation in the Ubiquitin Ligase RNF170 That Causes Autosomal Dominant Sensory Ataxia Destabilizes the Protein and Impairs Inositol 1,4,5-Trisphosphate Receptor-mediated Ca2+ Signaling

“…Use of the CRISPR/Cas9 system (24,25) allowed us to delete RNF170 and demonstrate, for the first time, that RNF170 catalyzes the addition of all ubiquitin conjugates to activated IP 3 R1. Intriguingly, this suggests that RNF170 interacts with multiple E2s, most likely Ubc13 and Ubc7, because Ubc13 is the only E2 known to build Lys 63 -linked chains (5, 32) and Ubc7, which builds Lys 48 -linked chains (5,32), is already strongly implicated in mediating IP 3 R1 ubiquitination and degradation (33 A hint as to why these mutations are apparently destabilizing is provided by the fact that they also cause RNF170 to migrate more rapidly on SDS-PAGE, indicative of a structural change in the protein that either causes more compact folding and/or alters interactions with SDS. The structural change appears to be subtle, as the Arg 198 to Cys mutation did not significantly affect TM2 and TM3 insertion into the ER membrane, or interaction with the erlin1/2 complex, but could still be sufficient to trigger autoubiquitination and targeting for ERAD.…”

SPFH2 Mediates the Endoplasmic Reticulum-associated Degradation of Inositol 1,4,5-Trisphosphate Receptors and Other Substrates in Mammalian Cells

“…2 -associated degradation (ERAD) pathway is responsible for the degradation of aberrant proteins in the ER (1) and, in addition to this "quality control" function, also accounts for the degradation of several metabolically regulated, native ER proteins (2,3). The essential features of the ERAD pathway are substrate recognition, polyubiquitination, and delivery to the 26 S proteasome, which is located in the cytosol (1).…”

“…Remarkably, activation of endogenous IP 3 Rs leads to their rapid polyubiquitination and subsequent degradation by the 26 S proteasome (34 -36), a phenomenon that has been demonstrated in many mammalian cell types, including cholecystokinin-stimulated pancreatic acinar cells (37), gonadotropin-releasing hormone (GnRH)-stimulated ␣T3-1 mouse pituitary gonadotropes (38), and endothelin 1 (ET1)-stimulated Rat-1 primary fibroblasts (39). The ERAD pathway seems to be responsible for this process, because the ubiquitin-conjugating enzyme that ubiquitinates IP 3 Rs is mam Ubc7 (3), an enzyme widely implicated in ERAD (2,40), and the p97-Ufd1-Npl4 complex mediates the degradation of polyubiquitinated IP 3 Rs (39). Importantly, endogenous IP 3 Rs represent a unique tool for studying ERAD, because activation almost instantaneously converts them from their native form into ERAD substrates (36,39).…”