Vigneron Nathalie, Abi Habib Joanna, Van den Eynde Benoît J
Ludwig Institute for Cancer Research, B-1200 Brussels, Belgium; Walloon Excellence in Life Sciences and Biotechnology (WELBIO), B-1200 Brussels, Belgium; de Duve Institute, Université catholique de Louvain, B-1200 Brussels, Belgium.
Ludwig Institute for Cancer Research, B-1200 Brussels, Belgium; Walloon Excellence in Life Sciences and Biotechnology (WELBIO), B-1200 Brussels, Belgium; de Duve Institute, Université catholique de Louvain, B-1200 Brussels, Belgium.
Anal Biochem. 2015 Aug 1;482:7-15. doi: 10.1016/j.ab.2015.04.019. Epub 2015 Apr 23.
Because of its crucial role in various cellular processes, the proteasome is the focus of intensive research for the development of proteasome inhibitors to treat cancer and autoimmune diseases. Here, we describe a new and easy assay to measure the different proteasome activities in vitro (chymotrypsin-like, caspase-like, and trypsin-like) based on proteasome capture on antibody-coated plates, namely the capture proteasome assay (CAPA). Applying the CAPA to lysates from cells expressing standard proteasome, immunoproteasome, or intermediate proteasomes β5i or β1i-β5i, we can monitor the activity of the four proteasome subtypes. The CAPA provided similar results as the standard whole-cell proteasome-Glo assay without the problem of contaminating proteases requiring inhibitors. However, the profile of trypsin-like activity differed between the two assays. This could be partly explained by the presence of MgSO4 in the proteasome-Glo buffer, which inhibits the trypsin-like activity of the proteasome. The CAPA does not need MgSO4 and, therefore, provides a more precise measurement of the trypsin-like activity. The CAPA provides a quick and accurate method to measure proteasome activity in vitro in a very specific manner and should be useful for the development of proteasome inhibitors.
由于蛋白酶体在各种细胞过程中发挥着关键作用，它是开发用于治疗癌症和自身免疫性疾病的蛋白酶体抑制剂的密集研究焦点。在此，我们描述了一种新的简便检测方法，基于蛋白酶体在抗体包被平板上的捕获，即捕获蛋白酶体检测法（CAPA），用于在体外测量不同的蛋白酶体活性（类胰凝乳蛋白酶活性、类半胱天冬酶活性和类胰蛋白酶活性）。将CAPA应用于表达标准蛋白酶体、免疫蛋白酶体或中间蛋白酶体β5i或β1i-β5i的细胞裂解物，我们可以监测四种蛋白酶体亚型的活性。CAPA提供的结果与标准全细胞蛋白酶体-Glo检测法相似，且不存在需要抑制剂来防止污染蛋白酶的问题。然而，两种检测方法中类胰蛋白酶活性的情况有所不同。这可能部分是由于蛋白酶体-Glo缓冲液中存在MgSO4，它会抑制蛋白酶体的类胰蛋白酶活性。CAPA不需要MgSO4，因此能更精确地测量类胰蛋白酶活性。CAPA提供了一种快速且准确的方法，以非常特异的方式在体外测量蛋白酶体活性，应该对蛋白酶体抑制剂的开发有用。
