Identification of CLN6 as a molecular entity of endoplasmic reticulum-driven anti-aggregate activity
Identification of CLN6 as a molecular entity of endoplasmic reticulum-driven anti-aggregate activity
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鉴定 CLN6 作为内质网驱动的抗聚集活性的分子实体
DOI：
10.1016/j.bbrc.2017.05.002
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发表时间：
2017
影响因子：
3.1
通讯作者：
Yamazaki Tetsuo
中科院分区：
文献类型：
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作者：
Yamashita Arisa;Hiraki Yuri;Yamazaki Tetsuo
α B-晶状体蛋白（αB-crystallin，αBC）是一种小分子热休克蛋白。αBC基因突变与α-stac病相关，α-stac病是一种遗传性肌病，组织学特征为细胞内蛋白质聚集体积聚。致病的R120 G αBC突变体，在120位具有甘氨酸到甘氨酸的替换，是一种易于聚集的蛋白质。我们以前的研究表明，R120 G突变体在HeLa细胞中的聚集是通过在内质网（ER）上强制表达αBC来阻止的。为了阐明对R120 G突变体的预防作用的分子本质，我们分离了与ER锚定的αBC（TMαBC）结合的蛋白质。ER跨膜CLN 6蛋白被鉴定为TMαBC的结合剂。HeLa细胞中CLN 6的敲低减弱了TMαBC对R120 G突变体的抗聚集活性。相反，CLN 6过表达增强了活性，表明CLN 6作为TMαBC的下游效应物起作用。CLN 6与R120 G突变体发生物理相互作用，并抑制其在HeLa细胞中的聚集，即使TMαBC不共表达。此外，CLN 6对R120 G突变体的拮抗作用在用溶酶体抑制剂处理后受损，表明CLN 6需要完整的自噬-溶酶体系统来防止R120 G突变体聚集。因此，我们得出结论，CLN 6不仅是使用TMαBC的ER操纵赋予的抗聚集活性的分子实体，而且还作为治疗干预的潜在靶点。
αB-crystallin (αBC) is a small heat shock protein. Mutations in the αBC gene are linked to α-crystallinopathy, a hereditary myopathy histologically characterized by intracellular accumulation of protein aggregates. The disease-causing R120G αBC mutant, harboring an arginine-to-glycine replacement at position 120, is an aggregate-prone protein. We previously showed that the R120G mutant's aggregation in HeLa cells was prevented by enforced expression of αBC on the endoplasmic reticulum (ER). To elucidate the molecular nature of the preventive effect on the R120G mutant, we isolated proteins binding to ER-anchored αBC (TMαBC). The ER transmembrane CLN6 protein was identified as a TMαBC's binder. CLN6 knockdown in HeLa cells attenuated TMαBC's anti-aggregate activity against the R120G mutant. Conversely, CLN6 overexpression enhanced the activity, indicating that CLN6 operates as a downstream effector of TMαBC. CLN6 physically interacted with the R120G mutant, and repressed its aggregation in HeLa cells even when TMαBC was not co-expressed. Furthermore, CLN6's antagonizing effect on the R120G mutant was compromised upon treatment with a lysosomal inhibitor, suggesting CLN6 requires the intact autophagy-lysosome system to prevent the R120G mutant from aggregating. We hence conclude that CLN6 is not only a molecular entity of the anti-aggregate activity conferred by the ER manipulation using TMαBC, but also serves as a potential target of therapeutic interventions.