Rao Geeta, Nkepang Gregory, Xu Jian, Yari Hooman, Houson Hailey, Teng Chengwen, Awasthi Vibhudutta
Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States.
Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States.
Front Chem. 2018 Sep 10;6:392. doi: 10.3389/fchem.2018.00392. eCollection 2018.
The proteasome is a validated target in drug discovery for diseases associated with unusual proteasomal activity. Here we report that two diphenyldihaloketones, CLEFMA and EF24, inhibit the peptidase activity of the 26S proteasome. The objective of this study was to investigate interaction of these compounds with the proteasome and identify a putative target within the protein components of the 26S proteasome. We employed standard fluorogenic peptide-based proteasome activity assay for trypsin-like, chymotrypsin-like, and caspase-like activities of human purified 26S proteasome in cell-free conditions. GFPu-1 and HUVEC cells were used as proteasome reporter cells. Direct binding studies used purified 19S, 20S, 26S, and recombinant RPN13-Pru for interaction with biotinylated analogs of CLEFMA and EF24. The reaction mixtures were subjected to horizontal gel electrophoresis, streptavidin-blotting, pull-down assays, and immunoblotting. The identity of the interacting protein was determined by 2D gel electrophoresis and LC-MS/MS. Drug affinity responsive target stability technique was utilized to examine if CLEFMA binding confers protection to RPN13 against thermolysin-catalyzed proteolysis. We found that trypsin-and chymotrypsin-like activities of the 26S proteasome were reduced significantly by both compounds. The compounds also reduced the proteolytic activity in GFPu-1 and HUVEC cells, resulting in accumulation of ubiquitinated proteins without affecting the autophagy process. From direct binding assays a 43 kDa protein in the 26S proteasome was found to be the interacting partner. This protein was identified by tandem mass spectroscopy as regulatory particle subunit 13 (RPN13), a ubiquitin receptor in the 19S regulatory particle. Furthermore, binding of CLEFMA to RPN13 did not protect latter from thermolysin-mediated proteolysis. Together, this study showed diphenyldihaloketones as potential proteasome inhibitors for treatment of diseases with perturbed proteasome function. The results also unraveled RPN13 as a unique target of CLEFMA and EF24. As a result, these compounds inhibit both trypsin-like and chymotrypsin-like proteasome activities.
蛋白酶体是与异常蛋白酶体活性相关疾病药物研发中一个已得到验证的靶点。在此我们报告，两种二苯基二卤代酮，CLEFMA和EF24，可抑制26S蛋白酶体的肽酶活性。本研究的目的是探究这些化合物与蛋白酶体的相互作用，并在26S蛋白酶体的蛋白质组分中鉴定一个假定靶点。我们在无细胞条件下，采用基于荧光肽的标准蛋白酶体活性测定法检测人纯化26S蛋白酶体的胰蛋白酶样、糜蛋白酶样和半胱天冬酶样活性。GFPu-1细胞和人脐静脉内皮细胞（HUVEC）用作蛋白酶体报告细胞。直接结合研究使用纯化的19S、20S、26S以及重组RPN13-Pru与CLEFMA和EF24的生物素化类似物进行相互作用。反应混合物进行水平凝胶电泳、链霉亲和素印迹、下拉试验和免疫印迹。通过二维凝胶电泳和液相色谱-串联质谱法（LC-MS/MS）确定相互作用蛋白的身份。利用药物亲和力响应靶点稳定性技术检测CLEFMA的结合是否赋予RPN13对嗜热菌蛋白酶催化的蛋白水解的保护作用。我们发现这两种化合物均显著降低了26S蛋白酶体的胰蛋白酶样和糜蛋白酶样活性。这些化合物还降低了GFPu-1细胞和HUVEC细胞中的蛋白水解活性，导致泛素化蛋白积累，而不影响自噬过程。从直接结合试验中发现26S蛋白酶体中的一种43 kDa蛋白是相互作用伙伴。通过串联质谱法将该蛋白鉴定为调节颗粒亚基13（RPN13），它是19S调节颗粒中的一种泛素受体。此外，CLEFMA与RPN13的结合并未保护后者免受嗜热菌蛋白酶介导的蛋白水解。总之，本研究表明二苯基二卤代酮是治疗蛋白酶体功能紊乱疾病的潜在蛋白酶体抑制剂。研究结果还揭示RPN13是CLEFMA和EF24的独特靶点。因此，这些化合物可抑制胰蛋白酶样和糜蛋白酶样蛋白酶体活性。
