Molecular identification of adrenal inner zone antigen as a heme-binding protein

Department of Molecular Physiological Chemistry, Graduate School of Medicine, Osaka University, Japan.
FEBS Journal (Impact Factor: 4). 12/2005; 272(22):5832-43. DOI: 10.1111/j.1742-4658.2005.04977.x
Source: PubMed


The adrenal inner zone antigen (IZA), which reacts specifically with a monoclonal antibody raised against the fasciculata and reticularis zones of the rat adrenal, was previously found to be identical with a protein variously named 25-Dx and membrane-associated progesterone receptor. IZA was purified as a glutathione S-transferase-fused or His(6)-fused protein, and its molecular properties were studied. The UV-visible absorption and EPR spectra of the purified protein showed that IZA bound a heme chromophore in high-spin type. Analysis of the heme indicated that it is of the b type. Site-directed mutagenesis studies were performed to identify the amino-acid residues that bind the heme to the protein. The results suggest that two Tyr residues, Tyr107 and Tyr113, and a peptide stretch, D99-K102, were important for anchoring the heme into a hydrophobic pocket. The effect of IZA on the steroid 21-hydroxylation reaction was investigated in COS-7 cell expression systems. The results suggest that the coexistence of IZA with CYP21 enhances 21-hydroxylase activity.

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Available from: Gavin P Vinson, Sep 24, 2014
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    • "Identical residues are indicated (*). The D86 residue of CYB5D2 and the conserved D120 residue in PGRMC1 are indicated (arrow), along with conserved tyrosine residues (Y73, Y79, Y127 in CYB5D2) previously implicated in PGRMC1 and Dap1 mediated heme-binding [6]–[8], [12], [25]. B) 293T cells were transiently transfected with empty vector (EV), CYB5D2, and the indicated CYB5D2 mutants. "
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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.
    PLoS ONE 01/2014; 9(1):e86435. DOI:10.1371/journal.pone.0086435 · 3.23 Impact Factor
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    • "However, the idea that PGRMC1 alone binds P4 is not universally accepted (Rohe et al., 2009). For example, Min et al. found that GST-tagged rat inner zone antigen [found to be identical to PGRMC1; see (Cahill, 2007)] expressed in E. coli did not bind P4 in pull-down assays (Min et al., 2005). "
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    ABSTRACT: Progesterone (P4) regulates a wide range of neural functions and likely acts through multiple receptors. Over the past 30 years, most studies investigating neural effects of P4 focused on genomic and non-genomic actions of the classical progestin receptor (PGR). More recently the focus has widened to include two groups of non-classical P4 signaling molecules. Members of the Class II progestin and adipoQ receptor (PAQR) family are called membrane progestin receptors (mPRs) and include: mPRα (PAQR7), mPRβ (PAQR8), mPRγ (PAQR5), mPRδ (PAQR6), and mPRε (PAQR9). Members of the b5-like heme/steroid-binding protein family include progesterone receptor membrane component 1 (PGRMC1), PGRMC2, neudesin, and neuferricin. Results of our recent mapping studies show that members of the PGRMC1/S2R family, but not mPRs, are quite abundant in forebrain structures important for neuroendocrine regulation and other non-genomic effects of P4. Herein we describe the structures, neuroanatomical localization, and signaling mechanisms of these molecules. We also discuss possible roles for Pgrmc1/S2R in gonadotropin release, feminine sexual behaviors, fluid balance and neuroprotection, as well as catamenial epilepsy.
    Frontiers in Neuroscience 09/2013; 7(7):164. DOI:10.3389/fnins.2013.00164 · 3.66 Impact Factor
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    • "Moreover, it has been reported that progesterone binds directly to PGRMC1 (Peluso et al., 2008). On the other hand, other reports suggest that PGRMC1 is indirectly related to progesterone activity because of adrenal steroidogenesis, but it does not bind directly to progesterone (Min et al., 2005). Nevertheless, PGRMC1 is closely involved in cellular responses to progesterone. "
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    ABSTRACT: Neudesin (neuron-derived neurotrophic factor; NENF) was identified as a neurotrophic factor that is involved in neuronal differentiation and survival. It is abundantly expressed in the central nervous system, and its neurotrophic activity is exerted via the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. Neudesin is also an anorexigenic factor that suppresses food intake in the hypothalamus. It is a member of the membrane-associated progesterone receptor (MAPR) family and shares key structural motifs with the cytochrome b5-like heme/steroid-binding domain. Progesterone receptor membrane component 1 (PGRMC1), the first to be discovered among the MAPR family, binds progesterone to induce "rapid non-genomic effects" in biological responses that are unrelated to the nuclear progesterone receptors (PRs). Hence, neudesin may also be involved in the rapid non-genomic actions of progesterone. In this review, we summarize the identification, structure, and activity of neudesin in the central nervous system, and present an essential overview of the current understanding of its physiological roles and the prospect of elucidating its non-genomic progesterone effects.
    Frontiers in Neuroscience 06/2013; 7(7):111. DOI:10.3389/fnins.2013.00111 · 3.66 Impact Factor
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