Purification And Biochemical Characterization Of LRSAM1, A LRR And RING Domain E3 Ubiquitin Ligase

Human leucine rich repeat and sterile alpha motif containing 1(LRSAM1) is an E3 ubiquitin ligase to founction in many ways, including regulation of the cargo sorting, signal pathway, cell adhesion and antibacterial autophagy, which all depend its E3 ligase activity. LRSAM1 is characterized by a conserved C-terminal RING finger domain and N-terminal LRR domain for its E3 activity and interaction.Ubiquitination is one of the most important protein posttranslational modifications in all eukaryote organisms. Ubiquitination involves three enzymes, E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes and E3 ubiquitin ligasing enzymes, and is ATP-, Mg2+-dependent. Ubiquitination plays vital role in many ways, such as cell proliferation, signal transduction, protein cellular localization, antivirus, repair of DNA damage, autophagy, growth and development, apoptosis and so on.Most recently, it had been reported that ubiquitin system played a key role in mediating the autophagy degradation of ubiquitin-coated bacteria. At the same time, LRSAM1 was identified as the crucial E3 ligase responsible for ubiquitin-dependent autophagy of intracellular pathogenic bacteria. However, it is little known about the biochemical characterization and function of LRSAM1. To search the pathogenic bacterial protein(s) which can interact with LRSAM1 is the ulterior object. Its purification strategy with high E3 ligase activity was never reported. Therefor, we cloned, expressed and purified LRSAM1 using prokaryotic expression system firstly, optimized its activity and researched its biochemical characterization. The results about our research follow:The expressed LRSAM1 protein in E. coli was almost in the pellet of the bacterial cell lysate in the form of inclusion bodies. The different fusion tags, 6*His, GST, Sumo and pelB, were used to enhance its solubility but had no effect on the solubility. Urea was added to denature the inclusion bodies and enhance the solubility. After renaturation, the LRSAM1 protein existed in the supernatant. However, the affinity tags could not bind with the GST beads or NI-NTA beads because of the misfolding and intricate spatial structure. No recombinant proteins can be purified successfully by ion-exchange column chromatography in denaturation or renaturation. The emergence of these problems enforced us to find new ways for high solubility and purity of LRSAM1. Therefor, no tag construction was considered for its expression and purification without the potential interference in its activition. Through the process of denaturation, renaturation and ammonium sulfate precipitation method, the purity of LRSAM1 was greater than 95% as assessed by SDS-PAGE.For LRSAM1 E3 activity was bad when we conducted the assay in vitro, we carried out experiments to compare the induction temperature, induction concentration, bacterial strains, buffer with different components for purification, the p H of buffer for purification for high E3 activity. And in this study we found that the activity of LRSAM1 stayed closely with the pH value of buffer.LRSAM1 is classified as the typical RING finger E3 ubiquitin ligase with zinc finger motif. Through sequence alignment, we ascertained its active sites in the zinc finger motif. Site-directed mutagenesis technology was utilized to research the sites for its E3 activity and the results showed that the two sites, Cys675 and His692, were the vital active sites.LRSAM1 is an E3 ubiquitin ligase with the ability of autoubiquitination. A lot of researches showed the seven lysine residues were identified for different ubiquitination chains with different functions. For example, monoubiquitination plays a role in intracellular location and regulation, Lys48-linked polyubiquitin chains modulate the proteasomal degradation of target proteins and Lys63-linked polyubiquitin chains played a part in DNA repair, stress response, endocytosis and so on. Therefor, we studied the types of autopolyubiquitin chains of LRSAM1. The results indicated that LRSAM1-driven ubiquitin chains favored Lys6-, Lys27-, Lys29- and Lys48-linkages.Multiple different E2 s can function with one E3 for the ubiquitination. So we tested many E2 s for the autoubiquitination of LRSAM1. It had been reported that UbcH5 b and UbcH5 c as the specific E2 taked part in the process of autoubiquitination of LRSAM1. As a faminly, UbcH5 type E2 posseses three members, Ubc H5 a, UbcH5 b and UbcH5 c. The results indicated that for its structure and function simility with UbcH5 b and UbcH5 c, UbcH5 a also had the ability to participated in this process. However, other E2 s, UbcH3 b, UbcH6, UbcH7 and UbcH10, tested in this research failed to mediate the LRSAM1’s E3 activity.Ubiquitination is the process by which the Gly carboxyl in the C-terminue of ubiquitin molecule attachs with the Lys ε-amino in the target substrates for amide linkage. The lysines in substrates are the key molecule for ubiquitination. Through sequence alignment, we confirmed that four lysines in the C-terminue of LRSAM1 maybe the sites for isopeptide bond formation and further experimrnts showed that the Lys491 in LRSAM1 was the mainly autoubiquitination site. The formation and removal of ubiquitin molecule are a unequally reversible process. The former process needs three steps with three enzymes, E1, E2 and E3, and the latter was modulated by deubiquitinating enzymes. Sse L, an effector protein, secreted by Salmonella, function as a deubiquitinase with no target substrates. Here, we purified activited Sse L and found it deubiquitinate the polyubiquitin chains formed by LRSAM1.In conclusion, LRSAM1, as a human E3 ubiquitin ligase, was expressed in E.coli as inclusion bodies and purified with high purity through the process of denaturation, renaturation and ammonium sulfate precipitation method. For its stable activity, we confirmed that the pH value in buffer used for the purification had a vital influence on the LRSAM1 activity. The two sites, Cys675 and His692, in the consensus sequence with conservation for zinc binding were necessary for its activity. For the identification of ubiquitin chains driven by LRSAM1, the ubiquitin assays showed that LRSAM1 primarily drived Lys6-, Lys27-, Lys29- and Lys48-linked polyubiquitin chains. Among seven E2 s tested, UbcH5 family E2, UbcH5 a, UbcH5 b and UbcH5 c, played the role in the process of LRSAM1 autoubiquitination. The Lys491 in LRSAM1 was identified as the main autoubiquitination site by comparing the four conserved lysine residues. SseL, as a deubiquitinase enzyme of Salmonella, acted to deubiquitinate ubiquitin chains formed by LRSAM1. In this research, we presented the strategy to gain LRSAM1 with high purity and activity, which laied the foundation for searching the unknown proteins interacting with LRSAM1 in vitro and confirmed its biochemical characterization.