The Cryo-EM Structure Of The Monomer Of Human Proteasome Regulatory Particle PA200 And Its Proteasome Complex

The proteasome is a large multi-subunit protease complex composed of 20S core particles(CP)and different types of regulatory particles(RP).The proteasome regulatory particles are the important components of the proteasome,participating in the regulation of protein degradation in many vital signal pathways,and playing an key role in maintaining cell homeostasis.At present,three types of proteasome regulatory particles and their proteasome complex structures with 20S CP have been solved.The structural information provide important reference information for in-depth understanding of the mechanism of proteasome and drug design in the field of cancer therapy.The research object of this article,human proteasome regulatory particle 200(PA200),is a single-chain ATP-independent proteasome regulatory particle that can combine with human 20S CP to form a proteasome complex and catalyze the acetylation of histone degradation,plays an important role in DNA repair and spermatogenesis.In this paper,PA200 was overexpressed through an insect expression system,and PA200 monomer protein was purified.Then,The PA200 was assembled with human 20S CP in vitro.The enzyme activity test proved the 20S CP and PA200-20S CP complex used in this experiment have certain chymotrypsin activity,and the chymotrypsin activity of PA200-20S CP complex is higher than that of 20S CP,indicating that PA200 as a proteasome regulating particle can enhance the chymotrypsin activity of 20S CP;To further explore the molecular mechanism of PA200 regulating the activity of 20S CP and the substrate recognition mechanism of PA200,we present cryo-electron microscopy(cryo-EM)structures of the human PA200-20S CP complex and PA200 at 2.72?and 3.75?,respectively.On the whole,PA200 presents a dome-like structure,covering one end of20S CP;based on the analysis of the structure of the PA200-20S CP complex,this paper analyzes the interaction interface amino acid(aa)residues between PA200 and 20S CP.It clarified the molecular mechanism of PA200 inducing the opening of the 20S CP channel.PA200 uses the C-terminal Hb YX motif(YYA)to induce 20S CP?-loop rearrangement to open the channel at the end of the 20S CP binding to PA200;In addition,through the comparison with the structure of the PA200 and its yeast homologous protein Blm10,we found that there are two unique openings composed of positively charged amino acid residues on PA200,and these two openings are combined with additional electron cloud density,according to their unique electron cloud density we infer that they may be inositol phosphate 5,6-[PP]₂-Ins P4 and Ins P6.Further high performance liquid chromatography-mass spectrometry(HPLC-MS)experiments showed that the small molecules bound by the two openings are indeed 5,6-[PP]₂-Ins P4 and Ins P6.These small molecules play an important role in the process of histone deacetylation.Therefore,we speculate that the ability of PA200 to bind to such substances may be related to the recognizing acetylated H4 histones of bromine-like domain(BRDL),there is a certain correlation between PA200-20S CP complex degrading acetylated histones,which also provides an idea for the subsequent disclosure that PA200-20S CP proteasome complex recognizes and degrades acetylated histones,and it is inferred that these two openings are likely to be the passage for the substrate to enter the 20S CP through the PA200.At the same time,in order to analyze the acetylation recognition mechanism of PA200,this paper first compares the BRDL structure and sequence of PA200 with the typical human BRD family.It is found that the BRDL of PA200 has only 82 amino acid residues,and the ZA loop is significantly shorter.Different from the typical human BRD family,PA200 can not be divided into eight typical human BRD families.This study further explored the interaction between PA200 and H4 histone peptides with different numbers of acetylation sites.It is preliminarily speculated that histones require more than one Kac site to bind to PA200 in an in vitro environment,and may require additional post-translational modification helps to combine the two.In summary,the results of this study provide important insights for studying the molecular mechanism of 20S CP channel opening during the substrates degradation of human PA200-mediated and exploring the mechanism of PA200 recognizing H4 histones during acetylation-mediated degradation of the proteasome.