In Vitro Characterization Of Serine 47 Phosphorylated Cytochrome C

Cytochrome c is a 12.4kDa ubiquitously expressed protein known to be involved in many physiological processes of the cell such as respiration and apoptosis. The goal of our lab is to increase our knowledge of the regulation of cytochrome c in these opposite activities, and our working model posits that cytochrome c is decisively regulated by phosphorylation. When phosphorylated, cytochrome c leads to an optimal functioning in the electron transport chain by lowering electron flux, preventing harmful high mitochondrial membrane potentials and thus ROS production under healthy conditions. However, under cellular stress cytochrome c might be dephosphorylated favoring high mitochondrial membrane potentials and ROS and its participation in apoptosis. Our lab has previously published two phosphorylation sites in cow, namely Y48 in the liver and Y97 in the heart. The aim of my thesis was to identify phosphorylation site(s) on kidney cytochrome c and to perform functional characterization of the cow kidney protein. Kidney cytochrome c was found to be phosphorylated on S47 and based on this, suitable cytochrome c variants were over expressed in a prokaryotic system. These cytochrome c variants were used to study the effect of phosphorylation on the most common activities of cytochrome c protein i.e., cellular respiration and apoptosis. The results of the in vitro study revealed that the phosphomimetic mutant Ser47Glu has lower rates of respiration compared to wild type as well as S47A mutant which is in line with the working model of our lab. In addition, any mutation of the Ser47 residue resulted in almost fully diminished caspase activity when compared to wild type, suggesting that this residue might be key to the regulation of the apoptotic activity of cytochrome c

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: Implications for AMP Kinase

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc iso- lated from kidneys is phosphorylated on Thr28, leading to a par- tial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing supe- rior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type un- phosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (����m), and ROS levels are reduced compared with wild type. As we show by high resolu- tion crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a cen- tral position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kid- ney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue- specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ����m hyperpolarization, a known cause of ROS and trigger of apoptosis

Cytochrome <i>c</i> release is inhibited by insulin administration.

<p>(A) Sham operated control animals (Sham, n = 3) show faint detectable amounts of Cyt<i>c</i> in the cytosolic fraction of hippocampal CA1 neurons. After 8 min of ischemia followed by 24 h of reperfusion (R24, n = 5) cytosolic Cyt<i>c</i> increases ∼4 fold (p < 0.05) and there is a trend toward mitochondrial Cyt<i>c</i> decrease (p = 0.08), both indicative of mitochondrial Cyt<i>c</i> release. Animals exposed to 8 min of ischemia followed by 24 h of reperfusion with a single bolus of IV insulin at the onset of reperfusion (T24, n = 5) demonstrate a reduction of cytosolic Cyt<i>c</i> (p < 0.05) and increased mitochondrial Cyt<i>c</i> compared to the R24 controls. (B and C) Densitometric analyses of Western blot data (Mean +/−SEM, *p < 0.05).</p

Serine‐47 phosphorylation of cytochrome c

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Insulin prevents neuronal cell death in the CA1 hippocampus following brain ischemia.

<p>Cresyl violet stained sections (top row) show CA1 hippocampus densely populated with pyramidal neurons in sham-operated controls (Sham), and triple-label immunofluorescence (bottom row) shows these cells to be NeuN-positive (red). After 8 min of global brain ischemia followed by 14 days of reperfusion (I/R, n = 5) there is a 90% loss of CA1 neurons and an increase in Iba-1-positive microglia and GFAP-positive astrocytes (green and magenta, respectively). Animals exposed to 8 min of ischemia followed by 14 days of reperfusion with a single bolus of insulin given at the onset of reperfusion (I/R + Ins, n = 4) demonstrate a 49% increase in NeuN-positive neurons (p < 0.05) while Iba-1 and GFAP-positive cells remain unchanged.</p

Nano-LC/ESI-MS/MS spectrum of EDLIApYLKKATNE.

<p>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.</p

Insulin treatment leads to phosphorylation of brain cytochrome <i>c</i>.

<p>Ischemic pig brain tissue was treated +/− insulin following Cyt<i>c</i> purification under conditions that preserve protein phosphorylation. (A) Western analysis with an anti-phosphotyrosine antibody (4G10) indicates tyrosine phosphorylation of Cyt<i>c</i> after insulin treatment (lane 4), whereas Cyt<i>c</i> isolated without insulin treatment (lane 5) or obtained from a commercial source (lane 3, Sigma Cyt<i>c</i>) does not produce any signal. Lane M, protein size marker (kDa); lane 1, EGF stimulated A431 cell lysate (positive control for Western analysis); lane 2, ovalbumin (negative control for Western analysis). Western analysis with anti-phosphoserine and anti-phosphothreonine antibodies did not show any signal (not shown). (B) Control Western blot with an anti-Cyt<i>c</i> antibody shows similar loading. Samples in lanes 1–5 as denoted in A. Note that additional bands in Sigma Cyt<i>c</i> correspond to the Cyt<i>c</i> dimer and trimer that are sometimes observed depending on the batch.</p

In vivo induction of cytochrome <i>c</i> Tyr97 phosphorylation in rat brain by insulin treatment.

<p>(A) Cyt<i>c</i> was isolated from rat brain after global brain ischemia (lane 1), from untreated control rats (lane 2), from sham-operated animals after insulin treatment (lane 3), and from rat brain after ischemia with insulin treatment (lane 4). Top, Western blot with an anti-phosphotyrosine antibody (4G10) reveals no detectable tyrosine phosphorylation of brain Cyt<i>c</i> under control conditions and after global brain ischemia (lanes 2 and 1, respectively), whereas tyrosine phosphorylation of Cyt<i>c</i> is strongly induced after insulin treatment (lane 3), but slightly reduced by ischemic stress (lane 4). Bottom, Coomassie gel shows equal loading (1 µg per lane) and purity of the isolated Cyt<i>c</i> species. (B) Nano-LC/ESI-MS/MS analysis of rat brain Cyt<i>c</i> after insulin treatment (corresponding to lane 3 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y5, and y6. The sequence of the peptide was definitively assigned by b3, b4, y2, y3, y4, y5, and y6. (C) Nano-LC/ESI-MS/MS analysis of rat brain Cyt<i>c</i> after global brain ischemia and insulin treatment (corresponding to lane 4 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y6, and y7. The sequence of the peptide was definitively assigned by b3, b4, y3, y4, y6, and y7.</p

Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity

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