Proteomic Identification of Cathepsin B and Nucleophosmin as Novel UVA-Targets in Human Skin Fibroblasts
Proteomic Identification of Cathepsin B and Nucleophosmin as Novel UVA-Targets in Human Skin Fibroblasts
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DOI：
10.1111/j.1751-1097.2010.00818.x
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发表时间：
2010-11-01
影响因子：
3.3
通讯作者：
Wondrak, Georg T.
中科院分区：
文献类型：
--
作者：
Lamore, Sarah D.;Qiao, Shuxi;Wondrak, Georg T.
太阳UVA暴露在皮肤光老化和光致癌中起着致病作用。在这里，我们描述了蛋白质组学鉴定新的UVA目标在人类皮肤成纤维细胞的二维差异凝胶电泳（2D-DIGE）的方法。成纤维细胞暴露于非细胞毒性剂量的UVA或不处理，总蛋白提取物进行CyDye标记，然后通过2D-DIGE/质谱鉴定差异表达的蛋白质，通过免疫检测独立确认。显示最明显的UVA诱导上调的蛋白质被确定为核仁蛋白nucleophosmin。经历最明显的UVA诱导的下调的蛋白质被鉴定为组织蛋白酶B，一种溶酶体半胱氨酸蛋白酶，在细胞UVA暴露后显示酶活性丧失和成熟改变。广泛的溶酶体积累的脂褐素样自发荧光和嗜锇物质发生在UVA暴露的成纤维细胞共聚焦荧光显微镜和透射电子显微镜，分别检测。阵列分析表明UVA诱导的氧化应激反应基因表达上调，UVA诱导的成纤维细胞组织蛋白酶B酶活性的丧失被抗氧化剂干预抑制。使用CA 074 Me的药理学组织蛋白酶B抑制模拟UVA诱导的溶酶体自体荧光积累和组织蛋白酶B成熟缺陷。总之，这些数据支持的假设，即组织蛋白酶B是UVA诱导的光氧化应激的一个重要目标，通过损害溶酶体清除脂褐素的原因参与皮肤光损伤。
Solar UVA exposure plays a causative role in skin photoaging and photocarcinogenesis. Here, we describe the proteomic identification of novel UVA-targets in human dermal fibroblasts following a two-dimensional-difference-gel-electrophoresis (2D-DIGE) approach. Fibroblasts were exposed to noncytotoxic doses of UVA or left untreated, and total protein extracts underwent CyDye-labeling followed by 2D-DIGE/mass-spectrometric identification of differentially expressed proteins, confirmed independently by immunodetection. The protein displaying the most pronounced UVA-induced upregulation was identified as the nucleolar protein nucleophosmin. The protein undergoing the most pronounced UVA-induced downregulation was identified as cathepsin B, a lysosomal cysteine-protease displaying loss of enzymatic activity and altered maturation after cellular UVA exposure. Extensive lysosomal accumulation of lipofuscin-like autofluorescence and osmiophilic material occurred in UVA-exposed fibroblasts as detected by confocal fluorescence microscopy and transmission electron microscopy, respectively. Array analysis indicated UVA-induced upregulation of oxidative stress response gene expression, and UVA-induced loss of cathepsin B enzymatic activity in fibroblasts was suppressed by antioxidant intervention. Pharmacological cathepsin B inhibition using CA074Me mimicked UVA-induced accumulation of lysosomal autofluorescence and deficient cathepsin B maturation. Taken together, these data support the hypothesis that cathepsin B is a crucial target of UVA-induced photo-oxidative stress causatively involved in dermal photodamage through the impairment of lysosomal removal of lipofuscin.