Measurement of the Heme Affinity for Yeast Dap1p, and Its Importance in Cellular Function

Thompson, Alisha; Reddi, Amit R.; Shi, Xiaoli; Goldbeck, Robert A.; Moënne‐Loccoz, Pierre; Gibney, Brian R.; Holman, Theodore R.

doi:10.1021/bi7013739

Cited by 29 publications

(46 citation statements)

References 48 publications

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“…Nonetheless, on the basis of the Nernst plot shown in Figure 1A, we estimated the redox potential of the WT protein to be −331 mV, which is similar to that reported for Dap1 (−307 mV). 24 The relatively low redox potentials of hPGRMC1 are consistent with the assignment of its proximal heme ligand to a negatively charged ligand, a tyrosinate. Intriguingly, the Y95H mutation only leads to ~+50 mV increase in the redox potential (−331 → −284 mV; Figure 1B), suggesting an axial ligand with a negative charge, possibly a hydroxide ion ( vide inf ra ).…”

Section: ■ Results and Discussionsupporting

confidence: 63%

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Spectroscopic and Mutagenesis Studies of Human PGRMC1

et al. 2015

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Progesterone receptor membrane component 1 (PGRMC1) is a 25 kDa protein with an N-terminal transmembrane domain and a putative C-terminal cytochrome b5 domain. Heme-binding activity of PGRMC1 has been shown in various homologues of PGRMC1. Although the general definition of PGRMC1 is as a progesterone receptor, progesterone-binding activity has not been directly demonstrated in any of the purified PGRMC1 proteins fully loaded with heme. Here, we show that the human homologue of PGRMC1 (hPGRMC1) binds heme in a five-coordinate (5C) high-spin (HS) configuration, with an axial tyrosinate ligand, likely Y95. The negatively charged tyrosinate ligand leads to a relatively low redox potential of approximately −331 mV. The Y95C or Y95F mutation dramatically reduces the ability of the protein to bind heme, supporting the assignment of the axial heme ligand to Y95. On the other hand, the Y95H mutation retains ~90% of the heme-binding activity. The heme in Y95H is also 5CHS, but it has a hydroxide axial ligand, conceivably stabilized by the engineered-in H95 via an H-bond; CO binding to the distal ligand-binding site leads to an exchange of the axial ligand to a histidine, possibly H95. We show that progesterone binds to hPGRMC1 and introduces spectral changes that manifest conformational changes to the heme. Our data offer the first direct evidence supporting progesterone-binding activity of PGRMC1.

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Section: ■ Results and Discussionsupporting

confidence: 63%

“…In general, the UV–vis spectra of hPGRMC1 are similar to those of the yeast analogue, Dap1. 14,24 More intriguingly, they are also analogous to a group of recently discovered heme-transport proteins with a tyrosinate as the sole axial heme ligand, such as ShuT. 14,26 …”

Section: ■ Results and Discussionmentioning

confidence: 99%

Spectroscopic and Mutagenesis Studies of Human PGRMC1

et al. 2015

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“…Pgrmc1 is required for key functions in tumor growth, promoting survival in cancer cells (21) particularly after damage from chemotherapeutic drugs (11,22). The damage resistance function of Pgrmc1 is conserved with its yeast homologues (23)(24)(25).Pgrmc1 promotes multiple phenotypes in cancer cells, including apoptotic resistance, anchorage-independent growth, invasion, tumor growth, and metastasis (21,26,27). In some cases progesterone signaling promotes the anti-apoptotic activity of Pgrmc1 (21), and Pgrmc1 was originally named Hpr6.6 (human membrane progesterone receptor) and mPR (membrane progesterone receptor) (29).…”

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confidence: 99%

“…Furthermore, in lung cancer cells progesterone inhibits growth, and Pgrmc1 antagonizes this activity(27). However, Pgrmc1 is required for some aspects of progesterone signaling through an unidentified mechanism, which may include binding to the RNA-binding protein PAIR-BP1 (plasminogen activator inhibitor 1 mRNA-binding protein (32)].Pgrmc1 is related to cytochrome b 5 and binds to heme (23,25,33,34). Pgrmc1 is highly expressed in the liver, where it likely binds to P450 proteins (34) and proteins that regulate cholesterol synthesis (35).…”

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confidence: 99%

Pgrmc1 (Progesterone Receptor Membrane Component 1) Associates with Epidermal Growth Factor Receptor and Regulates Erlotinib Sensitivity

Ahmed

Rohe

Twist

et al. 2010

Journal of Biological Chemistry

125

132

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Tumorigenesis requires the concerted action of multiple pathways, including pathways that stimulate proliferation and metabolism. Epidermal growth factor receptor (EGFR) is a transmembrane receptor-tyrosine kinase that is associated with cancer progression, and the EGFR inhibitors erlotinib/tarceva and tyrphostin/AG-1478 are potent anti-cancer therapeutics. Pgrmc1 (progesterone receptor membrane component 1) is a cytochrome b 5 -related protein that is up-regulated in tumors and promotes cancer growth. Pgrmc1 and its homologues have been implicated in cell signaling, and we show here that Pgrmc1 increases susceptibility to AG-1478 and erlotinib, increases plasma membrane EGFR levels, and co-precipitates with EGFR. Pgrmc1 co-localizes with EGFR in cytoplasmic vesicles and cofractionates with EGFR in high density microsomes. The findings have therapeutic potential because a Pgrmc1 small molecule ligand, which inhibits growth in a variety of cancer cell types, de-stabilized EGFR in multiple tumor cell lines. EGFR is one of the most potent receptor-tyrosine kinases driving tumorigenesis, and our data support a role for Pgrmc1 in promoting several cancer phenotypes at least in part by binding EGFR and stabilizing plasma membrane pools of the receptor.The proliferation of a number of cancers is driven by receptor-tyrosine kinases, which span the cell membrane and transmit signals from polypeptide hormones. Activation of the epidermal growth factor receptor (EGFR) 2 -tyrosine kinase has been linked to increased proliferation, angiogenesis, metastasis, and decreased apoptosis (1). EGFR is up-regulated in a variety of tumors (2), and EGFR and HER2/neu overexpression are associated with a poor prognosis in multiple tumor types (3). EGFR is inhibited by a growing number of drugs, including antibodies such as cetuximab and small molecule inhibitors such as erlotinib (Tarceva/OSI-774) and gefitinib (3, 4). However, the degree to which patients respond favorably to these drugs varies markedly between those expressing wild-type EGFR and mutants (5-7). One of the emerging themes in targeting kinases is the importance of processing events, intracellular transport, and interaction partners among the kinases (8,9). In the present study we demonstrate an association between EGFR and progesterone receptor membrane component 1 (Pgrmc1).Pgrmc1 is related to cytochrome b 5 and has binding sites for Src homology 2 (SH2) and SH3 domain-containing proteins and consensus phosphorylation sites for tyrosine kinases (10). Pgrmc1 is induced in a variety of cancers, including breast, thyroid, colon, ovary, and lung cancer (11-16). Furthermore, Pgrmc1 expression increases with tumor stage in ovarian cancer (11), and Pgrmc1 is elevated in estrogen receptor-negative breast tumors (17, 18). Pgrmc1 was originally identified by its induction by dioxin during liver tumorigenesis (19) and is one of six genes in a signature predicting non-genotoxic carcinogens (20). Pgrmc1 is required for key functions in tumor growth, promoting survival in cancer cells...

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“…These last two features can dominate heme affinity. For example, soluble guanylate cyclase, which has an axial histidine ligand, loses heme upon oxidation from the ferrous to the ferric state [6], whereas Dap1p, which has an axial tyrosine ligand, exhibits enhanced heme loss upon reduction from the ferric state to the ferrous state [7]. Various enzymes experience heme loss upon exposure to nitric oxide (NO • ) [8].…”

Section: Introductionmentioning

confidence: 99%

Introduction of a covalent histidine–heme linkage in a hemoglobin: A promising tool for heme protein engineering

Rice

Preimesberger

Johnson

et al. 2014

Journal of Inorganic Biochemistry

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The hemoglobins of the cyanobacteria Synechococcus and Synechocystis (GlbNs) are capable of spontaneous and irreversible attachment of the b heme to the protein matrix. The reaction, which saturates the heme 2-vinyl by addition of a histidine residue, is reproduced in vitro by preparing the recombinant apoprotein, adding ferric heme, and reducing the iron to the ferrous state. Spontaneous covalent attachment of the heme is potentially useful for protein engineering purposes. Thus, to explore whether the histidine–heme linkage can serve in such applications, we attempted to introduce it in a test protein. We selected as our target the heme domain of Chlamydomonas eugametos LI637 (CtrHb), a eukaryotic globin that exhibits less than 50% sequence identity with the cyanobacterial GlbNs. We chose two positions, 75 in the FG corner and 111 in the H helix, to situate a histidine near a vinyl group. We characterized the proteins with gel electrophoresis, absorbance spectroscopy, and NMR analysis. Both T111H and L75H CtrHbs reacted upon reduction of the ferric starting material containing cyanide as the distal ligand to the iron. With L75H CtrHb, nearly complete (> 90%) crosslinking was observed to the 4-vinyl as expected from the X-ray structure of wild-type CtrHb. Reaction of T111H CtrHb also occurred at the 4-vinyl in a 60% yield, indicating a preference for the flipped heme orientation in the starting material. The work suggests that the His–heme modification will be applicable to the design of proteins with a non-dissociable heme group.

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Measurement of the Heme Affinity for Yeast Dap1p, and Its Importance in Cellular Function

Cited by 29 publications

References 48 publications

Spectroscopic and Mutagenesis Studies of Human PGRMC1

Spectroscopic and Mutagenesis Studies of Human PGRMC1

Pgrmc1 (Progesterone Receptor Membrane Component 1) Associates with Epidermal Growth Factor Receptor and Regulates Erlotinib Sensitivity

Introduction of a covalent histidine–heme linkage in a hemoglobin: A promising tool for heme protein engineering

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