Dap1/PGRMC1 binds and regulates cytochrome P450 enzymes

Cytochrome P450 enzymes are heme-dependent monoxygenases that play a central role in human physiology. Despite the numerous physiological processes that P450 enzymes impact, the electron donors P450 oxidoreductase and cytochrome b5 are the only proteins known to interact with and modulate the activity of ER microsomal P450s. Here, we report that Dap1/PGRMC1 is required for ER P450 function in yeast and humans. We show that S. pombe Dap1 is a hemoprotein that binds and positively regulates Cyp51A1 and Cyp61A1, two P450s required for sterol biosynthesis. Similarly, loss of human PGRMC1 reduces activity of Cyp51A1, blocking cholesterol synthesis and increasing production of toxic sterol intermediates. PGRMC1 stably binds Cyp51A1 and human P450s from three additional families including Cyp3A4, which metabolizes pharmaceutical compounds. These findings demonstrate that PGRMC1 is required for P450 activity and suggest that interindividual variation in PGRMC1 function may impact multiple biochemical pathways and drug metabolism.     

Electrochemical measurement of intraprotein and interprotein electron transfer

Intramolecular and intermolecular direct (unmediated) electron transfer was studied by electrochemical techniques in a flavohemoprotein cytochrome P450 BM3 (CYP102A1 from Bacillius megaterium) and between cytochromes b ₅ and c. P450 BM3 was immobilized on a screen printed graphite electrode modified with a biocompatible nanocomposite material based on didodecyldimethylammonium bromide (DDAB) and gold nanoparticles. Analytical characteristics of SPG/DDAB/Au/P450 BM3 electrodes were studied with cyclic voltammetry and square wave voltammetry. The electron transport chain in P450 BM3 immobilized on the nanostructured electrode is: electrode → FAD → FMN → heme; i.e., electron transfer takes place inside the cytochrome, in evidence of functional interaction between its diflavin and heme domains. The effects of substrate (lauric acid) or inhibitor (metyrapone or imidazole) binding on the electro-chemical parameters of P450 BM3 were assessed. Electrochemical analysis has also demonstrated intermolecular electron transfer between electrode-immobilized and soluble cytochromes properly differing in redox potentials.     

Steroidogenic electron transport in adrenal cortex mitochondria

Summary The flavoprotein NADPH-adrenodoxin reductase and the iron sulfur protein adrenodoxin function as a short electron transport chain which donates electrons one-at-a-time to adrenal cortex mitochondrial cytochromes P-450. The soluble adrenodoxin acts as a mobile one-electron shuttle, forming a complex first with NADPH-reduced adrenodoxin reductase from which it accepts an electron, then dissociating, and finally reassociating with and donating an electron to the membrane-bound cytochrome P-450 (Fig. 9). Dissociation and reassociation with flavoprotein then allows a second cycle of electron transfers. A complex set of factors govern the sequential protein-protein interactions which comprise this adrenodoxin shuttle mechanism; among these factors, reduction of the iron sulfur center by the flavin weakens the adrenodoxinadrenodoxin reductase interaction, thus promoting dissociation of this complex to yield free reduced adrenodoxin. Substrate (cholesterol) binding to cytochrome P-450_scc both promotes the binding of the free adrenodoxin to the cytochrome, and alters the oxidation-reduction potential of the heme so as to favor reduction by adrenodoxin. The cholesterol binding site on cytochrome P-450_scc appears to be in direct communication with the hydrophobic phospholipid milieu in which this substrate is dissolved. Specific effects of both phospholipid headgroups and fatty acyl side-chains regulate the interaction of cholesterol with its binding side. Cardiolipin is an extremely potent positive effector for cholesterol binding, and evidence supports the existence of a specific effector lipid binding site on cytochrome P.450_scc to which this phospho-lipid binds.     

Functional characterization of the C-terminal domain of the cytochrome c maturation protein CcmE

CcmE is a heme chaperone involved in the periplasmic maturation of c-type cytochromes in many bacteria and plant mitochondria. It binds heme covalently and subsequently transfers it to the apo form of cytochromes c. To examine the role of the C-terminal domain of CcmE in the binding of heme, in vitro heme binding to the apo form of a truncated (immediately before Pro-136) version of the periplasmic domain of the heme chaperone from Escherichia coli was studied. Removal of the C-terminal domain dramatically altered the ligation of non-covalently bound heme in CcmE' (the soluble form lacking the membrane anchor) but only slightly affected its affinity for protoporphyrin IX and 8-anilino-1-naphthalenesulfonate. This finding has significant mechanistic implications for in vivo holo-CcmE formation and indicates that the C-terminal region is not required for the recruitment and docking of heme into its binding site but is likely to contain amino acid(s) involved in heme iron axial coordination. Removal of the C-domain significantly impaired in vivo heme binding to CcmE and conversion of apocytochrome to holoprotein by a similar factor, suggesting that the C-terminal domain of the chaperone is primarily involved in heme binding to CcmE rather than in heme transfer to the apo cytochrome.     

The one-electron autoxidation of human cytochrome P450 3A4

Monomeric cytochrome P450 3A4 (CYP3A4), the most prevalent cytochrome P450 in human liver, can simultaneously bind one, two, or three molecules of substrates and effectors. The difference in the functional properties of such binding intermediates gives rise to homotropic and heterotropic cooperative kinetics of this enzyme. To understand the overall kinetic processes operating in CYP3A4, we documented the kinetics of autoxidation of the oxy-ferrous intermediate of CYP3A4 as a function of testosterone concentration. The rate of autoxidation in the presence of testosterone was significantly lower than that observed with no substrate present. Stability of the oxy-ferrous complex in CYP3A4 and the amplitude of the geminate CO rebinding increased significantly as a result of binding of just one testosterone molecule. In contrast, the slow phase in the kinetics of cyanide binding to the ferric CYP3A4 correlated with a shift of the heme iron spin state, which is only caused by the association of a second molecule of testosterone. Our results show that the first substrate binding event prevents the escape of diatomic ligands from the distal heme binding pocket, stabilizes the oxy-ferrous complex, and thus serves as an important modulator of the uncoupling channel in the cytochromes P450.     

The role of cytochrome b 5 structural domains in interaction with cytochromes P450

To understand the role of the structural elements of cytochrome b ₅ in its interaction with cytochrome P450 and the catalysis performed by this heme protein, we carried out comparative structural and functional analysis of the two major mammalian forms of membrane-bound cytochrome b ₅ — microsomal and mitochondrial, designed chimeric forms of the heme proteins in which the hydrophilic domain of one heme protein is replaced by the hydrophilic domain of another one, and investigated the effect of the highly purified native and chimeric heme proteins on the enzymatic activity of recombinant cytochromes P4503A4 and P45017A1 (CYP3A4 and CYP17A1). We show that the presence of a hydrophobic domain in the structure of cytochrome b ₅ is necessary for its effective interaction with its redox partners, while the nature of the hydrophobic domain has no significant effect on the ability of cytochrome b ₅ to stimulate the activity of cytochrome P450-catalyzed reactions. Thus, the functional properties of cytochrome b ₅ are mainly determined by the structure of the hemebinding domain.     

Orientation of cytochromes P450 in the endoplasmic reticulum

The orientation of eukaryotic cytochromes P450, with respect to the membrane of the endoplasmic reticulum, has been investigated. There is now good evidence that the tertiary structure of these proteins is essentially the same as that of the soluble bacterial isoenzyme cytochrome P450CI, with the exception of an extension at the N-terminus which is thought to form a membrane-anchoring sequence. The remainder of the molecule protrudes from the cytosolic face of the membrane so that it can interact with substrates and electron-donating proteins. Two models based on this structure have been considered, in which the plane of the heme of cytochrome P450 is oriented either parallel with or perpendicular to the plane of the membrane of the endoplasmic reticulum. The validity of these models has been assessed from the results of studies involving the binding of antipeptide antibodies directed toward known regions of cytochromes P450, modeling of the interaction of cytochrome P450 with cytochrome b5, proposed intramolecular movements of cytochrome P450 during its catalytic cycle, and the partitioning of substrates for cytochrome P450 between the cytosol and membrane. It is concluded that cytochrome P450 is most likely oriented such that the heme is not fixed horizontal to the plane of the membrane of the endoplasmic reticulum and may well lie with the heme perpendicular to the membrane.     

Specificity of Isatin Interaction with Cytochromes P450

Cytochrome P450-dependent monooxygenase systems exist basically in all living organisms, where they perform various important functions. The coordinated functioning of these systems involves many proteins participating in different protein-protein interactions (PPI). Previously, we have found that the endogenous non-peptide bioregulator isatin (indoledione-2,3), synthesized from indole by means of certain cytochromes P450 (e.g. P450 2E1, P450 2C19, P450 2A6) regulates affinity of some PPI. In this study, an attempt has been undertaken to register a direct interaction of isatin with a set of different proteins related to the functioning of cytochrome P450-dependent monooxygenase systems: five isoforms of cytochromes P450, two isoforms of cytochrome b₅, cytochrome P450 reductase, adrenodoxin, adrenodoxin reductase and ferrochelatase. The study has shown high affinity specific binding of isatin only to cytochromes P450 (the equilibrium dissociation constant (K_d) is about 10^–8 M).     

Progesterone receptor membrane component 1/Sigma-2 receptor associates with MAP1LC3B and promotes autophagy

Autophagy resembles a recycling process in which proteins, organelles, or regions of the cytoplasm are enveloped and degraded. We have found that two of the central autophagy proteins, MAP1LC3 (microtubule-associated protein 1 light chain 3, also described as LC3) and UVRAG (UV radiation resistance associated/UV radiation associated gene), complex with PGRMC1/S2R (progesterone receptor membrane component 1, also known as sigma-2 receptor). PGRMC1 is a cytochrome that is induced in cancer and is essential for tumor formation, invasion, and metastasis. Autophagy contributes to the turnover of long-lived and/or ubiquitinated proteins and the clearance of damaged organelles, and we have shown that PGRMC1 promotes both processes. Inhibition of PGRMC1 by RNAi or small molecule inhibitors causes autophagy substrates to increase and aberrant mitochondria to accumulate. We propose that this disruption of autophagy upon PGRMC1 inhibition increases AMPK activation, elevating the levels of TSC1 (tuberous sclerosis complex) and TSC2 and inactivating MTOR and RPS6KB/p70S6K, causing cleaved MAP1LC3B levels to increase. Thus, PGRMC1 binds to key components of the autophagy machinery and is required for the degradative activity of autophagy.     

The C-terminal flexible domain of the heme chaperone CcmE is important but not essential for its function

CcmE is a heme chaperone active in the cytochrome c maturation pathway of Escherichia coli. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome c. The recently solved structure of soluble CcmE revealed a compact core consisting of a beta-barrel and a flexible C-terminal domain with a short alpha-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type c cytochromes, only one additional residue, D131, was required, and for efficient cytochrome c maturation, the seven-residue sequence (131)DENYTPP(137) was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome c maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE.     

Mammalian cytochromes P450—Importance of tissue specificity

Mammals express multiple cytochromes P450 simultaneously in a variety of tissues, including the liver, kidney, lung, adrenal, gonads, brain, and most others. For cytochromes P450 that are expressed in many tissues or cell types, the tissue/cell type-specific expression might be associated with their special physiological roles. Several cytochrome P450 enzymes are found not only in different cell types and tissues, but also in different subcellular compartments. Generally, all mammalian cytochrome P450 enzymes are membrane bound. The two major groups are represented by microsomal cytochromes P450 that reside in the endoplasmic reticulum, and mitochondrial cytochromes P450, that reside in the inner mitochondrial membrane. However, the outer nuclear membrane, different Golgi compartments, peroxisomes and the plasma membrane are also sites where cytochromes P450 were observed. For example, CYP51 is an ER enzyme in majority of tissues but in male germ cells it trafficks through the Golgi to acrosome, where it is stabilized for several weeks. Surprisingly, in brains of heme synthesis deficient mice, a soluble form of CYP1A1 was detected whose activity has been restored by the addition of heme. In the majority of cases each cytochrome P450 enzyme resides in a single subcellular compartment in a certain cell, however, examples of simultaneous localization in different subcellular compartments have also been described, such as endoplasmic reticulum, Golgi and plasma membrane for CYP2E1. This review will focus on the physiological importance of mammalian cytochrome P450 expression and localization in different tissues or cell types and subcellular compartments.     

Mammalian cytochromes P450--importance of tissue specificity

Mammals express multiple cytochromes P450 simultaneously in a variety of tissues, including the liver, kidney, lung, adrenal, gonads, brain, and most others. For cytochromes P450 that are expressed in many tissues or cell types, the tissue/cell type-specific expression might be associated with their special physiological roles. Several cytochrome P450 enzymes are found not only in different cell types and tissues, but also in different subcellular compartments. Generally, all mammalian cytochrome P450 enzymes are membrane bound. The two major groups are represented by microsomal cytochromes P450 that reside in the endoplasmic reticulum, and mitochondrial cytochromes P450, that reside in the inner mitochondrial membrane. However, the outer nuclear membrane, different Golgi compartments, peroxisomes and the plasma membrane are also sites where cytochromes P450 were observed. For example, CYP51 is an ER enzyme in majority of tissues but in male germ cells it trafficks through the Golgi to acrosome, where it is stabilized for several weeks. Surprisingly, in brains of heme synthesis deficient mice, a soluble form of CYP1A1 was detected whose activity has been restored by the addition of heme. In the majority of cases each cytochrome P450 enzyme resides in a single subcellular compartment in a certain cell, however, examples of simultaneous localization in different subcellular compartments have also been described, such as endoplasmic reticulum, Golgi and plasma membrane for CYP2E1. This review will focus on the physiological importance of mammalian cytochrome P450 expression and localization in different tissues or cell types and subcellular compartments.     

Progesterone receptor membrane component 1: an integrative review

Progesterone receptor membrane component 1 (PGRMC1) contains a cytochrome b5 domain fold and belongs to the so-called membrane-associated progesterone receptor (MAPR) protein family that is widespread in eukaryotes. PGRMC1 and the related PGRMC2 mammalian family member diverged sometime after the evolution of segmented metazoan body plan and the appearance of vertebrates. Therefore PGRMC1 might be expected to be involved in some ancient eukaryotic processes, as well as more modern functions related to multicellularity and tissue interactions. Perhaps this explains the perplexing diversity of contexts where PGRMC1 has been observed, apparently being involved in different cellular processes at various sub-cellular locations. This review attempts to collate and interpret these observations. Ironically, despite being the archetypal member of the MAPR family, it has yet to be demonstrated that PGRMC1 exhibits specific progesterone binding. Potential roles of heme and steroid/sterol ligands are reviewed, as well as the implications of apparent target sequences within PGRMC1 for binding by SH2- and SH3-domain proteins as well as kinases. These motifs are modelled using the cytochrome b5 domain NMR structure of the Arabidopsis protein 1J03, implicating a possible function for PGRMC1 as an adaptor protein involved in regulating protein interactions and intracellular signal transduction and/or membrane trafficking. This interpretation is supported by the apparent presence of immunoreceptor tyrosine-based activation motif/ITAM sequences that are involved in endocytosis and vesicle targeting, and the colocalisation of PGRMC1 with caveolin and at the cytoplasmic membrane. Evidence for roles in disease, especially cancer, is also discussed.     

The interaction of covalently bound heme with the cytochrome c maturation protein CcmE

The heme chaperone CcmE is a novel protein that binds heme covalently via a histidine residue as part of its essential function in the process of cytochrome c biogenesis in many bacteria as well as plant mitochondria. In the continued absence of a structure of the holoform of CcmE, identification of the heme ligands is an important step in understanding the molecular function of this protein and the role of covalent heme binding to CcmE during the maturation of c-type cytochromes. In this work, we present spectroscopic data that provide insight into the ligation of the heme iron in the soluble domain of CcmE from Escherichia coli. Resonance Raman spectra demonstrated that one of the heme axial ligands is a histidine residue and that the other is likely to be Tyr134. In addition, the properties of the heme resonances of the holo-protein as compared with those of a form of CcmE with non-covalently bound heme provide evidence for the modification of one of the heme vinyl side chains by the protein, most likely the 2-vinyl group.     

Characterization of the heme of cytochrome P-450 using gas chromatography-mass spectrometry

The heme moieties of cytochromes P-450 and P₁-450 (P-448) have been characterized. CO-binding particles, devoid of cytochrome b₅, were prepared from normal or 3-methylcholanthrene-treated animals. Heme was removed by acid-acetone treatment of the CO-binding particles and crystallized. Heme isolated from hemoglobin of the corresponding animals served as a control. Reductive degradation (hydriodic acid) followed by gas chromatography/mass spectrometry analysis indicated the presence of opso-, crypto-, hemo-, and phyllopyrrole. Visible spectra of the iron-free tetrapyrroles isolated from microsomal heme and hemoglobin were identical and showed typical aetioporphyrin spectra. Finally, the mass spectra of the tetrapyrrole dimethyl esters isolated from microsomal heme and hemoglobin were identical to authentic protoporphyrin IX dimethyl ester. These data strongly suggest that the heme of cytochrome P-450 and P₁-450 are identical and are the same the same as that of hemoglobin, namely protoporphyrin IX.     

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

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 b₅-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 co-fractionates 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.     

Purification and characterization of a cytosolic cytochrome P450 from the yeast Trichosporon cutaneum

A soluble cytochrome P450 from the yeast Trichosporon cutaneum was purified to homogeneity, using ammonium sulfate fractionation followed by fast protein liquid chromatography (FPLC) with DEAE-cellulose and phenyl-Sepharose columns. This procedure resulted in a 45-fold increase in specific activity with an activity yield of 6.8%. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified enzyme was homogeneous and had a molecular mass of 45 kDa. The purified enzyme contained a heme group and had a characteristic absorption peak at 448 nm in the reduced carbon monoxide difference spectrum. This enzyme was a monomeric protein and catalyzed the conversion of salicylic acid to catechol in the presence of NADH or NADPH. The N-terminal amino acid sequence indicated that the Trichosporon cutaneum cytochrome P450 did not show homology to most eukaryotic cytochromes P450, but had a high degree of homology to one cytochrome P450, the nitric oxide reductase, of Fusarium oxysporum.     

Candida albicans NADPH-P450 reductase: Expression, purification, and characterization of recombinant protein

Candida albicans is responsible for serious fungal infections in humans. Analysis of its genome identified NCP1 gene coding for a putative NADPH-P450 reductase (NPR) enzyme. This enzyme appears to supply reducing equivalents to cytochrome P450 or heme oxygenase enzymes for fungal survival and virulence. In this study, we report the characterization of the functional features of NADPH-P450 reductase from C. albicans. The recombinant C. albicans NPR protein harboring a 6×(His)-tag was expressed heterologously in Escherichia coli, and was purified. Purified C. albicans NPR has an absorption maximum at 453 nm, indicating the feature of an oxidized flavin cofactor, which was decreased by the addition of NADPH. It also evidenced NADPH-dependent cytochrome c or nitroblue tetrazolium reducing activity. This purified reductase protein was successfully able to substitute for purified mammalian NPR in the reconstitution of the human P450 1A2-catalyzed O-deethylation of 7-ethoxyresorufin. These results indicate that purified C. albicans NPR is an orthologous reductase protein that supports cytochrome P450 or heme oxygenase enzymes in C. albicans.