Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II)

Abstract

Dysferlin is a type-II transmembrane protein and the causative gene of limb girdle muscular dystrophy type 2B and Miyoshi myopathy (LGMD2B/MM), in which specific loss of dysferlin labeling has been frequently observed. Recently, a novel mutant (L1341P) dysferlin has been shown to aggregate in the muscle of the patient. Little is known about the relationship between degradation of dysferlin and pathogenesis of LGMD2B/MM. Here, we examined the degradation of normal and mutant (L1341P) dysferlin. Wild-type (wt) dysferlin mainly localized to the ER/Golgi, associated with retrotranslocon, Sec61alpha, and VCP(p97), and was degraded by endoplasmic reticulum (ER)-associated degradation system (ERAD) composed of ubiquitin/proteasome. In contrast, mutant dysferlin spontaneously aggregated in the ER and induced eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation and LC3 conversion, a key step for autophagosome formation, and finally, ER stress cell death. Unlike proteasome inhibitor, E64d/pepstatin A, inhibitors of lysosomal proteases did not stimulate the accumulation of the wt-dysferlin, but stimulated aggregation of mutant dysferlin in the ER. Furthermore, deficiency of Atg5 and dephosphorylation of eIF2alpha, key molecules for LC3 conversion, also stimulated the mutant dysferlin aggregation in the ER. Rapamycin, which induces eIF2alpha phosphorylation-mediated LC3 conversion, inhibited mutant dysferlin aggregation in the ER. Thus, mutant dysferlin aggregates in the ER-stimulated autophagosome formation to engulf them via activation of ER stress-eIF2alpha phosphorylation pathway. We propose two ERAD models for dysferlin degradation, ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Mutant dysferlin aggregates on the ER are degraded by the autophagy/lysosome ERAD(II), as an alternative to ERAD(I), when retrotranslocon/ERAD(I) system is impaired by these mutant aggregates.

Enhanced Lysosomal Activity Is Involved in Bax Inhibitor-1-induced Regulation of the Endoplasmic Reticulum (ER) Stress Response and Cell Death against ER Stress

“…6, C and D). Lysosomal activity-associated protein degradation also functions as a cytoplasmic quality control mechanism for the elimination of protein aggregates and damaged organelles (8,27). Similar to the role of bafilomycin in this experiment, defects in the ERAD II system can cause the accumulation of cytoplasmic inclusion bodies and protein aggregates in the cytoplasm, leading to toxicity (28).…”

“…Lysosomal activity is an ERAD II pathway, whereas ERAD I is a proteasome/ubiquitination pathway (7). The ERAD mechanism increases the protein folding capacity by reducing protein folding loads (7,8), implying that ERAD is a physiological pathway that can regulate ER stress responses (8,9).…”

Protein Folding and Quality Control in the ER

“…1E) (Ishida et al 2009). When ERAF/ERAD or ubiquitin proteasome activities are compromised, autophagy is triggered via signaling pathways that usually involve unfolded protein response (UPR)-dependent elements (Ogata et al 2006;Fujita et al 2007;Hosokawa et al 2007;Kouroku et al 2007;Yorimitsu and Klionsky 2007b). Additionally, the ER membrane itself may be a source of lipids for autophagosome formation (HayashiNishino et al 2009;Yla-Anttila et al 2009).…”

The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells

“…29,30 Similarly, autophagy induced by a misfolded dysferlin mutant that aggregates and accumulates in the ER was also inhibited in eIF2aA/A knockin MEFs. 31 These studies imply that the PERK/eIF2a phosphorylation pathway is critical for ER stress-induced autophagy. On the other hand, in Ire1-deficient MEFs or wild-type MEFs treated with a JNK inhibitor, the ER stress-induced autophagy was inhibited.…”