Redundant and Antagonistic Roles of XTP3B and OS9 in Decoding Glycan and Non-glycan Degrons in ER-Associated Degradation.

Glycoproteins engaged in unproductive folding in the ER are marked for degradation by a signal generated by progressive demannosylation of substrate N-glycans that is decoded by ER lectins, but how the two lectins, OS9 and XTP3B, contribute to non-glycosylated protein triage is unknown. We generated cell lines with homozygous deletions of both lectins individually and in combination. We found that OS9 and XTP3B redundantly promote glycoprotein degradation and stabilize the SEL1L/HRD1 dislocon complex, that XTP3B profoundly inhibits the degradation of non-glycosylated proteins, and that OS9 antagonizes this inhibition. The relative expression of OS9 and XTP3B and the distribution of glycan and non-glycan degrons within the same protein contribute to the fidelity and processivity of glycoprotein triage and, therefore, determine the fates of newly synthesized proteins in the early secretory pathway.

Prion-Like Characteristics of Polyglutamine-Containing Proteins.

Transmissible spongiform encephalopathies are infectious neurodegenerative diseases caused by the conversion of prion protein (PrP) into a self-replicating conformation that spreads via templated conversion of natively folded PrP molecules within or between cells. Recent studies provide compelling evidence that prion-like behavior is a general property of most protein aggregates associated with neurodegenerative diseases. Many of these disorders are associated with spontaneous protein aggregation, but genetic mutations can increase the aggregation propensity of specific proteins, including expansion of polyglutamine (polyQ) tracts, which is causative of nine inherited neurodegenerative diseases. Aggregates formed by polyQ-expanded huntingtin (Htt) in Huntington's disease can transfer between cells and seed the aggregation of cytoplasmic wild-type Htt in a prion-like manner. Additionally, prion-like properties of glutamine-rich proteins underlie nonpathological processes in yeast and higher eukaryotes. Here, we review current evidence supporting prion-like characteristics of polyQ and glutamine-rich proteins.

Unassembled CD147 is an endogenous endoplasmic reticulum-associated degradation substrate.

Degradation of folding- or assembly-defective proteins by the endoplasmic reticulum-associated degradation (ERAD) ubiquitin ligase, Hrd1, is facilitated by a process that involves recognition of demannosylated N-glycans by the lectin OS-9/XTP3-B via the adaptor protein SEL1L. Most of our knowledge of the machinery that commits proteins to this fate in metazoans comes from studies of overexpressed mutant proteins in heterologous cells. In this study, we used mass spectrometry to identify core-glycoslyated CD147 (CD147(CG)) as an endogenous substrate of the ERAD system that accumulates in a complex with OS-9 following SEL1L depletion. CD147 is an obligatory assembly factor for monocarboxylate transporters. The majority of newly synthesized endogenous CD147(CG) was degraded by the proteasome in a Hrd1-dependent manner. CD147(CG) turnover was blocked by kifunensine, and interaction of OS-9 and XTP3-B with CD147(CG) was inhibited by mutations to conserved residues in their lectin domains. These data establish unassembled CD147(CG) as an endogenous, constitutive ERAD substrate of the OS-9/SEL1L/Hrd1 pathway.

Differential regulation of HMG-CoA reductase and Insig-1 by enzymes of the ubiquitin-proteasome system.

The endoplasmic reticulum (ER)-resident enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyzes the rate-limiting step in sterol production and is the therapeutic target of statins. Understanding HMG-CoA reductase regulation has tremendous implications for atherosclerosis. HMG-CoA reductase levels are regulated in response to sterols both transcriptionally, through a complex regulatory loop involving the ER Insig proteins, and posttranslationally, by Insig-dependent protein degradation by the ubiquitin-proteasome system. The ubiquitin ligase (E3) gp78 has been implicated in the sterol-regulated degradation of HMG-CoA reductase and Insig-1 through ER-associated degradation (ERAD). More recently, a second ERAD E3, TRC8, has also been reported to play a role in the sterol-accelerated degradation of HMG-CoA reductase. We interrogated this network in gp78(-/-) mouse embryonic fibroblasts and also assessed two fibroblast cell lines using RNA interference. Although we consistently observe involvement of gp78 in Insig-1 degradation, we find no substantive evidence to support roles for either gp78 or TRC8 in the robust sterol-accelerated degradation of HMG-CoA reductase. We discuss factors that might lead to such discrepant findings. Our results suggest a need for additional studies before definitive mechanistic conclusions are drawn that might set the stage for development of drugs to manipulate gp78 function in metabolic disorders.

An endoplasmic reticulum (ER) membrane complex composed of SPFH1 and SPFH2 mediates the ER-associated degradation of inositol 1,4,5-trisphosphate receptors.

How endoplasmic reticulum (ER) proteins that are substrates for the ER-associated degradation (ERAD) pathway are recognized for polyubiquitination and proteasomal degradation is largely unresolved. Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) form tetrameric calcium channels in ER membranes, whose primary role is to control the release of ER calcium stores, but whose levels are also regulated, in an activation-dependent manner, by the ERAD pathway. Here we report that the ER membrane protein SPFH1 and its homolog SPFH2 form a heteromeric approximately 2 MDa complex that binds to IP(3)R tetramers immediately after their activation and is required for their processing. The complex is ring-shaped (diameter approximately 250A(),) and RNA interference-mediated depletion of SPFH1 and SPFH2 blocks IP(3)R polyubiquitination and degradation. We propose that this novel SPFH1/2 complex is a recognition factor that targets IP(3)Rs and perhaps other substrates for ERAD.

SPFH2 mediates the endoplasmic reticulum-associated degradation of inositol 1,4,5-trisphosphate receptors and other substrates in mammalian cells.

Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum (ER) membrane calcium channels that, upon activation, become substrates for the ER-associated degradation (ERAD) pathway. Although it is clear that IP(3) receptors are polyubiquitinated upon activation and are transferred to the proteasome by a p97-based complex, currently nothing is known about the proteins that initially select activated IP(3) receptors for ERAD. Here, we sought to identify novel proteins that associate with and mediate the ERAD of endogenous activated IP(3) receptors. SPFH2, an uncharacterized SPFH domain-containing protein, rapidly associated with IP(3) receptors in a manner that preceded significant polyubiquitination and the association of p97 and related proteins. SPFH2 was found to be an ER membrane protein largely residing within the ER lumen and in resting and stimulated cells was linked to ERAD pathway components, apparently via endogenous substrates undergoing degradation. Suppression of SPFH2 expression by RNA interference markedly inhibited IP(3) receptor polyubiquitination and degradation and the processing of other ERAD substrates. Overall, these studies identify SPFH2 as a key ERAD pathway component and suggest that it may act as a substrate recognition factor.