Dataset Information

Microtubules methylation LC-MSMS

ABSTRACT: Microtubules are critical for mitosis, cell motility, and protein and organelle transport, and are a validated target for anticancer drugs. However, tubulin regulation and recruitment in these cellular processes is less understood. Post-translational modifications of tubulin are proposed to regulate microtubule functions and dynamics. Although many such modifications have been investigated, tubulin methylations and enzymes responsible for methylation have only recently begun to be described. Here we report that N-lysine methyl transferase KMT5A (SET8/PR‑Set7), which methylates histone H4K20, also methylates α‑tubulin. Furthermore, the transcription factor LSF binds both tubulin and SET8, and enhances α-tubulin methylation in vitro, countered by FQI1, a specific small molecule inhibitor of LSF. Thus, the three SET8, LSF, and tubulin, all essential for mitotic progression, interact with each other. Overall, these results point to dual functions for both SET8 and LSF not only in chromatin regulation, but also for cytoskeletal modification.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Brain, T Cell, Epithelial Cell, Fibroblast

SUBMITTER: HANG GYEONG CHIN

LAB HEAD: Sriharsa Pradhan

PROVIDER: PXD014257 | Pride | 2020-02-28

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Ctrl_1_2_30min_Top10_n1000_QE_120315-2.2.0.388.pdResult	Other
	Ctrl_1_2_30min_Top10_n1000_QE_120315.pep.xml	Pepxml
	Ctrl_1_30min_Top10_n1000_QE_120315.mzML	Mzml
	Ctrl_1_30min_Top10_n1000_QE_120315.raw	Raw
	Ctrl_2_30min_Top10_n1000_QE_120315.mzML	Mzml

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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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Microtubules methylation LC-MSMS

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