Preliminary Structural Analysis Of The REG?-substrate Protein Complex

In eukaryotic cells,80%-90% of proteins are degraded by proteasomes.The proteasomes regulate important physiological processes such as cell cycle,cell death and immune response by degrading mutant,misfolded or other proteins.REG?,as a proteasome regulatory granule,forms a ubiquitin-independent and ATP-independent proteasomal degradation system with the 20 S core granule.Previously,the researches on REG?-20 S proteasome mainly focused on its substrate proteins and related physiological functions.The systematic research on the substrate recognition mechanism of REG? has not been reported.This study intended to explain the recognition mechanism from biochemistry and protein structure two levels.In this study,we successfully purified REG? protein using the prokaryotic E.coli expression system and the eukaryotic Sf9 insect cell expression system.In the early stage,we have mastered the purification method of REG? in prokaryotic E.coli.And "upgrading" it to the eukaryotic Sf9 insect cell level is not only an inheritance,but also an innovation.Next,we used prokaryotic and eukaryotic systems to purify the substrate proteins HCV-core(HCV core protein)and p21.However,most of the proteins appeared in "inclusion bodies",and it was difficult to obtain high-quality proteins.We therefore focused our attention on the key part of HCV-core and p21 that binds to REG?.Subsequent experiments such as binding of REG? with biotin-p21(140-164)peptide in vitro,Avidin pull-down,BLI and other experiments verified that the key site for binding of the peptide to REG? is the R/K repeat region.We further obtained the electron microscopic structure of the REG?-substrate protein complex by negative staining and cryo-electron microscopy.This preliminarily explained the recognition mechanism of REG? and substrate proteins from the molecular structure level.In conclusion,this study initially explored the recognition and binding mechanism of REG? and substrate proteins from biochemistry and molecular structure two levels.It is not only a reverse verification of the REG? degron theory in our laboratory,but also provides a theoretical basis for a comprehensive understanding of the physiological and pathological functions of REG?.