Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response.

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.

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Policy information about availability of computer code Data collection Data analysis Dalia Barsyte-Lovejoy Apr 13, 2020 The MmPRMT7_ SGC8158 dataset was collected at the 24ID-E beamline at the Advanced Photon Source (APS).
The IC50 values and statistical significance (student and multiple t-test, one-way Anova), were determined, where applicable, using GraphPad Prism 7 software. Kinetic curves for SPR analysis were fitted using a 1:1 binding model and the Biacore T200 Evaluation software (Ver.3.1, GE Health Sciences Inc.). Band intensities for western blot analysis were determined using Image Studio Ver 5.2 (Licor). Apoptosis levels and cell confluency were analyzed with IncuCyte™ ZOOM (2015A) software.
For intracellular compounds concentration MassLynx 4.1 software from Waters was used for data analysis with the QuanLynx module. Standard curves were generated by using the linear fit of mass peak areas and the known concentrations of SGC8158 and SGC8158N.
For Mass Spectrometric analysis of arginine monomethylation the raw files were searched and quantified using MaxQuant version 1.6.2.1 and using the UP000005640 UniPprot Release 2018_08 human database (Swiss-Prot reference containing 20,352 protein entries, downloaded on 24 October, 2018). PTM scores for monomethylarginine were generated using the MaxQuant platform as previously described and site level occupancy was calculated by the ratio of modified peptide in two samples, the unmodified peptide version and the protein ratio. P-values from four independent replicates calculated by empirical Bayes moderated t-tests and adjusted using the Benjamini-Hochberg procedure as implemented in the Bioconductor package limma (v3.38.3). Known methylation sites were referenced from PhosphoSitePlus® v6.5.8 for Figure 2c and Supplementary Table 4. Gene ontology enrichment analysis was performed using clusterProfiler (ver. 3.10.1).

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All studies must disclose on these points even when the disclosure is negative.  Table 4  Resolution cutoff was applied to the MmPRMT7_SGC8158 X-ray diffraction data using both CC1/2 and (1/delta) pre-established criteria. reference (PMID: 23793146) The data were generated from at least 3 technical replicates. In most cases the experiments were successfully repeated on separate dates. All the details are indicated in the manuscript.
No specific randomization method was applied. Batches of cultured cells were randomly allocated to treatment groups and control groups.
Subsets of X-ray diffraction amplitudes were withheld for calculation of Rfree values.