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    • A biochemical characterisation of this functionally crucial

    2022-08-09

    A biochemical characterisation of this functionally crucial LSD1-p53 interaction is the focus of this study. Using several complementary assays [29], [30], [31], [32], we find that a well-defined segment of p53-CTD is capable of binding to LSD1 active site, thereby inhibiting the enzymatic activity of the demethylase. This association is mostly electrostatic in nature and likely involves an active-site region that has recently been demonstrated to be targeted by a new class of LSD1 inhibitors.
    Materials and methods
    Results Our studies started from the reported observations that histone demethylase LSD1 and tumour suppressor p53 interact for reciprocal regulation [17], [28]. Upon binding of LSD1 to p53-CTD, lysine 370 of p53 would become demethylated, resulting in a down-regulation of p53 activity through a weakened interaction with 53BP1. LSD1 is in return redirected to specific lysophosphatidic acid synthesis loci, triggering gene expression modulation via H3Lys4 demethylation. Lysine 370 is part of p53-CTD, which is characterized by an enrichment of positively charged residues and represents one of the two disordered regions within the tumour suppressor protein (Fig. 1A) [33]. We first wanted to explore the catalytic activity of LSD1 in complex with its co-repressor CoREST1 on this p53-Lys370 residue in vitro. To this aim, we employed established enzymatic assays with three CTD-derived peptides dimethylated at Lys370 (residues 353–388; 363–388; 353–378). Although these same peptides were used by other authors for pull-down and co-immunoprecipitation experiments [28], in our hands no evident catalytic Lys370-demethylation activity was observed. We employed two spectroscopic assays that measure hydrogen peroxide and formaldehyde production, respectively. In either case no detectable change in absorbance was observed over a time of 20 min with substrate concentrations up to 400 μM (i.e. estimated lower limit of detection is kcat ∼0.001 min−1) [34]. This result was replicated using no less than five different LSD1-CoREST1 enzyme preparations which were fully active in H3Lys4 demethylation (see Fig. 1B, black curves). Nonetheless, we found that the incubation with both dimethylLys370 353–388 and 363–388 peptides as well as unmodified 363–388 and 363–393 peptides lead to a 2 °C increase in the unfolding temperature as measured by a thermal shift assay [30], suggesting binding to the enzyme. Consistently, we observed that these peptides inhibit the demethylase activity on methylLys4-H3 peptide (data shown in Fig. 1B and Fig. S1 and summarized in Table 1 and Supplementary Tables 1 and 2). These findings supported the idea that, though not a direct LSD1 substrate, p53 can physically interact with LSD1-CoREST1 through the CTD residues, hampering the histone demethylation catalytic activity. We then aimed at characterizing a minimal unit within p53-CTD for interaction with LSD1-CoREST1 and the factors that may modulate this interaction. To do so, we expanded the set of peptides to a total of twelve CTD-derived sequences and probed their ability to compete with histone H3 tail to bind to the LSD1 active site, therefore inhibiting its enzymatic activity (Fig. 1B and Table 1). These peptides varied in sequence length, covering differing segments within residues 358–393 of p53-CTD, and bore different mutations or amino acid modifications that possibly modulate binding properties [35], [36], [37]. With respect to modifications, we analyzed Ser phosphorylation, Lys methylations, and Lys acetylations which are known to occur in the post-translationally modified p53 as detailed in Table 1 (Fig. 1A) [21], [22]. We found that the strongest binding was obtained with the peptide covering the amino acids 363–388 (Ki of 6.4 μM) and that the overall lengths of the peptides do not greatly influence binding affinity as long as the sequence 379–388 is retained (Fig. 1A and Table 1). Indeed, peptide 353–378 does not show detectable inhibition of LSD1-CoREST1 whereas the 379–388 peptide retains an affinity similar to that comprising residues 371–388 (Ki of 22 μM). Consistent with the fact that the 379–388 segment is the key player for the association of p53-CTD to LSD1-COREST1, Lys370 methylation does not strongly affect binding causing only a two-fold increase in Ki from 6.4 μM to 11.4 Conversely, significant (though not drastic) effects were observed for side chain modifications targeting the core segment. Binding is decreased by Lys382 and Lys386 acetylation, Ser378 phosphorylation, and Lys381Met mutation.