Abstract
Cytochrome c (Cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc1 complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that Cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry. We generated and overexpressed wild-type, phosphomimetic T58E, and two controls, T58A and T58I Cytc; the latter replacement is found in human and testis-specific Cytc. In vitro, COX activity, caspase-3 activity, and heme degradation in the presence of H2O2 were decreased with phosphomimetic Cytc compared to wild-type. Cytc-knockout cells expressing T58E or T58I Cytc showed a reduction in intact cell respiration, mitochondrial membrane potential (∆Ψm), ROS production, and apoptotic activity compared to wild-type. We propose that, under physiological conditions, Cytc is phosphorylated, which controls mitochondrial respiration and apoptosis. Under conditions of stress Cytc phosphorylations are lost leading to maximal respiration rates, ∆Ψm hyperpolarization, ROS production, and apoptosis.
| Original language | English (US) |
|---|---|
| Article number | 15815 |
| Journal | Scientific reports |
| Volume | 9 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 1 2019 |
| Externally published | Yes |
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Regulation of Respiration and Apoptosis by Cytochrome c Threonine 58 Phosphorylation. / Wan, Junmei; Kalpage, Hasini A.; Vaishnav, Asmita; Liu, Jenney; Lee, Icksoo; Mahapatra, Gargi; Turner, Alice A.; Zurek, Matthew P.; Ji, Qinqin; Moraes, Carlos T.; Recanati, Maurice Andre; Grossman, Lawrence I.; Salomon, Arthur R.; Edwards, Brian F.P.; Hüttemann, Maik.
In: Scientific reports, Vol. 9, No. 1, 15815, 01.12.2019.Research output: Contribution to journal › Article
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TY - JOUR
T1 - Regulation of Respiration and Apoptosis by Cytochrome c Threonine 58 Phosphorylation
AU - Wan, Junmei
AU - Kalpage, Hasini A.
AU - Vaishnav, Asmita
AU - Liu, Jenney
AU - Lee, Icksoo
AU - Mahapatra, Gargi
AU - Turner, Alice A.
AU - Zurek, Matthew P.
AU - Ji, Qinqin
AU - Moraes, Carlos T.
AU - Recanati, Maurice Andre
AU - Grossman, Lawrence I.
AU - Salomon, Arthur R.
AU - Edwards, Brian F.P.
AU - Hüttemann, Maik
PY - 2019/12/1
Y1 - 2019/12/1
N2 - Cytochrome c (Cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc1 complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that Cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry. We generated and overexpressed wild-type, phosphomimetic T58E, and two controls, T58A and T58I Cytc; the latter replacement is found in human and testis-specific Cytc. In vitro, COX activity, caspase-3 activity, and heme degradation in the presence of H2O2 were decreased with phosphomimetic Cytc compared to wild-type. Cytc-knockout cells expressing T58E or T58I Cytc showed a reduction in intact cell respiration, mitochondrial membrane potential (∆Ψm), ROS production, and apoptotic activity compared to wild-type. We propose that, under physiological conditions, Cytc is phosphorylated, which controls mitochondrial respiration and apoptosis. Under conditions of stress Cytc phosphorylations are lost leading to maximal respiration rates, ∆Ψm hyperpolarization, ROS production, and apoptosis.
AB - Cytochrome c (Cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc1 complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that Cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry. We generated and overexpressed wild-type, phosphomimetic T58E, and two controls, T58A and T58I Cytc; the latter replacement is found in human and testis-specific Cytc. In vitro, COX activity, caspase-3 activity, and heme degradation in the presence of H2O2 were decreased with phosphomimetic Cytc compared to wild-type. Cytc-knockout cells expressing T58E or T58I Cytc showed a reduction in intact cell respiration, mitochondrial membrane potential (∆Ψm), ROS production, and apoptotic activity compared to wild-type. We propose that, under physiological conditions, Cytc is phosphorylated, which controls mitochondrial respiration and apoptosis. Under conditions of stress Cytc phosphorylations are lost leading to maximal respiration rates, ∆Ψm hyperpolarization, ROS production, and apoptosis.
UR - http://www.scopus.com/inward/record.url?scp=85074253242&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85074253242&partnerID=8YFLogxK
U2 - 10.1038/s41598-019-52101-z
DO - 10.1038/s41598-019-52101-z
M3 - Article
C2 - 31676852
AN - SCOPUS:85074253242
VL - 9
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
IS - 1
M1 - 15815
ER -