CYB5D2 Requires Heme-Binding to Regulate HeLa Cell Growth and Confer Survival from Chemotherapeutic Agents

Anthony Bruce,Adrian P Rybak,Irina V Lebedeva

doi:10.1371/journal.pone.0086435

Anthony Bruce, Adrian P Rybak + Show 1 more

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https://doi.org/10.1371/journal.pone.0086435

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Abstract

The cytochrome b5 domain containing 2 (CYB5D2; Neuferricin) protein has been reported to bind heme, however, the critical residues responsible for heme-binding are undefined. Furthermore, the relationship between heme-binding and CYB5D2-mediated intracellular functions remains unknown. Previous studies examining heme-binding in two cytochrome b5 heme-binding domain-containing proteins, damage-associated protein 1 (Dap1; Saccharomyces cerevisiae) and human progesterone receptor membrane component 1 (PGRMC1), have revealed that conserved tyrosine (Y) 73, Y79, aspartic acid (D) 86, and Y127 residues present in human CYB5D2 may be involved in heme-binding. CYB5D2 binds to type b heme, however, only the substitution of glycine (G) at D86 (D86G) within its cytochrome b5 heme-binding (cyt-b5) domain abolished its heme-binding ability. Both CYB5D2 and CYB5D2(D86G) localize to the endoplasmic reticulum. Ectopic CYB5D2 expression inhibited cell proliferation and anchorage-independent colony growth of HeLa cells. Conversely, CYB5D2 knockdown and ectopic CYB5D2(D86G) expression increased cell proliferation and colony growth. As PGRMC1 has been reported to regulate the expression and activities of cytochrome P450 proteins (CYPs), we examined the role of CYB5D2 in regulating the activities of CYPs involved in sterol synthesis (CYP51A1) and drug metabolism (CYP3A4). CYB5D2 co-localizes with cytochrome P450 reductase (CYPOR), while CYB5D2 knockdown reduced lanosterol demethylase (CYP51A1) levels and rendered HeLa cells sensitive to mevalonate. Additionally, knockdown of CYB5D2 reduced CYP3A4 activity. Lastly, CYB5D2 expression conferred HeLa cell survival from chemotherapeutic agents (paclitaxel, cisplatin and doxorubicin), with its ability to promote survival being dependent on its heme-binding ability. Taken together, this study provides evidence that heme-binding is critical for CYB5D2 in regulating HeLa cell growth and survival, with endogenous CYB5D2 being required to modulate CYP activities.

Highlights

Progesterone receptor membrane component 1 (PGRMC1) is the most extensively investigated member of the membrane associated progesterone receptor (MAPR) family
Upon structural analysis of the 1J03 protein in Arabidopsis thaliana [23,24], it was revealed that key residues and structural elements within its cyt-b5 domain are conserved with PGRMC1 [2]
We demonstrate that the D86 residue, a highly conserved amino acid within the cyt-b5 region, is required for CYB5D2 to bind heme

Summary

Introduction

Progesterone receptor membrane component 1 (PGRMC1) is the most extensively investigated member of the membrane associated progesterone receptor (MAPR) family. The yeast homolog of PGRMC1, damage associated protein 1 (Dap1), a cytochrome b5 heme-binding (cytb5) protein, is required for survival from the DNA methylating agent, methyl methane-sulfonate (MMS) [4,5]. Substitution of the conserved D91 residue with G prevents Dap from association with heme and Dap1(D91G) is incapable of protecting yeast from MMS-induced toxicity [6]. UV-visible absorption and electron paramagnetic resonance (ESR) spectra were used to demonstrate that PGRMC1 binds to b-type heme [8]. The hydrophobic hemebinding pocket of PGRMCI was affected by mutating two conserved tyrosine (Y107, Y113) residues [8], while mutating the conserved aspartic acid residue at position 120 to glycine (D120G) in PGRMC1 resulted in the loss of its heme-binding ability [7]

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