The Self-association And Activity Regulation Of LRSAM1 E3 Ligase

Ubiquitination is one important process of protein post-translational modifications.Eukaryotic cel s employ a wide repertoire of ubiquitin signals to regulate processes like DNA damage repair,signal transduction pathways,and innate immune responses,autophagy,apoptosis and so on.Ubiquitin modification is a process in a series of reactions including ubiquitin activation by an ubiquitin-activating enzyme(E1),followed by ubiquitin transfer to an ubiquitin-conjugating enzyme(E2)and attachment of ubiquitin to the target protein by an ubiquitin-protein ligase(E3).The large E3 family of ubiquitinprotein ligases has a crucial role in recognition of specific substrates.There are more than 600 E3 s in humans.Currently,E3 ligases can be classified in three main types depending on the presence of characteristic domains and on the mechanism of ubiquitin transfer to the substrate protein.(1)the HECT domain family,(2)the RING-finger domain(including RING-like E3 ligases,e.g.,U-Box and PHD domains)family and(3)the RBR family.The ful-length LRSAM1 with 723 amino acids is composed of an N-terminal leucine-rich repeat(LRR)domains,two coiled-coil(CC)domains,one sterile-alpha motif(SAM),two Pro-(Ser/Thr)-Ala-Pro(PTAP)tetrapeptide motifs and a C-terminal RING-finger domain.However,the functions of these domains and their intermolecular or intramolecular regulation were not well characterized except that the RING functions as the domain related with the E3 activity.Compared with the other E3 ligases,LRSAM1 protein structure,function and active control,positioning and signal transduction remained to be further study.In particular,the study of LRSAM1 activity self-regulation is the base of other function or structure research.Because it has not been reported about LRSAM1 protein active regulation,the study of the E3 activity regulation is imperative.We prepared and purified the polyclonal antibody,detected the specificity and titer,and used the self-made antibody in the following research.Then we started from its structural domains,used in vitro ubiquitination assays and in vivo ubiquitination assays,Western blot and other experimental measures to analyze the regulation of LRSAM1 E3 activity.Main results for this study are as follows:We developed the purification of recombinant no tag LRSAM1 with excel ent activity using a bacterial expression system.We used freund's adjuvant to emulsified the LRSAM1 protein as an antigen.Rabbits were immunized with emulsion products.After a basic immune system and strengthen the immune twice,got the antiserum.Used CNBractivated sepharose to crosslink LRSAM1 antigen protein,through the affinity chromatography purified LRSAM1 polyclonal antibody.Tests showed the high sensitivity and specificity,which laid a foundation for later study.Some E3 ligases can form intermolecular oligomerization to regulate their E3 activity and function.By chemical cross-linking with Glutaraldehyde,we found LRSAM1 protein could self-associated to promote intermolecular ubiquitination.Western blot analyses revealed that LRSAM1?RING,C-6*His could be modified by active LRSAM1,but this inactive mutant could not efficiently ubiquitinate itself.When HAand 6*His-tagged LRSAM1 proteins are co-expressed in 293 T cel s,HA-LRSAM1 can be detected after a His tag pull down assay,indicating the self-association of LRSAM1 in vivo.We found the minimize units of LRSAM1 in intermolecular interactions is LRR and CC1 domain,LRR domain can be mono-ubiquitinated and CC1 domain can be polyubiquitinated by other LRSAM1.There are 13 lysine residues in LRR domain,so we mutated these lysine residues to arginine residues to determine the ubiquitination site.But these data showed that none of the 13 single mutants abolished the mono-ubiquitination.Recent reports confirmed that other residues,including serine,threonine,cysteine and the N-terminal NH2 group of the substrates,can be conjugated with ubiquitin.We chemical y modified the NH2 groups to determine where LRSAM1 ubiquitinated LRSAM1 LRR.Finally,we proved that LRSAM1 can promote the N-terminal mono-ubiquitination of LRSAM1 LRR with two independent blocked ?-NH2 tests.When we detected the activity of truncated LRSAM1 protein,we found deletion of the N-terminal domains gradually reduced the E3 ligase activation capacity.Especially,the activity of LRSAM1 RING was rarely detected,unless the protein concentration used in the ubiquitination assay was 20-fold higher than LRSAM1 WT.Tethered RING domain with the N-terminal domains can enhance its activity obviously.Compared with the polyubiquitin chains synthesized by the other 3 mutants and LRSAM1 WT,the ubiquitinated products formed by LRSAM1 RING were mainly the multi-ubiquitin chains in the ladder.We examined different truncations to further explore the reason in down-regulation of activity.The results showed that LRSAM1 SAM and LRSAM1CC2-SAM had an inhibitory effect on LRSAM1,whereas LRSAM1CC1-CC2-SAM lost this inhibition,indicating that the CC1 domain counteracted the CC2-SAM-mediated inhibition of LRSAM1 E3 activity.We used tethered mutants and LRSAM1 WT in vitro ubiquitination assays,we found it is the tethered CC1 domain with CC2-SAM that counteract CC2-SAM-mediated inhibition of LRSAM1 E3 activity rather than the individual CC1 domain.That showed the linker between CC1 and CC2-SAM is the key to the active control.Finally,we repeated the experiment in mammalian cel s,and get the same conclusion.In conclusion,we expressed and purified the high quality LRSAM1 antigen protein in bacterial expression system.Then we prepared the polyclonal antibody with antigen protein,tested its high sensitivity and specificity and used for further experiments.Although some E3 protein can self-inhibited because of their LRR domain,our data showed tethered RING domain with the N-terminal domains can enhance its activity obviously.LRSAM1 protein could self-associated to promote intermolecular ubiquitination.In other words,LRSAM1 can interact with each other to promote intermolecular ubiquitination.In the next research,we found the additional CC2-SAM domain can inhibit LRSAM1 E3 activity significantly,and only the tethered CC1 domain with CC2-SAM that counteract CC2-SAM-mediated inhibition of LRSAM1 E3 activity rather than the individual CC1 domain.At the same time,we found K491 is the main poly-ubiquitination site,and LRR domain can be mono-ubiquitinated in N-terminal by intermolecular transfer of ubiquitin molecules.