The FAM86 domain of FAM86A confers substrate specificity to promote EEF2-Lys525 methylation.

FAM86A is a class I lysine methyltransferase (KMT) that generates trimethylation on the eukaryotic translation elongation factor 2 (EEF2) at Lys525. Publicly available data from The Cancer Dependency Map project indicate high dependence of hundreds of human cancer cell lines on FAM86A expression. This classifies FAM86A among numerous other KMTs as potential targets for future anticancer therapies. However, selective inhibition of KMTs by small molecules can be challenging due to high conservation within the S-adenosyl methionine (SAM) cofactor binding domain among KMT subfamilies. Therefore, understanding the unique interactions within each KMT-substrate pair can facilitate developing highly specific inhibitors. The FAM86A gene encodes an N-terminal FAM86 domain of unknown function in addition to its C-terminal methyltransferase domain. Here, we used a combination of X-ray crystallography, the AlphaFold algorithms, and experimental biochemistry to identify an essential role of the FAM86 domain in mediating EEF2 methylation by FAM86A. To facilitate our studies, we also generated a selective EEF2K525 methyl antibody. Overall, this is the first report of a biological function for the FAM86 structural domain in any species and an example of a noncatalytic domain participating in protein lysine methylation. The interaction between the FAM86 domain and EEF2 provides a new strategy for developing a specific FAM86A small molecule inhibitor, and our results provide an example in which modeling a protein-protein interaction with AlphaFold expedites experimental biology.

Structure of DNMT3B homo-oligomer reveals vulnerability to impairment by ICF mutations.

DNA methyltransferase DNMT3B plays an essential role in establishment of DNA methylation during embryogenesis. Mutations of DNMT3B are associated with human diseases, notably the immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome. How ICF mutations affect DNMT3B activity is not fully understood. Here we report the homo-oligomeric structure of DNMT3B methyltransferase domain, providing insight into DNMT3B-mediated DNA methylation in embryonic stem cells where the functional regulator DNMT3L is dispensable. The interplay between one of the oligomer interfaces (FF interface) and the catalytic loop renders DNMT3B homo-oligomer a conformation and activity distinct from the DNMT3B-DNMT3L heterotetramer, and a greater vulnerability to certain ICF mutations. Biochemical and cellular analyses further reveal that the ICF mutations of FF interface impair the DNA binding and heterochromatin targeting of DNMT3B, leading to reduced DNA methylation in cells. Together, this study provides a mechanistic understanding of DNMT3B-mediated DNA methylation and its dysregulation in disease.

Identification of novel epitopes targeting non-structural protein 2 of PRRSV using monoclonal antibodies.

Porcine reproductive and respiratory syndrome virus (PRRSV) is leading to huge losses in the swine industry worldwide. Its nonstructural protein 2 (Nsp2), with a cysteine protease domain (PL2), is crucial for virus replication and as a trigger to host innate immune regulation. In this study, three monoclonal antibodies (mAbs) to Nsp2, designated 4A12, 4G8, and 8H11, were generated. Subsequently, a sequence of recombinant peptides with partial overlap was utilized to determine the epitopes using these mAbs. We found three novel minimal linear Nsp2 B cell epitopes, 188ELSDDSNRPV197, 42HLKRYSPPAE51, and 54CGWHCISA61, which were identified by the antibodies 4A12, 4G8, and 8H11, respectively. Structure analysis indicates that 42HLKRYSPPAE51 and 188ELSDDSNRPV197 are located separately in hypervariable region 1 and hypervariable region 2 of Nsp2. Interestingly, 54CGWHCISA61 is located in the PL2 region, which is highly conserved in all arteriviruses, particularly at the expected conserved catalytic site at Cys54. Importantly, 54CGWHCISA61 is located in the inner region of the expected 3D structure of Nsp2, which reveals that the epitope is cryptic. These findings not only provide valuable insight for vaccine design and hold diagnostic potential for the identified epitopes, but also reveal a protective mechanism against variation under selective pressure in an important epitope.

Secondary Haemophilus parasuis infection enhances highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) infection-mediated inflammatory responses.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) infection often predisposes pigs to secondary bacterial infection, which induces robust inflammatory responses. However, whether the secondary bacterial infection synergizes HP-PRRSV infection and enhances inflammatory responses is not fully understood. Here, we characterized HP-PRRSV infection-mediated secondary bacterial infection and robust inflammatory responses. HP-PRRSV infection induced higher levels of cytokines (IL-1ß, IL-18, IL-6 and TNF-α) in the sera in piglets and bacterial loads of 11 bacterial species in the lung were increased after HP-PRRSV infection, including Mycoplasma hyorhinis, Haemophilus parasuis and Escherichia coli. Concurrent infection with HP-PRRSV and H. parasuis model showed that inflammatory cytokines expression and secretion in porcine alveolar macrophages (PAMs) were increased in comparison with PAMs infected with HP-PRRSV or H. parasuis alone. Additionally, we found that H. parasuis RNA plays an important role in the robust inflammatory response enhancement in HP-PRRSV-infected PAMs. Taken together, our findings suggest that bacterial RNA transfection enhanced HP-PRRSV-mediated inflammatory responses in HP-PRRSV and H. parasuis (HPS) concurrent infection, which provides an important clue for comprehensive understanding of HP-PRRSV and bacterial coinfection-mediated pathology.

Identification of a linear B-cell epitope on non-structural protein 12 of porcine reproductive and respiratory syndrome virus, using a monoclonal antibody.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused tremendous economic losses and continues to be a serious problem to the swine industry worldwide. The structure and function of PRRSV nonstructural protein 12 (NSP12) is still unknown. In this study, we produced a monoclonal antibody, named as 1E5, against the NSP12 protein of HP (highly pathogenic) -PRRSV strain HuN4. A series of partially overlapping recombinant NSP12 truncations and synthesized peptides were used to define the epitope recognized by 1E5. We found that 130KANATSMRFH139 is the minimal linear epitope and that it is highly conserved among some HP-PRRSV isolates of type 2 PRRSV, but not the classical isolates of type 2 PRRSV or the isolates of type 1 PRRSV. Therefore, 1E5 can be used to establish a valuable tool to distinguish infections with HP-PRRSV isolates of type 2 PRRSV from the classical isolates of type 2 PRRSV and type 1 PRRSV.