Fujita Eriko, Kouroku Yoriko, Isoai Atsushi, Kumagai Hiromichi, Misutani Akifumi, Matsuda Chie, Hayashi Yukiko K, Momoi Takashi
Divisions of Development and Differentiation, Department of Human Inherited Metabolic Disease, Yokohama, Kanagawa, Japan.
Hum Mol Genet. 2007 Mar 15;16(6):618-29. doi: 10.1093/hmg/ddm002. Epub 2007 Mar 1.
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.
肌膜蛋白是一种II型跨膜蛋白，是2B型肢带型肌营养不良症和宫下肌病（LGMD2B/MM）的致病基因，在这些疾病中经常观察到肌膜蛋白标记的特异性缺失。最近，一种新型突变体（L1341P）肌膜蛋白已被证明在患者肌肉中聚集。关于肌膜蛋白降解与LGMD2B/MM发病机制之间的关系知之甚少。在这里，我们研究了正常和突变体（L1341P）肌膜蛋白的降解。野生型（wt）肌膜蛋白主要定位于内质网/高尔基体，与逆转位蛋白、Sec61α和VCP（p97）相关，并通过由泛素/蛋白酶体组成的内质网（ER）相关降解系统（ERAD）降解。相比之下，突变体肌膜蛋白在内质网中自发聚集，诱导真核翻译起始因子2α（eIF2α）磷酸化和LC3转化，这是自噬体形成的关键步骤，最终导致内质网应激细胞死亡。与蛋白酶体抑制剂不同，溶酶体蛋白酶抑制剂E64d/胃蛋白酶抑制剂A不会刺激wt-肌膜蛋白的积累，但会刺激突变体肌膜蛋白在内质网中的聚集。此外，Atg5的缺乏和eIF2α的去磷酸化，这是LC3转化的关键分子，也会刺激突变体肌膜蛋白在内质网中的聚集。雷帕霉素可诱导eIF2α磷酸化介导的LC3转化，抑制突变体肌膜蛋白在内质网中的聚集。因此，突变体肌膜蛋白在内质网中聚集，通过激活内质网应激-eIF2α磷酸化途径刺激自噬体形成以吞噬它们。我们提出了两种肌膜蛋白降解的ERAD模型，泛素/蛋白酶体ERAD（I）和自噬/溶酶体ERAD（II）。当逆转位蛋白/ERAD（I）系统因这些突变体聚集而受损时，内质网上聚集的突变体肌膜蛋白可通过自噬/溶酶体ERAD（II）降解，作为ERAD（I）的替代途径。
