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PRMT1 controls the Senescence Associated Secretory Phenotype in Replication Stress Response

ABSTRACT: The protein arginine methyltransferase PRMT1 is overexpressed in various human cancers and linked to poor response to therapy. Although various inhibitors targeting its activity are under development, we still do not fully understand how PRMT1 is involved in the cellular processes underpinning tumorigenesis and chemoresistance. Mass spectrometryÃ¢â‚¬â€œbased proteomics revealed PRMT1 as a regulator of global arginine methylation changes in response to replicative stress in cancer cells. We show that, upon cisplatin, DNA-dependent protein kinase binds to- and phosphorylates PRMT1, inducing its chromatin recruitment and redirecting its enzymatic activity from soluble protein targets towards the histone substrate Arg3 of histone H4 (H4R3). On chromatin, DNA-PK/PRMT1 axis induces the Senescence-Associated Secretory Phenotype through the deposition of H4R3me2a at the pro-inflammatory gene promoters and by sustaining p65 binding to chromatin. Finally, PRMT1 inhibition reduced the clonogenic outgrowth of ovarian cancer cells exposed to low doses of CDDP and sensitized them to apoptosis. While unravelling a novel role of PRMT1 in replication stress response, our findings suggest the opportunity of targeting PRMT1 to sensitize cancer cells to genotoxic chemotherapeutics.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Ovary Cancer Cell Line

SUBMITTER: Enrico Massignani

LAB HEAD: Tiziana Bonaldi

PROVIDER: PXD014799 | Pride | 2020-01-15

REPOSITORIES: Pride

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Project description:Protein methylation has been implicated in many important biological contexts including signaling, metabolism, and transcriptional control. Despite the importance of this post- translational modification, the global analysis of protein methylation by mass spectrometry-based proteomics has not been extensively studied due to the lack of robust, well-characterized techniques for methyl-peptidemethyl peptide enrichment. Here, to better investigate protein methylation, we optimized and compared two methods for methyl-peptidemethyl peptide enrichment: immunoaffinity purification (IAP) and high pH strong cation exchange (SCX). Comparison of these methods revealed that they are largely orthogonal for monomethyl arginine (MMA), suggesting that the usage of both techniques is required to provide a global view of protein methylation. Using both IAP and SCX, we investigated changes in protein methylation downstream of protein arginine methyltransferase 1 (PRMT1). Together, these techniques allowed us to quantify over ~1,000 arginine methylation sites on 407 proteins. Of these methylation sites, PRMT1 knockdown resulted in significant changes to 110 arginine methylation sites on 59 proteins. In contrast, zero lysine methylation sites were significantly changed upon PRMT1 knockdown. In PRMT1 knockdown cells, 24 MMA sites exhibited both significant downregulation (ie, putative PRMT1-mediated MMA targets) and 63 significant upregulation (ie, putative PRMT1-mediated ADMA targets). PRMT1 knockdown induced significant changes in asymmetric dimethyl arginine (ADMA), consistent with being PRMT1 targets. Additionally, We also observed that PRMT1 knockdown induced significant increases in symmetric dimethyl arginine (SDMA) methylation, suggesting that loss of PRMT1 activity allows scavenging of PRMT1 substrates by Type II PRMTs. Analysis of the subcellular localization and protein function of the significantly changing methyl-proteins revealed that the majority of PRMT1 substrates are localized to the nucleus and involved in RNA and DNA binding. Taken together, our results suggest that deep protein methylation profiling by mass spectrometry requires orthogonal enrichment techniques, identify novel PRMT1 methylation targets, and highlight in order to understand the dynamic interplay between methyltransferases in mammalian cells.

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PRMT1 controls the Senescence Associated Secretory Phenotype in Replication Stress Response

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