SPOTing Acetyl-Lysine Dependent Interactions

Picaud, S.; Filippakopoulos, P.

doi:10.3390/microarrays4030370

Cited by 14 publications

(9 citation statements)

References 29 publications

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“…Threonine 32 has been found to be phosphorylated in a proteomic screen (Kettenbach et al., 2011); however, its biological role remains elusive. We interrogated binding by employing SPOT peptide arrays (Picaud and Filippakopoulos, 2015). Using 20-amino acid (aa)-long histone H3 peptides, we observed binding to most combinations containing a central K14ac modification (Figure 2A; Table S1).…”

Section: Resultsmentioning

confidence: 99%

Multivalent Histone and DNA Engagement by a PHD/BRD/PWWP Triple Reader Cassette Recruits ZMYND8 to K14ac-Rich Chromatin

et al. 2016

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SummaryElucidation of interactions involving DNA and histone post-translational-modifications (PTMs) is essential for providing insights into complex biological functions. Reader assemblies connected by flexible linkages facilitate avidity and increase affinity; however, little is known about the contribution to the recognition process of multiple PTMs because of rigidity in the absence of conformational flexibility. Here, we resolve the crystal structure of the triple reader module (PHD-BRD-PWWP) of ZMYND8, which forms a stable unit capable of simultaneously recognizing multiple histone PTMs while presenting a charged platform for association with DNA. Single domain disruptions destroy the functional network of interactions initiated by ZMYND8, impairing recruitment to sites of DNA damage. Our data establish a proof of principle that rigidity can be compensated by concomitant DNA and histone PTM interactions, maintaining multivalent engagement of transient chromatin states. Thus, our findings demonstrate an important role for rigid multivalent reader modules in nucleosome binding and chromatin function.

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Section: Resultsmentioning

confidence: 99%

Multivalent Histone and DNA Engagement by a PHD/BRD/PWWP Triple Reader Cassette Recruits ZMYND8 to K14ac-Rich Chromatin

et al. 2016

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“…Cellulose-bound peptide arrays were prepared using standard Fmoc solid phase peptide synthesis on a MultiPep-RSi-Spotter (INTAVIS, Köln, Germany) according to the SPOT synthesis method provided by the manufacturer, as previously described (Picaud and Filippakopoulos, 2015). Peptides were synthesized on amino-functionalized cellulose membranes (Whatman Chromatography paper Grade 1CHR, GE Healthcare Life Sciences #3001-878) and the presence of SPOTed peptides was confirmed by ultraviolet light (UV, λ = 280 nM).…”

Section: Methodsmentioning

confidence: 99%

Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains

et al. 2019

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Summary Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.

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“…Cellulose‐bound peptide arrays were prepared employing standard Fmoc solid‐phase peptide synthesis using a MultiPep‐RSi‐Spotter (INTAVIS, Köln, Germany) as previously described (Picaud & Filippakopoulos, ). The assay was performed using recombinant 6xHIS‐GST‐BIR2 domain of XIAP protein, and bound protein was detected using anti‐His antibody HRP‐conjugated.…”

Section: Methodsmentioning

confidence: 99%

Small molecule inhibitors reveal an indispensable scaffolding role of RIPK 2 in NOD 2 signaling

et al. 2018

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RIPK2 mediates inflammatory signaling by the bacteria‐sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD‐mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP‐binding and XIAP‐mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between β2 and β3 of the N‐lobe of the kinase, which is in close proximity to the ATP‐binding pocket. Through characterization of a new series of ATP pocket‐binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2‐XIAP interaction, and of cellular and in vivo NOD2 signaling. Our study exemplifies how targeting of the ATP‐binding pocket in RIPK2 can be exploited to interfere with the RIPK2‐XIAP interaction for modulation of NOD signaling.

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SPOTing Acetyl-Lysine Dependent Interactions

Cited by 14 publications

References 29 publications

Multivalent Histone and DNA Engagement by a PHD/BRD/PWWP Triple Reader Cassette Recruits ZMYND8 to K14ac-Rich Chromatin

Multivalent Histone and DNA Engagement by a PHD/BRD/PWWP Triple Reader Cassette Recruits ZMYND8 to K14ac-Rich Chromatin

Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains

Small molecule inhibitors reveal an indispensable scaffolding role of RIPK 2 in NOD 2 signaling

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