Protein Arginine Methyltransferase 5 Catalyzes Substrate Dimethylation in a Distributive Fashion

Abstract

Protein arginine methyltransferase 5 (PRMT5) is a histone-modifying enzyme whose activity is aberrantly upregulated in various cancers and thereby contributes to a progrowth phenotype. Indeed, knockdown of PRMT5 leads to growth arrest and apoptosis, suggesting that inhibitors targeting this enzyme may have therapeutic utility in oncology. To aid the development of inhibitors targeting PRMT5, we initiated mechanistic studies geared to understand how PRMT5 selectively catalyzes the symmetric dimethylation of its substrates. Toward that end, we characterized the regiospecificity and processivity of bacterially expressed Caenorhabditis elegans PRMT5 (cPRMT5), insect cell-expressed human PRMT5 (hPRMT5), and human PRMT5 complexed with methylosome protein 50 (MEP50), i.e., the PRMT5·MEP50 complex. Our studies confirm that arginine 3 is the only site of methylation in both histone H4 and H4 tail peptide analogues and that sites distal to the site of methylation promote the efficient symmetric dimethylation of PRMT5 substrates by increasing the affinity of the monomethylated substrate for the enzyme. Additionally, we show for the first time that both cPRMT5 and the hPRMT5·MEP50 complex catalyze substrate dimethylation in a distributive manner, which is assisted by long-range interactions. Finally, our data confirm that MEP50 plays a key role in substrate recognition and activates PRMT5 activity by increasing its affinity for protein substrates. In total, our results suggest that it may be possible to allosterically inhibit PRMT5 by targeting binding pockets outside the active site.

Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism

“…Although we were the first group to identify that SAM concentration affects the level of PRMT processivity, this study is not the first published example. Other studies of PRMT1 and PRMT5 have shown that higher SAM concentrations can lead to more aDMA and sDMA, respectively, even though SAM was not implicated as a possible cause . Similarly, although we were the first to explicitly show that enzyme concentration affects processivity, our conclusion is actually well represented in published studies that have classified different PRMT enzymes as acting processively or distributively .…”

“…A processive enzyme, in this instance, does not release the substrate prior to the second methylation in aDMA or sDMA formation, whereas a distributive enzyme releases substrate after the first methylation to give MMA as the predominant product. Several studies offer compelling data and rationales in support of processive or distributive mechanisms for PRMT1, PRMT2, PRMT3, CARM1, PRMT5, PRMT6, PRMT7, and PRMT9 …”

Chemical Biology of Protein Arginine Modifications in Epigenetic Regulation

“…124 This stands in contrast to the PADs, where long-range interactions are unimportant. 19 These distal elements are typically positively charged and are thought to interact via ionic interactions with several negatively charged patches found on PRMT1 (Figure 25B).…”

A TGFβ-PRMT5-MEP50 axis regulates cancer cell invasion through histone H3 and H4 arginine methylation coupled transcriptional activation and repression

“…PRMT5 is always complexed with the WD-repeat protein MEP50 to bind and orient substrate to the catalytic site to preferentially produce monomethylarginine 4, 22, 52, 53 . Elevated PRMT5 and MEP50 are found in many solid and blood cancers and often correlated with enhanced tumor growth and poor disease prognosis 2, 9, 23, 32, 43, 47 .…”