Identification of in vitro JMJD lysine demethylase candidate substrates via systematic determination of substrate preference

Hoekstra, Matthew; Biggar, Kyle K.

doi:10.1016/j.ab.2021.114429

Cited by 7 publications

(7 citation statements)

References 40 publications

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“…The data represents the average of three luminescent readings and error bars represent the SEM (n=3). Data for KDM5A was previously published ( Hoekstra and Biggar, 2021 ). …”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…Data represents the mean luminescence and standard deviation (n=3) normalized to the max signal. Data for KDM5A was previously published ( Hoekstra and Biggar, 2021 ). …”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…Systematically designed peptide libraries containing methylated lysine residues are used to characterize enzyme-substrate preference and identify new candidate substrates in vitro . For complete details on the use and execution of this protocol, please refer to Hoekstra and Biggar (2021) .…”

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confidence: 99%

“…For complete details on the use and execution of this protocol, please refer to Hoekstra and Biggar (2021) .…”

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Evaluation of Jumonji C lysine demethylase substrate preference to guide identification of in vitro substrates

et al. 2022

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“…The data represents the average of three luminescent readings and error bars represent the SEM (n=3). Data for KDM5A was previously published ( Hoekstra and Biggar, 2021 ). …”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…Data represents the mean luminescence and standard deviation (n=3) normalized to the max signal. Data for KDM5A was previously published ( Hoekstra and Biggar, 2021 ). …”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

mentioning

confidence: 99%

“…For complete details on the use and execution of this protocol, please refer to Hoekstra and Biggar (2021) .…”

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Evaluation of Jumonji C lysine demethylase substrate preference to guide identification of in vitro substrates

et al. 2022

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“…In turn, these candidate recognition motifs can then be used to score and prioritize the methyllysine proteome for candidate substrates. Figure for KDM5A was adapted from 124 . Reprinted with permission of Star Protocols -Cell Press.…”

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confidence: 99%

Characterization of lysine demethylase KDM5 family: Substrate specificity and identification of potential novel non-histone substrates

Hoekstra¹

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A major regulatory influence over cell biology is lysine methylation and demethylation within histone proteins. The KDM5/JARID1 sub-family are 2oxoglutarate and Fe(II)-dependent lysine-specific histone demethylases that are characterized by their Jumonji catalytic domains. This enzyme family is known to facilitate the removal of tri-/di-methyl modifications from lysine 4 of histone H3 (i.e., H3-K4me3/2), a mark associated with active gene expression. As a result, studies to date have revolved around KDM5's influence on disease through their ability to regulate H3-K4me2/3. Recently, evidence has demonstrated that KDM5's may influence disease beyond H3-K4 demethylation, making it critical to further investigate KDM5 demethylation of non-histone proteins. In efforts to help identify potential non-histone substrates for the KDM5 family, we developed a library of 180 permutated peptide substrates (PPS), with sequences that are systematically altered from the WT H3-K4me3 sequence. From this library, we characterized recombinant KDM5A/B/C/D substrate preference. Subsequently we developed recognition motifs for each KDM5 demethylase and used them to predict potential substrates for KDM5A/B/C/D. Demethylation activity was then profiled to generate a list of high/medium/low-ranking substrates for further in vitro validation for each of KDM5A/B/C/D. Through this approach, we analyzed prediction success rate and identified 66 high-ranking substrates in which KDM5 demethylases displayed significant in vitro activity towards. We further shown the ability to monitor changes in cellular methylation in a handful of the 66 high ranking candidate substrates in response to KDM5 inhibition. Specifically, we iii focused validation efforts on a high-ranking KDM5A novel substrate: p53-K370me3. We demonstrated significant recombinant KDM5A1-588ΔAP and KDM5A1-801 activity towards the p53-K370me3 substrate in vitro. We then monitored KDM5A-mediated demethylation of the p53-K370me3/2 substrate in HCT 116 cells using a combination of wild-type KDM5A and inactive-mutant KDM5AH483A overexpression plasmids, along with immunoblotting, (co-) immunoprecipitation and mass spectrometry analysis. Furthermore, we have shown that KDM5A expression influences the established p53-53BP1 interaction.Finally, we identified a novel p53-TAF5 interaction dominated through the p53-K370me3 state and how KDM5A activity might affect this interaction. Ultimately, we have provided the first evidence of a KDM5 demethylase targeting a nonhistone substrate for demethylation, via the novel KDM5A demethylation of the p53-K370me3 substrate. iv PrefaceThe following articles included in this thesis were either published or submitted to a scientific journal for means of publishing at the time the thesis was completed.Chapter 2 contains the following paper that was published in Star Protocols-Cell Press that was reproduced in part: Hoekstra, M., Chopra, A., Willmore, W.G., Biggar, K.K. ( 2022). Evaluation of jumonji C lysine demethylase substrate preference to guide identifica...

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Origins of Catalysis in Non‐Heme Fe(II)/2‐Oxoglutarate‐Dependent Histone Lysine Demethylase KDM4A with Differently Methylated Histone H3 Peptides

Devadas,

Thomas,

Rifayee

et al. 2024

Chemistry A European J

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Histone lysine demethylase 4A (KDM4A), a non‐heme Fe(II)/2‐oxoglutarate (2OG) dependent oxygenase that catalyzes the demethylation of tri‐methylated lysine residues at the 9, 27, and 36 positions of histone H3 (H3K9me3, H3K27me3, and H3K36me3). These methylated residues show contrasting transcriptional roles; therefore, understanding KDM4A’s catalytic mechanisms with these substrates is essential to explain the factors that control the different sequence‐dependent demethylations. In this study, we use molecular dynamics (MD)‐based combined quantum mechanics/molecular mechanics (QM/MM) methods to investigate determinants of KDM4A catalysis with H3K9me3, H3K27me3 and H3K36me3 substrates. In KDM4A‐H3(5‐14)K9me3 and KDM4A‐H3(23‐32)K27me3 ferryl complexes, the O‐H distance positively correlates with the activation barrier of the rate‐limiting step, however in the KDM4A‐H3(32‐41)K36me3, no direct one‐to‐one relationship was found implying that the synergistic effects between the geometric parameters, second sphere interactions and the intrinsic electric field contribute for the effective catalysis for this substrate. The intrinsic electric field along the Fe‐O bond changes between the three complexes and shows a positive correlation with the HAT activation barrier, suggesting that modulating electric field can be used for fine engineering KDM catalysis with a specific substrate. The results reveal how KDM4A uses a combination of strategies to enable near equally efficient demethylation of different H3Kme3 residues.

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Identification of in vitro JMJD lysine demethylase candidate substrates via systematic determination of substrate preference

Cited by 7 publications

References 40 publications

Evaluation of Jumonji C lysine demethylase substrate preference to guide identification of in vitro substrates

Evaluation of Jumonji C lysine demethylase substrate preference to guide identification of in vitro substrates

Characterization of lysine demethylase KDM5 family: Substrate specificity and identification of potential novel non-histone substrates

Origins of Catalysis in Non‐Heme Fe(II)/2‐Oxoglutarate‐Dependent Histone Lysine Demethylase KDM4A with Differently Methylated Histone H3 Peptides

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