Lu Haibin, Chandrasekar Balakumaran, Oeljeklaus Julian, Misas-Villamil Johana C, Wang Zheming, Shindo Takayuki, Bogyo Matthew, Kaiser Markus, van der Hoorn Renier A L
Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.L., B.C., J.C.M.-V., R.A.L.v.d.H.);Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (H.L., B.C., J.C.M.-V., T.S., R.A.L.v.d.H.);Center for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany (J.O., Z.W., M.K.); andDepartment of Pathology, Stanford School for Medicine, Stanford, California 94305-5324 (M.B.).
Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.L., B.C., J.C.M.-V., R.A.L.v.d.H.);Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (H.L., B.C., J.C.M.-V., T.S., R.A.L.v.d.H.);Center for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany (J.O., Z.W., M.K.); andDepartment of Pathology, Stanford School for Medicine, Stanford, California 94305-5324 (M.B.)
Plant Physiol. 2015 Aug;168(4):1462-75. doi: 10.1104/pp.114.254466. Epub 2015 Jun 5.
Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins.
半胱氨酸蛋白酶是一类重要的酶，参与植物发育和防御相关的程序性细胞死亡以及其他生物学过程。由于有数十种半胱氨酸蛋白酶受到翻译后加工、环境条件和抑制剂的调控，因此需要新的方法来单独研究这些关键酶。在这里，我们介绍基于荧光活性的探针，这些探针特异性靶向三个不同的半胱氨酸蛋白酶亚家族：类菠萝蛋白酶、组织蛋白酶B样蛋白酶和液泡加工酶。我们将这些新探针用于拟南芥蛋白酶敲除系和农杆菌浸润叶片的蛋白酶活性分析，以鉴定探针靶点，并用于其他植物物种以证明其广泛适用性。这些探针表明，大多数市售蛋白酶抑制剂靶向植物中意想不到的蛋白酶。当应用于萌发种子时，这些探针揭示了类菠萝蛋白酶、组织蛋白酶B样蛋白酶和液泡加工酶的动态活性，这与种子储存蛋白的再利用相吻合。
