20S proteasome hydrolysis of LLVY substrates to determine preferences for moieties in its primed substrate channel.
20S proteasome hydrolysis of LLVY substrates to determine preferences for moieties in its primed substrate channel.
复制标题
LLVY 底物的 20S 蛋白酶体水解以确定其底物通道中部分的偏好。
DOI：
10.1016/j.bmcl.2023.129233
复制
发表时间：
2023
影响因子：
2.7
通讯作者：
Trader,DarciJ
中科院分区：
文献类型：
--
作者：
Muli,ChristineS;Trader,DarciJ
蛋白酶体是细胞中一种必需的多催化酶，其负责以泛素依赖性或非依赖性机制降解蛋白质。许多基于活性的探针、抑制剂和刺激剂已被开发用于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底物通道的氨基酸的相互作用，该通道在催化活性苏氨酸残基之前。如蛋白酶体抑制剂belactosin所证明的，在催化性苏氨酸之后，存在与底物的正相互作用以增加β5底物通道的选择性或裂解速率的潜力。为了研究蛋白酶体在其引发的底物通道中可以接受哪些部分，我们开发了液相色谱-质谱（LC-MS）方法来定量纯化的人蛋白酶体对底物的切割。这种方法使我们能够快速评估含有可与β5蛋白酶体通道的S1′位点相互作用的部分的蛋白酶体底物。我们能够确定在S1′底物位置的极性部分的偏好。我们相信这些信息可以用于设计未来的蛋白酶体抑制剂或基于活性的探针。
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.