Is cytochrome P‐450<sub>scc</sub> a transmembrane protein?

Usanov, Sergey A.; Chernogolov, Alexey; Chashchin, Vadim L.

doi:10.1016/0014-5793(90)81432-n

Cited by 16 publications

(12 citation statements)

References 6 publications

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“…Immunochemical identification of the recombinant proteins was determined by immunoblotting analysis (21). The concentration of AdR and Adx was determined using molar extinction coefficients of 11 mM -1 cm -1 at 450 nm, and 10 mM -1 cm -1 at 414 nm, respectively (22,23).…”

Section: Methodsmentioning

confidence: 99%

“…The mutant proteins were able to be reduced and bind CO as shown by the characteristic 450 nm spectral maximum suggesting an absence of serious changes in the conformation. A problem was encountered during the purification of the R426Q mutant because it did not bind to immobilized Adx, which is the method we typically use to affinity purify wild-type P450scc (14,21). Therefore, we replaced the Adx affinity chromatography procedure with hydrophobic chromatography using cholateSepharose 4B (immobilized cholic acid).…”

Section: Selection Of P450scc Residues Potentially Involved In Adxmentioning

confidence: 99%

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Probing the Interaction of Bovine Cytochrome P450scc (CYP11A1) with Adrenodoxin: Evaluating Site-Directed Mutations by Molecular Modeling

et al. 2002

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The present study was undertaken to evaluate the role of positively charged amino acid residues proposed to reside on the proximal surface of bovine cytochrome P450 cholesterol side chain cleavage (P450scc, CYP11A1) and to determine which residues may be involved in protein-protein interactions with the electron carrier adrenodoxin (Adx). In previous studies, nine different lysine residues were identified by chemical and immunological cross-linking experiments as potentially interacting with Adx, while in the present study, two arginine residues have been identified from sequence alignments. From these 11 residues, 13 different P450scc mutants were made of which only seven were able to be expressed and characterized. Each of the seven mutants were evaluated for their ability to bind Adx, to be reduced, and for their enzymatic activity. Among these, K403Q and K405Q showed a consistent decrease in Adx binding, the ability to be reduced by Adx, and enzymatic activity, with K405Q being affected to a much greater extent. More dramatic was the complete loss of Adx binding by R426Q, while still retaining its ability to be chemically reduced and bind carbon monoxide. Independently, a homology model of P450scc was constructed and docked with the structure of Adx. Four potential sites of interaction were identified: P450scc:K403 with Adx:D76, P450scc:K405 with Adx:D72; P450scc:R426 with Adx:E73, and P450scc:K267 with Adx:E47. Thus, the biochemical and molecular modeling studies together support the hypothesis that K267, K403, K405, and R426 participate in the electrostatic interaction of P450scc with Adx.

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Section: Methodsmentioning

confidence: 99%

Section: Selection Of P450scc Residues Potentially Involved In Adxmentioning

confidence: 99%

Probing the Interaction of Bovine Cytochrome P450scc (CYP11A1) with Adrenodoxin: Evaluating Site-Directed Mutations by Molecular Modeling

et al. 2002

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“…tease-Sonication of adrenocortical mitochondria in the presence of trypsin results in the clipping of endogenous cytochrome P450scc in two fragments of 26 and 29 kDa, which is considered to indicate a native conformation of the protein in the inner membrane (56,57). Newly imported Su9(112)-P450scc, however, was completely degraded by trypsin when mitochondria were lysed by sonication (data not shown).…”

Section: Fig 5 Pim1-mediated Proteolysis Of Newly Imported P450sccmentioning

confidence: 99%

ATP-dependent Proteolysis in Mitochondria

Savel'ev¹,

Novikova²,

Kovaleva³

et al. 1998

Journal of Biological Chemistry

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To analyze protein degradation in mitochondria and the role of molecular chaperone proteins in this process, bovine apocytochrome P450scc was employed as a model protein. When imported into isolated yeast mitochondria, P450scc was mislocalized to the matrix and rapidly degraded. This proteolytic breakdown was mediated by the ATP-dependent PIM1 protease, a Lon-like protease in the mitochondrial matrix, in cooperation with the mtHsp70 system. In addition, a derivative of P450scc was studied to which a heterologous transmembrane region was fused at the amino terminus. This protein became anchored to the inner membrane upon import and was degraded by the membrane-embedded, ATP-dependent m-AAA protease. Again, degradation depended on the mtHsp70 system; it was inhibited at nonpermissive temperature in mitochondria carrying temperature-sensitive mutant forms of Ssc1p, Mdj1p, or Mge1p. These results demonstrate overlapping substrate specificities of PIM1 and the m-AAA protease, and they assign a central role to the mtHsp70 system during the degradation of misfolded polypeptides by both proteases.Molecular chaperone proteins bind non-native protein structures and stabilize them against aggregation (1). By this means, they ensure proper folding of newly synthesized proteins, provide protection against heat denaturation, and mediate the vectorial translocation of polypeptides across biological membranes (2-7). Furthermore, evidence is accumulating that chaperone proteins play a pivotal role in ATP-dependent proteolytic processes (8 -10). Chaperone and proteolytic activities thereby constitute a quality control system which prevents the possibly deleterious accumulation of misfolded polypeptides in the cell. For instance, the degradation of misfolded polypeptides by Lon-like proteases in Escherichia coli or mitochondria depends on Hsp70 proteins which prevent the aggregation of substrate polypeptides (11)(12)(13)(14)(15). In addition to classical chaperone proteins that cooperate with ATP-dependent proteases during proteolysis, intrinsic chaperone-like properties have been assigned to some ATP-dependent proteases themselves which may be crucial for the degradation of non-native polypeptides (9, 10). The best studied cases are the hetero-oligomeric Clp proteases of prokaryotes whose regulatory subunits exert ATP-dependent chaperone activity (16,17).Several ATP-dependent proteases have been identified in mitochondria, which mediate the selective degradation of proteins in this organelle (10, 18,19). These proteases are required for maintenance of the respiratory competence of yeast cells suggesting important regulatory functions during the biogenesis of mitochondria. An ATP-dependent protease, highly homologous to Escherichia coli Lon protease, has been identified in the mitochondrial matrix space (20, 21). The corresponding genes from humans (22, 23) and yeast (24, 25) were cloned and termed PIM1 (for proteolysis in mitochondria) or LON. PIM1-mediated proteolysis is required for the maintenance of mitochondrial genome integ...

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“…Usanov et al . studied the interaction of the mitochondrion-associated CYP11A1 with antibodies to the whole enzyme and its fragments (7). We investigated CYPs 27A1 and 11A1 by combining heterologous expression in E. coli and site-directed mutagenesis (8, 11).…”

Section: Introductionmentioning

confidence: 99%

Studies of Membrane Topology of Mitochondrial Cholesterol Hydroxylases CYPs 27A1 and 11A1

Pikuleva

Mast

Liao

et al. 2008

Lipids

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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.

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Is cytochrome P‐450_scc a transmembrane protein?

Cited by 16 publications

References 6 publications

Probing the Interaction of Bovine Cytochrome P450scc (CYP11A1) with Adrenodoxin: Evaluating Site-Directed Mutations by Molecular Modeling

Probing the Interaction of Bovine Cytochrome P450scc (CYP11A1) with Adrenodoxin: Evaluating Site-Directed Mutations by Molecular Modeling

ATP-dependent Proteolysis in Mitochondria

Studies of Membrane Topology of Mitochondrial Cholesterol Hydroxylases CYPs 27A1 and 11A1

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Is cytochrome P‐450scc a transmembrane protein?

Cited by 16 publications

References 6 publications

Probing the Interaction of Bovine Cytochrome P450scc (CYP11A1) with Adrenodoxin: Evaluating Site-Directed Mutations by Molecular Modeling

Probing the Interaction of Bovine Cytochrome P450scc (CYP11A1) with Adrenodoxin: Evaluating Site-Directed Mutations by Molecular Modeling

ATP-dependent Proteolysis in Mitochondria

Studies of Membrane Topology of Mitochondrial Cholesterol Hydroxylases CYPs 27A1 and 11A1

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Is cytochrome P‐450_scc a transmembrane protein?