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

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Eriko Fujita, Yoriko Kouroku, Atsushi Isoai, Hiromichi Kumagai, Akifumi Misutani, Chie Matsuda, Yukiko K. Hayashi, Takashi Momoi, Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II), Human Molecular Genetics, Volume 16, Issue 6, 15 March 2007, Pages 618–629, https://doi.org/10.1093/hmg/ddm002
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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, Sec61α, 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 2α (eIF2α) 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 eIF2α, key molecules for LC3 conversion, also stimulated the mutant dysferlin aggregation in the ER. Rapamycin, which induces eIF2α 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-eIF2α 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.

INTRODUCTION

Mutations of the gene encoding dysferlin cause the allelic autosomal recessive disorders limb girdle muscular dystrophy type 2B and Miyoshi myopathy (LGMD2B/MM) (1,2). The predicted amino acid sequence of dysferlin possesses seven intracellular C2 (Ca++-binding) domains and a carboxy-terminal transmembrane region (3). Dysferlin protein is a type-II transmembrane protein and is expressed in various tissues, including muscle, and is located primarily in muscle sarcolemma (4,5). It is implicated in membrane repair of disrupted surface membranes of muscle (6,7). The specific loss of dysferlin labeling has been frequently observed in the muscle tissues of LGMD2B/MM patients (1,2). Little is known about the degradation of wild-type (wt) and mutant dysferlin.

Endoplasmic reticulum (ER) quality-control system recognizes and removes nascent proteins that fail to fold or assemble properly. The aberrant proteins such as misfolded and malfolded proteins activate stress-signaling pathways to rescue the cells via ER stress-sensor proteins such as PERK [protein kinase regulated by RNA (PKR)-like ER kinase] (8,9). The aberrant proteins are extracted and retrotranslocated to the cytoplasm where they are degraded by the ER-associated egradation (ERAD) system composed of ubiquitin/proteasome (10–12). Valosin-containing protein p97 [VCP(p97)] and Sec61 are involved in the retrotranslocation for the extraction of aberrant luminal and membrane proteins from the ER (13). If insufficiently degraded, the accumulated aberrant proteins in the ER are assumed to activate the ER stress-mediated cell death pathway via activation of the c-Jun N-terminal kinase (JNK) and/or caspase-12 located at the outer layer of the ER (14,15). ER stress is closely related with the pathogenesis of conformation diseases such as Parkinson's disease and polyglutamine (polyQ) disease (15,16). Recently, some novel variants of LGMD2B/MM have shown patchy sarcolemmal immunostaining or intracellular aggregates of dysferlin in the muscle fibers of patients (17,18). A relationship between ER stress and mutant (L1341P)-dysferlin aggregates has also been suggested.

Macroautophagy (hereafter referred to as autophagy) engulfs bulk cytosolic material and organelles within double-membrane vesicles, known as autophagosomes, and then degrades them to liberate nutrients via fusion with the lysosome (this pathway is hereafter referred to as autophagy/lysosome). The molecular mechanism of autophagy formation has been intensely studied in yeast and mammalian cells (19,20); many autophagy-related genes (Atgs) are involved in the autophagy formation. In addition to ubiquitin/proteasome, constitutive autophagy has an important role in protein-quality control; _Atg7_- and/or _Atg5_-deficient mice show the accumulation of ubiquitinated proteins in the liver and brain (21–23).

Very recently, we have shown that cytoplasmic polyQ-induced autophagy formation is linked to ER stress-mediated PERK/eIF2α (eukaryotic translation initiation factor 2α) phosphorylation and that cytoplasmic polyQ aggregates, which are not sufficiently degraded by ubiquitin/proteasome system, are degraded by autophagy/lysosome (15,24). Thus, constitutive autophagy is stimulated to degrade the cytoplasmic mutant protein aggregates by their aggregate-mediated stress-induced eIF2α phosphorylation. However, it is not clear how aberrant membrane protein aggregates located in the ER are degraded when retrotranslocation or ubiquitin/proteasome ERAD is impaired.

However, since it is technically difficult to distinguish normally folded and misfolded state in the wt-dysferlin, we examined the difference between the effects of wt-dysferlin and mutant (L1341P)-dysferlin with malfolded states on their possible degradation systems, ubiquitin/proteasome and autophagy/lysosome. We demonstrate that wt-dysferlin is localized to the ER and is a substrate for ubiquitin/proteasome ERAD(I), while mutant dysferlin aggregates are additionally degraded by the autophagy/lysosome ERAD(II).

RESULTS

Subcellular localization of dysferlin

We examined the subcellular distribution of dysferlin in C2C5 cells by immunostaining with several subcellular markers (Fig. 1). Although dysferlin is expressed ubiquitously in various cells, its levels are undetectable by immunostaining except in muscle tissues. Therefore, we prepared C2C5 cells expressing tetracycline (Tet)-inducible wt-dysferlin gene (Tet-wt-dysferlin C2C5 cells), controlled the expression of wt-dysferlin using this Tet-on/off system, and examined its subcellular localization after induction. Tet-wt-dysferlin partially localized to the ER (anti-KDEL reactivity) and to the Golgi (anti-GM130 reactivity), but not to the lysosome (anti-cathepsin D reactivity) or mitochondria (anti-F0ATPase reactivity) (Fig. 1A). Approximately, 4% of the cells showed Tet-wt-dysferlin accumulation in the ER/Golgi. The accumulated Tet-wt-dysferlin colocalized with Sec61α and partly colocalized with VCP(p97), components of retrotranslocon on the ER (Fig. 1B and C). Furthermore, wt-dysferlin was associated with Sec61α and VCP(p97) in COS cells (Fig. 1D).

Figure 1.

Intracellular localization of Tet-induced dysferlin and its association with components of retrotranslocon. (A) C2C5 cells expressing pcDNA4/TO/myc-His-wt-dysferlin (Tet-wt-dysferlin C2C5 cells) were incubated in medium containing tetracycline (Tet). Dysferlin localization was examined by immunostaining using anti-His (green) and various subcellular makers such as anti-KDEL (red) for ER, anti-GM130 (red) for Golgi, anti-FoATPase (red) for mitochondria, and anti-cathepsin D (red) for lysosome at 24 h after Tet-on. (B) Colocalization of Tet-wt-dysferlin with VCP(p97) in C2C5 cells. Localization of dysferlin and VCP(p97) were detected by immunostaining using anti-His (green) and anti-VCP(p97) (red), respectively. Yellow; merged. (C) Colocalization of Tet-wt-dysferlin with Sec61α in C2C5 cells. Localization of dysferlin and Sec61α was detected by immunostaining using anti-His (green) and anti-Sec61α (red), respectively. Yellow; merged. (D) Interactions between GFP-dysferlin and VCP(p97) or Sec61α. Interaction between dysferlin and VCP(p97) or Sec61α was detected by immunoprecipitation and immunoblot analysis using anti-GFP and anti-VCP(p97) or anti-Sec61α, respectively. Scale bars: 15 µm.

Relationship between dysferlin accumulation in the ER and impaired ERAD

We examined the two possible degradation systems for wt-dysferlin; ubiquitin/proteasome ERAD and the lysosome. Recently, we showed that cytoplasmic polyQ aggregates, which inhibit the function of the ERAD and retrotranslocon, stimulate PERK/eIF2α phosphorylation, thereby activating the autophagic pathway as well as the apoptotic pathway (24,25). Cytoplasmic polyQ aggregates stimulated Tet-wt-dysferlin accumulation in the ER (Fig. 2A); the population of cells showing this accumulation was increased by about 5-fold with the presence of the cytoplasmic polyQ aggregates. The proteasome inhibitor, benzyloxycarbonyl-l- leucyl-l-leucyl-leucinal [z-LLL-CHO (MG132); LLL], also induced the accumulation of Tet-wt-dysferlin in the ER. LLL (5 µm) treatment induced dysferlin accumulation in a Triton-insoluble fraction within 12–24 h (Fig. 2B). LLL (5 µm) as well as polyQ aggregates stimulated Tet-wt-dysferlin accumulation in the ER (Fig. 2C). The accumulated Tet-wt-dysferlin co-aggregated with anti-polyubiquitin reactivity (Fig. 2D). In contrast, lysosomal protease inhibitors, E64d (10 µg/ml), membrane permeable inhibitor of cathepsin A, H and L, and pepstatin A (10 µg/ml), an inhibitor of cathepsin D and E, did not stimulate the Tet-wt-dysferlin accumulation in the ER (Fig. 2B and C). Thus, wt-dysferlin seems to be mainly degraded in the ubiquitin/proteasome ERAD but not in the lysosome.

Figure 2.

LLL and cytoplasmic polyQ aggregates induced accumulation of Tet-wt-dysferlin in the ER. Tet-wt-dysferlin C2C5 cells were incubated in medium containing tetracycline (Tet) and transfected by pEGFP-72CAG. (A) PolyQ aggregates-induced dysferlin aggregation in the ER. Immunofluorescence images of polyQ72 aggregates and dysferlin aggregates (left) and percentages of cells showing dysferlin aggregates in cells expressing GFP-polyQ72 and in non-expressing cells (right). Left panel shows lower magnification (a–d: Scale bar, 25 µm) and higher magnification (e and f: Scale bar, 15 µm). Insets (a–d) show cells which were not transfected GFP-polyQ72. Anti-His (dysferlin; red) and GFP-polyQ72 (green). The white and blue broken lines show outlines of the cell and the nucleus, respectively. The percentages were determined by counting 100–200 cells expressing GFP-polyQ72 at 48 h after transfection. The values are averages of the percentages of the number of cells obtained in three experiments. Error bars indicate standard deviation. (B) Immunoblot analysis of the effect of LLL and E64d/pepstatin A on the level of Tet-wt-dysferlin in the soluble or insoluble fraction. Tet-wt-dysferlin C2C5 cells were incubated in medium containing Tet and LLL (5 µM) or E64d/pepstatin A (10 µg/ml) for the indicated periods. Triton-soluble and insoluble dysferlin were detected by immunoblot analysis using an anti-His antibody at the indicated time. (C) Effect of LLL and E64d/pepstatin A on the Tet-wt-dysferlin aggregates. Dysferlin aggregates were shown by immunostaining using an anti-His antibody (green). Scale bar, 25 µm. (D) Colocalization of Tet-wt-dysferlin with polyubiquitinates. LLL-treated Tet-wt-dysferlin C2C5 cells were immunostained with anti-His and anti-polyubiquitin (red) antibody (green). Yellow; merged. Scale bar, 15 µm. (E) Effect of various ER stress stimuli on the Tet-wt-dysferlin aggregates. Dysferlin aggregates were shown by immunostaining using an anti-His antibody (green). Scale bar, 25 µm.

Brefeldin, an inhibitor for _trans_-Golgi network, as well as other ER stress stimuli such as thapsigargin and tunicamycin slightly stimulated the accumulation of Tet-wt-dysferlin in the ER (Fig. 2E), suggesting that only a small population of Tet-wt-dysferlin as well as endogenous dysferlin is transported to the cell surface but a large population of Tet-wt-dysferlin remains in the ER/Golgi. Thus, wt-dysferlin is preferentially recognized by the retrotranslocon and more easily degraded by ubiquitin/proteasome ERAD when compared with other ER proteins.

Dysferlin aggregation in the ER and ER stress cell death

In contrast with wt-dysferlin, overexpressed mutant dysferlins (L1341P and W999C) (18,26) spontaneously formed patchy aggregations in the ER (Fig. 3A and Supplementary Material, Fig. S1); mutant (L1341P)-dysferlin had patchy aggregation in 28 h, and formed large aggregates, which led to apoptotic features in 32 h, while wt-dysferlin did not show the spontaneous aggregates and apoptotic feature at 32 h after transfection (Fig. 3B).

Figure 3.

The relationship between accumulation of mutant dysferlin in the ER and cell death. (A) Spontaneous aggregation of mutant (L1341P) in C2C5 cells. C2C5 cells were transfected with pCI-neo-wt-dysferlin or mutant (L1341P). Mutant dysferlin spontaneously formed patchy aggregation and induced cell shrinkage with apoptotic features (arrows). Scale bar, 25 µm. (B) Mutant (L1341P)-dysferlin-induced apoptosis. Wt- and mutant dysferlin were transfected into C2C5 cells and their accumulations in the ER were monitored. Mutant dysferlin (green) formed patchy aggregates at 28 h and cells expressing mutant dysferlin aggregates showed cell shrinkage with apoptotic morphological features in 32 h after transfection. Nucleus (Blue). Scale bar, 15 µm. (C) Mutant dysferlin-induced ER stress cell death. Mutant dysferlin-induced (green) ER stress cell death with c-Jun phosphorylation (anti-c-Jun-p reactivity) (red) in 28 h, activation of caspase-12 (anti-active caspase-12 reactivity) (red) in 32 h, Chop-upregulation (red) in 28 h, and ubiquitin (red) in 32 h after transfection. Scale bar, 15 µm.

Defects in ERAD, inhibition of proteasome activity and impaired retrotranslocation causes the accumulation of ER proteins, which subsequently induces ER stress-induced cell death. Like LLL treatment, the overexpression of the mutant (L1341P)-dysferlin induced C/EBP homologous protein (Chop) up-regulation and c-Jun phosphorylation (anti-Jun-p positive) in 28 h and caspase-12 activation (anti-active caspase-12-positive) in 32 h (Fig. 3C and Supplementary Material, Fig. S2). The patchy aggregates colocalized with ubiquitin immunoreactivity (Fig. 3C and Supplementary Material, Fig. S3). Thus, the mutant dysferlin accumulates and spontaneously aggregates in the ER and has a potential ability to induce the ER stress cell death.

Degradation of mutant dysferlin aggregates in lysosome

We compared the degradation of Tet-wt and -mutant (L1341P) dysferlin. LLL stimulated both Tet-wt and -mutant dysferlin accumulation and aggregation in the ER (Fig. 4A upper and lower), and increased the number of cells expressing dysferlin aggregates (Fig. 4A upper and middle); the population of cells showing wt- and mutant-dysferlin aggregates were increased from 2–3% to 67% and 39–91% by LLL treatment, respectively. E64d/pepstatin A also stimulated the accumulation and aggregation of Tet-mutant dysferlin in the ER but did not Tet-wt-dysferlin (Fig. 4B upper and lower). The population of cells showing the mutant dysferlin aggregates was increased from 33 to 68% by E64d/pepstatin A treatment, but the population of cells showing wt-dysferlin aggregates was not (Fig. 4B upper and middle). Thus, in contrast with Tet-wt-dysferlin data, Tet-mutant dysferlin seems to be degraded not only by the ubiquitin/proteasome but also by the lysosome.

Figure 4.

Degradation of mutant dysferlin in proteasome and lysosome. Tet-wt- and mutant (L1341P)-dysferlin cells were incubated in the presence or absence of LLL (5 µm) (A) or E64d/pepstatin A (10 µg/ml) (B). Accumulation of wt- or mutant dysferlin was detected by immunostaining (upper panels), by counting the number of cells showing dysferlin aggregates (middle panels). Arrowheads indicate cells showing the mutant dysferlin aggregates and/or cell shrinkage with apoptotic features. Triton-insoluble dysferlin was detected by immunoblot analysis using anti-dysferlin (lower panels). Scale bars, 25 µm. Error bars indicate standard deviation.

Involvement of ER stress-induced eIF2α-mediated autophagy pathway in the lysosomal degradation of mutant dysferlin

We examined the degradation pathway of the mutant (L1341P)-dysferlin aggregated in the ER to the lysosome. There are two possible pathways for lysosomal degradation: the Golgi-endosome pathway (27) and autophagy pathway (20). Brefeldin A (10 µm) did not significantly stimulate mutant dysferlin aggregation in the ER (Supplementary Material, Fig. S4), suggesting that mutant dysferlin preferentially localizes to the ER/Golgi. Therefore, the Golgi-endosome pathway cannot be the major pathway by which mutant dysferlin undergoes lysosomal degradation. We next examined the possible involvement of the autophagy pathway in the lysosomal degradation of mutant dysferlin.

eIF2α phosphorylation stimulates autophagosome formation (28) and the Atg5_–_Atg12 complex-dependent conversion of LC3 [Atg8; microtubule-associated protein 1 (MAP1) light chain 3] (24), LC3-I (18 kDa; free form) and LC3-II (16 kDa; membrane-bound form), are involved in key steps of autophagosome formation (19,20). We previously demonstrated that ER stress stimuli such as tunicamycin and thapsigargin, which stimulate eIF2α phosphorylation, induce the expression of Atg12 gene and LC3 conversion (24). Dominant negative (DN) PERK (29) and dephosphorylation of eIF2α (30) inhibited the thapsigargin-induced LC3 conversion (Fig. 5A and B). Thus, ER stress stimulated the autophagosome formation by activating the PERK–eIF2α phosphorylation pathway.

Figure 5.

Degradation of mutant dysferlin by eIF2α-mediated autophagy. Involvement of PERK pathway (A) and eIF2α phosphorylation (B) in thapsigargin-induced LC3 conversion. C2C5 cells and Tet-myc-tagged DN-PERK [PERK (K618A)] C2C5 cells (24,29) (A) and eIF2α S/S (WT) and A/A (mutation) MEF cells (30) (B) were incubated in the presence or absence of thapsigargin, and LC3 conversion and eIF2α phosphorylation were examined by immunoblot analysis. (C) Effect of LLL on the LC3 conversion and eIF2α phosphorylation of the Tet-wt- and mutant (L1341P)-dysferlin C2C5 cells. induced LC3 conversion and eIF2α phosphorylation. (D) Effect of overexpressed wt- and mutant (L1341P)-dysferlin on the LC3 conversion and eIF2α phosphorylation of C2C5 cells. (E) Immunostaining analysis of the LC3 conversion and eIF2α phosphorylation induced by wt- and mutant (L1341P)-dysferlin. Anti-dysferlin (green), anti-LC3 (red) and anti-eIF2α-p (red), and merged (yellow). Scale bar, 15 µm.

We examined the involvement of the autophagy/lysosome pathway in the degradation of the mutant dysferlin aggregates in the ER. In contrast with wt-dysferlin, overexpression of mutant (L1341P)-dysferlin as well as LLL-induced LC3 conversion and eIF2α phosphorylation (Fig. 5C and D). LC3-I is diffusely immunostained by anti-LC3 in the cytoplasm of cells with no stress, while LC3-II, which is tightly associated with the autophagosomal membrane, shows intense vesicular and granular anti-LC3 immunostaining (31,32). Immunostaining analysis showed that most cells expressing mutant dysferlin aggregates were eIF2α phosphorylation-positive and vesicular anti-LC3-positive, while cells expressing wt-dysferlin were negative (Fig. 5E). Most of the patchy aggregates of mutant dysferlin colocalized with vesicular anti-LC3 reactivity.

The deficiency of Atg5 stimulated the accumulation of Triton-insoluble mutant (L1341P) dysferlin and increased the population of cells containing aggregated mutant dysferlin (Fig. 6A); the population of cells displaying wt- and mutant dysferlin was 13 and 24% in wild-type (Atg5+/+) mouse embryonic fibroblast (MEF) cells, respectively, and 20 and 53% in _Atg5_−/− MEF cells (33), respectively. Thus, autophagy mediates the transfer of mutant dysferlin rather than wt-dysferlin from the ER to the lysosome. eIF2α dephosphorylation, which inhibits autophagosome formation (24,28), also stimulated the accumulation of Triton-insoluble mutant (L1341P)-dysferlin and its aggregation, and increased the population of cells expressing mutant dysferlin aggregates (Fig. 6B); the population of cells displaying wt- and mutant dysferlin were 10 and 17% in wild-type (eIF2α S/S) MEF cells, respectively, and 22 and 46% in eIF2α A/A MEF cells (30), respectively. These results suggest that mutant dysferlin aggregation in the ER stimulates autophagosome formation by activation of ER stress-eIF2α phosphorylation pathway.

Figure 6.

Effect of Atg5 deficiency and eIF2α phosphorylation on the degradation of wt- and mutant dysferlin aggregation. Wt- and mutant (L1341P)-dysferlin were transfected into Atg5+/+ and _Atg5_−/− MEF cells (33) (A), and eIF2α S/S (WT) and A/A (mutation) MEF cells (30) (B). Accumulation of wt- and mutant dysferlin were detected by immunostaining (upper panels) and by counting the number of cells containing dysferlin aggregates (middle panels). Triton-insoluble mutant dysferlin was examined by immunoblot analysis using anti-dysferlin (lower panels). Arrowheads indicate cells showing the mutant dysferlin aggregates and/or cell shrinkage with apoptotic features. Scale bars, 25 µm. Error bars indicate standard deviation.

Stimulation of the degradation of mutant dysferlin by rapamycin

Rapamycin stimulates autophagy formation via mammalian target of rapamycin (34). It has also been shown to promote the degradation of polyQ aggregates thereby inhibiting cell death (24,35,36). Rapamycin also stimulates GCN2-mediated eIF2α phosphorylation in yeast (37) and LC3 conversion via PERK-independent eIF2α phosphorylation (24). Rapamycin (0.5–10 µg/ml) induced eIF2α phosphorylation and LC3 conversion (Fig. 7A). Consequently, rapamycin inhibited the accumulation of the Triton-insoluble form of the mutant (L1341P) dysferlin and its aggregation, and decreased the population of cells displaying mutant dysferlin aggregates from 38 to 18% (Fig. 7B), while 3-methyladenine (3-MA), which stimulates the autophagy formation, increased them from 36 to 44% (Fig. 7C).

Figure 7.

Effect of rapamycin or 3-MA on the mutant dysferlin aggregates. (A) Rapamycin-induced LC3 conversion and eIF2α phosphorylation. C2C5 cells were incubated with rapamycin at the indicated concentration (0.5–10 µg/ml). Stimulation of eIF2α phosphorylation and LC3 conversion by rapamycin was examined. Effect of rapamycin (B) or 3-MA (C) on the mutant dysferlin aggregation. Mutant (L1341P)-dysferlin was transfected into C2C5 cells and incubated in the presence or absence of rapamycin (10 µg/ml) or 3-MA (10 mm). Accumulation of mutant dysferin was detected by immunostaining (left), by counting the number of cells showing mutant dysferlin aggregates using anti-dysferlin (right and upper panel). Triton insoluble-mutant dysferlin was detected by immunoblot analysis (right and lower panel). Arrowheads indicate cells showing the mutant dysferlin aggregates and/or cell shrinkage with apoptotic features. Scale bar, 25 µm. Error bars indicate standard deviation.

Collectively, we propose two ERAD models for dysferlin degradation, ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II) (Fig. 8).

Figure 8.

Model for the autophagy/lysosome ERAD system. Wt-dysferlin is located in the ER and Golgi and some of them are transported to the surface membrane and play a role in the vesicular membrane fusion system. Usually, wt- and mutant dysferlin are degraded by ubiquitin/proteasome ERAD system. However, when mutant dysferlin is not sufficiently degraded by ubiquitin/proteasome ERAD, they aggregate in the ER and stimulate autophagy formation, LC3 conversion, via the ER stress-PERK-eIF2α phosphorylation. The mutant dysferlin aggregates on the ER membrane could be recognized by the elongating membrane to form the autophagosome and then degraded by autolysosome on the fusion with lysosome. Thus, autophagy/lysosome pathway acts as ERAD( II), an alternative ERAD, for the degradation of the mutant dysferlin aggregates when the ubiquitin/proteasome ERAD(I) is impaired. However, if autophagy/lysosome ERAD(II) insufficiently degrades the mutant dysferlin aggregates, cells may choose the ER stress-mediated cell death.

DISCUSSION

Ubiquitin/proteasome ERAD for dysferlin degradation and pathogenesis of LGMD2B/MM

Dysferlin is ubiquitously expressed in many tissues but only detectable in muscle sarcolemma (4,5). In the most of LGMD2B/MM patient muscle, however, loss of dysferlin has been shown (1,2). Most of overexpressed dysferlin were located in the ER/Golgi but not located at the plasma membrane (Fig. 1), suggesting that only small amount of dysferlin is necessary for cells, and unnecessary or misfolded dysferlin is checked by protein quality control system for degradation. LLL as well as polyQ aggregates induces the accumulation of Tet-wt-dysferlin in the ER and the co-aggregation with polyubiquitination (Fig. 2), suggesting that wt-dysferlin is a substrate for ubiquitin/proteasome ERAD. VCP(p97) functions to extract aberrant proteins from the retrotranslocon composed of Sec61 and direct them towards ubiquitin/proteasome ERAD (13,38,39). Association with Sec61α or VCP(p97) (Fig. 1) may cause the preferential extraction of excess wt- and some mutant-dysferlin from the ER in order for degradation via the ubiquitin/proteasome ERAD can occur. Otherwise, wt-dysferlin may be more susceptible to the degradation by ubiquitin/proteasome than proteins located in the lumen of the ER, because dysferlin with the structure of type II membrane protein and a carboxy-terminal transmembrane region is assumed to be located on the ER membrane exposing its most part to the cytoplasm. This may be another reason that the specific loss of the mutant dysferlin is frequently observed in the muscle of the LGMD2B/MM patients.

In contrast with wt-dysferlin, dysferlin with mutation (L1341P) in the C2 domain, which shows the intracellular aggregation in muscle fibers of the patient (18), spontaneously aggregated in the ER (Fig. 4). Intracellular C2 domains of dysferlin show homology to C2A of synaptotagmin (3), suggesting that these domains are involved in the Ca++-dependent fusion process. L1341 is one of the most highly conserved positions, implying an important structural role for this amino acid. One possible explanation for the aggregation of the mutant (L1341P)-dysferlin in the ER is that the loss of function of the C2 domain may cause a defect of association between the mutant dysferlin and the retrotranslocon or VCP(p97). Alternatively, the mutation (L1341P) may cause deleterious effects on the secondary and tertiary structure of dysferlin, resulting in a resistance to the ubiquitin/proteasome ERAD and a tendency to spontaneously aggregate in the ER. Furthermore, these mutant dysferlin aggregates induced the coaggregation of the GFP-polyQ11 located in the cytoplasm with polyubiquitination (Supplementary Material, Fig. S3) and polyQ72 aggregates induced the wt-dysferlin aggregation on the ER (Fig. 2A), suggesting another possibility that mutant dysferlin aggregates inhibit the proteasome activity in the cytoplasm, causing the accumulation of mutant dysferlin and stimulating their further aggregation on the ER. Detailed analysis of the relationship between the structures of various mutant dysferlins and their susceptibilities to the degradation and their abilities to the inhibition of the proteasome activity remains to be performed.

The autophagy/lysosome degradation pathway as an alternative ERAD

LLL treatment induces the accumulation of the misfolded states in the wt-dysferlin in the ER by inhibiting the degradation of misfolded proteins, while mutant dysferlin has higher tendency to spontaneously aggregate in the ER. It is highly likely that the accumulation of misfolded proteins including misfolded dysferlin caused by LLL treatment or malfolded mutant dysferlin induces ER stress and stimulates LC3 conversion and the autophagic pathway to eliminate these aberrant aggregates (Figs 3 and 4). E64d/pepstatin A, lysosomal protease inhibitors, did not stimulate the prominent accumulation of wt-dysferlin (Figs 2 and 4), but stimulated mutant dysferlin accumulation (Fig. 4), suggesting that wt-dysferlin is mainly degraded by ubiquitin/proteasome ERAD, while mutant dysferlin is degraded in the lysosome in addition to ubiquitin/proteasome ERAD.

Autophagy usually plays a role in the degradation of cellular organs under critical conditions. However, constitutive autophagy, which occurs independent of nutrient stress, has been recently shown to play an important role in maintaining the homeostasis of non-dividing cells including hepatocytes and a subset of neural cells; the ubiquitinated proteins are accumulated in the liver and brain of the _Atg7_- and/or _Atg5_-deficient mice (21–23). In the yeast, Atg6/Beclin, which is involved in vesicle nucleation for autophagy by associating with PI3K as a type I complex (40), inhibits the aggregation of the α-protease inhibitor (A1PiZ) in the ER and rescued the ERAD defect (41). An α-proteinase inhibitor extracted from the ER is degraded by two functionally distinct degradation pathways in yeast: the soluble inhibitor is degraded by the ubiquitin/proteasome system, whereas the inhibitor that aggregated due to an ERAD defect is degraded by autophagy/lysosome.

Inhibition of lysosomal proteases, E64d/pepstatin A (42), and Atg5 deficiency stimulated mutant (L1341P)-dysferlin aggregation in the mammalian cells (Figs 4 and 6), suggesting that the autophagy/lysosome pathway, an alternative ERAD system, is further stimulated by mutant dysferlin aggregates in the ER for their degradation; i.e. when mutant or aberrant dysferlins are not sufficiently degraded by ubiquitin/proteasome ERAD, they spontaneously aggregate on the ER membrane and may stimulate the autophagy/lysosome pathway as an alternative ERAD to engulf them for further degradation in lysosome (Fig. 8).

Interestingly, Atg5 deficiency also stimulated the aggregation of wt-dysferlin (Fig. 6A and Supplementary Material, Fig. S5); the population of cells displaying wt-dysferlin aggregation was increased from 13 to 20% by Atg5 deficiency. Thus, the constitutive autophagy seems to play a role in the clearance of some of the misfolded dysferlin included in wt-dysferlin as a protein-quality control system although this contribution to the degradation of wt-dysferlin is much less than ubiquitin/proteasome (Fig. 4A). In contrast, E64d/pepstatin A does not increase the accumulation of the wt-dysferlin (Figs 2 and 4B), suggesting that constitutive autophagy may not be always associated with lysosomal degradation but may be associated with clearance system or wt-dysferlin may be degraded by the E64d/pepstatin A-insensitive proteases.

Regulation of autophagy formation by eIF2α phosphorylation

eIF2α phosphorylation is known to be involved in the stress-induced autophagy (24,28); starvation stimulates autophagy formation via eIF2α phosphorylation (28). Recently, we showed that serum deprivation and rapamycin-induced LC3 conversion via a PERK-independent eIF2α phosphorylation-dependent pathway (15,24). As we describe here, ER stress inducing stimuli such as thapsigargin as well as polyQ aggregates induces LC3 conversion via PERK-eIF2α phosphorylation pathway (Fig. 5A and B) (24). Thus, it is likely that the mutant dysferlin aggregates in the ER may induce the autophagosome formation for the activation of the alternative ERAD pathway, via stimulation of the ER stress-PERK/eIF2α phosphorylation pathway (Fig. 8). Furthermore, eIF2α dephosphorylation and Atg5 deficiency stimulated the accumulation of mutant (L1341P)-dysferlin (Fig. 6), suggesting that ER stress-eIF2α phosphorylation may also regulate the autolysosome formation, the fusion process of autophagosome with lysosome. Rapamycin seems to be helpful for the degradation of the aberrant protein aggregates including mutant dysferlin in the autophagy/lysosome (Fig. 7) and for cell survival because it stimulates autophagosome formation via non-ER stress-eIF2α phosphorylation pathway (24).

In conclusion, we propose two ERAD models for dysferlin degradation, the ubiquitin/proteasome ERAD(I) and the autophagy/lysosome ERAD(II) (Fig. 8). Autophagy/lysosome ERAD(II) is an alternative ERAD system for the degradation of excess mutant dysferlin when the ubiquitin/proteasome ERAD(I) is impaired. Mutant (L1341P) dysferlin aggregates on the ER membrane stimulate autophagosome formation via activating ER stress-eIF2α phosphorylation. It is formally possible that autophagy/lysosome ERAD(II) degrades other aberrant proteins when ubiquitin/proteasome ERAD(I) is impaired.

MATERIALS AND METHODS

Cell culture

C2C5 cells were P19 EC cells constitutively expressing c-jun (43). MEF cells from eIF2α S/S (WT) and eIF2α A/A (mutant) knock-in mouse embryos were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS), 0.1 mm non-essential amino acids solution (Gibco), 1 × amino acids solution (Gibco) and 100 U/ml penicillin/streptomycin (Gibco) at 37°C in a humidified atmosphere of 5% CO2 (30). MEFs from _Atg5_−/− and Atg5+/+ mouse embryos (33) CO5 cells, and C2C5 cells were cultured in α-minimum essential medium (α-MEM; Sigma, St Louis, MO, USA) supplemented with 10% FCS at 37°C in humidified atmosphere of 5% CO2. The C2C5 cells expressing Tet-DN-PERK(K618A) were prepared as described previously (24).

Site-directed mutagenesis

To generate the dysferlin mutants [L1341P; T4022C (18), W999C; G3370T (26)], site-directed mutagenesis were performed using QuikChange II Site-Directed Mutagenesis kit (Stratagene, Ceder Creek, TX, USA) (44), with the following primers: L1341P sense 5′-ATCCTGGCATGGGGCC_C_GC GGAACATGAAGAGT-3′, antisense 5′-ACTCTTCATGTTCCGCG_G_GCCCCATGCCAGGAT-3′; W999C sense 5′-GGAAGATGAGGAATG_C_TCCACAGACCTCAAC-3′, antisense 5′-GTTGAGGTCTGTGGA_G_CATTCCTCATCTTCC-3′. Italicized nucleotides were mutated sites. Mutations were confirmed by nucleotide sequencing analysis.

Plasmid construction and establishment of C2C5 cell line expressing tetracycline-inducible dysferlin

Human dysferlin gene was inserted into the _Eco_RI site of the pcDNA4/TO/myc-His expression vector (Invitrogen, Carlsbad, CA, USA), and then transfected into C2C5 cells using the calcium-phosphate method (45), and clones expressing dysferlin [Tet-wt-dysferlin, Tet-mutant (L1341P) or (W999C) dysferlin] were selected by incubation with 50 µg/ml of zeocine and 25 µg/ml blastin (Invitrogen). Cells were cultured in α-MEM supplemented with 10% tetracycline (Tet) system-approved FCS (BD Bioscience Clontech, Palo Alto, CA, USA) at 37°C in a humidified atmosphere of 5% CO2 (Tet-off). Cells were cultured in α-MEM supplemented with 10% FCS containing tetracycline at 37°C for 12 h in a humidified atmosphere of 5% CO2 (Tet-on). The expression of dysferlin was examined by immunostaining and immunoblotting using an anti-His (Santa Cruz Biotech., Santa Cruz, CA, USA).

DNA ladder

Fragmented DNA isolation was performed according to Prigent et al. (46).

Immunoblot analysis

pCI-neo-dysferlin and pCI-neo-mutant dysferlin (L1341P and W999C) (8 µg) were transfected into C2C5 cells and/or MEF cells (6 cm dish) using the calcium-phosphate method (45) and/or Lipofectamine 2000-manufactor's manual (Invitrogen). Dysferlin-transfected C2C5 cells, Tet-wt- and mutant- dysferlin C2C5 cells, Tet-DN-PERK (K618)-C2C5 cells (24,29) and MEF cells were incubated for the indicated period in the presence or absence of LLL (5 µm) (Bostonbiochem, Cambridge, MA, USA), brefeldin A (2 µg/ml) (Biomol, Plymouth Meeting, PA, USA), thapsigargin (2 µm) (Sigma), rapamycin (10 µg/ml) (Wako Pure Chemical, Osaka, Japan), 3-MA (10 mm) (Sigma) or E64d (10 µg/ml) (Calbiochem, La Jolla, CA, USA) and pepstatin A (10 µg/ml) (ICN Biochemicals, Aurora, OH, USA). Cells were lysed with phosphate-buffered saline (PBS) containing 1% Triton X-100.

After centrifugation at 10 000_g_ for 20 min, the cell extracts (50 µg protein) were subjected to SDS–PAGE (7.5–12%) and immunoblot analysis as described previously (24). Insoluble fractions were lysed into SDS sample buffer and subjected to SDS–PAGE and immunoblot analysis. Proteins in the gels were electrophoretically transferred to nitrocellulose filters. After filters were incubated with anti-LC3 (42,47), anti-tubulin (Sigma), anti-c-Jun, anti-c-Jun-p, anti-eIF2α, anti-eIF2α-p (Cell Signaling Technology, Danvers, MA, USA), anti-caspase-12 (Santa Cruz Biotech), anti-Chop (GADD153) (Santa Cruz Biotech), anti-Hamlet (Novocastra, UK), anti-Dysferlin (Spring, Fremont, CA, USA), anti-GFP (Boehringer Mannheim, Mannheim, Germany) and anti-His, the reactivities on the filters were detected by alkaline phosphatase-conjugated, goat anti-rabbit or anti-mouse immunoglobulin (Promega, Madison, WI, USA), respectively, and nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate.

Immunoprecipitation

pEGFP-dysferlin was transfected into COS cells. The cell pellets were lysed with RIPA buffer (PBS containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) on ice and centrifuged. The cell extracts were incubated with anti-GFP antibody at 4°C overnight, and the immunocomplexes were precipitated by protein A and G (Promega). Their immunoprecipitates were analyzed by immunoblot analysis using anti-GFP, anti-VCP(p97) (Progen, Heidelberg, Germany) and anti-Sec61α (Upstate, Lake Placid, NY, USA).

Immunostaining

C2C5 cells and MEF cells were transfected with pEGFP-dysferlin, pCI-neo-dysferlin and pCI-neo-mutant (L1341P, W999C) dysferlin using the calcium-phosphate method (41). C2C5-Tet-dysferlin cells were transfected with pEGFP-72CAG (25) by the calcium-phosphate method. C2C5 cells, Tet-wt-dysferlin C2C5 cells, and Tet-mutant-dysferlin C2C5 cells and MEF cells were incubated in the presence or absence of LLL (5 µm), thapsigargin (2 µm) (Sigma), tunicamycin (2 µg/ml) (Wako Pure Chemical), and brefeldin A (2 µg/ml), rapamycin (10 µg/ml), 3-MA (10 mm) or E64d (10 µg/ml) and pepstatin A (10 µg/ml), were fixed with 4% paraformaldehyde in PBS at the indicated time. They were incubated with antibodies; anti-casp12D341, antisera against the cleavage site of mouse caspase-12 at D341 (25), anti-c-Jun-p, anti-eIF2α-p anti-LC3 (42,47), anti-Chop, anti-Hamlet, anti-dysferlin, anti-polyubiquitin (MBL, Nagoya, Japan), anti-ubiquitin (Dako, Glostrup, Denmark), anti-KDEL (Stressgene, Victoria, Canada), anti-GM130 (Transduction laboratories, Lexington, KY, USA), anti-F0ATPase (48), anti-cathepsin D (49), anti-GFP, anti-VCP(p97), anti-Sec61α and anti-His for 24 h at 4°C and then incubated with FITC- and rhodamine-labeled goat anti-mouse or rabbit (Leinco Technologies, St Louis, MO, USA) for 1 h at 37°C. Their nuclei were labeled with Hoechst 33342 (Molecular Probes, Eugene, OR, USA). They were viewed with a confocal laser-scanning microscope (CSU-10, Yokogawa, Tokyo, Japan). Percentages of cells showing wt- and mutant dysferlin aggregates or dysferlin accumulation were determined by counting 100–200 cells expressing. The values are averages of the percentages of the number of cells obtained in three experiments.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

ACKNOWLEDGEMENTS

We thank Dr Randau J. Kaufmann, Dr Noboru Mizushima, Dr David Ron and Dr Eiki Kominami for kindly providing the eIF2αS/S and eIF2αA/A MEF cells, _Atg5_−/− MEF cells, DN-PERK, myc-tagged PERK (K618A), plasmid and antibodies, respectively, and Roberto Sitia for discussion. We offer one's sincere condolences for Dr Kichi Arahata, who collaborate on this study in his life. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (No. 18700333) from the Ministry of Education, Science, Sports, and Culture and by Research Grants 17A-10 for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare, and Research on Brain Science from the Ministry of Health and Welfare of Japan, the Human Science Foundation.

Conflict of Interest statement. None declared.

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Author notes

†

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

© 2007 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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