Hemelaar Joris, Borodovsky Anna, Kessler Benedikt M, Reverter David, Cook Julie, Kolli Nagamallesawari, Gan-Erdene Tudev, Wilkinson Keith D, Gill Grace, Lima Christopher D, Ploegh Hidde L, Ovaa Huib
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Cell Biol. 2004 Jan;24(1):84-95. doi: 10.1128/MCB.24.1.84-95.2004.
Modification of proteins by ubiquitin (Ub)-like proteins (UBLs) plays an important role in many cellular processes, including cell cycle progression, nuclear transport, and autophagy. Protein modification occurs via UBL-conjugating and -deconjugating enzymes, which presumably exert a regulatory function by determining the conjugation status of the substrate proteins. To target and identify UBL-modifying enzymes, we produced Nedd8, ISG15, and SUMO-1 in Escherichia coli and equipped them with a C-terminal electrophilic trap (vinyl sulfone [VS]) via an intein-based method. These C-terminally modified UBL probes reacted with purified UBL-activating (E1), -conjugating (E2), and -deconjugating enzymes in a covalent fashion. Modified UBLs were radioiodinated and incubated with cell lysates prepared from mouse cell lines and tissues to allow visualization of polypeptides reactive with individual UBL probes. The cell type- and tissue-specific labeling patterns observed for the UBL probes reflect distinct expression profiles of active enzymes, indicating tissue-specific functions of UBLs. We identify Ub C-terminal hydrolase L1 (UCH-L1) and DEN1/NEDP1/SENP8, in addition to UCH-L3, as proteases with specificity for Nedd8. The Ub-specific protease isopeptidase T/USP5 is shown to react with ISG15-VS. Furthermore, we demonstrate that the desumoylation enzyme SuPr-1 can be modified by SUMO-1-VS, a modification that is dependent on the SuPr-1 active-site cysteine. The UBL probes described here will be valuable tools for the further characterization of the enzymatic pathways that govern modification by UBLs.
泛素样蛋白（UBL）对蛋白质的修饰在许多细胞过程中发挥着重要作用，包括细胞周期进程、核运输和自噬。蛋白质修饰通过UBL结合酶和去结合酶发生，这些酶可能通过确定底物蛋白的结合状态发挥调节功能。为了靶向和鉴定UBL修饰酶，我们在大肠杆菌中表达了Nedd8、ISG15和SUMO-1，并通过基于内含肽的方法在其C末端配备了亲电捕获基团（乙烯砜[VS]）。这些C末端修饰的UBL探针与纯化的UBL激活酶（E1）、结合酶（E2）和去结合酶发生共价反应。修饰后的UBL进行放射性碘化，并与从小鼠细胞系和组织制备的细胞裂解物孵育，以可视化与单个UBL探针反应的多肽。观察到的UBL探针的细胞类型和组织特异性标记模式反映了活性酶的不同表达谱，表明UBL具有组织特异性功能。除了UCH-L3，我们还鉴定出泛素C末端水解酶L1（UCH-L1）和DEN1/NEDP1/SENP8为对Nedd8具有特异性的蛋白酶。泛素特异性蛋白酶异肽酶T/USP5被证明与ISG15-VS反应。此外，我们证明去SUMO化酶SuPr-1可以被SUMO-1-VS修饰，这种修饰依赖于SuPr-1活性位点的半胱氨酸。本文所述的UBL探针将成为进一步表征UBL修饰酶促途径的有价值工具。
