Characterization of the Ubiquitin-Modified Proteome Regulated by Transient Forebrain Ischemia

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-e-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-e-GG. Sixty-six proteins were highly ubiquitylated, with 10 or more K-e-GG peptides. Based on selection criteria of greater than fivefold increase and Po0.001, 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.

Aging Is Associated With Impaired Activation of Protein Homeostasis‐Related Pathways After Cardiac Arrest in Mice

“…Previously, we and others have provided evidence that these pathways are activated in brains of young mice subjected to ischemic stress 19, 20, 21, 22, 23, 24, 25, 26. Because CA results in whole‐body ischemia, we expected these conserved stress response pathways to be activated in all organs investigated after CA/CPR.…”

Regulation of mitophagy in ischemic brain injury

“…This is temporally consistent with the peak of autophagic fl ux [15] , implying that Parkin-labeled mitochondria can be eliminated by autophagy. Besides, ubiquitinated proteins are also present in the same model [76] , r egardless of whether Parkin is responsible for the ubiquitination or not, and this is now under discussion.…”

“…This is temporally consistent with the peak of autophagic fl ux [15] , implying that Parkin-labeled mitochondria can be eliminated by autophagy. Besides, ubiquitinated proteins are also present in the same model [76] , r egardless of whether Parkin is responsible for the ubiquitination or not, and this is now under discussion.Parkin is known to ubiquitylate several important substrates in cardiac injury [77] , indicating that it may participate in ischemic brain injury by activating mitophagy. In ischemic preconditioning (IPC) of the rat heart, Parkin is translocated to mitochondria, and its ablation abolishes the cardioprotective effects of IPC [27] , suggesting that Parkinmediated mitophagy is required in this process.…”

O-linked β-N-acetylglucosamine modification of proteins is activated in post-ischemic brains of young but not aged mice: Implications for impaired functional recovery from ischemic stress

“…[7][8][9] We evaluated the effect of age on ischemia-induced changes in global ubiquitin conjugation and polyubiquitin with K48-and K63-linkage ( Figure 1c and d, Figure S3). Global ubiquitin conjugation and polyubiquitin with K48 and K63 linkage were significantly increased after ischemia in young mice but this stress response was significantly suppressed in aged mice.…”