Structure Characterization Of The Branched K11/K48 Tri-ubiquitin

Ubiquitin modification plays an important role in cell,which can regulate many physiological processes such as protein degradation,DNA repair,signal transduction and so on.The disorder of ubiquitin system may cause many diseases,such as cancer,neurological diseases and so on.The complexity and functionality of ubiquitin system are that ubiquitin can form a variety of polyubiquitin chains to achieve diverse physiological functions.Early studies mainly focused on the homotypic of ubiquitin chain,but recently found that there are a wide range of heterotype ubiquitin chain in cells.If two or more residues of a ubiquitin are modified at the same time,it is called branched ubiquitin chain.Branched ubiquitin chains have more connection forms and can transmit more information.In the past,it was generally believed that K48 ubiquitin chain is the main type of ubiquitin chain that mediates proteasome degradation of substrate.However,in recent years,it has been found that branched K11/K48 ubiquitin chain plays an important role in regulating protein degradation during cell mitosis and in protein quality control system and the rate of substrate protein degradation mediated by branched K11/K48 ubiquitin chain is faster.It has been found that branched K11/K48 ubiquitin chain modified proteins are associated with the pathogenesis of neurodegenerative diseases such as Huntington's disease.The physiological process of branched K11/K48 ubiquitin chain in cells has been studied comprehensively,but there are few studies on the branched K11/K48 ubiquitin chain.The research on these problems is still unclear and needs further exploration.In this thesis,branched K11/K48-triub was selected as the research subject,and the branched K11/K48-triub was synthesized by in vitro enzyme linked method.In this paper,we first studied the structure of branched K11/K48-triub by NMR,and found that there may be a special noncovalent interaction between the two distal subunits,can lead to the average distance getting farther.Single molecular fluorescence resonance energy transfer(sm FRET)method was used to study the dynamic conformational changes between K48 distal subunit and proximal subunit.It was found that the proportion of medium FRET state(open state)of branched K11/K48-triub was significantly increased compared with that of K48-diub.We also found that the ubiquitin receptors Rpn1 and Rpn10 mainly bind to the open state of K48 diubiquitin.Through fluorescence polarization experiment,we found that the binding of branched K11/K48-triub to ubiquitin receptors Rpn1 and Rpn10 was significantly stronger than that of K48-diub,but the binding of K11-diub to ubiquitin receptors Rpn1 and Rpn10 was very weak.These results indicate that proportion of open state of K48-diub in branched K11/K48-tri Ub is increased which enhances its binding to ubiquitin receptors Rpn1 and Rpn10.Deubiquitinase(DUB)can also regulate the degradation of substrate by26 S proteasome.We selected two typical DUBs(OTUB1 and UCH37)to degrade branched K11/K48-triub and K48 diubiquitin respectively.We found that branched K11/K48-triub could not inhibit the degradation of OTUB1,but UCH37 could accelerate the degradation of branched K11/K48-triub.These results indicate that the branched K11/K48 ubiquitin chain can promote the degradation of ubiquitin substrate by proteasome through enhancing the binding with ubiquitin receptor and being efficiently degraded by DUB.In conclusion,we used a variety of biophysical methods to study the dynamic structure of branched K11/K48-triub,and revealed the molecular mechanism of branched K11/K48 ubiquitin chain relative to K48 ubiquitin chain to enhance substrate degradation by proteasome.By studying the structure of ubiquitin chain and the molecular mechanism of regulating physiological function,we can understand the complex ubiquitin code and reveal the basic principle of information transmission in cells.