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

Hasini A. Kalpage; Asmita Vaishnav; Jenney Liu; Ashwathy Varughese; Junmei Wan; Alice A. Turner; Qinqin Ji; Matthew P. Zurek; Alexandr A. Kapralov; Valerian E. Kagan; Joseph S. Brunzelle; Maurice Andre Recanati; Lawrence I. Grossman; Thomas H. Sanderson; Icksoo Lee; Arthur R. Salomon; Brian F.P. Edwards; Maik Hüttemann

doi:10.1096/fj.201901120R

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

Hasini A. Kalpage, Asmita Vaishnav, Jenney Liu, Ashwathy Varughese, Junmei Wan, Alice A. Turner, Qinqin Ji, Matthew P. Zurek, Alexandr A. Kapralov, Valerian E. Kagan, Joseph S. Brunzelle, Maurice Andre Recanati, Lawrence I. Grossman, Thomas H. Sanderson, Icksoo Lee, Arthur R. Salomon, Brian F.P. Edwards, Maik Hüttemann

Pharmacology

Research output: Contribution to journal › Article › peer-review

31 Scopus citations

Abstract

Cytochrome c (Cytc) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cytc in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cytc purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects in vitro using phosphorylated Cytc purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cytc at 1.55 Å and suggest that it spatially matches S47-phosphorylated Cytc, making it a good model system. Both S47-phosphorylated and phosphomimetic Cytc showed a lower oxygen consumption rate in reaction with isolated Cytc oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cytc showed lower caspase-3 activity. Furthermore, phosphomimetic Cytc had decreased cardiolipin peroxidase activity and is more stable in the presence of H₂O₂. Our data suggest that S47 phosphorylation of Cytc is tissue protective and promotes cell survival in the brain.—Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.-A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity. FASEB J. 33, 13503-13514 (2019). www.fasebj.org.

Original language	English (US)
Pages (from-to)	13503-13514
Number of pages	12
Journal	FASEB Journal
Volume	33
Issue number	12
DOIs	https://doi.org/10.1096/fj.201901120R
State	Published - Dec 2019

Funding

This work was supported by the U.S. National Institutes of Health (NIH) National Institute of General Medical Sciences Grants R01 GM116807 and R01 GM113908, NIH National Institute of Neurological Disorders and Stroke Grants R01 NS091242 and R42 NS105238, and by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program under Award W81XWH‐16‐1‐0175. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the funding agencies including the Department of Defense or the NIH. This research used resources of the Advanced Photon Source, a U.S. Department of Energy Office of Science User Facility operated for the Department of Energy Office of Science by Argonne National Laboratory under Contract DE‐AC02‐ 06CH11357. Use of the Life Sciences Collaborative Access Team (LS‐CAT) Sector 21 was supported by the Michigan Economic Development Corp., the Michigan Technology Tri‐Corridor (Grant 085P1000817), and Wayne State University Office of the Vice‐President for Research. The authors declare no conflicts of interest.

Keywords

apoptosis
electron transport chain
ischemia
respiration
signaling

ASJC Scopus subject areas

Biotechnology
Biochemistry
Molecular Biology
Genetics

Access to Document

10.1096/fj.201901120R

Cite this

Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M. A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P., & Hüttemann, M. (2019). Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity. FASEB Journal, 33(12), 13503-13514. https://doi.org/10.1096/fj.201901120R

@article{0a72571080cc44449af43cf258634715,

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

abstract = "Cytochrome c (Cytc) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cytc in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cytc purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects in vitro using phosphorylated Cytc purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cytc at 1.55 {\AA} and suggest that it spatially matches S47-phosphorylated Cytc, making it a good model system. Both S47-phosphorylated and phosphomimetic Cytc showed a lower oxygen consumption rate in reaction with isolated Cytc oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cytc showed lower caspase-3 activity. Furthermore, phosphomimetic Cytc had decreased cardiolipin peroxidase activity and is more stable in the presence of H2O2. Our data suggest that S47 phosphorylation of Cytc is tissue protective and promotes cell survival in the brain.—Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.-A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, H{\"u}ttemann, M. Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity. FASEB J. 33, 13503-13514 (2019). www.fasebj.org.",

keywords = "apoptosis, electron transport chain, ischemia, respiration, signaling",

author = "Kalpage, {Hasini A.} and Asmita Vaishnav and Jenney Liu and Ashwathy Varughese and Junmei Wan and Turner, {Alice A.} and Qinqin Ji and Zurek, {Matthew P.} and Kapralov, {Alexandr A.} and Kagan, {Valerian E.} and Brunzelle, {Joseph S.} and Recanati, {Maurice Andre} and Grossman, {Lawrence I.} and Sanderson, {Thomas H.} and Icksoo Lee and Salomon, {Arthur R.} and Edwards, {Brian F.P.} and Maik H{\"u}ttemann",

note = "Funding Information: This work was supported by the U.S. National Institutes of Health (NIH) National Institute of General Medical Sciences Grants R01 GM116807 and R01 GM113908, NIH National Institute of Neurological Disorders and Stroke Grants R01 NS091242 and R42 NS105238, and by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program under Award W81XWH‐16‐1‐0175. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the funding agencies including the Department of Defense or the NIH. This research used resources of the Advanced Photon Source, a U.S. Department of Energy Office of Science User Facility operated for the Department of Energy Office of Science by Argonne National Laboratory under Contract DE‐AC02‐ 06CH11357. Use of the Life Sciences Collaborative Access Team (LS‐CAT) Sector 21 was supported by the Michigan Economic Development Corp., the Michigan Technology Tri‐Corridor (Grant 085P1000817), and Wayne State University Office of the Vice‐President for Research. The authors declare no conflicts of interest. Publisher Copyright: {\textcopyright} FASEB.",

year = "2019",

month = dec,

doi = "10.1096/fj.201901120R",

language = "English (US)",

volume = "33",

pages = "13503--13514",

journal = "FASEB Journal",

issn = "0892-6638",

publisher = "John Wiley & Sons Inc.",

number = "12",

}

Kalpage, HA, Vaishnav, A, Liu, J, Varughese, A, Wan, J, Turner, AA, Ji, Q, Zurek, MP, Kapralov, AA, Kagan, VE, Brunzelle, JS, Recanati, MA, Grossman, LI, Sanderson, TH, Lee, I, Salomon, AR, Edwards, BFP & Hüttemann, M 2019, 'Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity', FASEB Journal, vol. 33, no. 12, pp. 13503-13514. https://doi.org/10.1096/fj.201901120R

TY - JOUR

T1 - Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity

AU - Kalpage, Hasini A.

AU - Vaishnav, Asmita

AU - Liu, Jenney

AU - Varughese, Ashwathy

AU - Wan, Junmei

AU - Turner, Alice A.

AU - Ji, Qinqin

AU - Zurek, Matthew P.

AU - Kapralov, Alexandr A.

AU - Kagan, Valerian E.

AU - Brunzelle, Joseph S.

AU - Recanati, Maurice Andre

AU - Grossman, Lawrence I.

AU - Sanderson, Thomas H.

AU - Lee, Icksoo

AU - Salomon, Arthur R.

AU - Edwards, Brian F.P.

AU - Hüttemann, Maik

N1 - Funding Information: This work was supported by the U.S. National Institutes of Health (NIH) National Institute of General Medical Sciences Grants R01 GM116807 and R01 GM113908, NIH National Institute of Neurological Disorders and Stroke Grants R01 NS091242 and R42 NS105238, and by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program under Award W81XWH‐16‐1‐0175. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the funding agencies including the Department of Defense or the NIH. This research used resources of the Advanced Photon Source, a U.S. Department of Energy Office of Science User Facility operated for the Department of Energy Office of Science by Argonne National Laboratory under Contract DE‐AC02‐ 06CH11357. Use of the Life Sciences Collaborative Access Team (LS‐CAT) Sector 21 was supported by the Michigan Economic Development Corp., the Michigan Technology Tri‐Corridor (Grant 085P1000817), and Wayne State University Office of the Vice‐President for Research. The authors declare no conflicts of interest. Publisher Copyright: © FASEB.

PY - 2019/12

Y1 - 2019/12

N2 - Cytochrome c (Cytc) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cytc in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cytc purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects in vitro using phosphorylated Cytc purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cytc at 1.55 Å and suggest that it spatially matches S47-phosphorylated Cytc, making it a good model system. Both S47-phosphorylated and phosphomimetic Cytc showed a lower oxygen consumption rate in reaction with isolated Cytc oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cytc showed lower caspase-3 activity. Furthermore, phosphomimetic Cytc had decreased cardiolipin peroxidase activity and is more stable in the presence of H2O2. Our data suggest that S47 phosphorylation of Cytc is tissue protective and promotes cell survival in the brain.—Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.-A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity. FASEB J. 33, 13503-13514 (2019). www.fasebj.org.

AB - Cytochrome c (Cytc) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cytc in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cytc purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects in vitro using phosphorylated Cytc purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cytc at 1.55 Å and suggest that it spatially matches S47-phosphorylated Cytc, making it a good model system. Both S47-phosphorylated and phosphomimetic Cytc showed a lower oxygen consumption rate in reaction with isolated Cytc oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cytc showed lower caspase-3 activity. Furthermore, phosphomimetic Cytc had decreased cardiolipin peroxidase activity and is more stable in the presence of H2O2. Our data suggest that S47 phosphorylation of Cytc is tissue protective and promotes cell survival in the brain.—Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.-A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity. FASEB J. 33, 13503-13514 (2019). www.fasebj.org.

KW - apoptosis

KW - electron transport chain

KW - ischemia

KW - respiration

KW - signaling

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

Abstract

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Oxidized rat cytochrome c mutant (S47E)

The structure of oxidized rat cytochrome c at 1.13 angstroms resolution

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

Abstract

Funding

Keywords

ASJC Scopus subject areas

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Oxidized rat cytochrome c mutant (S47E)

The structure of oxidized rat cytochrome c at 1.13 angstroms resolution

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