Research and academic discussion on ubiquitination traditionally revolve around three pivotal aspects: the E3 ligase, the type of ubiquitination, and the specific ubiquitination sites. Through exemplary analysis of the article "PSMD12-Mediated M1 Ubiquitination of Influenza A Virus at K102 Regulates Viral Replication," this paper will delve into an in-depth exploration of the critical roles of these three elements in understanding the complexities of protein ubiquitination.
Identification of E3 Ubiquitin Ligase
In the process of identifying E3 ubiquitin ligase, IP-MS is a widely adopted efficient methodology. In this study, researchers firstly overexpressed the M1 protein of the H5N6 influenza virus within DF-1 cells, and some candidate proteins that interact with the M1 protein were screened out using IP-MS technology. Subsequently, these candidate genes were cloned into plasmids, and they were overexpressed in DF-1 cells again through H5N6 influenza virus infection. The experimental results revealed that 3 proteins could promote the replication of the H5N6 influenza virus, while 5 proteins exhibited inhibitory effects (Figure 1). Among all the proteins identified, PSMD12 demonstrated the most potent promotional effect, thus the researchers selected PSMD12 as the target for the next step of validation.
In light of the observation that overexpression of PSMD12 promoted the proliferation of H5N6 influenza virus, the authors carried out PSMD12 knockdown experiments to validate this conclusion. They designed three pairs of siRNAs and knocked down PSMD12 in DF-1 cells. Through western blot and viral titration growth curve assays, it was discovered that PSMD12 knockdown could inhibit the proliferation of H5N6 influenza virus. This result was further corroborated in A549 cells.
As revealed by the IP-MS identification results in Figure 1, the authors establish the interactive relationship between PSMD12 and M1. To further substantiate this finding, the authors conducted Co-IP experiments in DF-1 and 293T cells, which reconfirmed the interaction between PSMD12 and M1. In addition, the authors observed the co-localization of PSMD12 and M1 in DF-1, HeLa, and A549 cells through the utilization of Indirect Immunofluorescence (IFA) techniques. The collective data from these experiments consistently underscore the reciprocal interaction between PSMD12 and M1.
Determining the ubiquitination type of the substrate
The E3 ubiquitin ligase PSMD12, capable of interacting with M1 of the influenza virus, has already been identified. The natural next step is to verify the ubiquitination of M1 by PSMD12 and subsequently ascertain the type of ubiquitination involved with M1. The authors initially co-transfected M1 and Ub, utilizing co-immunoprecipitation (Co-IP) to illustrate the ubiquitination modification of M1. Overexpressing PSMD12 enhanced M1's ubiquitination modification. Considering the ubiquitin (Ub) molecule harbors seven K situses for substrate linkage: K6, K11, K27, K29, K33, K48, and K63, the authors independently co-transfected each type of Ub with M1. They found ubiquitination modifications of M1 occurred in K6, K11, K29, K33, K48, and K63. Subsequently, the authors co-transfected each type of Ub with M1 and PSMD12. They discovered that PSMD12 fosters K63 ubiquitination of M1. Thus, it is evident that PSMD12 stimulates K63 ubiquitination modification of M1.
As a note, M1 can undergo six types of ubiquitination modifications, implying at least six distinct E3 enzymes can interact with M1 to facilitate its ubiquitination. Moreover, multiple lysine (K) residues exist on M1, enhancing the scope for ubiquitin-like modifications such as Sumoylation, Neddylation, and ISGylation, and the occurrence of acetylated modifications. In those aspects not yet addressed, there remains room for further discussion.
Determine the ubiquitination site
Upon completion of the first and second stages, the authors identified that the E3 ubiquitin ligase PSMD12 could facilitate the K63 ubiquitination modification of the influenza virus M1. In the third step of determining the ubiquitination sites on the substrate, if there are fewer lysine residues (K) on the substrate, direct mutation of each K to R is feasible. However, in cases where the substrate contains a surplus of K, prediction becomes a necessity, serving to reduce the workload.
For the prediction of potential ubiquitination sites in M1, the authors employed BDM-PUB (http://bdmpub.biocuckoo.org/results.php) and UBPRED (http://www.ubpred.org/) software. The results revealed that lysine residues at positions K21, K35, K98, K101, K102, K113, K187, and K242 in M1 could be potential sites for ubiquitination modification. Accordingly, the authors constructed individual K to R mutants at these eight sites.
Subsequently, the authors conducted Co-IP experiments following standard procedures: co-transfecting Ub, M1, as well as the K-R mutants and PSMD12. The findings revealed that PSMD12 could not enhance the ubiquitin modification of the M1-K102R mutant. Additionally, after co-transfecting Ub-K63, M1, the M1-K102R mutant, and PSMD12 and carrying out another Co-IP experiment, it was discovered that PSMD12 also couldn't stimulate the K63 ubiquitin modification of the M1-K102R mutant.
In conclusion, through Co-IP and IFA (indirect immunofluorescence assay) experiments, the authors further authenticated that the interaction and co-localisation between the M1-K102R mutant and PSMD12 were weakened. These experimental results not only corroborate the authors' initial predictions but also provide more detailed information about the ubiquitination modifications of the influenza virus M1 protein.
Building on the findings so far, the authors have been able to explore three critical aspects of ubiquitination. Through the interaction between PSMD12 and the influenza virus M1, they discovered that the K102 site of M1 undergoes K63-type ubiquitination. Subsequent conventional phenotype verification further confirmed the impact of this modification on viral replication and pathogenicity. Further details of this subject are beyond the scope of this current summary.
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