Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 17 de 17
Filter
Add more filters










Publication year range
1.
Sci Rep ; 14(1): 12976, 2024 06 05.
Article in English | MEDLINE | ID: mdl-38839792

ABSTRACT

Crystal structures of human long-chain acyl-CoA dehydrogenase (LCAD) and the catalytically inactive Glu291Gln mutant, have been determined. These structures suggest that LCAD harbors functions beyond its historically defined role in mitochondrial ß-oxidation of long and medium-chain fatty acids. LCAD is a homotetramer containing one FAD per 43 kDa subunit with Glu291 as the catalytic base. The substrate binding cavity of LCAD reveals key differences which makes it specific for longer and branched chain substrates. The presence of Pro132 near the start of the E helix leads to helix unwinding that, together with adjacent smaller residues, permits binding of bulky substrates such as 3α, 7α, l2α-trihydroxy-5ß-cholestan-26-oyl-CoA. This structural element is also utilized by ACAD11, a eucaryotic ACAD of unknown function, as well as bacterial ACADs known to metabolize sterol substrates. Sequence comparison suggests that ACAD10, another ACAD of unknown function, may also share this substrate specificity. These results suggest that LCAD, ACAD10, ACAD11 constitute a distinct class of eucaryotic acyl CoA dehydrogenases.


Subject(s)
Acyl-CoA Dehydrogenase, Long-Chain , Models, Molecular , Substrate Specificity , Humans , Acyl-CoA Dehydrogenase, Long-Chain/metabolism , Acyl-CoA Dehydrogenase, Long-Chain/genetics , Acyl-CoA Dehydrogenase, Long-Chain/chemistry , Crystallography, X-Ray , Catalytic Domain , Acyl-CoA Dehydrogenases/metabolism , Acyl-CoA Dehydrogenases/genetics , Acyl-CoA Dehydrogenases/chemistry , Protein Conformation , Amino Acid Sequence
2.
Heliyon ; 10(7): e28231, 2024 Apr 15.
Article in English | MEDLINE | ID: mdl-38590848

ABSTRACT

Human familial isolated pituitary adenoma (FIPA) has been linked to germline heterozygous mutations in the gene encoding the aryl hydrocarbon receptor-interacting protein (AIP, also known as ARA9, XAP2, FKBP16, or FKBP37). To investigate the hypothesis that AIP is a pituitary adenoma tumor suppressor via its role in aryl hydrocarbon receptor (AHR) signaling, we have compared the pituitary phenotype of our global null Aip (AipΔC) mouse model with that of a conditional null Aip model (Aipfx/fx) carrying the same deletion, as well as pituitary phenotypes of Ahr global null and Arnt conditional null animals. We demonstrate that germline AipΔC heterozygosity results in a high incidence of pituitary tumors in both sexes, primarily somatotropinomas, at 16 months of age. Biallelic deletion of Aip in Pit-1 cells (Aipfx/fx:rGHRHRcre) increased pituitary tumor incidence and also accelerated tumor progression, supporting a loss-of-function/loss-of-heterozygosity model of tumorigenesis. Tumor development exhibited sexual dimorphism in wildtype and Aipfx/fx:rGHRHRcre animals. Despite the role of AHR as a tumor suppressor in other cancers, the observation that animals lacking AHR in all tissues, or ARNT in Pit-1 cells, do not develop somatotropinomas argues against the hypothesis that pituitary tumorigenesis in AIP-associated FIPA is related to decreased activities of either the Ahr or Arnt gene products.

3.
Res Sq ; 2024 Feb 29.
Article in English | MEDLINE | ID: mdl-38464032

ABSTRACT

Crystal structures of human long-chain acyl-CoA dehydrogenase (LCAD) and the E291Q mutant, have been determined. These structures suggest that LCAD harbors functions beyond its historically defined role in mitochondrial ß-oxidation of long and medium-chain fatty acids. LCAD is a homotetramer containing one FAD per 43kDa subunit with Glu291 as the catalytic base. The substrate binding cavity of LCAD reveals key differences which makes it specific for longer and branched chain substrates. The presence of Pro132 near the start of the E helix leads to helix unwinding that, together with adjacent smaller residues, permits binding of bulky substrates such as 3α, 7α, l2α-trihydroxy-5ß-cholestan-26-oyl-CoA. This structural element is also utilized by ACAD11, a eucaryotic ACAD of unknown function, as well as bacterial ACADs known to metabolize sterol substrates. Sequence comparison suggests that ACAD10, another ACAD of unknown function, may also share this substrate specificity. These results suggest that LCAD, ACAD10, ACAD11 constitute a distinct class of eucaryotic acyl CoA dehydrogenases.

4.
Toxicol Rep ; 9: 1-11, 2022.
Article in English | MEDLINE | ID: mdl-34950569

ABSTRACT

Proteins containing PER-ARNT-SIM (PAS) domains are commonly associated with environmental adaptation in a variety of organisms. The PAS domain is found in proteins throughout Archaea, Bacteria, and Eukarya and often binds small-molecules, supports protein-protein interactions, and transduces input signals to mediate an adaptive physiological response. Signaling events mediated by PAS sensors can occur through induced phosphorelays or genomic events that are often dependent upon PAS domain interactions. In this perspective, we briefly discuss the diversity of PAS domain containing proteins, with particular emphasis on the prototype member, the aryl hydrocarbon receptor (AHR). This ligand-activated transcription factor acts as a sensor of the chemical environment in humans and many chordates. We conclude with the idea that since mammalian PAS proteins often act through PAS-PAS dimers, undocumented interactions of this type may link biological processes that we currently think of as independent. To support this idea, we present a framework to guide future experiments aimed at fully elucidating the spectrum of PAS-PAS interactions with an eye towards understanding how they might influence environmental sensing in human and wildlife populations.

5.
iScience ; 24(10): 103153, 2021 Oct 22.
Article in English | MEDLINE | ID: mdl-34646991

ABSTRACT

The dual function protein ACAD9 catalyzes α,ß-dehydrogenation of fatty acyl-CoA thioesters in fatty acid ß-oxidation and is an essential chaperone for mitochondrial respiratory complex I (CI) assembly. ACAD9, ECSIT, and NDUFAF1 interact to form the core mitochondrial CI assembly complex. Current studies examine the molecular mechanism of ACAD9/ECSIT/NDUFAF1interactions. ACAD9 binds to the carboxy-terminal half and NDUFAF1 to the amino-terminal half of ECSIT. Binary complexes are unstable and aggregate easily, while the ACAD9/ECSIT/NDUFAF1 ternary complex is soluble and highly stable. Molecular modeling and small-angle X-ray scattering studies identified intra-complex interaction sites and binding sites for other assembly factors. Binding of ECSIT at the ETF binding site in the amino-terminal domain of ACAD9 is consistent with observed loss of FAD and enzymatic activity and demonstrates that the two functions of ACAD9 are mutually exclusive. Mapping of 42 known pathogenic mutations onto the homology-modeled ACAD9 structure provides structural insights into pathomechanisms of CI deficiency.

6.
Arch Biochem Biophys ; 701: 108792, 2021 04 15.
Article in English | MEDLINE | ID: mdl-33556357

ABSTRACT

The influence of the side chains and positioning of the carboxy-terminal residues of NADPH-cytochrome P450 oxidoreductase (CYPOR) on catalytic activity, structure of the carboxy terminus, and interaction with cofactors has been investigated. A tandem deletion of residues Asp675 and Val676, that was expected to shift the position of the functionally important Trp677, resulted in higher cytochrome c reductase activity than that expected from previous studies on the importance of Asp675 and Trp677 in catalysis. Crystallographic determination of the structure of this variant revealed two conformations of the carboxy terminus. In one conformation (Mol A), the last α-helix is partially unwound, resulting in repositioning of all subsequent residues in ß-strand 21, from Arg671 to Leu674 (corresponding to Ser673 and Val676 in the wild type structure). This results in the two C-terminal residues, Trp677 and Ser678, being maintained in their wild type positions, with the indole ring of Trp677 stacked against the isoalloxazine ring of FAD as seen in the wild type structure, and Ser673 occupying a similar position to the catalytic residue, Asp675. The other, more disordered conformation is a mixture of the Mol A conformation and one in which the last α-helix is not unwound and the nicotinamide ring is in one of two conformations, out towards the protein surface as observed in the wild type structure (1AMO), or stacked against the flavin ring, similar to that seen in the W677X structure that lacks Trp677 and Ser678 (1JA0). Further kinetic analysis on additional variants showed deletion or substitution of alanine or glycine for Trp677 in conjunction with deletion of Ser678 produced alterations in interactions of CYPOR with NADP+, 2'5'-ADP, and 2'-AMP, as well as the pH dependence of cytochrome c reductase activity. We postulate that deletion of bulky residues at the carboxy terminus permits increased mobility leading to decreased affinity for the 2'5'-ADP and 2'-AMP moieties of NADP+ and subsequent domain movement.


Subject(s)
Adenosine Diphosphate/chemistry , Adenosine Monophosphate/chemistry , Flavin-Adenine Dinucleotide/chemistry , NADPH-Ferrihemoprotein Reductase/chemistry , NADP/chemistry , Binding Sites , Crystallography, X-Ray , Kinetics , NADPH-Ferrihemoprotein Reductase/genetics , Protein Conformation, alpha-Helical , Structure-Activity Relationship
7.
Arch Toxicol ; 93(7): 1917-1926, 2019 07.
Article in English | MEDLINE | ID: mdl-31111189

ABSTRACT

The Toll-like receptor 7 agonist imiquimod (IMQ) is an approved drug for the topical treatment of various skin diseases that, in addition, is currently tested in multiple clinical trials for the immunotherapy of various types of cancers. As all of these trials include application of IMQ to the skin and evidence exists that exposure to environmental pollutants, i.e., tobacco smoke, affects its therapeutic efficacy, the current study aims to elucidate the cutaneous metabolism of the drug. Treatment of human keratinocytes with 2.5 µM benzo[a]pyrene (BaP), a tobacco smoke constituent and aryl hydrocarbon receptor (AHR) agonist, for 24 h induced cytochrome P450 (CYP) 1A enzyme activity. The addition of IMQ 30 min prior measurement resulted in a dose-dependent inhibition of CYP1A activity, indicating that IMQ is either a substrate or inhibitor of CYP1A isoforms. Incubation of 21 recombinant human CYP enzymes with 0.5 µM IMQ and subsequent LC-MS analyses, in fact, identified CYP1A1 and CYP1A2 as being predominantly responsible for IMQ metabolism. Accordingly, treatment of keratinocytes with BaP accelerated IMQ clearance and the associated formation of monohydroxylated IMQ metabolites. A co-incubation with 5 µM 7-hydroxyflavone, a potent inhibitor of human CYP1A isoforms, abolished basal as well as BaP-induced IMQ metabolism. Further studies with hepatic microsomes from CD-1 as well as solvent- and ß-naphthoflavone-treated CYP1A1/CYP1A2 double knock-out and respective control mice confirmed the critical contribution of CYP1A isoforms to IMQ metabolism. Hence, an exposure to life style-related, dietary, and environmental AHR ligands may affect the pharmacokinetics and, thus, treatment efficacy of IMQ.


Subject(s)
Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1A2/metabolism , Imiquimod/metabolism , Keratinocytes/metabolism , Adult , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/metabolism , Cells, Cultured , Chromatography, Liquid , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A2/genetics , Dose-Response Relationship, Drug , Female , Humans , Imiquimod/administration & dosage , Male , Mass Spectrometry , Mice , Mice, Inbred C57BL , Mice, Knockout , Microsomes, Liver/metabolism , Middle Aged , Receptors, Aryl Hydrocarbon/metabolism
8.
Biochemistry ; 58(19): 2408-2418, 2019 05 14.
Article in English | MEDLINE | ID: mdl-31009206

ABSTRACT

NADPH-cytochrome P450 oxidoreductase (CYPOR), the essential flavoprotein of the microsomal cytochrome P450 monooxygenase system, is anchored in the phospholipid bilayer by its amino-terminal membrane-binding domain (MBD), which is necessary for efficient electron transfer to cytochrome P450. Although crystallographic and kinetic studies have established the structure of the soluble catalytic domain and the role of conformational motions in the control of electron transfer, the role of the MBD is largely unknown. We examined the role of the MBD in P450 catalysis through studies of amino-terminal deletion mutants and site-directed spin labeling. We show that the MBD spans the membrane and present a model for the orientation of CYPOR on the membrane capable of forming a complex with cytochrome P450. EPR power saturation measurements of CYPOR mutants in liposomes containing a lipid/Ni(II) chelate identified a region of the soluble domain interacting with the membrane. The deletion of more than 29 residues from the N-terminus of CYPOR decreases cytochrome P450 activity concomitant with alterations in electrophoretic mobility and an increased resistance to protease digestion. The altered kinetic properties of these mutants are consistent with electron transfer through random collisions rather than via formation of a stable CYPOR-P450 complex. Purified MBD binds weakly to cytochrome P450, suggesting that other interactions are also required for CYPOR-P450 complex formation. We propose that the MBD and flexible tether region of CYPOR, residues 51-63, play an important role in facilitating the movement of the soluble domain relative to the membrane and in promoting multiple orientations that permit specific interactions of CYPOR with its varied partners.


Subject(s)
Cell Membrane/metabolism , NADPH-Ferrihemoprotein Reductase/metabolism , Catalytic Domain , Crystallography, X-Ray , Cysteine/chemistry , Cytochrome P-450 Enzyme System/metabolism , Electron Transport , Escherichia coli/cytology , Flavoproteins/metabolism , Hydrophobic and Hydrophilic Interactions , Kinetics , Lipid Bilayers/metabolism , Liposomes/metabolism , NADP/metabolism , Oxidoreductases, N-Demethylating/metabolism , Plasmids/genetics , Protein Structure, Tertiary , Sequence Analysis, Protein
9.
Biochemistry ; 57(6): 945-962, 2018 02 13.
Article in English | MEDLINE | ID: mdl-29308883

ABSTRACT

Conformational changes in NADPH-cytochrome P450 oxidoreductase (CYPOR) associated with electron transfer from NADPH to electron acceptors via FAD and FMN have been investigated via structural studies of the four-electron-reduced NADP+-bound enzyme and kinetic and structural studies of mutants that affect the conformation of the mobile Gly631-Asn635 loop (Asp632 loop). The structure of four-electron-reduced, NADP+-bound wild type CYPOR shows the plane of the nicotinamide ring positioned perpendicular to the FAD isoalloxazine with its carboxamide group forming H-bonds with N1 of the flavin ring and the Thr535 hydroxyl group. In the reduced enzyme, the C8-C8 atoms of the two flavin rings are ∼1 Šcloser than in the fully oxidized and one-electron-reduced structures, which suggests that flavin reduction facilitates interflavin electron transfer. Structural and kinetic studies of mutants Asp632Ala, Asp632Phe, Asp632Asn, and Asp632Glu demonstrate that the carboxyl group of Asp632 is important for stabilizing the Asp632 loop in a retracted position that is required for the binding of the NADPH ribityl-nicotinamide in a hydride-transfer-competent conformation. Structures of the mutants and reduced wild type CYPOR permit us to identify a possible pathway for NADP(H) binding to and release from CYPOR. Asp632 mutants unable to form stable H-bonds with the backbone amides of Arg634, Asn635, and Met636 exhibit decreased catalytic activity and severely impaired hydride transfer from NADPH to FAD, but leave interflavin electron transfer intact. Intriguingly, the Arg634Ala mutation slightly increases the cytochrome P450 2B4 activity. We propose that Asp632 loop movement, in addition to facilitating NADP(H) binding and release, participates in domain movements modulating interflavin electron transfer.


Subject(s)
NADPH-Ferrihemoprotein Reductase/chemistry , NADPH-Ferrihemoprotein Reductase/metabolism , NADP/metabolism , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Amino Acid Substitution , Animals , Crystallography, X-Ray , Electron Transport , Flavin Mononucleotide/chemistry , Flavin Mononucleotide/metabolism , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Kinetics , Models, Molecular , NADP/chemistry , NADPH-Ferrihemoprotein Reductase/genetics , Oxidation-Reduction , Point Mutation , Protein Binding , Protein Conformation , Rats
10.
PLoS One ; 12(10): e0185094, 2017.
Article in English | MEDLINE | ID: mdl-28981549

ABSTRACT

Retinal phenotypes of the PPCD1 mouse, a mouse model of posterior polymorphous corneal dystrophy, have been characterized. PPCD1 mice on the DBA/2J background (D2.Ppcd1) have previously been reported to develop an enlarged anterior chamber due to epithelialization and proliferation of the corneal endothelium and subsequent blockage of the iridocorneal angle. Results presented here show that D2.Ppcd1 mice develop increased intraocular pressure (IOP), with measurements at three months of age revealing significant increases in IOP. Significant retinal ganglion cell layer cell loss is observed at five months of age. D2.Ppcd1 animals also exhibit marked degeneration of the outer nuclear layer in association with hyperplasia of the retinal pigment epithelium. Evidence of retinal detachment is present as early as three weeks of age. By 3.5 months of age, focal areas of outer nuclear layer loss are observed. Although the GpnmbR150X mutation leads to increased IOP and glaucoma in DBA/2J mice, development of anterior segment and retinal defects in D2.Ppcd1 animals does not depend upon presence of the GpnmbR150X mutation.


Subject(s)
Corneal Dystrophies, Hereditary/physiopathology , Retina/pathology , Animals , Corneal Dystrophies, Hereditary/genetics , Intraocular Pressure , Mice , Mice, Inbred DBA , Retinal Detachment/pathology , Retinal Ganglion Cells/pathology
11.
PLoS One ; 11(6): e0157577, 2016.
Article in English | MEDLINE | ID: mdl-27310661

ABSTRACT

We have previously described a mouse model of human posterior polymorphous corneal dystrophy (PPCD) and localized the causative mutation to a 6.2 Mbp region of chromosome 2, termed Ppcd1. We now show that the gene rearrangement linked to mouse Ppcd1 is a 3.9 Mbp chromosomal inversion flanked by 81 Kbp and 542 bp deletions. This recombination event leads to deletion of Csrp2bp Exons 8 through 11, Dzank1 Exons 20 and 21, and the pseudogene Znf133. In addition, we identified translocation of novel downstream sequences to positions adjacent to Csrp2bp Exon 7 and Dzank1 Exon 20. Twelve novel fusion transcripts involving Csrp2bp or Dzank1 linked to downstream sequences have been identified. Eight are expressed at detectable levels in PPCD1 but not wildtype eyes. Upregulation of two Csrp2bp fusion transcripts, as well as upregulation of the adjacent gene, Ovol2, was observed. Absence of the PPCD1 phenotype in animals haploinsufficient for Csrp2bp or both Csrp2bp and Dzank1 rules out haploinsufficiency of these genes as a cause of mouse PPCD1. Complementation experiments confirm that PPCD1 embryonic lethality is due to disruption of Csrp2bp expression. The ocular expression pattern of Csrp2bp is consistent with a role for this protein in corneal development and pathogenesis of PPCD1.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Carrier Proteins/genetics , Chromosomes, Mammalian/chemistry , Corneal Dystrophies, Hereditary/genetics , Gene Rearrangement , Histone Acetyltransferases/genetics , RNA, Messenger/genetics , Transcription Factors/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Base Sequence , Carrier Proteins/metabolism , Chromosome Mapping , Cornea/metabolism , Cornea/pathology , Corneal Dystrophies, Hereditary/metabolism , Corneal Dystrophies, Hereditary/pathology , Disease Models, Animal , Exons , Genetic Association Studies , Genetic Complementation Test , Histone Acetyltransferases/metabolism , Humans , Introns , Mice , RNA, Messenger/metabolism , Sequence Deletion , Transcription Factors/metabolism
12.
Proc Natl Acad Sci U S A ; 111(52): 18757-62, 2014 Dec 30.
Article in English | MEDLINE | ID: mdl-25512522

ABSTRACT

The diurnal variation in acetaminophen (APAP) hepatotoxicity (chronotoxicity) reportedly is driven by oscillations in metabolism that are influenced by the circadian phases of feeding and fasting. To determine the relative contributions of the central clock and the hepatocyte circadian clock in modulating the chronotoxicity of APAP, we used a conditional null allele of brain and muscle Arnt-like 1 (Bmal1, aka Mop3 or Arntl) allowing deletion of the clock from hepatocytes while keeping the central and other peripheral clocks (e.g., the clocks controlling food intake) intact. We show that deletion of the hepatocyte clock dramatically reduces APAP bioactivation and toxicity in vivo and in vitro because of a reduction in NADPH-cytochrome P450 oxidoreductase gene expression, protein, and activity.


Subject(s)
Acetaminophen/pharmacokinetics , Analgesics, Non-Narcotic/pharmacokinetics , Circadian Rhythm , Cytochrome P-450 Enzyme System/biosynthesis , Gene Expression Regulation, Developmental , Hepatocytes/enzymology , Acetaminophen/adverse effects , Acetaminophen/pharmacology , Analgesics, Non-Narcotic/adverse effects , Analgesics, Non-Narcotic/pharmacology , Animals , Circadian Rhythm Signaling Peptides and Proteins/genetics , Circadian Rhythm Signaling Peptides and Proteins/metabolism , Cytochrome P-450 Enzyme System/genetics , Hepatocytes/pathology , Mice , Mice, Transgenic
13.
Proc Natl Acad Sci U S A ; 111(39): 14295-300, 2014 Sep 30.
Article in English | MEDLINE | ID: mdl-25225411

ABSTRACT

The circadian clock plays a significant role in many aspects of female reproductive biology, including estrous cycling, ovulation, embryonic implantation, onset of puberty, and parturition. In an effort to link cell-specific circadian clocks to their specific roles in female reproduction, we used the promoter that controls expression of Steroidogenic Factor-1 (SF1) to drive Cre-recombinase-mediated deletion of the brain muscle arnt-like 1 (Bmal1) gene, known to encode an essential component of the circadian clock (SF1-Bmal1(-/-)). The resultant SF1-Bmal1(-/-) females display embryonic implantation failure, which is rescued by progesterone supplementation, or bilateral or unilateral transplantation of wild-type ovaries into SF1-Bmal1(-/-) dams. The observation that the central clock, and many other peripheral clocks, are fully functional in this model allows the assignment of the implantation phenotype to the clock in ovarian steroidogenic cells and distinguishes it from more general circadian related systemic pathology (e.g., early onset arthropathy, premature aging, ovulation, late onset of puberty, and abnormal estrous cycle). Our ovarian transcriptome analysis reveals that deletion of ovarian Bmal1 disrupts expression of transcripts associated with the circadian machinery and also genes critical for regulation of progesterone production, such as steroidogenic acute regulatory factor (Star). Overall, these data provide a powerful model to probe the interlocking and synergistic network of the circadian clock and reproductive systems.


Subject(s)
ARNTL Transcription Factors/deficiency , ARNTL Transcription Factors/physiology , Embryo Implantation/physiology , Ovary/cytology , Ovary/physiology , Steroids/biosynthesis , ARNTL Transcription Factors/genetics , Aging/genetics , Aging/physiology , Animals , Circadian Rhythm/genetics , Circadian Rhythm/physiology , Embryo Implantation/drug effects , Embryo Implantation/genetics , Estrus/genetics , Estrus/physiology , Female , Gene Expression Profiling , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Ovary/transplantation , Pregnancy , Progesterone/administration & dosage , Promoter Regions, Genetic , Sexual Maturation/genetics , Sexual Maturation/physiology , Steroidogenic Factor 1/genetics
14.
J Biol Chem ; 286(18): 16246-60, 2011 May 06.
Article in English | MEDLINE | ID: mdl-21345800

ABSTRACT

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp(147) and Arg(514) in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP(+) revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP(+) shows movement of the Gly(631)-Asn(635) loop. In the NADP(+)-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP(+) shows movement of the Gly(631)-Asn(635) loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly(631)-Asn(635) loop movement controls NADPH binding and NADP(+) release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.


Subject(s)
Flavin Mononucleotide/chemistry , NADPH-Ferrihemoprotein Reductase/chemistry , NADP/chemistry , Amino Acid Substitution , Animals , Crystallography, X-Ray , Electron Transport , Flavin Mononucleotide/metabolism , Mutation, Missense , NADP/metabolism , NADPH-Ferrihemoprotein Reductase/genetics , NADPH-Ferrihemoprotein Reductase/metabolism , Oxidation-Reduction , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Rats
15.
PLoS One ; 5(8): e12213, 2010 Aug 16.
Article in English | MEDLINE | ID: mdl-20808945

ABSTRACT

The PPCD1 mouse, a spontaneous mutant that arose in our mouse colony, is characterized by an enlarged anterior chamber resulting from metaplasia of the corneal endothelium and blockage of the iridocorneal angle by epithelialized corneal endothelial cells. The presence of stratified multilayered corneal endothelial cells with abnormal patterns of cytokeratin expression are remarkably similar to those observed in human posterior polymorphous corneal dystrophy (PPCD) and the sporadic condition, iridocorneal endothelial syndrome. Affected eyes exhibit epithelialized corneal endothelial cells, with inappropriate cytokeratin expression and proliferation over the iridocorneal angle and posterior cornea. We have termed this the "mouse PPCD1" phenotype and mapped the mouse locus for this phenotype, designated "Ppcd1", to a 6.1 Mbp interval on Chromosome 2, which is syntenic to the human Chromosome 20 PPCD1 interval. Inheritance of the mouse PPCD1 phenotype is autosomal dominant, with complete penetrance on the sensitive DBA/2J background and decreased penetrance on the C57BL/6J background. Comparative genome hybridization has identified a hemizygous 78 Kbp duplication in the mapped interval. The endpoints of the duplication are located in positions that disrupt the genes Csrp2bp and 6330439K17Rik and lead to duplication of the pseudogene LOC100043552. Quantitative reverse transcriptase-PCR indicates that expression levels of Csrp2bp and 6330439K17Rik are decreased in eyes of PPCD1 mice. Based on the observations of decreased gene expression levels, association with ZEB1-related pathways, and the report of corneal opacities in Csrp2bp(tm1a(KOMP)Wtsi) heterozygotes and embryonic lethality in nulls, we postulate that duplication of the 78 Kbp segment leading to haploinsufficiency of Csrp2bp is responsible for the mouse PPCD1 phenotype. Similarly, CSRP2BP haploinsufficiency may lead to human PPCD.


Subject(s)
Corneal Diseases/genetics , Disease Models, Animal , Animals , Chromosomes, Mammalian/genetics , Comparative Genomic Hybridization , Corneal Diseases/pathology , Epithelium, Corneal/pathology , Humans , Male , Mice , Phenotype
16.
Biochemistry ; 42(22): 6804-13, 2003 Jun 10.
Article in English | MEDLINE | ID: mdl-12779335

ABSTRACT

NADPH-cytochrome P450 reductase is a flavoprotein which contains both an FAD and FMN cofactor. Since the distribution of electrons is governed solely by the redox potentials of the cofactors, there are nine different ways the electrons can be distributed and hence nine possible unique forms of the protein. More than one species of reductase will exist at a given level of oxidation except when the protein is either totally reduced or oxidized. In an attempt to unambiguously characterize the redox properties of the physiologically relevant FMNH(2) form of the reductase, the T491V mutant of NADPH-cytochrome P450 reductase has been reconstituted with 5'-deazaFAD which binds to the FAD-binding site of the reductase with a K(d) of 94 nM. The 5'-deazaFAD cofactor does not undergo oxidation or reduction under our experimental conditions. The molar ratio of FMN to 5'-deazaFAD in the reconstituted reductase was 1.1. Residual FAD accounted for less than 5% of the total flavins. Addition of 2 electron equivalents to the 5'-deazaFAD T491V reductase from dithionite generated a stoichiometric amount of the FMN hydroquinone form of the protein. The 5'-deazaFAD moiety remained oxidized under these conditions due to its low redox potential (-650 mV). The 2-electron-reduced 5'-deazaFAD reductase was capable of transferring only a single electron from its FMN domain to its redox partners, ferric cytochrome c and cytochrome b(5). Reduction of the cytochromes and oxidation of the reductase occurred simultaneously. The FMNH(2) in the 5'-deazaFAD reductase autoxidizes with a first-order rate constant of 0.007 s(-)(1). Availability of a stable NADPH-cytochrome P450 reductase capable of donating only a single electron to its redox partners provides a unique tool for investigating the electron-transfer properties of an intact reductase molecule.


Subject(s)
Flavin-Adenine Dinucleotide/analogs & derivatives , Flavin-Adenine Dinucleotide/chemistry , NADPH-Ferrihemoprotein Reductase/chemistry , NADPH-Ferrihemoprotein Reductase/genetics , Amino Acid Substitution , Animals , Binding Sites , Dithionite/chemistry , Escherichia coli/metabolism , Flavin Mononucleotide/chemistry , Kinetics , NADPH-Ferrihemoprotein Reductase/metabolism , Oxidation-Reduction , Protein Structure, Tertiary , Rats , Spectrophotometry, Ultraviolet
17.
J Biol Chem ; 277(8): 6536-41, 2002 Feb 22.
Article in English | MEDLINE | ID: mdl-11742006

ABSTRACT

The microsomal flavoprotein NADPH-cytochrome P450 oxidoreductase (CYPOR) is believed to function as the primary, if not sole, electron donor for the microsomal cytochrome P450 mixed-function oxidase system. Development of the mammalian embryo is dependent upon temporally and spatially regulated expression of signaling factors, many of which are synthesized and/or degraded via the cytochromes P450 and other pathways involving NADPH-cytochrome P450 oxidoreductase as the electron donor. Expression of CYPOR as early as the two-cell stage of embryonic development (The Institute for Genomic Research Mouse Gene Index, version 5.0, www.tigr.org/tdb/mgi) suggests that CYPOR is essential for normal cellular functions and/or early embryogenesis. Targeted deletion of the translation start site and membrane-binding domain of CYPOR abolished microsomal CYPOR expression and led to production of a truncated, 66-kDa protein localized to the cytoplasm. Although early embryogenesis was not affected, a variety of embryonic defects was observable by day 10.5 of gestation, leading to lethality by day 13.5. Furthermore, a deficiency of heterozygotes was observed in 2-week-old mice as well as late gestational age embryos, suggesting that loss of one CYPOR allele produced some embryonic lethality. CYPOR -/- embryos displayed a marked friability, consistent with defects in cell adhesion. Ninety percent of CYPOR -/- embryos isolated at days 10.5 or 11.5 of gestation could be classified as either Type I, characterized by grossly normal somite formation but having neural tube, cardiac, eye, and limb abnormalities, or Type II, characterized by a generalized retardation of development after approximately day 8.5 of gestation. No CYPOR -/- embryos were observed after day 13.5 of gestation. These studies demonstrate that loss of microsomal CYPOR does not block early embryonic development but is essential for progression past mid-gestation.


Subject(s)
Abnormalities, Multiple/enzymology , Fetal Death , NADPH-Ferrihemoprotein Reductase/genetics , Sequence Deletion , Abnormalities, Multiple/genetics , Animals , Crosses, Genetic , Gestational Age , Heart Defects, Congenital/embryology , Heart Defects, Congenital/enzymology , Heart Defects, Congenital/genetics , Mice , Mice, Knockout , NADPH-Ferrihemoprotein Reductase/deficiency , Neural Tube Defects/embryology , Neural Tube Defects/enzymology , Neural Tube Defects/genetics , Phenotype , Restriction Mapping
SELECTION OF CITATIONS
SEARCH DETAIL
...