Full-length myocilin (MYOC) protein is purified from mammalian cells as a dimer

Myocilin (MYOC) is the gene with mutations most commonly observed in glaucoma patients. MYOC protein collected from patients and analyzed under non-reducing conditions suggests that MYOC is not normally found in a monomeric form. MYOC was first reported almost 20 years ago; however, a technical challenge still faced by researchers is an inability to isolate full-length MYOC protein for experimental purposes. Herein we describe two methods by which to isolate sufficient quantities of full-length MYOC protein from mammalian cells. Analytical size exclusion chromatography of isolated full-length MYOC protein indicated that it is predominantly dimeric and we propose a structure for the MYOC dimer. By sharing methods to obtain full-length MYOC protein, we hope to provide researchers with a tool that has previously not been available. The ultimate goal of MYOC research is to understand this target so we can help the patient that carries a MYOC mutation retain vision and maintain quality of life

Characterization of the Enzymatic Activity of SETDB1 and Its 1:1 Complex with ATF7IP

The protein methyltransferase (PMT) SETDB1 is a strong candidate oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine 9 of histone 3 (H3K9), utilizing <i>S</i>-adenosylmethionine (SAM) as the methyl donor and its catalytic activity, has been reported to be regulated by a partner protein ATF7IP. Here, we examine the contribution of ATF7IP to the <i>in vitro</i> activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed and 1:1 stoichiometric complexes were purified for comparison against SETDB1 enzyme alone. We employed both radiometric flashplate-based and SAMDI mass spectrometry assays to follow methylation on histone H3 15-mer peptides, where lysine 9 was either unmodified, monomethylated, or dimethylated. Results show that SETDB1 and the SETDB1:ATF7IP complex efficiently catalyze both monomethylation and dimethylation of H3K9 peptide substrates. The activity of the binary complex was 4-fold lower than SETDB1 alone. This difference was due to a decrease in the value of <i>k</i>_cat as the substrate <i>K</i>_M values were comparable between SETDB1 and the SETDB1:ATF7IP complex. H3K9 methylation by SETDB1 occurred in a distributive manner, and this too was unaffected by the presence of ATF7IP. This finding is important as H3K9 can be methylated by HMTs other than SETDB1 and a distributive mechanism would allow for interplay between multiple HMTs on H3K9. Our results indicate that ATF7IP does not directly modulate SETDB1 catalytic activity, suggesting alternate roles, such as affecting cellular localization or mediating interaction with additional binding partners

Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex

The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15–RBM39 interaction, we solved the DCAF15–DDB1–DDA1–indisulam–RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets

The structural basis of Indisulam-mediated recruitment of RBM39 to the DCAF15-DDB1-DDA1 E3 ligase complex

AbstractThe anti-cancer agent Indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which Indisulam mediates DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-Indisulam-RBM39(RRM2) complex structure to 2.3 Å. DCAF15 has a novel topology which embraces the RBM39(RRM2) domain largely via nonpolar interactions, and Indisulam binds between DCAF15 and RBM39(RRM2) and coordinates additional interactions between the two proteins. Studies with RBM39 point mutants and Indisulam analogs validated the structural model and defined the RBM39 alpha-helical degron motif. The degron is found only in RBM23 and RBM39 and only these proteins were detectably downregulated in Indisulam-treated HCT116 cells. This work further explains how Indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade novel targets.</jats:p