Restrepo Santiago Yori, Martin Andreas
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
California Institute for Quantitative Biosciences (QB3), University of California at Berkeley, Berkeley, CA 94720, USA.
bioRxiv. 2024 Dec 17:2024.12.16.628829. doi: 10.1101/2024.12.16.628829.
The 26S proteasome complex is the hub for regulated protein degradation in the cell. It is composed of two biochemically distinct complexes: the 20S core particle with proteolytic active sites in an internal chamber and the 19S regulatory particle, consisting of a lid and base subcomplex. The base contains ubiquitin receptors and an AAA+ (ATPases associated with various cellular activities) motor that unfolds substrates prior to degradation. The base subcomplex can be expressed recombinantly in and reconstituted into functional 26S proteasomes , which allows the introduction of unnatural amino acids with novel functions or other mutations that may not be permissible . Here, we present a method for the introduction of the photo-induced crosslinking amino acid p-benzoyl-L-phenylalanine into the proteasomal base subcomplex. This approach has exciting implications for the study of protein-protein interactions of this complex that mediates the degradation of an incredibly diverse protein pool.
26S蛋白酶体复合物是细胞中蛋白质降解调控的核心。它由两个生化性质不同的复合物组成：20S核心颗粒，其内部腔室具有蛋白水解活性位点；以及19S调节颗粒，由盖子和底座亚复合物组成。底座含有泛素受体和一个AAA+（与各种细胞活动相关的ATP酶）马达，该马达在底物降解之前将其展开。底座亚复合物可以在体外重组表达并重新组装成功能性的26S蛋白酶体，这使得具有新功能的非天然氨基酸或其他可能不被允许的突变得以引入。在这里，我们提出了一种将光诱导交联氨基酸对苯甲酰-L-苯丙氨酸引入蛋白酶体底座亚复合物的方法。这种方法对于研究介导极其多样的蛋白质池降解的该复合物的蛋白质-蛋白质相互作用具有令人兴奋的意义。
