Altered ca2+ homeostasis in red blood cells of polycythemia vera patients following disturbed organelle sorting during terminal erythropoiesis

Ralfs Buks; Tracy Dagher; Maria Giustina Rotordam; David Monedero Alonso; Sylvie Cochet; Emilie Fleur Gautier; Philippe Chafey; Bruno Cassinat; Jean Jacques Kiladjian; Nadine Becker; Isabelle Plo; Stéphane Egée; Wassim El Nemer

doi:10.3390/cells11010049

Altered ca²⁺ homeostasis in red blood cells of polycythemia vera patients following disturbed organelle sorting during terminal erythropoiesis

Ralfs Buks, Tracy Dagher, Maria Giustina Rotordam, David Monedero Alonso, Sylvie Cochet, Emilie Fleur Gautier, Philippe Chafey, Bruno Cassinat, Jean Jacques Kiladjian, Nadine Becker, Isabelle Plo, Stéphane Egée, Wassim El Nemer^* (Corresponding Author)

^*Corresponding author for this work

Aberdeen Centre For Environmental Sustainability

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

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Abstract

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca²⁺ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2^V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2^V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2^V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2^V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

Original language	English
Article number	49
Number of pages	23
Journal	Cells
Volume	11
Issue number	1
Early online date	24 Dec 2021
DOIs	https://doi.org/10.3390/cells11010049
Publication status	Published - 1 Jan 2022

Bibliographical note

Funding Information:
Funding: The work was supported by Institut National de la Santé et de la Recherche Médicale (Inserm); Institut National de la Transfusion Sanguine (INTS); the University of Paris; and grants from Laboratory of Excellence (Labex) GR-Ex, reference No. ANR-11-LABX-0051. The Labex GREx is funded by the IdEx program “Investissements d’avenir” of the French National Research Agency, reference No. ANR-11-IDEX-0005-02 and ANR-18-IDEX-0001. R.B., M.G.R., and D.M.A. were funded by the European Union’s Horizon 2020 Research and Innovation Program under grant agreement No. 675115-RELEVANCE-H2020-MSCA-ITN-2015. R.B. also received financial support from Société Française d’Hématologie (SFH) and Club du Globule Rouge et du Fer (CGRF). R.B. is currently funded by the Innovate UK Research and Innovation Knowledge Transfer Partnership (KTP) between University of Aberdeen and Vertebrate Antibodies Ltd. (Partnership No. KTP12327). T.D. was supported by PhD grants from Université Paris Saclay MESR (Ministère Enseignement Supérieur et de la Recherche) and then FRM (Fondation recherche médicale). The Orbitrap Fusion mass spectrometer was acquired with funds from Fonds Europeen de Developpement Regional (FEDER) through the Operational Program for Competitiveness Factors and Employment 2007-2013 and from the Canceropole Ile de France.

Data Availability Statement

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [63] partner repository with the dataset identifier PXD029965. The data are available via ProteomeXchange with identifier PXD029965.

Keywords

Ca2+
Enucleation
JAK2V617F
Organelle sorting
Polycythemia vera
Red blood cells
Reticulocytes

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Cite this

Buks, R., Dagher, T., Rotordam, M. G., Alonso, D. M., Cochet, S., Gautier, E. F., Chafey, P., Cassinat, B., Kiladjian, J. J., Becker, N., Plo, I., Egée, S., & El Nemer, W. (2022). Altered ca²⁺ homeostasis in red blood cells of polycythemia vera patients following disturbed organelle sorting during terminal erythropoiesis. Cells, 11(1), Article 49. https://doi.org/10.3390/cells11010049

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title = "Altered ca2+ homeostasis in red blood cells of polycythemia vera patients following disturbed organelle sorting during terminal erythropoiesis",

abstract = "Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the G{\'a}rdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.",

keywords = "Ca2+, Enucleation, JAK2V617F, Organelle sorting, Polycythemia vera, Red blood cells, Reticulocytes",

author = "Ralfs Buks and Tracy Dagher and Rotordam, {Maria Giustina} and Alonso, {David Monedero} and Sylvie Cochet and Gautier, {Emilie Fleur} and Philippe Chafey and Bruno Cassinat and Kiladjian, {Jean Jacques} and Nadine Becker and Isabelle Plo and St{\'e}phane Eg{\'e}e and {El Nemer}, Wassim",

note = "Funding Information: Funding: The work was supported by Institut National de la Sant{\'e} et de la Recherche M{\'e}dicale (Inserm); Institut National de la Transfusion Sanguine (INTS); the University of Paris; and grants from Laboratory of Excellence (Labex) GR-Ex, reference No. ANR-11-LABX-0051. The Labex GREx is funded by the IdEx program “Investissements d{\textquoteright}avenir” of the French National Research Agency, reference No. ANR-11-IDEX-0005-02 and ANR-18-IDEX-0001. R.B., M.G.R., and D.M.A. were funded by the European Union{\textquoteright}s Horizon 2020 Research and Innovation Program under grant agreement No. 675115-RELEVANCE-H2020-MSCA-ITN-2015. R.B. also received financial support from Soci{\'e}t{\'e} Fran{\c c}aise d{\textquoteright}H{\'e}matologie (SFH) and Club du Globule Rouge et du Fer (CGRF). R.B. is currently funded by the Innovate UK Research and Innovation Knowledge Transfer Partnership (KTP) between University of Aberdeen and Vertebrate Antibodies Ltd. (Partnership No. KTP12327). T.D. was supported by PhD grants from Universit{\'e} Paris Saclay MESR (Minist{\`e}re Enseignement Sup{\'e}rieur et de la Recherche) and then FRM (Fondation recherche m{\'e}dicale). The Orbitrap Fusion mass spectrometer was acquired with funds from Fonds Europeen de Developpement Regional (FEDER) through the Operational Program for Competitiveness Factors and Employment 2007-2013 and from the Canceropole Ile de France. ",

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AU - Alonso, David Monedero

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AU - Egée, Stéphane

AU - El Nemer, Wassim

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N2 - Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

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