Natalia Mast - Academia.edu (original) (raw)

Papers by Natalia Mast

Neurotherapeutics

Cholesterol and 24-hydroxycholesterol are the most abundant brain sterols and represent the subst... more Cholesterol and 24-hydroxycholesterol are the most abundant brain sterols and represent the substrate and product, respectively, of cytochrome P450 46A1 (CYP46A1), a CNS-specific enzyme. CYP46A1 controls cholesterol elimination and turnover in the brain, the two processes that determine the rate of brain sterol flux through the plasma membranes and thereby the properties of these membranes. Brain sterol flux is decreased in Cyp46a1−/− mice compared to wild-type mice and increased in 5XFAD mice (a model of Alzheimer’s disease) when they are treated with a small dose of efavirenz, a CYP46A1 activator. Herein, we first assessed the brain proteome (synaptosomal fractions) and phospho-proteome (synaptosomal fractions and brain homogenates) of efavirenz-treated and control 5XFAD mice. Then, based on the pattern of protein abundance change, we conducted acetyl-CoA measurements (brain homogenates and mitochondria) and metabolic profiling (brain homogenates). The phospho-proteomics datasets ...

Biology

Cholesterol homeostasis in the retina, a sensory organ in the back of the eye, has been studied i... more Cholesterol homeostasis in the retina, a sensory organ in the back of the eye, has been studied in mice but not hamsters, despite the latter being more similar to humans than mice with respect to their whole-body cholesterol maintenance. The goal of this study was to begin to assess hamster retina and conduct initial interspecies comparisons. First, young (3-month old) and mature (6-month old) Syrian (golden) hamsters were compared with 3- and 6-month old mice for ocular biometrics and retinal appearance on optical coherence tomography and fluorescein angiography. Of the 30 evaluated hamsters, seven had retinal structural abnormalities and all had increased permeability of retinal blood vessels. However, hamsters did not carry the mutations causing retinal degenerations 1 and 8, had normal blood glucose levels, and only slightly elevated hemoglobin A1c content. Cholesterol and six other sterols were quantified in hamster retina and compared with sterol profiles in mouse and human re...

PLOS ONE

Cytochrome P450 46A1 (CYP46A1) converts cholesterol to 24-hydroxycholesterol and thereby controls... more Cytochrome P450 46A1 (CYP46A1) converts cholesterol to 24-hydroxycholesterol and thereby controls the major pathways of cholesterol removal from the brain. Cyp46a1-/mice have a reduction in the rate of cholesterol biosynthesis in the brain and significant impairments to memory and learning. To gain insights into the mechanisms underlying Cyp46a1-/phenotype, we used Cyp46a1-/mice and quantified their brain sterol levels and the expression of the genes pertinent to cholesterol homeostasis. We also compared the Cyp46a1-/and wild type brains for protein phosphorylation and ubiquitination. The data obtained enable the following inferences. First, there seems to be a compensatory upregulation in the Cyp46a1-/brain of the pathways of cholesterol storage and CYP46A1-independent removal. Second, transcriptional regulation of the brain cholesterol biosynthesis via sterol regulatory element binding transcription factors is not significantly activated in the Cyp46a1-/brain to explain a compensatory decrease in cholesterol biosynthesis. Third, some of the liver X receptor target genes (Abca1) are paradoxically upregulated in the Cyp46a1-/brain, possibly due to a reduced activation of the small GTPases RAB8, CDC42, and RAC as a result of a reduced phosphorylation of RAB3IP and PAK1. Fourth, the phosphorylation of many other proteins (a total of 146) is altered in the Cyp46a1-/brain, including microtubule associated and neurofilament proteins (the MAP and NEF families) along with proteins related to synaptic vesicles and synaptic neurotransmission (e.g., SLCs, SHANKs, and BSN). Fifth, the extent of protein ubiquitination is increased in the Cyp46a1-/brain, and the affected proteins pertain to ubiquitination (UBE2N), cognition (STX1B and ATP1A2), cytoskeleton function (TUBA1A and YWHAZ), and energy production (ATP1A2 and ALDOA). The present study demonstrates the diverse potential effects of CYP46A1 deficiency on brain functions and identifies important proteins that could be affected by this deficiency.

Journal of lipid research, Jan 17, 2018

Apolipoprotein E (APOE) is a component of lipid-transporting particles and a recognition ligand f... more Apolipoprotein E (APOE) is a component of lipid-transporting particles and a recognition ligand for receptors, which bind these particles. The APOE isoform 2 is a risk factor for age-related macular degeneration; nevertheless APOE absence in humans and mice does not significantly affect the retina. We found that retinal cholesterol biosynthesis and the levels of retinal cholesterol were increased in mice, whereas cholesterol elimination by metabolism was decreased. No focal cholesterol deposits were observed in the retina. Retinal proteomics identified the most abundant cholesterol-related proteins in wild type mice and revealed that of these cholesterol-related proteins, only APOA4 had increased expression in the retina. In addition, there were changes in retinal abundance of proteins involved in pro- and anti-inflammatory responses, cellular cytoskeleton maintenance, vesicular traffic, and retinal iron homeostasis. The data obtained indicate that when APOE is absent, particles con...

The Journal of biological chemistry, Aug 22, 2017

Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase) is the enzyme responsible for the majo... more Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase) is the enzyme responsible for the majority of cholesterol elimination from the brain. Previously, we found that the anti-HIV drug efavirenz (EFV) can pharmacologically activate CYP46A1 in mice. Herein, we investigated whether CYP46A1 could also be activated by endogenous compounds, including major neurotransmitters. In vitro experiments with purified recombinant CYP46A1 indicated that CYP46A1 is activated by L-glutamate (L-Glu), L-aspartate, γ-aminobutyric acid, and acetylcholine, with L-Glu eliciting the highest increase (3-fold) in CYP46A1-mediated cholesterol 24-hydroxylation. We also found that L-Glu and other activating neurotransmitters bind to the same site on the CYP46A1 surface, which differs from the EFV-binding site. The other principal differences between EFV and L-Glu in CYP46A1 activation include an apparent lack of L-Glu binding to the P450 active site and different pathways of signal transduction from the all...

The Journal of biological chemistry, Mar 11, 2017

Cytochrome P450 27A1 (CYP27A1 or sterol 27-hydroxylase) is a ubiquitous, multifunctional enzyme c... more Cytochrome P450 27A1 (CYP27A1 or sterol 27-hydroxylase) is a ubiquitous, multifunctional enzyme catalyzing regio- and stereo-specific hydroxylation of different sterols. In humans, complete CYP27A1 deficiency leads to cerebrotendinous xanthomatosis or nodule formation in tendons and brain (preferentially in the cerebellum) rich in cholesterol and cholestanol, the 5α-saturated analog of cholesterol. In Cyp27a1(-/-) mice, xanthomas are not formed, despite a significant cholestanol increase in the brain and cerebellum. The mechanism behind cholestanol production has been clarified, yet little is known about its metabolism, except that CYP27A1 might metabolize cholestanol. It also is unclear why CYP27A1 deficiency results in preferential cholestanol accumulation in the cerebellum. We hypothesized that cholestanol might be metabolized by CYP46A1, the principal cholesterol 24-hydroxylase in the brain. We quantified sterols along with CYP27A1 and CYP46A1 in mouse models (Cyp27a1(-/-) , Cyp...

Archives of Biochemistry and Biophysics, Aug 1, 2004

Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic ... more Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic and substrate-binding properties of four truncated cytochromes P450 46A1 were investigated in the present study. All four lacked the N-terminal transmembrane region (residues 3-27), and, in addition, D46A1H had a 4Â His-tag fused to the C-terminus; HD46A1 had the N-terminal 4Â His-tag; HD46A1D had a 4Â His-tag at the N-terminus and did not contain a proline-rich region at the C-terminus (residues 494-499); and D46A1D lacked the C-terminal proline-rich region. The truncated enzymes were expressed at 390-650 nmol/L culture levels, distributed at about a 1:1 ratio between the membrane fraction and the cytosol in low ionic strength buffer, and were predominantly monomers in detergent-free buffer. They had moderately decreased catalytic efficiencies for either cholesterol or 24S-hydroxycholesterol or both, whereas their substrate-binding constants were either unchanged or decreased 2-fold. The two forms, D46A1D and HD46A1D, both lacking the C-terminal proline-rich region seem to be good candidates for future crystallographic studies because they contain only 0.3-0.8% of high molecular weight aggregates and their catalytic efficiencies are decreased no more than 2.3-fold.

Biochemistry (Moscow)

Addition of an amphipathic bee venom peptide, melittin, to sarcoplasmic reticulum (SR) vesicles i... more Addition of an amphipathic bee venom peptide, melittin, to sarcoplasmic reticulum (SR) vesicles isolated from rabbit skeletal muscles resulted in a fast (<1 min) blue shift in the fluorescence maximum of the melittin--SR membrane complex. Over the following 45 min the position of the fluorescence maximum did not change, but the fluorescence intensity of the melittin--SR membrane complex decreased by approximately 35% with rate constant 0.14 min-1. Melittin rapidly quenched the isotropic signal in the EPR spectrum of spin-labeled stearic acid added to SR membranes. Further changes in the spectral parameters of the spin probe bound to SR membranes in the presence of melittin indicated an increase of the viscosity of the probe microenvironment (empiric parameter T/eta was decreased by approximately 35% with rate constant 0.11 min-1). The surface potential of SR membranes measured using a pH-sensitive dye, neutral red, decreased after melittin addition from -60 to -30 mV. It was demonstrated with the use of a cross-linking agent, cupric o-phenanthroline, that melittin induced slow aggregation of Ca-ATPase protein in SR membranes; the content of enzyme in the monomeric form decreased with rate constant 0.14 min-1. It is concluded that melittin binds rapidly to SR membranes, inducing slow changes in Ca-ATPase conformation and oligomeric state as well as structural transitions in the lipid bilayer of SR membranes.

Biochemistry (Moscow)

The inhibition of Na,K-ATPase from duck salt gland and Ca-ATPase from rabbit skeletal muscle sarc... more The inhibition of Na,K-ATPase from duck salt gland and Ca-ATPase from rabbit skeletal muscle sarcoplasmic reticulum by melittin, a 26-residue peptide from bee venom, was studied. Melittin irreversibly inhibits both enzymes. At melittin/ATPase molar ratio (30-50):1, the time dependence of the inhibition is described by the sum of two exponential curves. At pH 7.0, the fast phase of the inhibition provides for about 50% of total loss of activity with pseudo-first order rate constants of 1.52 +/- 0.17 and 1.20 +/- 0.21 min-1 for Na,K- and Ca-ATPase, respectively. The corresponding pseudo-first order rate constants for the slow phase were 0.12 +/- 0.02 and 0.09 +/- 0.02 min-1. The inhibition of both enzymes by melittin depends upon pH; the inhibition increases when the pH is increased from 6.0 to 8.5. The enhancement of the inhibition concomitant to increase in pH is mainly due to an increase in the rate constant of the fast phase. ATP protects both enzymes from the inhibition by melitt...

Biochemistry. Biokhimii͡a, 2003

Proteins interacting with alpha 1 beta 1-type of Na,K-ATPase were revealed in pig kidney outer me... more Proteins interacting with alpha 1 beta 1-type of Na,K-ATPase were revealed in pig kidney outer medulla and duck salt glands using three different methods (immunoprecipitation, protein overlay, and chemical cross-linking). Immunoprecipitation was performed after solubilization of protein homogenate with Triton X-100 so that both membrane and cytosol proteins bound to Na,K-ATPase could be revealed. Two other methods were used to study the interaction of cytosol proteins with purified Na,K-ATPase. The sets of proteins revealed by each method in outer medulla of pig kidney were different. Proteins interacting with Na,K-ATPase that have molecular masses 10, 15, 70, 75, 105, 120, and 190 kD were found using the immunoprecipitation method. The chemical cross-linking method revealed proteins with molecular masses 25, 35, 40, 58, 68-70, and 86-88 kD. The protein overlay method revealed in the same tissue proteins with molecular masses 38, 42, 43, 60, 62, 66, 70, and 94 kD.

Biochemistry, 2005

The conversion of cholesterol to 7alpha-hydroxycholesterol catalyzed by cytochrome P450 7A1 (CYP7... more The conversion of cholesterol to 7alpha-hydroxycholesterol catalyzed by cytochrome P450 7A1 (CYP7A1) initiates the major pathway for cholesterol elimination in mammals. In the present work we focused on identification of determinants of the CYP7A1 substrate specificity inside the active site using a homology model with a novel P450-fold, site-directed mutagenesis, and substrate-binding and kinetic studies. Forty-one mutants, encompassing twenty-six amino acid residues, were generated and characterized, and of these, seven residues appear to determine cholesterol binding in the active site. In addition, four cholesterol derivatives were used as active site probes in the wild type and the seven mutant enzymes, and the spectral binding constants and products were analyzed. It was concluded that Asn288 in the I helix plays a key role in the P450-cholesterol contacts by hydrogen bonding to the steroid 3beta-hydroxyl, while Val280 and Ala284 are beside and the Trp283 is above the steroid nucleus orienting the cholesterol molecule. Leu360 and Ala358 between the K helix and the beta1-4 strand and Leu485 in the beta4 sheet-turn appear to define the size of the active site over the heme pyrrole ring A, thus limiting the orientation and size of the substrate at the steroid A ring. Additionally, the A358V mutant was found to form two new products, one being 7beta-hydroxycholesterol. Our data indicate that a tight fit of cholesterol in the enzyme active site is in part responsible for the high efficiency of cholesterol turnover by CYP7A1.

Biochemistry, 2003

The known activity of cytochrome P450 46A1 (P450 46A1) is 24(S)-hydroxylation of cholesterol. Thi... more The known activity of cytochrome P450 46A1 (P450 46A1) is 24(S)-hydroxylation of cholesterol. This reaction produces biologically active oxysterol, 24(S)-hydroxycholesterol, and is also the first step in enzymatic degradation of cholesterol in the brain. We report here that P450 46A1 can further metabolize 24(S)-hydroxycholesterol, giving 24,25- and 24,27-dihydroxycholesterols in both the cell cultures transfected with P450 46A1 cDNA and the in vitro reconstituted system with recombinant enzyme. In addition, P450 46A1 was able to carry out side chain hydroxylations of two endogenous C27-steroids with and without a double bond between C5-C6 (7alpha-hydroxycholesterol and cholestanol, respectively) and introduce a hydroxyl group on the steroid nucleus of the C21-steroid hormones with the C4-C5 double bond (progesterone and testosterone). Also, P450 46A1 was found to metabolize xenobiotics carrying out dextromethorphan O- and N-demethylations, diclofenac 4'-hydroxylation, and phenacetin O-deethylation. Thus, substrate specificities of P450 46A1 are not limited to cholesterol and include a number of structurally diverse compounds. Activities of P450 46A1 suggest that, in addition to the involvement in cholesterol homeostasis in the brain, this enzyme may participate in metabolism of neurosteroids and drugs that can cross the blood-brain barrier and are targeted to the central nervous system.

Biochemistry, 1997

Incubation of melittin with sarcoplasmic reticulum membranes at pH 7.0 and different melittin: Ca... more Incubation of melittin with sarcoplasmic reticulum membranes at pH 7.0 and different melittin: Ca-ATPase molar ratios results in the progressive loss of enzyme activity. At high melittin:Ca-ATPase molar ratios (10:1 and 30:1), enzyme inhibition may be described by a biexponential curve. At pH 7.0, the values of the pseudo-first-order rate constants are 1.0 and 0.1 min -1 for the fast and slow phases of inhibition, respectively, at a melittin:Ca-ATPase molar ratio of 30:1. At pH 6.0 and a melittin:Ca-ATPase molar ratio of 30:1, melittin does not inhibit Ca-ATPase. Melittin-induced aggregation of Ca-ATPase molecules was studied using cupric phenanthroline as a chemical cross-linking agent. At a melittin:Ca-ATPase molar ratio of 5:1, aggregation of Ca-ATPase protein was not observed; however, the loss of enzyme activity was about 30% after 30 min. At melittin:Ca-ATPase molar ratios of 10:1 and 30:1, significant aggregation of Ca-ATPase protein takes place. The rate of Ca-ATPase aggregation is much lower than the rate of enzyme inhibition. At melittin:Ca-ATPase molar ratios of 10:1 and 30:1, the rate of Ca-ATPase protein aggregation is close to that for the slow phase of enzyme inhibition. At pH 6.0 and a melittin:Ca-ATPase molar ratio of 30:1, significant aggregation of Ca-ATPase occurs. It is concluded that melittin induces both Ca-ATPase inhibition and aggregation. These two processes may occur simultaneously, but under some conditions either inhibition or aggregation takes place independently of each other. Therefore, the aggregation of Ca-ATPase induced by melittin is not necessary for enzyme inhibition. adenine dinucleotide, reduced form; BSA, bovine serum albumin.

Archives of Biochemistry and Biophysics, 2004

Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic ... more Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic and substrate-binding properties of four truncated cytochromes P450 46A1 were investigated in the present study. All four lacked the N-terminal transmembrane region (residues 3-27), and, in addition, D46A1H had a 4Â His-tag fused to the C-terminus; HD46A1 had the N-terminal 4Â His-tag; HD46A1D had a 4Â His-tag at the N-terminus and did not contain a proline-rich region at the C-terminus (residues 494-499); and D46A1D lacked the C-terminal proline-rich region. The truncated enzymes were expressed at 390-650 nmol/L culture levels, distributed at about a 1:1 ratio between the membrane fraction and the cytosol in low ionic strength buffer, and were predominantly monomers in detergent-free buffer. They had moderately decreased catalytic efficiencies for either cholesterol or 24S-hydroxycholesterol or both, whereas their substrate-binding constants were either unchanged or decreased 2-fold. The two forms, D46A1D and HD46A1D, both lacking the C-terminal proline-rich region seem to be good candidates for future crystallographic studies because they contain only 0.3-0.8% of high molecular weight aggregates and their catalytic efficiencies are decreased no more than 2.3-fold.

The Journal of biological chemistry, Jan 23, 2016

The process of vision is impossible without the photoreceptor cells, which have a unique structur... more The process of vision is impossible without the photoreceptor cells, which have a unique structure and specific maintenance of cholesterol. Herein we report on the previously unrecognized cholesterol-related pathway in the retina discovered during follow-up characterizations of Cyp27a1(-/-)Cyp46a1(-/-) mice. These animals have retinal hypercholesterolemia and convert excess retinal cholesterol into cholesterol esters, normally present in the retina in very small amounts. We established that in the Cyp27a1(-/-)Cyp46a1(-/-) retina, cholesterol esters are generated by and accumulate in the photoreceptor outer segments (OS), which is the retinal layer with the lowest cholesterol content. Mouse OS were also found to express the cholesterol-esterifying enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT1), but not lecithin-cholesterol acyltransferase (LCAT), and to differ from humans in retinal expression of ACAT1. Nevertheless, cholesterol esters were discovered to be abundant in hu...

Acta Crystallogr D Biol Cryst, 2009

Human cytochrome P450 46A1 (CYP46A1) is one of the key enzymes in cholesterol homeostasis in the ... more Human cytochrome P450 46A1 (CYP46A1) is one of the key enzymes in cholesterol homeostasis in the brain. The crystallization and heavy-atom structure solution of an active truncated CYP46A1 in complex with the high-affinity substrate analogue cholesterol-3-sulfate (CH-3S) is reported. The 2.6 angstroms structure of CYP46A1-CH-3S was solved using both anion and cation heavy-atom salts. In addition to the native anomalous signal from the haem iron, an NaI anion halide salt derivative and a complementary CsCl alkali-metal cation salt derivative were used. The general implications of the use of halide and alkali-metal quick soaks are discussed. The importance of using isoionic strength buffers, the titration of heavy-atom salts into different ionic species and the role of concentration are considered. It was observed that cation/anion-binding sites will occasionally overlap, which could negatively impact upon mixed RbBr soaks used for multiple anomalous scatterer MAD (MMAD). The use of complementary cation and anion heavy-atom salt derivatives is a convenient and powerful tool for MIR(AS) structure solution.

The Journal of biological chemistry, Jan 27, 2016

Cytochrome P450 46A1 (CYP46A1) is a microsomal enzyme and cholesterol 24-hydroxylase that control... more Cytochrome P450 46A1 (CYP46A1) is a microsomal enzyme and cholesterol 24-hydroxylase that controls cholesterol elimination from the brain. This P450 is also a potential target for Alzheimer disease because it can be activated pharmacologically by some marketed drugs, as exemplified by efavirenz, the anti-HIV medication. Previously, we suggested that pharmaceuticals activate CYP46A1 allosterically through binding to a site on the cytosolic protein surface, which is different from the enzyme active site facing the membrane. Here we identified this allosteric site for efavirenz on CYP46A1 by using a combination of hydrogen-deuterium exchange coupled to MS, computational modeling, site-directed mutagenesis, and analysis of the CYP46A1 crystal structure. We also mapped the binding region for the CYP46A1 redox partner oxidoreductase and found that the allosteric and redox partner binding sites share a common border. On the basis of the data obtained, we propose the mechanism of CYP46A1 al...

Lipids, Sep 13, 2008

Mitochondrial cytochrome P450 enzymes (CYP or P450, EC 1.14.13.15) play an important role in meta... more Mitochondrial cytochrome P450 enzymes (CYP or P450, EC 1.14.13.15) play an important role in metabolism of cholesterol. CYP27A1 hydroxylates cholesterol at position 27 and, thus, initiates cholesterol removal from many extrahepatic tissues. CYP11A1 is a steroidogenic P450 that converts cholesterol to pregnenolone, the first step in the biosynthesis of all steroid hormones. We utilized a new approach to study membrane topology of CYPs 27A1 and 11A1. This approach involves heterologous expression of membrane-bound P450 in E. coli, isolation of the P450-containing E. coli membranes, treatment of the membranes with protease, removal of the digested soluble portion and extraction of the membrane-associated peptides, which are then identified by mass spectrometry. By using this approach, we found four membrane-interacting peptides in CYP27A1, and two peptides in CYP11A1. Peptides in CYP27A1 represent a contiguous portion of the polypeptide chain (residues 210-251) corresponding to the putative F-G loop and adjacent portions of the F and G helices. Peptides in CYP11A1 are from the putative F-G loop (residues 218-225) and the C-terminal portion of the G helix (residues 238-250). This data is consistent with those obtained previously by us and others and provide new information about membrane topology of CYPs 27A1 and 11A1.

The Journal of Biological Chemistry, 2011

Mitochondrial cytochrome P450 11A1 (CYP11A1 or P450 11A1) is the only known enzyme that cleaves t... more Mitochondrial cytochrome P450 11A1 (CYP11A1 or P450 11A1) is the only known enzyme that cleaves the side chain of cholesterol, yielding pregnenolone, the precursor of all steroid hormones. Pregnenolone is formed via three sequential monooxygenation reactions that involve the progressive production of 22R-hydroxycholesterol (22HC) and 20␣,22R-dihydroxycholesterol, followed by the cleavage of the C20-C22 bond. Herein, we present the 2.5-Å crystal structure of CYP11A1 in complex with the first reaction intermediate, 22HC. The active site cavity in CYP11A1 represents a long curved tube that extends from the protein surface to the heme group, the site of catalysis. 22HC occupies two-thirds of the cavity with the 22R-hydroxyl group nearest the heme, 2.56 Å from the iron. The space at the entrance to the active site is not taken up by 22HC but filled with ordered water molecules. The network formed by these water molecules allows the "soft" recognition of the 22HC 3␤-hydroxyl. Such a mode of 22HC binding suggests shuttling of the sterol intermediates between the active site entrance and the heme group during the three-step reaction. Translational freedom of 22HC and torsional motion of its aliphatic tail are supported by solution studies. The CYP11A1-22HC co-complex also provides insight into the structural basis of the strict substrate specificity and high catalytic efficiency of the enzyme and highlights conserved structural motifs involved in redox partner interactions by mitochondrial P450s.

Neurotherapeutics

Cholesterol and 24-hydroxycholesterol are the most abundant brain sterols and represent the subst... more Cholesterol and 24-hydroxycholesterol are the most abundant brain sterols and represent the substrate and product, respectively, of cytochrome P450 46A1 (CYP46A1), a CNS-specific enzyme. CYP46A1 controls cholesterol elimination and turnover in the brain, the two processes that determine the rate of brain sterol flux through the plasma membranes and thereby the properties of these membranes. Brain sterol flux is decreased in Cyp46a1−/− mice compared to wild-type mice and increased in 5XFAD mice (a model of Alzheimer’s disease) when they are treated with a small dose of efavirenz, a CYP46A1 activator. Herein, we first assessed the brain proteome (synaptosomal fractions) and phospho-proteome (synaptosomal fractions and brain homogenates) of efavirenz-treated and control 5XFAD mice. Then, based on the pattern of protein abundance change, we conducted acetyl-CoA measurements (brain homogenates and mitochondria) and metabolic profiling (brain homogenates). The phospho-proteomics datasets ...

Biology

Cholesterol homeostasis in the retina, a sensory organ in the back of the eye, has been studied i... more Cholesterol homeostasis in the retina, a sensory organ in the back of the eye, has been studied in mice but not hamsters, despite the latter being more similar to humans than mice with respect to their whole-body cholesterol maintenance. The goal of this study was to begin to assess hamster retina and conduct initial interspecies comparisons. First, young (3-month old) and mature (6-month old) Syrian (golden) hamsters were compared with 3- and 6-month old mice for ocular biometrics and retinal appearance on optical coherence tomography and fluorescein angiography. Of the 30 evaluated hamsters, seven had retinal structural abnormalities and all had increased permeability of retinal blood vessels. However, hamsters did not carry the mutations causing retinal degenerations 1 and 8, had normal blood glucose levels, and only slightly elevated hemoglobin A1c content. Cholesterol and six other sterols were quantified in hamster retina and compared with sterol profiles in mouse and human re...

PLOS ONE

Cytochrome P450 46A1 (CYP46A1) converts cholesterol to 24-hydroxycholesterol and thereby controls... more Cytochrome P450 46A1 (CYP46A1) converts cholesterol to 24-hydroxycholesterol and thereby controls the major pathways of cholesterol removal from the brain. Cyp46a1-/mice have a reduction in the rate of cholesterol biosynthesis in the brain and significant impairments to memory and learning. To gain insights into the mechanisms underlying Cyp46a1-/phenotype, we used Cyp46a1-/mice and quantified their brain sterol levels and the expression of the genes pertinent to cholesterol homeostasis. We also compared the Cyp46a1-/and wild type brains for protein phosphorylation and ubiquitination. The data obtained enable the following inferences. First, there seems to be a compensatory upregulation in the Cyp46a1-/brain of the pathways of cholesterol storage and CYP46A1-independent removal. Second, transcriptional regulation of the brain cholesterol biosynthesis via sterol regulatory element binding transcription factors is not significantly activated in the Cyp46a1-/brain to explain a compensatory decrease in cholesterol biosynthesis. Third, some of the liver X receptor target genes (Abca1) are paradoxically upregulated in the Cyp46a1-/brain, possibly due to a reduced activation of the small GTPases RAB8, CDC42, and RAC as a result of a reduced phosphorylation of RAB3IP and PAK1. Fourth, the phosphorylation of many other proteins (a total of 146) is altered in the Cyp46a1-/brain, including microtubule associated and neurofilament proteins (the MAP and NEF families) along with proteins related to synaptic vesicles and synaptic neurotransmission (e.g., SLCs, SHANKs, and BSN). Fifth, the extent of protein ubiquitination is increased in the Cyp46a1-/brain, and the affected proteins pertain to ubiquitination (UBE2N), cognition (STX1B and ATP1A2), cytoskeleton function (TUBA1A and YWHAZ), and energy production (ATP1A2 and ALDOA). The present study demonstrates the diverse potential effects of CYP46A1 deficiency on brain functions and identifies important proteins that could be affected by this deficiency.

Journal of lipid research, Jan 17, 2018

Apolipoprotein E (APOE) is a component of lipid-transporting particles and a recognition ligand f... more Apolipoprotein E (APOE) is a component of lipid-transporting particles and a recognition ligand for receptors, which bind these particles. The APOE isoform 2 is a risk factor for age-related macular degeneration; nevertheless APOE absence in humans and mice does not significantly affect the retina. We found that retinal cholesterol biosynthesis and the levels of retinal cholesterol were increased in mice, whereas cholesterol elimination by metabolism was decreased. No focal cholesterol deposits were observed in the retina. Retinal proteomics identified the most abundant cholesterol-related proteins in wild type mice and revealed that of these cholesterol-related proteins, only APOA4 had increased expression in the retina. In addition, there were changes in retinal abundance of proteins involved in pro- and anti-inflammatory responses, cellular cytoskeleton maintenance, vesicular traffic, and retinal iron homeostasis. The data obtained indicate that when APOE is absent, particles con...

The Journal of biological chemistry, Aug 22, 2017

Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase) is the enzyme responsible for the majo... more Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase) is the enzyme responsible for the majority of cholesterol elimination from the brain. Previously, we found that the anti-HIV drug efavirenz (EFV) can pharmacologically activate CYP46A1 in mice. Herein, we investigated whether CYP46A1 could also be activated by endogenous compounds, including major neurotransmitters. In vitro experiments with purified recombinant CYP46A1 indicated that CYP46A1 is activated by L-glutamate (L-Glu), L-aspartate, γ-aminobutyric acid, and acetylcholine, with L-Glu eliciting the highest increase (3-fold) in CYP46A1-mediated cholesterol 24-hydroxylation. We also found that L-Glu and other activating neurotransmitters bind to the same site on the CYP46A1 surface, which differs from the EFV-binding site. The other principal differences between EFV and L-Glu in CYP46A1 activation include an apparent lack of L-Glu binding to the P450 active site and different pathways of signal transduction from the all...

The Journal of biological chemistry, Mar 11, 2017

Cytochrome P450 27A1 (CYP27A1 or sterol 27-hydroxylase) is a ubiquitous, multifunctional enzyme c... more Cytochrome P450 27A1 (CYP27A1 or sterol 27-hydroxylase) is a ubiquitous, multifunctional enzyme catalyzing regio- and stereo-specific hydroxylation of different sterols. In humans, complete CYP27A1 deficiency leads to cerebrotendinous xanthomatosis or nodule formation in tendons and brain (preferentially in the cerebellum) rich in cholesterol and cholestanol, the 5α-saturated analog of cholesterol. In Cyp27a1(-/-) mice, xanthomas are not formed, despite a significant cholestanol increase in the brain and cerebellum. The mechanism behind cholestanol production has been clarified, yet little is known about its metabolism, except that CYP27A1 might metabolize cholestanol. It also is unclear why CYP27A1 deficiency results in preferential cholestanol accumulation in the cerebellum. We hypothesized that cholestanol might be metabolized by CYP46A1, the principal cholesterol 24-hydroxylase in the brain. We quantified sterols along with CYP27A1 and CYP46A1 in mouse models (Cyp27a1(-/-) , Cyp...

Archives of Biochemistry and Biophysics, Aug 1, 2004

Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic ... more Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic and substrate-binding properties of four truncated cytochromes P450 46A1 were investigated in the present study. All four lacked the N-terminal transmembrane region (residues 3-27), and, in addition, D46A1H had a 4Â His-tag fused to the C-terminus; HD46A1 had the N-terminal 4Â His-tag; HD46A1D had a 4Â His-tag at the N-terminus and did not contain a proline-rich region at the C-terminus (residues 494-499); and D46A1D lacked the C-terminal proline-rich region. The truncated enzymes were expressed at 390-650 nmol/L culture levels, distributed at about a 1:1 ratio between the membrane fraction and the cytosol in low ionic strength buffer, and were predominantly monomers in detergent-free buffer. They had moderately decreased catalytic efficiencies for either cholesterol or 24S-hydroxycholesterol or both, whereas their substrate-binding constants were either unchanged or decreased 2-fold. The two forms, D46A1D and HD46A1D, both lacking the C-terminal proline-rich region seem to be good candidates for future crystallographic studies because they contain only 0.3-0.8% of high molecular weight aggregates and their catalytic efficiencies are decreased no more than 2.3-fold.

Biochemistry (Moscow)

Addition of an amphipathic bee venom peptide, melittin, to sarcoplasmic reticulum (SR) vesicles i... more Addition of an amphipathic bee venom peptide, melittin, to sarcoplasmic reticulum (SR) vesicles isolated from rabbit skeletal muscles resulted in a fast (<1 min) blue shift in the fluorescence maximum of the melittin--SR membrane complex. Over the following 45 min the position of the fluorescence maximum did not change, but the fluorescence intensity of the melittin--SR membrane complex decreased by approximately 35% with rate constant 0.14 min-1. Melittin rapidly quenched the isotropic signal in the EPR spectrum of spin-labeled stearic acid added to SR membranes. Further changes in the spectral parameters of the spin probe bound to SR membranes in the presence of melittin indicated an increase of the viscosity of the probe microenvironment (empiric parameter T/eta was decreased by approximately 35% with rate constant 0.11 min-1). The surface potential of SR membranes measured using a pH-sensitive dye, neutral red, decreased after melittin addition from -60 to -30 mV. It was demonstrated with the use of a cross-linking agent, cupric o-phenanthroline, that melittin induced slow aggregation of Ca-ATPase protein in SR membranes; the content of enzyme in the monomeric form decreased with rate constant 0.14 min-1. It is concluded that melittin binds rapidly to SR membranes, inducing slow changes in Ca-ATPase conformation and oligomeric state as well as structural transitions in the lipid bilayer of SR membranes.

Biochemistry (Moscow)

The inhibition of Na,K-ATPase from duck salt gland and Ca-ATPase from rabbit skeletal muscle sarc... more The inhibition of Na,K-ATPase from duck salt gland and Ca-ATPase from rabbit skeletal muscle sarcoplasmic reticulum by melittin, a 26-residue peptide from bee venom, was studied. Melittin irreversibly inhibits both enzymes. At melittin/ATPase molar ratio (30-50):1, the time dependence of the inhibition is described by the sum of two exponential curves. At pH 7.0, the fast phase of the inhibition provides for about 50% of total loss of activity with pseudo-first order rate constants of 1.52 +/- 0.17 and 1.20 +/- 0.21 min-1 for Na,K- and Ca-ATPase, respectively. The corresponding pseudo-first order rate constants for the slow phase were 0.12 +/- 0.02 and 0.09 +/- 0.02 min-1. The inhibition of both enzymes by melittin depends upon pH; the inhibition increases when the pH is increased from 6.0 to 8.5. The enhancement of the inhibition concomitant to increase in pH is mainly due to an increase in the rate constant of the fast phase. ATP protects both enzymes from the inhibition by melitt...

Biochemistry. Biokhimii͡a, 2003

Proteins interacting with alpha 1 beta 1-type of Na,K-ATPase were revealed in pig kidney outer me... more Proteins interacting with alpha 1 beta 1-type of Na,K-ATPase were revealed in pig kidney outer medulla and duck salt glands using three different methods (immunoprecipitation, protein overlay, and chemical cross-linking). Immunoprecipitation was performed after solubilization of protein homogenate with Triton X-100 so that both membrane and cytosol proteins bound to Na,K-ATPase could be revealed. Two other methods were used to study the interaction of cytosol proteins with purified Na,K-ATPase. The sets of proteins revealed by each method in outer medulla of pig kidney were different. Proteins interacting with Na,K-ATPase that have molecular masses 10, 15, 70, 75, 105, 120, and 190 kD were found using the immunoprecipitation method. The chemical cross-linking method revealed proteins with molecular masses 25, 35, 40, 58, 68-70, and 86-88 kD. The protein overlay method revealed in the same tissue proteins with molecular masses 38, 42, 43, 60, 62, 66, 70, and 94 kD.

Biochemistry, 2005

The conversion of cholesterol to 7alpha-hydroxycholesterol catalyzed by cytochrome P450 7A1 (CYP7... more The conversion of cholesterol to 7alpha-hydroxycholesterol catalyzed by cytochrome P450 7A1 (CYP7A1) initiates the major pathway for cholesterol elimination in mammals. In the present work we focused on identification of determinants of the CYP7A1 substrate specificity inside the active site using a homology model with a novel P450-fold, site-directed mutagenesis, and substrate-binding and kinetic studies. Forty-one mutants, encompassing twenty-six amino acid residues, were generated and characterized, and of these, seven residues appear to determine cholesterol binding in the active site. In addition, four cholesterol derivatives were used as active site probes in the wild type and the seven mutant enzymes, and the spectral binding constants and products were analyzed. It was concluded that Asn288 in the I helix plays a key role in the P450-cholesterol contacts by hydrogen bonding to the steroid 3beta-hydroxyl, while Val280 and Ala284 are beside and the Trp283 is above the steroid nucleus orienting the cholesterol molecule. Leu360 and Ala358 between the K helix and the beta1-4 strand and Leu485 in the beta4 sheet-turn appear to define the size of the active site over the heme pyrrole ring A, thus limiting the orientation and size of the substrate at the steroid A ring. Additionally, the A358V mutant was found to form two new products, one being 7beta-hydroxycholesterol. Our data indicate that a tight fit of cholesterol in the enzyme active site is in part responsible for the high efficiency of cholesterol turnover by CYP7A1.

Biochemistry, 2003

The known activity of cytochrome P450 46A1 (P450 46A1) is 24(S)-hydroxylation of cholesterol. Thi... more The known activity of cytochrome P450 46A1 (P450 46A1) is 24(S)-hydroxylation of cholesterol. This reaction produces biologically active oxysterol, 24(S)-hydroxycholesterol, and is also the first step in enzymatic degradation of cholesterol in the brain. We report here that P450 46A1 can further metabolize 24(S)-hydroxycholesterol, giving 24,25- and 24,27-dihydroxycholesterols in both the cell cultures transfected with P450 46A1 cDNA and the in vitro reconstituted system with recombinant enzyme. In addition, P450 46A1 was able to carry out side chain hydroxylations of two endogenous C27-steroids with and without a double bond between C5-C6 (7alpha-hydroxycholesterol and cholestanol, respectively) and introduce a hydroxyl group on the steroid nucleus of the C21-steroid hormones with the C4-C5 double bond (progesterone and testosterone). Also, P450 46A1 was found to metabolize xenobiotics carrying out dextromethorphan O- and N-demethylations, diclofenac 4'-hydroxylation, and phenacetin O-deethylation. Thus, substrate specificities of P450 46A1 are not limited to cholesterol and include a number of structurally diverse compounds. Activities of P450 46A1 suggest that, in addition to the involvement in cholesterol homeostasis in the brain, this enzyme may participate in metabolism of neurosteroids and drugs that can cross the blood-brain barrier and are targeted to the central nervous system.

Biochemistry, 1997

Incubation of melittin with sarcoplasmic reticulum membranes at pH 7.0 and different melittin: Ca... more Incubation of melittin with sarcoplasmic reticulum membranes at pH 7.0 and different melittin: Ca-ATPase molar ratios results in the progressive loss of enzyme activity. At high melittin:Ca-ATPase molar ratios (10:1 and 30:1), enzyme inhibition may be described by a biexponential curve. At pH 7.0, the values of the pseudo-first-order rate constants are 1.0 and 0.1 min -1 for the fast and slow phases of inhibition, respectively, at a melittin:Ca-ATPase molar ratio of 30:1. At pH 6.0 and a melittin:Ca-ATPase molar ratio of 30:1, melittin does not inhibit Ca-ATPase. Melittin-induced aggregation of Ca-ATPase molecules was studied using cupric phenanthroline as a chemical cross-linking agent. At a melittin:Ca-ATPase molar ratio of 5:1, aggregation of Ca-ATPase protein was not observed; however, the loss of enzyme activity was about 30% after 30 min. At melittin:Ca-ATPase molar ratios of 10:1 and 30:1, significant aggregation of Ca-ATPase protein takes place. The rate of Ca-ATPase aggregation is much lower than the rate of enzyme inhibition. At melittin:Ca-ATPase molar ratios of 10:1 and 30:1, the rate of Ca-ATPase protein aggregation is close to that for the slow phase of enzyme inhibition. At pH 6.0 and a melittin:Ca-ATPase molar ratio of 30:1, significant aggregation of Ca-ATPase occurs. It is concluded that melittin induces both Ca-ATPase inhibition and aggregation. These two processes may occur simultaneously, but under some conditions either inhibition or aggregation takes place independently of each other. Therefore, the aggregation of Ca-ATPase induced by melittin is not necessary for enzyme inhibition. adenine dinucleotide, reduced form; BSA, bovine serum albumin.

Archives of Biochemistry and Biophysics, 2004

Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic ... more Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic and substrate-binding properties of four truncated cytochromes P450 46A1 were investigated in the present study. All four lacked the N-terminal transmembrane region (residues 3-27), and, in addition, D46A1H had a 4Â His-tag fused to the C-terminus; HD46A1 had the N-terminal 4Â His-tag; HD46A1D had a 4Â His-tag at the N-terminus and did not contain a proline-rich region at the C-terminus (residues 494-499); and D46A1D lacked the C-terminal proline-rich region. The truncated enzymes were expressed at 390-650 nmol/L culture levels, distributed at about a 1:1 ratio between the membrane fraction and the cytosol in low ionic strength buffer, and were predominantly monomers in detergent-free buffer. They had moderately decreased catalytic efficiencies for either cholesterol or 24S-hydroxycholesterol or both, whereas their substrate-binding constants were either unchanged or decreased 2-fold. The two forms, D46A1D and HD46A1D, both lacking the C-terminal proline-rich region seem to be good candidates for future crystallographic studies because they contain only 0.3-0.8% of high molecular weight aggregates and their catalytic efficiencies are decreased no more than 2.3-fold.

The Journal of biological chemistry, Jan 23, 2016

The process of vision is impossible without the photoreceptor cells, which have a unique structur... more The process of vision is impossible without the photoreceptor cells, which have a unique structure and specific maintenance of cholesterol. Herein we report on the previously unrecognized cholesterol-related pathway in the retina discovered during follow-up characterizations of Cyp27a1(-/-)Cyp46a1(-/-) mice. These animals have retinal hypercholesterolemia and convert excess retinal cholesterol into cholesterol esters, normally present in the retina in very small amounts. We established that in the Cyp27a1(-/-)Cyp46a1(-/-) retina, cholesterol esters are generated by and accumulate in the photoreceptor outer segments (OS), which is the retinal layer with the lowest cholesterol content. Mouse OS were also found to express the cholesterol-esterifying enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT1), but not lecithin-cholesterol acyltransferase (LCAT), and to differ from humans in retinal expression of ACAT1. Nevertheless, cholesterol esters were discovered to be abundant in hu...

Acta Crystallogr D Biol Cryst, 2009

Human cytochrome P450 46A1 (CYP46A1) is one of the key enzymes in cholesterol homeostasis in the ... more Human cytochrome P450 46A1 (CYP46A1) is one of the key enzymes in cholesterol homeostasis in the brain. The crystallization and heavy-atom structure solution of an active truncated CYP46A1 in complex with the high-affinity substrate analogue cholesterol-3-sulfate (CH-3S) is reported. The 2.6 angstroms structure of CYP46A1-CH-3S was solved using both anion and cation heavy-atom salts. In addition to the native anomalous signal from the haem iron, an NaI anion halide salt derivative and a complementary CsCl alkali-metal cation salt derivative were used. The general implications of the use of halide and alkali-metal quick soaks are discussed. The importance of using isoionic strength buffers, the titration of heavy-atom salts into different ionic species and the role of concentration are considered. It was observed that cation/anion-binding sites will occasionally overlap, which could negatively impact upon mixed RbBr soaks used for multiple anomalous scatterer MAD (MMAD). The use of complementary cation and anion heavy-atom salt derivatives is a convenient and powerful tool for MIR(AS) structure solution.

The Journal of biological chemistry, Jan 27, 2016

Cytochrome P450 46A1 (CYP46A1) is a microsomal enzyme and cholesterol 24-hydroxylase that control... more Cytochrome P450 46A1 (CYP46A1) is a microsomal enzyme and cholesterol 24-hydroxylase that controls cholesterol elimination from the brain. This P450 is also a potential target for Alzheimer disease because it can be activated pharmacologically by some marketed drugs, as exemplified by efavirenz, the anti-HIV medication. Previously, we suggested that pharmaceuticals activate CYP46A1 allosterically through binding to a site on the cytosolic protein surface, which is different from the enzyme active site facing the membrane. Here we identified this allosteric site for efavirenz on CYP46A1 by using a combination of hydrogen-deuterium exchange coupled to MS, computational modeling, site-directed mutagenesis, and analysis of the CYP46A1 crystal structure. We also mapped the binding region for the CYP46A1 redox partner oxidoreductase and found that the allosteric and redox partner binding sites share a common border. On the basis of the data obtained, we propose the mechanism of CYP46A1 al...

Lipids, Sep 13, 2008

Mitochondrial cytochrome P450 enzymes (CYP or P450, EC 1.14.13.15) play an important role in meta... more Mitochondrial cytochrome P450 enzymes (CYP or P450, EC 1.14.13.15) play an important role in metabolism of cholesterol. CYP27A1 hydroxylates cholesterol at position 27 and, thus, initiates cholesterol removal from many extrahepatic tissues. CYP11A1 is a steroidogenic P450 that converts cholesterol to pregnenolone, the first step in the biosynthesis of all steroid hormones. We utilized a new approach to study membrane topology of CYPs 27A1 and 11A1. This approach involves heterologous expression of membrane-bound P450 in E. coli, isolation of the P450-containing E. coli membranes, treatment of the membranes with protease, removal of the digested soluble portion and extraction of the membrane-associated peptides, which are then identified by mass spectrometry. By using this approach, we found four membrane-interacting peptides in CYP27A1, and two peptides in CYP11A1. Peptides in CYP27A1 represent a contiguous portion of the polypeptide chain (residues 210-251) corresponding to the putative F-G loop and adjacent portions of the F and G helices. Peptides in CYP11A1 are from the putative F-G loop (residues 218-225) and the C-terminal portion of the G helix (residues 238-250). This data is consistent with those obtained previously by us and others and provide new information about membrane topology of CYPs 27A1 and 11A1.

The Journal of Biological Chemistry, 2011

Mitochondrial cytochrome P450 11A1 (CYP11A1 or P450 11A1) is the only known enzyme that cleaves t... more Mitochondrial cytochrome P450 11A1 (CYP11A1 or P450 11A1) is the only known enzyme that cleaves the side chain of cholesterol, yielding pregnenolone, the precursor of all steroid hormones. Pregnenolone is formed via three sequential monooxygenation reactions that involve the progressive production of 22R-hydroxycholesterol (22HC) and 20␣,22R-dihydroxycholesterol, followed by the cleavage of the C20-C22 bond. Herein, we present the 2.5-Å crystal structure of CYP11A1 in complex with the first reaction intermediate, 22HC. The active site cavity in CYP11A1 represents a long curved tube that extends from the protein surface to the heme group, the site of catalysis. 22HC occupies two-thirds of the cavity with the 22R-hydroxyl group nearest the heme, 2.56 Å from the iron. The space at the entrance to the active site is not taken up by 22HC but filled with ordered water molecules. The network formed by these water molecules allows the "soft" recognition of the 22HC 3␤-hydroxyl. Such a mode of 22HC binding suggests shuttling of the sterol intermediates between the active site entrance and the heme group during the three-step reaction. Translational freedom of 22HC and torsional motion of its aliphatic tail are supported by solution studies. The CYP11A1-22HC co-complex also provides insight into the structural basis of the strict substrate specificity and high catalytic efficiency of the enzyme and highlights conserved structural motifs involved in redox partner interactions by mitochondrial P450s.