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Interactome profiling of a lysine deacetylase trapping probe library uncovers crosstalk between HDAC6 and NF-κB signaling

ABSTRACT: Lysine or histone deacetylases (HDACs) remove acetyl groups from lysine residues of numerous proteins, thereby regulating their function, activity and localization. Putative HDAC6 substrate sites, uncovered in the acetylome of HDAC6 knockdown cells, served as basis for the design of an HDAC6-trapping peptide library containing hydroxamic acids. Most probes enriched HDAC6 stronger from cell lysates than HDAC1. Probe sequences derived from α-tubulin, CRTC3, HSP90 and PPIA were confirmed as HDAC6 substrates and proteomics profiling of the corresponding trapping probes revealed preferential enrichment of HDAC6 together with known and previously unknown binding proteins. These included proteins of the NF-κB signaling complex which were independently confirmed as HDAC6 binders. NF-κB protein p50 was deacetylated by HDAC6 counteracting enhanced transcription of NF-κB target genes induced by p50 acetylation. These findings indicate a potential anti-inflammatory function of HDAC6 in NF-κB signaling.

ORGANISM(S): Homo Sapiens (human) Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Iris Finkemeier

PROVIDER: PXD044143 | JPOST Repository | Wed Jul 02 00:00:00 BST 2025

REPOSITORIES: jPOST

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			Action	DRS
	160511_QHF_FH_JSI10i_1.raw	Raw
	160511_QHF_FH_JSI10i_2.raw	Raw
	160511_QHF_FH_JSI10i_3.raw	Raw
	160511_QHF_FH_JSI11i_1.raw	Raw
	160511_QHF_FH_JSI11i_2.raw	Raw

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Project description:Isopeptide crosslinked proteins can be the product of transglutaminase or of exposure to organophosphorus toxicants (OP). Transglutaminase links glutamine to lysine with loss of ammonia. OP toxicants induce a link between glutamic acid and lysine with loss of water. Our goal was to establish criteria to distinguish real from false isopeptide crosslinks reported by software searches of mass spectrometry data. We used fragmentation spectra of tryptic peptides from MAP-rich tubulin Sus scrofa as a test system for detection of naturally-occurring isopeptide crosslinks. Data were analyzed with Protein Prospector. Criteria for the assignments included the presence of at least 1 crosslink specific product ion, fragment ions from both peptides, Protein Prospector scores ≥20, and best fit of the MS/MS data to the crosslinked peptide as opposed to a linear peptide. Out of 301,364 spectra, 15 potential transglutaminase-type crosslinked peptide candidates were identified. Manual evaluation of these MS/MS spectra reduced the number to 1 valid crosslink between Q112 of NFH and K368 of Tau. Immunopurification with anti-isopeptide 81D1C2 confirmed that MAP-rich tubulin contained only one isopeptide. Support for this isopeptide bond was obtained by showing that transglutaminase was capable of incorporating dansyl-aminohexyl -QQIV into K368. A model of the KIETHK-QLEAHNR isopeptide was synthesized with the aid of transglutaminase. MS/MS spectra of the model validated our interpretation of the native isopeptide. An OP-induced isopeptide bond between K163 of tubulin alpha-1A and E158 of tubulin beta-4B was induced by treating MAP-rich tubulin with 100 µM chlorpyrifos oxon. This crosslink was supported by the criteria described above and by the presence of diethoxyphospho-lysine 163 in the tubulin alpha-1A peptide. The information obtained in this work is valuable for future studies that aim to understand why exposure to OP is associated with increased risk of neurodegenerative disease.

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Interactome profiling of a lysine deacetylase trapping probe library uncovers crosstalk between HDAC6 and NF-κB signaling

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