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Activity-based protein profiling of protein arginine methyltransferase 1 - PubMed

  • ️Sat Jan 01 2011

. 2011 Oct 21;6(10):1127-35.

doi: 10.1021/cb2001473. Epub 2011 Aug 23.

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Activity-based protein profiling of protein arginine methyltransferase 1

Obiamaka Obianyo et al. ACS Chem Biol. 2011.

Abstract

The protein arginine methyltransferases (PRMTs) are SAM-dependent enzymes that catalyze the mono- and dimethylation of peptidyl arginine residues. PRMT1 is the founding member of the PRMT family, and this isozyme is responsible for methylating ∼85% of the arginine residues in mammalian cells. Additionally, PRMT1 activity is aberrantly upregulated in heart disease and cancer. As a part of a program to develop isozyme-specific PRMT inhibitors, we recently described the design and synthesis of C21, a chloroacetamidine bearing histone H4 tail analogue that acts as an irreversible PRMT1 inhibitor. Given the covalent nature of the interaction, we set out to develop activity-based probes (ABPs) that could be used to characterize the physiological roles of PRMT1. Herein, we report the design, synthesis, and characterization of fluorescein-conjugated C21 (F-C21) and biotin-conjugated C21 (B-C21) as PRMT1-specific ABPs. Additionally, we provide the first evidence that PRMT1 activity is negatively regulated in a spatial and temporal fashion.

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Figures

Figure 1
Figure 1

Structures of Activity-Based Probes (ABPs). The reporter tags, FITC and biotin, were coupled to the N-terminus of C21 to generate fluorescein-conjugated C21 (F-C21) and biotin-conjugated C21 (B-C21).

Figure 2
Figure 2

F-C21 Labels PRMT1 in a Time and Concentration Dependent Manner. A. Time Course with 2 μM PRMT1 and 2 μM F-C21. B. Concentration dependence of F-C21with 2 μM PRMT1 and 30 minute reaction time. C. Concentration dependence of F-F21 with 2 μM PRMT1 and 30 minute reaction time. D. F-C21 limit of detection was determined to be 0.5 μM, which corresponds to 250 ng PRMT1. E. B-C21 limit of detection was determined to be 0.05 μM, which corresponds to 25 ng PRMT1.

Figure 3
Figure 3

Labeling PRMT1 in MCF-7 Whole Cell Extracts with F-C21. MCF-7 whole cell extracts (WCE) were incubated in the prescence or absence of recombinant PRMT1 and F-C21. F-C21 labeling was outcompeted with C21.

Figure 4
Figure 4

Labeling PRMT1 in MCF-7 Cell Lysates with B-C21. A. MCF-7 breast cancer whole cell extracts were labeled with increasing amounts of B-C21 for 30 minutes and subject to western blot analysis using a streptavidin-HRP conjugate. Proteins marked with (*) appear to be biotinylated proteins since they are present in the absence of B-C21. B. Selective Isolation of PRMT1 from MCF-7 Whole Cell Extracts with B-C21. MCF-7 WCE were incubated with the specified amount of B-C21 for 30 minutes, then bound to streptavidin agarose beads overnight at 4 °C. Beads were washed several times before bound proteins were eluted, separated by SDS-PAGE, and subject to western blot analysis with the appropriate antibody.

Figure 5
Figure 5

A. PRMT1 was isolated from MCF-7 WCE and its identity was confirmed via western blot analysis (top) and Coomassie staining (bottom). B. B-C21 labeled enzyme was excised from the gel, subjected to tryptic digestion and the resulting peptides were observed and analyzed using MALDI-TOF MS and MS/MS analyses. The identities of the peptides and the sequence coverage is depicted. C. The expected and observed y and b ions for a representative tryptic peptide (i.e., 325DLDFTIDLDFK335) that was subjected to MS/MS analysis is depicted; the peptide is composed of amino acids 325 to 335 of human PRMT1 and the observed ions are highlighted in red.

Figure 6
Figure 6

Labeling MCF-7 Cytoplasmic and Nuclear Extracts Following Estrogen Stimulation. A. MCF-7 cells were stimulated with estrogen for the indicated amount of time and then the cytoplasmic and nuclear extracts were incubated with B-C21 and bound to streptavidin-agarose beads overnight. Bound proteins were eluted from the beads, separated by SDS-PAGE and visualized by western blot analysis using an anti-PRMT1 antibody (upper panel). To demonstrate equal protein loading, a 10% loading control was loaded onto a separate gel and visualized by western blot analysis using the same antibody (lower panel). Representative data from one of three experiments is depicted. B. Quantification of the western blots in panel A.

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References

    1. Lin WJ, Gary JD, Yang MC, Clarke S, Herschman HR. The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a protein-arginine N-methyltransferase. J Biol Chem. 1996;271:15034–15044. - PubMed
    1. McBride AE, Silver PA. State of the arg: protein methylation at arginine comes of age. Cell. 2001;106:5–8. - PubMed
    1. Torres-Padilla ME, Parfitt DE, Kouzarides T, Zernicka-Goetz M. Histone arginine methylation regulates pluripotency in the early mouse embryo. Nature. 2007;445:214–218. - PMC - PubMed
    1. Friesen WJ, Massenet S, Paushkin S, Wyce A, Dreyfuss G. SMN, the product of the spinal muscular atrophy gene, binds preferentially to dimethylarginine-containing protein targets. Mol Cell. 2001;7:1111–1117. - PubMed
    1. Tran CT, Leiper JM, Vallance P. The DDAH/ADMA/NOS pathway. Atheroscler. 2003;(Suppl 4):33–40. - PubMed

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