Preliminary Study On The Activated Structure Of Humen E3 Ubiquitin Ligase ARIH1

Ubiquitination is a common phenomenon of post-translational modification in eukaryotic cells,which has multiple functions such as mediating protein degradation,localization,and activation.The ubiquitination process is catalyzed by a series of enzymes.E3 ubiquitin ligase is an enzyme that directly mediates the transfer of ubiquitin to the substrate protein and plays an important role in the specificity of ubiquitination.There are more than 600 kinds of E3 in humans,which regulate the ubiquitination of hundreds of substrate proteins and participate in a wide range of life activities.HECT,RING and RBR are the three types of ubiquitin ligase E3.Among them,RING type E3 has been studied the most and its mechanism is the most in-depth,while RBR E3 is the latest to be discovered and the least understood.The members with Ariadne characteristic domains in RBR E3 constitute the Ariadne RBR E3 subfamily.Human homolog of Drosophila Ariadne-1(ARIH1)is a human homolog of ariadne-1 in Drosophila,belonging to the Ariadne RBR E3 class.Prior to this article,previous studies have shown that: 1)When ARIH1 exists alone,its catalytically active sites cannot perform the ubiquitin transfer function due to being buried by the Ariadne domain,so they are in a state of self-inhibition;2)When ARIH1 is associated with When the ubiquitin protein NEDD8 modified Cullin-RING E3 ligases(CRLs)bind,their self-inhibition state is lifted and the E3 enzyme activity is stimulated.However,the structure of the activated state and the molecular mechanism of activation are not yet known.This subject has conducted preliminary research on the structural biology of NEDD8-CRL-ARIH1-Ubch7-Ub protein complex: 1)We have explored the prokaryotic or eukaryotic expression system and independent expression by constructing different recombinant plasmids or Co-expression mode of protein expression,and finally choose a more efficient prokaryotic,separate expression and purification method to obtain the component protein;2)On this basis,by screening purification tags and optimizing purification steps,we have obtained the purity and The yield of protein samples that meet the basic requirements;3)CUL1,2,3,and 4A were verified to have in vitro ubiquitination activity;4)The molecular sieve successfully verified the strong interaction between CRLs and ARIH1,and passed the protein cut The short verification of CRLs CTD and ARIH1 interaction is very important;5)In vitro verified that the complex has self-ubiquitination activity,at the same time further crystallographic screening of protein complex samples,unfortunately,due to the complex The components are numerous and relatively dynamic,and the crystallization is difficult,and suitable crystallization conditions have not been selected.Although this study failed to obtain the NEDD8-CRL-ARIH1-Ubch7-Ub protein complex crystals,the sample preparation method of the protein complex was optimized,which provided a basis for the subsequent study of the dynamic structure of the complex.