期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 108, 期 20, 页码 8269-8274出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1018887108
关键词
cysteine labeling; repeat domain; conformation; oxidation state; mechanotransduction
资金
- National Institutes of Health [R01HL062352, P01DK032094, R01HL038794]
- Interdisciplinary Cardiovascular T32
- Penn's National Science Foundation-Nano Science Engineering Center
- Human Frontiers Sciences Program
- Division Of Materials Research
- Direct For Mathematical & Physical Scien [1120901] Funding Source: National Science Foundation
Questions of if and when protein structures change within cells pervade biology and include questions of how the cytoskeleton sustains stresses on cells-particularly in mutant versus normal cells. Cysteine shotgun labeling with fluorophores is analyzed here with mass spectrometry of the spectrin-actin membrane skeleton in sheared red blood cell ghosts from normal and diseased mice. Sheared samples are compared to static samples at 37 degrees C in terms of cell membrane intensity in fluorescence microscopy, separated protein fluorescence, and tryptic peptide modification in liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spectrin labeling proves to be the most sensitive to shear, whereas binding partners ankyrin and actin exhibit shear thresholds in labeling and both the ankyrin-binding membrane protein band 3 and the spectrin-actin stabilizer 4.1R show minimal differential labeling. Cells from 4.1R-null mice differ significantly from normal in the shear-dependent labeling of spectrin, ankyrin, and band 3: Decreased labeling of spectrin reveals less stress on the mutant network as spectrin dissociates from actin. Mapping the stress-dependent labeling kinetics of alpha- and beta-spectrin by LC-MS/MS identifies Cys in these antiparallel chains that are either force-enhanced or force-independent in labeling, with structural analyses indicating the force-enhanced sites are sequestered either in spectrin's triplehelical domains or in interactions with actin or ankyrin. Shear-sensitive sites identified comprehensively here in both spectrin and ankyrin appear consistent with stress relief through forced unfolding followed by cytoskeletal disruption.
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