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Research ArticleMinireview

Alternative Splicing in the Cytochrome P450 Superfamily Expands Protein Diversity to Augment Gene Function and Redirect Human Drug Metabolism

Andrew J. Annalora, Craig B. Marcus and Patrick L. Iversen
Drug Metabolism and Disposition April 2017, 45 (4) 375-389; DOI: https://doi.org/10.1124/dmd.116.073254

Article Figures & Data

Figures

  • Fig. 1.
    Fig. 1.

    Comparative analysis of the complete human cytochrome P450 transcriptome and the total number of single-nucleotide polymorphisms. Radar plot comparison of the total number of (A) human P450 transcript variants and (B) SNPs for each of the 57 human cytochrome P450 genes are shown, on the basis of a meta-analysis of information of the NCBI’s Ensembl, PubMed, and AceView databases and the GeneCards.org website. Total transcript variants for individual P450 genes identified in this study are superimposed (in pink) over the current number listed in the AceView database alone (in purple). The expansion in variant numbers we identified highlights the challenge of predicting interindividual variability in polymorphic P450 gene splicing, predicated on existing transcript databases. Radar plot comparison of the total human SNPs associated (as reported by GeneCards.org) revealed CYP5A1 (or thromboxane synthase 1) as the most polymorphic P450 gene (5274), with CYPs 2C19 (2467), 7B1 (3400), 19A1 (2178), and 39A1 (2129) showing higher rates of polymorphism than the average P450 gene (∼850 mutations per gene). (C) Bar graph representation of alternative P450 transcript-to-SNP ratios for each gene is shown at the bottom. CYP2E1, with 24 transcript variants and the lowest number of SNPs (33), displays the highest alternative transcript-to-SNP ratio (0.73) among human P450 genes. CYPs 21A2 (0.33), 27B1 (0.15), and CYP2D6 (0.11) also skew above normal for this ratio (∼0.05 alternative transcripts per SNP). Improved understanding of the SNP-based mechanisms that alter P450 gene splicing will facilitate the identification of novel SNP biomarkers that are predictive of drug interactions and human disease.

  • Fig. 2.
    Fig. 2.

    Tissue-specific and transformation-sensitive alternative splicing of CYP1A1 in humans. CYP1A1 is inducible in virtually every tissue of the body; however, a brain-specific CYP1A1 splice variant has been identified that preferentially skips exon 6 (Kommaddi et al., 2007). This shortened form of the enzyme is spectrally active but does not metabolize benzo(a)pyrene to toxic metabolites in brain. Structural analysis of the CYP1A1 crystal structure (PDB: 4I8V) indicates that removal of exon 6 (in yellow ribbon) would: 1) expand the opening of the pw2b substrate access channel, 2) reshape the ligand binding pocket by altering the β1-4 sheet, and 3) alter the redox partner binding surface via elimination of the K′ helix. A similar pattern of tissue-specific, alternative exon 2 usage in CYP1A1 has been reported in tumor cells (Leung et al., 2005). Ovarian cancer cells skip an 84-bp cryptic intron in exon 2 of CYP1A1 but remain in-frame to produce a catalytically unique splice variant with diminished estradiol metabolism that localizes to the nucleus and mitochondria, rather than the ER. Cryptic intron removal in exon 2 eliminates 28-amino-acid residues among helices E, F, and the E–F loop (shown in transparent gold ribbon), which putatively expands the opening of the pw3 substrate access channels located above the heme center and helix I.

  • Fig. 3.
    Fig. 3.

    Alternative-splicing of the human CYP24A1 gene: tissue- and tumor-specific exons 1, 2, and 10 inclusion. CYP24A1 is a mitochondrial P450 composed of 11 coding exons that metabolizes the vitamin D hormone to regulate its role in calcium homeostasis, cell growth, and immunomodulation. Alternatively spliced transcript variants have been identified for this gene, including a CYP24A1 splice variant (CYP24sv) specifically expressed in macrophage (Ren et al., 2005), which skips exons 1 and 2. The 372-amino-acid variant protein (∼43 kDa) lacks the N-terminus, helices A′, A, B, and B′ and portions of the β1 sheet but probably retains the ability to bind heme and substrate in some capacity. Ren and coauthors concluded that CYP24sv represents a cytosolic variant with dominant-negative function that quarantines 25-hydroxyvitamin D3 and other hormonal forms of vitamin D, slowing the rate of their metabolism in the mitochondria or endoplasmic reticulum. Structural analysis of CYP24A1 (PDB: 3K9V) suggests that CYP24sv’s unique N-terminus, derived from intron 2 (shown in green), reshapes the pw2a substrate access channel and provides contacts for sealing the ligand binding pocket via interactions with helices F–G and the β1 and β4 sheets systems. It is notable that exons 1 and 2 exclusion in CYP24A1 is exacerbated in human tumors of the breast and colon, where N-terminally truncated splice variants of approximately 40, 42, and 44 kDa have been identified (Fischer et al., 2009a; Horváth et al., 2010; Scheible et al., 2014). An additional, prostate cancer-related, splice variant of CYP24A1 that cleanly skips exon 10 has also been described (Horvath et al., 2010; Muindi et al., 2007). Exon 10 encodes much of the protein’s proximal surface, including the β1–3 sheet, meander region, CYS loop, and portions of the L-helix involved in heme-thiolate bond formation. Variants lacking exon 10 may express defects in heme and redox partner binding, giving rise to an alternative dominant negative form that may retain membrane-binding features; loss of exon 10 could also refine the alternative functions of cytosolic splice variants lacking exons 1 and 2.

  • Fig. 4.
    Fig. 4.

    Endoxenobiotic crosstalk among cytochrome P450 and nuclear receptor genes coordinate alternative splicing and resemble a primitive immune system. Human tissues are subject to exposure from over 400 FDA-approved drugs, >10,000 xenobiotics, and untold numbers of endogenous substrates and their metabolites (x > 100,000). Cytochrome P450 genes participate in phase I detoxification of many of these compounds, including model substrates benzo[a]pyrene (via CYP3A4) and calcifediol (via CYP24A1). P450 genes are classically induced to silence endoxenobiotic signaling through cognate nuclear receptors, which modulate global gene expression and splicing events by “coloring” or modulating the composition of coregulatory factors that comprise both the transcription complex and the spliceosome complex, which ultimately alter the nature of both ribosome assembly and gene expression (Auboeuf et al., 2005). Model substrates are subject to metabolism by a finite population of P450s in a given tissue, however, and because each gene is sensitive to an infinite number of environmentally sensitive, alternative splicing events, each individual may express a unique, tissue-specific “P450 gene cloud” comprising both wild-type (WT) and splice-altered variant forms (e.g., SV1, SV2, etc.). P450 splice variants can: 1) display reduced ability to metabolize model substrates, 2) function as dominant negatives to sequester compounds from metabolism or potentiate basal NR-mediated signaling, or 3) function as a conformationally distinct protein with alternative metabolic function or cellular role. When coupled with existing paradigms of alternate P450 trafficking and membrane-associated cooperativity, an integrated network of crosstalk among 57 P450 and 48 NR genes begins to emerge, as novel P450 metabolites may engage NR signaling pathways in unique ways that reprogram gene splicing and expression to promote cellular homeostasis in the face of endocrine disruption. NR signaling cascades can alter both the transcriptome and epigenome of an individual, providing an elegant feedback mechanisms for adaptation to cellular stress created by unique personal history and disease status.

Tables

  • TABLE 1 

    Pathologies associated with P450 splice variants

    P450 IsoformVariant FormRelated DiseaseReference
    CYP1A1∆Exon 2 and 6Ovarian cancerChinta et al. (2005)
    Bauer et al. (2007)
    Leung et al. (2005)
    CYP1B1Partial ∆Exon 2GlaucomaStoilov et al. (1998)
    Li et al. (2014)
    CYP2A6DeletionsLung cancerLiu et al. (2013b)
    CYP2B6∆Exons 4–6Liver/colon cancerMiles et al. (1989)
    CYP8A1∆Exons 2 and 9Essential hypertensionNakayama et al. (2002b)
    Nakayama et al. (2002a)
    CYP11B2Trans-splicing to CYP11B1Congenital adrenal hyperplasiaHampf et al. (2001)
    CYP17A1∆Exons 2, 4, 6, or 817-α Hydroxylase deficiency, 
congenital adrenal hyperplasiaYamaguchi et al. (1998)
    Costa-Santos et al. (2004)
    Hwang et al. (2011)
    CYP19A1∆Exon 5Aromatase deficiencyPepe et al. (2007)
    CYP24A1∆Exons 9–11Breast, colon, and prostate cancerMuindi et al. (2007)
    CYP26B1∆Exon 2AtherosclerosisElmabsout et al. (2012)
    Oral cancerChen et al. (2014)
    CYP27A1∆Exons 4,6, 7Cerebrotendinous xanthomatosisGaruti et al. (1996)
    Verrips et al. (1997)
    Chen et al. (1998)
    CYP27B1∆Exons 4–7Glioblastoma, leukemia, melanoma, cervical cancer, ovarian cancerMaas et al. (2001)
    Flanagan et al. (2003)
    Fischer et al. (2007)
    Wu et al. (2007)
    Fischer et al. (2009b)
    CYP46A1Intron 2Alzheimer diseaseLi et al. (2013a)
    Li et al. (2013b)
  • TABLE 2 

    Summary of human P450 splice variants

    Enzyme FamilyTranscript SizeExonsAmino AcidsMol. WeightVariant RNAsSNPsDisease LinksRatio of Viable Protein
    Gene no.NucleotideskDaTotal no.Total no.Total
    CYP1 (3)2683–59883–7512–54358.2–60.849a10522030.64b (31)
    CYP2 (16)1681–50245–9491–59655.7–67.6281118534130.50 (141)
    CYP3 (4)1604–216713–14502–53457.1–61.31203029930.47 (56)
    CYP4 (12)1678–33955–16505–63859.1–71.31709725370.53 (90)
    CYP5 (1)20931359466.930527460.48 (14)
    CYP7 (2)2765–29506504–50657.7–58.343688350.80 (3)
    CYP8 (2)3974–56321–10501–52758.1–59.591752160.77 (7)
    CYP11 (3)2311–35589503–52157.6–60.1521807740.57 (30)
    CYP17 (1)2001850857.413179310.57 (7)
    CYP19 (1)44791050357.9472178450.57 (27)
    CYP20 (1)18111347053.421130810.54 (11)
    CYP21 (1)21531049556.03091350.52 (16)
    CYP24 (1)32661251458.917711190.46 (8)
    CYP26 (3)1569–45366–7497–52256.2–57.523858220.68 (16)
    CYP27 (3)2579–51178–9372–53142.6–60.2551582580.58 (32)
    CYP39 (1)26231246954.115217920.50 (8)
    CYP46 (1)21861550056.81195270.57 (6)
    CYP51 (1)2817686098.01838820.65 (12)
    Total 571569–56321–16372–86042.6–98.096548,38410970.534 (515)

    • ↵a Total number of variant RNA transcripts represents the total of all nonredundant transcripts identified for human P450 forms in the AceView, Ensembl, and PubMed databases. Transcripts with the same predicted protein molecular weight (in kDa) with a fewer than four base-pair differences in nucleotide transcript length were considered the same transcript. Data for SNPs was obtained from GeneCards.org, and Disease Links were obtained from the MalaCards.org.

    • ↵b Ratio of viable protein represents the fraction of human P450 variant transcripts predicted to code functional proteins using the highest confidence scoring metrics for the AceView (very good) and ENSEMBL (Protein coding/merged Ensembl/Havana). Total number of predicted variant proteins indicated in parenthesis.

Additional Files

  • Data Supplement

    Files in this Data Supplement:

    • Supplemental Data -

      Supplemental Discussion

      Supplemental References

      Supplemental Table 1 - CYP Transcript Variant Subcellular Localization

      Supplemental Table 2 - Therapeutic Applications of Splice-Switching Oligonucleotides

      Supplemental Figure 1 - Schematic Diagram of the Alternative Gene Splicing Process

      Supplemental Figure 2 - Alternative Splicing and Function of the CYP1B1 Gene

      Supplemental Figure 3 - Alternative-Splicing of the Human CYP19A1: Tissue-specific Exon 1 and 5 Inclusion

      Supplemental Figure 4 - Alternative Splicing of Human Drug Metabolizing P450s: CYP2C9 and CYP2D6

      Supplemental Figure 5 - Trans-Splicing of the Human CYP3A Gene Family: Cryptic Alternative Exon 1 Usage

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Research ArticleMinireview

Alternative P450 Splicing Redirects Human Drug Metabolism

Andrew J. Annalora, Craig B. Marcus and Patrick L. Iversen
Drug Metabolism and Disposition April 1, 2017, 45 (4) 375-389; DOI: https://doi.org/10.1124/dmd.116.073254
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