Double deficiency of cathepsins B and L results in massive secretome alterations and suggests a degradative cathepsin-MMP axis
Double deficiency of cathepsins B and L results in massive secretome alterations and suggests a degradative cathepsin-MMP axis
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DOI：
10.1007/s00018-013-1406-1
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发表时间：
2014-03-01
影响因子：
8
通讯作者：
Schilling, Oliver
中科院分区：
文献类型：
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作者：
Tholen, Stefan;Biniossek, Martin L.;Schilling, Oliver
内溶酶体半胱氨酸组织蛋白酶在功能上相互配合。组织蛋白酶B（Cts B）和L（Cts 1）双敲除小鼠在出生后4周死亡，伴随着神经元内的（自噬）溶酶体积累。在Ctsb和Ctsl缺陷的小鼠胚胎成纤维细胞（MEF）中也观察到这种积累。先前的研究显示Ctsl对MEF分泌组的强烈影响。在这里，我们表明，Ctsb单独只有轻微的影响细胞外蛋白质组组成。依赖于Ctsb的蛋白酶裂解位点通过底物的末端胺同位素标记（TAILS）鉴定，揭示了对细胞外蛋白水解裂解的突出但大多是间接的影响。为了研究Ctsb和Ctsl的合作，我们对野生型MEFs和Ctsb（-/-）Ctsl（-/-）MEFs进行了定量分泌组比较。两种组织蛋白酶的缺失导致分泌组组成的急剧变化，突出了合作功能。虽然许多蛋白质水平降低，但免疫检测证实基质金属蛋白酶（MMP）-2水平升高。Ctsl的再表达拯救MMP-2丰度。Ctsl和在较小程度上Ctsb能够在酸性和中性pH下降解MMP-2。向MEF分泌组添加活性MMP-2降解蛋白质，其水平也由Ctsb和Ctsl双重缺陷降低。这些结果表明降解性Ctsl-MMP-2轴，导致组织蛋白酶缺乏时MMP-2水平增加，随后分泌蛋白如胶原α-l（I）降解。
Endolysosomal cysteine cathepsins functionally cooperate. Cathepsin B (Ctsb) and L (Ctsl) double-knockout mice die 4 weeks after birth accompanied by (autophago-) lysosomal accumulations within neurons. Such accumulations are also observed in mouse embryonic fibroblasts (MEFs) deficient for Ctsb and Ctsl. Previous studies showed a strong impact of Ctsl on the MEF secretome. Here we show that Ctsb alone has only a mild influence on extracellular proteome composition. Protease cleavage sites dependent on Ctsb were identified by terminal amine isotopic labeling of substrates (TAILS), revealing a prominent yet mostly indirect impact on the extracellular proteolytic cleavages. To investigate the cooperation of Ctsb and Ctsl, we performed a quantitative secretome comparison of wild-type MEFs and Ctsb (-/-) Ctsl (-/-) MEFs. Deletion of both cathepsins led to drastic alterations in secretome composition, highlighting cooperative functionality. While many protein levels were decreased, immunodetection corroborated increased levels of matrix metalloproteinase (MMP)-2. Re-expression of Ctsl rescues MMP-2 abundance. Ctsl and to a much lesser extent Ctsb are able to degrade MMP-2 at acidic and neutral pH. Addition of active MMP-2 to the MEF secretome degrades proteins whose levels were also decreased by Ctsb and Ctsl double deficiency. These results suggest a degradative Ctsl-MMP-2 axis, resulting in increased MMP-2 levels upon cathepsin deficiency with subsequent degradation of secreted proteins such as collagen alpha-1 (I).