Instability of the human cytochrome P450 reductase A287P variant is the major contributor to its Antley-Bixler syndrome-like phenotype

Karen M. McCammon, Satya P. Panda, Chuanwu Xia, Jung Ja P Kim, Daniela Moutinho, Michel Kranendonk, Richard J. Auchus, Eileen M. Lafer, Debashis Ghosh, Pavel Martasek, Rekha Kar, Bettie Sue Masters, Linda J. Roman

Research output: Contribution to journalArticle

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Abstract

Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo. Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.

Original languageEnglish
Pages (from-to)20487-20502
Number of pages16
JournalJournal of Biological Chemistry
Volume291
Issue number39
DOIs
Publication statusPublished - 23 Sep 2016

Fingerprint

Antley-Bixler Syndrome Phenotype
NADPH-Ferrihemoprotein Reductase
Oxidoreductases
Differential Scanning Calorimetry
Proteins
Cytochrome P-450 Enzyme System
Mutation
Differential scanning calorimetry
Sexual Development
Protein Synthesis Inhibitors
Protein Stability
Osteoblasts
Xenobiotics
Mutant Proteins
Cycloheximide
Purification
Genes
Crystal structure
Cells
Cell Line

Keywords

  • cytochrome P450
  • flavoprotein
  • membrane protein
  • protein crystallization
  • protein stability
  • reductase
  • POR deficiency
  • cytochrome P450 oxidoreductase
  • diflavin oxidoreductase

Cite this

McCammon, Karen M. ; Panda, Satya P. ; Xia, Chuanwu ; Kim, Jung Ja P ; Moutinho, Daniela ; Kranendonk, Michel ; Auchus, Richard J. ; Lafer, Eileen M. ; Ghosh, Debashis ; Martasek, Pavel ; Kar, Rekha ; Masters, Bettie Sue ; Roman, Linda J. / Instability of the human cytochrome P450 reductase A287P variant is the major contributor to its Antley-Bixler syndrome-like phenotype. In: Journal of Biological Chemistry. 2016 ; Vol. 291, No. 39. pp. 20487-20502.
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abstract = "Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo. Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.",
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McCammon, KM, Panda, SP, Xia, C, Kim, JJP, Moutinho, D, Kranendonk, M, Auchus, RJ, Lafer, EM, Ghosh, D, Martasek, P, Kar, R, Masters, BS & Roman, LJ 2016, 'Instability of the human cytochrome P450 reductase A287P variant is the major contributor to its Antley-Bixler syndrome-like phenotype' Journal of Biological Chemistry, vol. 291, no. 39, pp. 20487-20502. https://doi.org/10.1074/jbc.M116.716019

Instability of the human cytochrome P450 reductase A287P variant is the major contributor to its Antley-Bixler syndrome-like phenotype. / McCammon, Karen M.; Panda, Satya P.; Xia, Chuanwu; Kim, Jung Ja P; Moutinho, Daniela; Kranendonk, Michel; Auchus, Richard J.; Lafer, Eileen M.; Ghosh, Debashis; Martasek, Pavel; Kar, Rekha; Masters, Bettie Sue; Roman, Linda J.

In: Journal of Biological Chemistry, Vol. 291, No. 39, 23.09.2016, p. 20487-20502.

Research output: Contribution to journalArticle

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T1 - Instability of the human cytochrome P450 reductase A287P variant is the major contributor to its Antley-Bixler syndrome-like phenotype

AU - McCammon, Karen M.

AU - Panda, Satya P.

AU - Xia, Chuanwu

AU - Kim, Jung Ja P

AU - Moutinho, Daniela

AU - Kranendonk, Michel

AU - Auchus, Richard J.

AU - Lafer, Eileen M.

AU - Ghosh, Debashis

AU - Martasek, Pavel

AU - Kar, Rekha

AU - Masters, Bettie Sue

AU - Roman, Linda J.

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N2 - Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo. Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.

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KW - flavoprotein

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KW - protein stability

KW - reductase

KW - POR deficiency

KW - cytochrome P450 oxidoreductase

KW - diflavin oxidoreductase

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