Dataset Information

X-link of KIF2C-tubulin complex with DSSO

ABSTRACT: KIF2-tubulin complex was crosslinked with DSSO, trypsinized, and analyzed by LC-MS.

ORGANISM(S): Sus Scrofa Mus Musculus (mouse)

SUBMITTER: Nobutaka Hirokawa

PROVIDER: PXD007142 | JPOST Repository | Wed Sep 13 00:00:00 BST 2017

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	DSSO_EThcD.raw	Raw
	EThcD.xls	Xls

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Publications

Mechanism of Catalytic Microtubule Depolymerization via KIF2-Tubulin Transitional Conformation.

Ogawa Tadayuki T Saijo Shinya S Shimizu Nobutaka N Jiang Xuguang X Hirokawa Nobutaka N

Cell reports 20170901 11

Microtubules (MTs) are dynamic structures that are fundamental for cell morphogenesis and motility. MT-associated motors work efficiently to perform their functions. Unlike other motile kinesins, KIF2 catalytically depolymerizes MTs from the peeled protofilament end during ATP hydrolysis. However, the detailed mechanism by which KIF2 drives processive MT depolymerization remains unknown. To elucidate the catalytic mechanism, the transitional KIF2-tubulin complex during MT depolymerization was an ...[more]

PMID: 28903043

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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.