Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs
Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs
复制标题
DOI：
10.1016/s1074-5521(00)00061-2
复制
发表时间：
2000-01-01
影响因子：
--
通讯作者：
Greenbaum, D
中科院分区：
文献类型：
--
作者：
Bogyo, M;Verhelst, S;Greenbaum, D
背景：木瓜酶家族的溶酶体半胱氨酸蛋白酶是研究最多的蛋白水解酶之一。小分子抑制剂和荧光底物模拟物已被用来探索这些酶的生理作用。家族成员之间的高度同源性和底物专一性的重叠使得阐明单个蛋白酶的功能、表达和活性变得困难。结果：以多肽乙烯基砜和环氧化物为模板，我们制备了可以用放射性碘标记的探针。所得化合物共价标记各种组织蛋白和几个可能是蛋白酶的未知多肽，MB-074被发现是组织蛋白酶B活性的高选择性探针。使用标记了几种组织蛋白的探针来检测CA-074家族抑制剂的特异性和细胞通透性。虽然CA-074据报道在体内起作用，但我们发现它不能穿透细胞，但酯化CA-074导致组织蛋白酶B的活性和特异性显著降低。该探针还用于监测原代人类肿瘤组织和人胎盘来源的细胞中的蛋白酶活性。结论：我们制备了一种高选择性的组织蛋白酶B探针和几种特异性较低的试剂，用于组织蛋白酶生物学的研究。与常用的荧光底物相比，这种试剂有几个优点，可以在细胞总酶池中识别抑制物靶标。我们已经使用这些探针来表明，在肿瘤组织中以及在胎盘来源的细胞滋养层细胞分化为侵袭细胞的过程中，组织蛋白酶活性受到调节，这是在发育中的胚胎中建立血液循环所必需的。
Background: The lysosomal cysteine proteases of the papain family are some of the best studied proteolytic enzymes. Small-molecule inhibitors and fluorogenic substrate mimics have been used to probe the physiological roles of these proteases. A high degree of homology between family members and overlap in substrate specificity have made elucidating individual protease function, expression and activity difficult.Results: Using peptide vinyl sulfones and epoxide as templates, we have generated probes that can be tagged with radioactive iodine. The resulting compounds covalently label various cathepsins and several unidentified polypeptides likely to be proteases, MB-074 was found to be a highly selective probe of cathepsin B activity. Probes that labeled several cathepsins were used to examine the specificity and cell permeability of the CA-074 family of inhibitors. Although CA-074 reportedly acts in vivo, we find it is unable to penetrate cells, Esterifying CA-074 resulted in a cell-permeable inhibitor with dramatically reduced activity and specificity for cathepsin B. The probes were also used to monitor protease activity in primary human tumor tissue and cells derived from human placenta.Conclusions: We have generated a highly selective cathepsin B probe and several less specific reagents for the study of cathepsin biology. The reagents have several advantages over commonly used fluorogenic substrates, allowing inhibitor targets to be identified in a pool of total cellular enzymes. We have used the probes to show that cathepsin activity is regulated in tumor tissues and during differentiation of placental-derived cytotrophoblasts to invasive cells required for establishing blood circulation in a developing embryo.