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Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

Sanderson TH, Mahapatra G, Pecina P, Ji Q, Yu K, Sinkler C, Varughese A, Kumar R, Bukowski MJ, Tousignant RN, Salomon AR, Lee I, Hüttemann M - PLoS ONE (2013)

Bottom Line: Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration.This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion.These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, Michigan, United States of America ; Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

ABSTRACT
Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

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Nano-LC/ESI-MS/MS spectrum of EDLIApYLKKATNE.Peptides were eluted into the mass spectrometer by applying a HPLC gradient of 0–70% 0.1 M acetic acid/acetonitrile in 30 minutes. The mass spectrometer acquired top 9 data dependent ESI MS/MS spectra. The phosphorylation site was unequivocally assigned by fragment ions b6, b8, b9, b10, and y3, y4, y6. The sequence of the peptide was definitively assigned by b3, b6, b8, b9, b10, b11, b12, and y2, y3, y4, y6, y9, y10, y11.
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pone-0078627-g004: Nano-LC/ESI-MS/MS spectrum of EDLIApYLKKATNE.Peptides were eluted into the mass spectrometer by applying a HPLC gradient of 0–70% 0.1 M acetic acid/acetonitrile in 30 minutes. The mass spectrometer acquired top 9 data dependent ESI MS/MS spectra. The phosphorylation site was unequivocally assigned by fragment ions b6, b8, b9, b10, and y3, y4, y6. The sequence of the peptide was definitively assigned by b3, b6, b8, b9, b10, b11, b12, and y2, y3, y4, y6, y9, y10, y11.

Mentions: To provide further proof that Cytc was tyrosine phosphorylated after insulin treatment we determined the specific phosphorylation site. Isolated pig brain Cytc was digested with trypsin and analyzed by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS/MS). This method first enriched phosphorylated peptides via a TiO2 resin, and the eluted peptides were then subjected to tandem-MS for fragment examination. This analysis unambiguously revealed that Tyr97 was phosphorylated on Cytc following insulin treatment on the peptide EDLIApYLKKATNE by fragment ions b6, b8, b9, b10, and y3, y4, y6 (Fig. 4). Importantly, no phosphorylation site was identified using control Cytc that was directly isolated from ischemic brain tissue without insulin treatment, supporting findings by Western analysis (Fig. 3).


Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

Sanderson TH, Mahapatra G, Pecina P, Ji Q, Yu K, Sinkler C, Varughese A, Kumar R, Bukowski MJ, Tousignant RN, Salomon AR, Lee I, Hüttemann M - PLoS ONE (2013)

Nano-LC/ESI-MS/MS spectrum of EDLIApYLKKATNE.Peptides were eluted into the mass spectrometer by applying a HPLC gradient of 0–70% 0.1 M acetic acid/acetonitrile in 30 minutes. The mass spectrometer acquired top 9 data dependent ESI MS/MS spectra. The phosphorylation site was unequivocally assigned by fragment ions b6, b8, b9, b10, and y3, y4, y6. The sequence of the peptide was definitively assigned by b3, b6, b8, b9, b10, b11, b12, and y2, y3, y4, y6, y9, y10, y11.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818486&req=5

pone-0078627-g004: Nano-LC/ESI-MS/MS spectrum of EDLIApYLKKATNE.Peptides were eluted into the mass spectrometer by applying a HPLC gradient of 0–70% 0.1 M acetic acid/acetonitrile in 30 minutes. The mass spectrometer acquired top 9 data dependent ESI MS/MS spectra. The phosphorylation site was unequivocally assigned by fragment ions b6, b8, b9, b10, and y3, y4, y6. The sequence of the peptide was definitively assigned by b3, b6, b8, b9, b10, b11, b12, and y2, y3, y4, y6, y9, y10, y11.
Mentions: To provide further proof that Cytc was tyrosine phosphorylated after insulin treatment we determined the specific phosphorylation site. Isolated pig brain Cytc was digested with trypsin and analyzed by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS/MS). This method first enriched phosphorylated peptides via a TiO2 resin, and the eluted peptides were then subjected to tandem-MS for fragment examination. This analysis unambiguously revealed that Tyr97 was phosphorylated on Cytc following insulin treatment on the peptide EDLIApYLKKATNE by fragment ions b6, b8, b9, b10, and y3, y4, y6 (Fig. 4). Importantly, no phosphorylation site was identified using control Cytc that was directly isolated from ischemic brain tissue without insulin treatment, supporting findings by Western analysis (Fig. 3).

Bottom Line: Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration.This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion.These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, Michigan, United States of America ; Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

ABSTRACT
Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Show MeSH
Related in: MedlinePlus