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J Cereb Blood Flow Metab. 2014 Mar;34(3):425-32. doi: 10.1038/jcbfm.2013.210. Epub 2013 Dec 4.

Characterization of the ubiquitin-modified proteome regulated by transient forebrain ischemia.

Author information

1
Multidisciplinary Neuroprotection Laboratories, Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina, USA.
2
Proteomics Core Facility, Duke Institute for Genome Science and Policy, Durham, North Carolina, USA.
3
Duke University Small Molecule Synthesis Facility, Duke University Medical Center, Durham, North Carolina, USA.

Abstract

Ubiquitylation is a posttranslational protein modification that modulates various cellular processes of key significance, including protein degradation and DNA damage repair. In animals subjected to transient cerebral ischemia, ubiquitin-conjugated proteins accumulate in Triton-insoluble aggregates. Although this process is widely considered to modulate the fate of postischemic neurons, few attempts have been made to characterize the ubiquitin-modified proteome in these aggregates. We performed proteomics analyses to identify ubiquitylated proteins in postischemic aggregates. Mice were subjected to 10 minutes of forebrain ischemia and 4 hours of reperfusion. The hippocampi were dissected, aggregates were isolated, and trypsin-digested after spiking with GG-BSA as internal standard. K-ɛ-GG-containing peptides were immunoprecipitated and analyzed by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. We identified 1,664 peptides to 520 proteins containing at least one K-ɛ-GG. Sixty-six proteins were highly ubiquitylated, with 10 or more K-ɛ-GG peptides. Based on selection criteria of greater than fivefold increase and P<0.001, 763 peptides to 272 proteins were highly enriched in postischemic aggregates. These included proteins involved in important neuronal functions and signaling pathways that are impaired after ischemia. Results of this study could serve as an important platform to uncover the mechanisms linking insoluble ubiquitin aggregates to the functions of postischemic neurons.

PMID:
24301296
PMCID:
PMC3948117
DOI:
10.1038/jcbfm.2013.210
[Indexed for MEDLINE]
Free PMC Article
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