Greenbaum D, Medzihradszky K F, Burlingame A, Bogyo M
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
Chem Biol. 2000 Aug;7(8):569-81. doi: 10.1016/s1074-5521(00)00014-4.
Analysis of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymatic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly determining activities of an entire class of enzymes on a genome-wide scale would be of great utility.
We have engineered chemical probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes containing a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivatives was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular extracts. Finally, the affinity tag of DCG-04 allowed purification of modified proteases and identification by mass spectrometry.
We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixtures. These probes were used to determine relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purification/identification of targets.
通过基于系统的基因组学和蛋白质组学方法分析基因转录和翻译的全局变化，只能提供关于蛋白质功能的间接信息。在许多情况下，酶活性与转录或翻译水平不相关。因此，一种在全基因组范围内广泛测定整个酶类活性的直接方法将具有很大的实用性。
我们设计了可用于广泛追踪半胱氨酸蛋白酶活性的化学探针。通用半胱氨酸蛋白酶抑制剂E-64的结构用作支架。通过改变核心肽识别部分，同时在远端位点添加亲和标签（生物素和放射性碘）来合成类似物。所得含有P2亮氨酸残基的探针（DCG-03和DCG-04）靶向与E-64相同的广泛的半胱氨酸蛋白酶，并用于在正常皮肤细胞向癌细胞进展过程中分析这些蛋白酶。构建了DCG-04衍生物文库，其中亮氨酸残基被所有天然氨基酸取代。该文库用于获得粗细胞提取物中多个蛋白酶靶标的抑制剂活性谱。最后，DCG-04的亲和标签允许纯化修饰的蛋白酶并通过质谱鉴定。
我们创建了一种简单灵活的方法，用于在功能上鉴定半胱氨酸蛋白酶，同时追踪它们在粗蛋白混合物中的相对活性水平。这些探针用于确定整个癌症进展定义模型系统中多种蛋白酶的相对活性。此外，从亲和探针文库获得的信息提供了一种无需事先纯化/鉴定靶标即可获得详细功能信息的快速方法。
