Intravitreal injection or topical eye-drop application of a μ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa
Intravitreal injection or topical eye-drop application of a μ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa
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DOI：
10.1016/j.bbadis.2012.07.018
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发表时间：
2012-11-01
影响因子：
6.2
通讯作者：
Ishiguro, Sei-ichi
中科院分区：
文献类型：
--
作者：
Ozaki, Taku;Nakazawa, Mitsuru;Ishiguro, Sei-ichi
线粒体钙蛋白酶在视网膜感光细胞变性中启动凋亡诱导因子（AIF）依赖性细胞凋亡。线粒体钙蛋白酶抑制剂可能是该疾病的治疗靶点。因此，我们试图鉴定线粒体钙蛋白酶抑制剂，并确定其在皇家外科学院（RCS）大鼠（视网膜色素变性（RP）动物模型）中的作用。我们合成了mu-calpain的c2样（C2L）结构域的20聚肽。两种mu-calpain肽N2和N9抑制线粒体mu-calpain活性（IC50；分别为892和498 nM），但对其他蛋白酶无抑制作用。Western blotting结果显示，50 μ M的两种mu-calpain肽可引起线粒体mu-calpain的特异性降解。calpain的三维结构表明，肽N2和N9对应于蛋白酶核心结构域2和C2L结构域之间形成盐桥的区域。我们用10-mers测定了mu-calpain肽N2和N9的抑制区域，其中一个肽N2-10-2抑制线粒体mu-calpain的活性（IC50: 112 nM）。接下来，我们将肽N2-10-2偶联到HIV-1的c端，这是一种细胞穿透肽。利用离体大鼠肝脏线粒体，50 μ M HIV-conjugated mu-calpain N2-10-2 peptide （HIV-N μ, IC50; 285 nM）显著抑制AIF截断。通过TUNEL染色检测，玻璃体内注射20 mM HIV-N μ可阻止RCS大鼠视网膜感光细胞凋亡，并保留视网膜电图检测的视网膜功能。局部应用40 mM HIV-N μ也可阻止RCS大鼠视网膜光感受器凋亡。我们的研究结果表明，HIV-N mu是一种线粒体mu-calpain的肽抑制剂，为治疗RP提供了一种新的方式。(C) 2012 Elsevier B.V.版权所有
Mitochondrial mu-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial mu-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of mu-calpain. Two mu-calpain peptides N2 and N9 inhibited mitochondrial mu-calpain activity (IC50; 892 and 498 nM, respectively), but not other proteases. Western blotting showed that 50 mu M of both mu-calpain peptides caused specific degradation of mitochondrial mu-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of mu-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial mu-calpain (IC50: 112 nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50 mu M HIV-conjugated mu-calpain N2-10-2 peptide (HIV-N mu, IC50; 285 nM) significantly inhibited AIF truncation. The intravitreal injection of 20 mM HIV-N mu also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40 mM HIV-N mu also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-N mu, a peptide inhibitor of mitochondrial mu-calpain, offers a new modality for treating RP. (C) 2012 Elsevier B.V. All rights reserved.