Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 West Stadium Avenue, West Lafayette, IN 47907, United States.
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 West Stadium Avenue, West Lafayette, IN 47907, United States.
Bioorg Med Chem Lett. 2023 Apr 1;85:129233. doi: 10.1016/j.bmcl.2023.129233. Epub 2023 Mar 9.
The proteasome is an essential multi-catalytic enzyme in cells that is responsible for degrading proteins with a ubiquitin-dependent or -independent mechanism. Many activity-based probes, inhibitors, and stimulators have been developed to study or modulate the activity of the proteasome. The development of these proteasome probes or inhibitors have been based on their interaction with the amino acids of the β5 substrate channel proceeding the catalytically active threonine residue. There is potential for positive interactions with a substrate to increase selectivity or cleavage rate with the β5 substrate channel after the catalytic threonine as evidenced by the proteasome inhibitor belactosin. To study what moieties the proteasome could accept in its primed substrate channel, we developed a liquid chromatography- mass spectrometry (LC-MS) method to quantitate the cleavage of substrates by purified human proteasome. This method allowed us to rapidly evaluate proteasome substrates that contain a moiety that could interact with the S1' site of the β5 proteasome channel. We were able to determine a preference for a polar moiety at the S1' substrate position. We believe this information can be used in the design of future inhibitors or activity-based probes for the proteasome.
蛋白酶体是细胞中一种必需的多催化酶，负责通过泛素依赖或非依赖的机制降解蛋白质。已经开发了许多基于活性的探针、抑制剂和激动剂来研究或调节蛋白酶体的活性。这些蛋白酶体探针或抑制剂的开发是基于它们与β5 底物通道中催化活性苏氨酸残基之前的氨基酸的相互作用。正相互作用与底物结合后，在催化苏氨酸之后，β5 底物通道的选择性或切割率增加，这一点已被蛋白酶体抑制剂 belactosin 所证明。为了研究蛋白酶体在其引发的底物通道中可以接受哪些基团，我们开发了一种液相色谱-质谱 (LC-MS) 方法来定量由纯化的人蛋白酶体切割底物。该方法使我们能够快速评估包含可与β5 蛋白酶体通道 S1' 位点相互作用的基团的蛋白酶体底物。我们确定了在 S1' 底物位置对极性基团的偏好。我们相信这些信息可用于设计未来的蛋白酶体抑制剂或基于活性的探针。
