Human protein arginine methyltransferases (PRMTs) can be optimally active under nonphysiological conditions

Lowe, Troy L.; Clarke, Steven

doi:10.1016/j.jbc.2022.102290

Cited by 7 publications

(13 citation statements)

References 41 publications

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“…In fact, as reported in Table , compound 1i exhibited a 3-fold reduction in potency compared to its next higher homologue 1a . To confirm the activity of the compounds against PRMT9, we then used a radioisotope-based assay as a secondary screening approach. , In these experiments, Hs PRMT9 was incubated with 3 H-SAM and SF3B2 (401–550) peptide with and without inhibitors 1a – c at the reported concentrations, and then the reaction products were analyzed by SDS gel electrophoresis followed by fluorography and densitometric analysis. 5′-Deoxy-5′-(methylthio)adenosine (methylthioadenosine, MTA), the polyamine byproduct in the methionine salvage pathway that is reported to be a SAM-competitive inhibitor of PRMTs, − and the PRMT5 inhibitor EPZ015666 (GSK3235025) , were used as reference drugs.…”

Section: Resultsmentioning

confidence: 99%

Identification of a Protein Arginine Methyltransferase 7 (PRMT7)/Protein Arginine Methyltransferase 9 (PRMT9) Inhibitor

Feoli,

Iannelli,

Cipriano

et al. 2023

J. Med. Chem.

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Less studied than the other protein arginine methyltransferase isoforms, PRMT7 and PRMT9 have recently been identified as important therapeutic targets. Yet, most of their biological roles and functions are still to be defined, as well as the structural requirements that could drive the identification of selective modulators of their activity. We recently described the structural requirements that led to the identification of potent and selective PRMT4 inhibitors spanning both the substrate and the cosubstrate pockets. The reanalysis of the data suggested a PRMT7 preferential binding for shorter derivatives and prompted us to extend these structural studies to PRMT9. Here, we report the identification of the first potent PRMT7/9 inhibitor and its binding mode to the two PRMT enzymes. Label-free quantification mass spectrometry confirmed significant inhibition of PRMT activity in cells. We also report the setup of an effective AlphaLISA assay to screen small molecule inhibitors of PRMT9.

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Section: Resultsmentioning

confidence: 99%

Identification of a Protein Arginine Methyltransferase 7 (PRMT7)/Protein Arginine Methyltransferase 9 (PRMT9) Inhibitor

Feoli,

Iannelli,

Cipriano

et al. 2023

J. Med. Chem.

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“…Previous studies indicate that GST- Hs PRMT7 activity with human H2B (23–37) peptide substrate decreases by half with the addition of 50 mM NaCl or KCl [ 9 ]. Comparatively, a previous study of ionic strength in human HEK293 cells estimates the intracellular concentration of potassium ions to be about 120–140 mM in human cells [ 23 , 24 ].…”

Section: Resultsmentioning

confidence: 99%

“…We designated this GST-human PRMT7 fusion protein as GST- Hs PRMT7. Transformed cells were lysed, followed by purification of GST- Hs PRMT7 via column chromatography with glutathione-Sepharose beads as described previously [ 9 ]. Recombinant full-length human histone H2B was purchased from New England Biolabs (NEB catalog no.…”

Section: Methodsmentioning

confidence: 99%

“…In vitro methylation assays with GST- Hs PRMT7, selected methyl-accepting substrates, and [ 3 H]-AdoMet were set up as described previously [ 9 ] and performed in triplicate. Following incubation at 25°C unless otherwise indicated for the time periods specified, all samples were quenched with 0.5 μL 100% TFA and centrifuged at 9,300 g for 10 seconds in an Eppendorf centrifuge (Model 5417C).…”

Section: Methodsmentioning

confidence: 99%

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The exquisite specificity of human protein arginine methyltransferase 7 (PRMT7) toward Arg-X-Arg sites

Bondoc,

Lowe,

Clarke

2023

PLoS ONE

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Mammalian protein arginine methyltransferase 7 (PRMT7) has been shown to target substrates with motifs containing two arginine residues separated by one other residue (RXR motifs). In particular, the repression domain of human histone H2B (29-RKRSR-33) has been a key substrate in determining PRMT7 activity. We show that incubating human PRMT7 and [3H]-AdoMet with full-length Xenopus laevis histone H2B, containing the substitutions K30R and R31K (RKRSR to RRKSR), results in greatly reduced methylation activity. Using synthetic peptides, we have now focused on the enzymology behind this specificity. We show for the human and Xenopus peptide sequences 23–37 the difference in activity results from changes in the Vmax rather than the apparent binding affinity of the enzyme for the substrates. We then characterized six additional peptides containing a single arginine or a pair of arginine residues flanked by glycine and lysine residues. We have corroborated previous findings that peptides with an RXR motif have much higher activity than peptides that contain only one Arg residue. We show that these peptides have similar apparent km values but significant differences in their Vmax values. Finally, we have examined the effect of ionic strength on these peptides. We found the inclusion of salt had little effect on the Vmax value but a considerable increase in the apparent km value, suggesting that the inhibitory effect of ionic strength on PRMT7 activity occurs largely by decreasing apparent substrate-enzyme binding affinity. In summary, we find that even subtle substitutions in the RXR recognition motif can dramatically affect PRMT7 catalysis.

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“…The ionic strength has a moderate impact on their LCST in the range tested. In typical physiological conditions, the ionic strength is 0.15 m and can suffer a fluctuation of ±0.01 m. [21] In those cases, the error in the measurement introduced by the variation of the LCST is ≈±0.09 °C, which is smaller than the uncertainty of the measurement (0.1 °C as indicated in Figure 3c). Hence PNIPAM-NGs can be considered non-biased OCTh probes.…”

Section: Dtmentioning

confidence: 96%

3D Optical Coherence Thermometry Using Polymeric Nanogels

Muñoz‐Ortiz,

Alayeto,

Lifante

et al. 2023

Advanced Materials

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In nanothermometry, the use of nanoparticles as thermal probes enables remote and minimally invasive sensing. In the biomedical context, nanothermometry has emerged as a powerful tool where traditional approaches, like infrared thermal sensing and contact thermometers, fall short. Despite the strides of this technology in preclinical settings, nanothermometry is not mature enough to be translated to the bedside. This is due to two major hurdles: the inability to perform 3D thermal imaging and the requirement for tools that are readily available in the clinics. This work simultaneously overcomes both limitations by proposing the technology of optical coherence thermometry (OCTh). This is achieved by combining thermoresponsive polymeric nanogels and optical coherence tomography (OCT)—a 3D imaging technology routinely used in clinical practice. The volume phase transition of the thermoresponsive nanogels causes marked changes in their refractive index, making them temperature‐sensitive OCT contrast agents. The ability of OCTh to provide 3D thermal images is demonstrated in tissue phantoms subjected to photothermal processes, and its reliability is corroborated by comparing experimental results with numerical simulations. The results included in this work set credible foundations for the implementation of nanothermometry in the form of OCTh in clinical practice.

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Human protein arginine methyltransferases (PRMTs) can be optimally active under nonphysiological conditions

Cited by 7 publications

References 41 publications

Identification of a Protein Arginine Methyltransferase 7 (PRMT7)/Protein Arginine Methyltransferase 9 (PRMT9) Inhibitor

Identification of a Protein Arginine Methyltransferase 7 (PRMT7)/Protein Arginine Methyltransferase 9 (PRMT9) Inhibitor

The exquisite specificity of human protein arginine methyltransferase 7 (PRMT7) toward Arg-X-Arg sites

3D Optical Coherence Thermometry Using Polymeric Nanogels

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