Abstract：
Protein homeostasis, including protein folding, refolding, and degradation, is thought to decline with aging. HSPB5 (also known as αB-crystallin) prevents target protein aggregation as a molecular chaperone and exhibits a cytoprotective function against various cell stresses. To elucidate the effect of HSPB5 on endoplasmic reticulum (ER) stress, we searched for novel binding proteins of HSPB5 using the proximity-dependent biotin labeling method. Proteins presumed to interact with HSPB5 in cells treated with the proteasome inhibitor MG132 were identified by a reversible biotin-binding capacity method combining tamavidin2-REV magnetic beads and mass spectrometry. We discovered a new binding protein for HSPB5, polo-like kinase 2 (PLK2), which is an apoptosis-related enzyme. The expression of PLK2 was upregulated by MG132 treatment, and it was co-localized with HSPB5 near the ER in L6 muscle cells. Inhibition of PLK2 decreased ER stress-induced phosphorylation of serine 19 in HSPB5 and increased apoptosis by activation of caspase 3 under ER stress. Overexpression of HSPB5 (WT) suppressed the ER stress-induced caspase 3 activity, but this was not observed with phospho-deficient HSPB5 (3A) mutants. These results clarify the role of HSPB5 phosphorylation during ER stress and suggest that the PLK2/HSPB5 pathway plays an essential role in cytoprotection against proteasome inhibition-induced ER stress.
摘要：
蛋白质稳态，包括蛋白质折叠，重新折叠,和退化，被认为会随着年龄的增长而下降。HSPB5（也称为αB-晶状体蛋白）作为分子伴侣防止靶蛋白聚集，并表现出针对各种细胞应激的细胞保护功能。为了阐明HSPB5对内质网（ER）应激的影响，我们使用邻近依赖的生物素标记方法搜索HSPB5的新型结合蛋白。通过结合tamavidin2-REV磁珠和质谱的可逆生物素结合能力方法鉴定了假定与蛋白酶体抑制剂MG132处理的细胞中的HSPB5相互作用的蛋白质。我们发现了一种新的HSPB5结合蛋白，polo样激酶2（PLK2），这是一种与细胞凋亡相关的酶。MG132处理上调PLK2的表达,它与HSPB5共定位在L6肌细胞的ER附近。PLK2的抑制降低了ER应激诱导的HSPB5中丝氨酸19的磷酸化，并通过在ER应激下激活caspase3增加了细胞凋亡。HSPB5（WT）的过表达抑制了ER胁迫诱导的caspase3活性，但这在磷酸缺陷型HSPB5(3A)突变体中没有观察到。这些结果阐明了HSPB5磷酸化在ER应激过程中的作用，并表明PLK2/HSPB5途径在针对蛋白酶体抑制诱导的ER应激的细胞保护中起着至关重要的作用。
