Expression and Functional Characterization of Breast Cancer-Associated Cytochrome P450 4Z1 inSaccharomyces cerevisiae

Abstract

CYP4Z1 is an "orphan" cytochrome P450 (P450) enzyme that has provoked interest because of its hypothesized role in breast cancer through formation of the signaling molecule 20-hydroxyeicosatetraenoic acid (20-HETE). We expressed human CYP4Z1 in and evaluated its catalytic capabilities toward arachidonic and lauric acids (AA and LA). Specific and sensitive mass spectrometry assays enabled discrimination of the regioselectivity of hydroxylation of these two fatty acids. CYP4Z1 generated 7-, 8-, 9-, 10-, and 11-hydroxy LA, whereas the 12-hydroxy metabolite was not detected. HET0016, the prototypic CYP4 inhibitor, only weakly inhibited laurate metabolite formation (IC ∼15 M). CYP4Z1 preferentially oxidized AA to the 14(S),15(R)-epoxide with high regioselectivity and stereoselectivity, a reaction that was also insensitive to HET0016, but neither 20-HETE nor 20-carboxy-AA were detectable metabolites. Docking of LA and AA into a CYP4Z1 homology model was consistent with this preference for internal fatty acid oxidation. Thus, human CYP4Z1 has an inhibitor profile and product regioselectivity distinct from most other CYP4 enzymes, consistent with CYP4Z1's lack of a covalently linked heme. These data suggest that, if CYP4Z1 modulates breast cancer progression, it does so by a mechanism other than direct production of 20-HETE.

Interrogation of CYP2D6 Structural Variant Alleles Improves the Correlation Between CYP2D6 Genotype and CYP2D6‐Mediated Metabolic Activity

“…Rare variants were characterized in vitro using a cytochrome P450 S. cerevisiae model (detailed in ) . Methods include construction of yeast expression plasmids: three controls (wild‐type, inactive, and intermediate) and seven rare coding CYP2D6 variants (P8S, S168A, D198N, P267L, V338M, A449D, and R474Q).…”

“…Rare variants were characterized in vitro using a cytochrome P450 S. cerevisiae model (detailed in Supplementary Methods). [37][38][39][40][41][42] Methods include construction of yeast expression plasmids: three controls (wild-type, inactive, and intermediate) and seven rare coding CYP2D6 variants (P8S, S168A, D198N, P267L, V338M, A449D, and R474Q). Each construct was functionally characterized using both a click chemistry-compatible ticlopidine 5′-carboxypropargyl amide probe that has specificity for CYP2D6 activity and a fluorogenic CYP2D6 substrate, Vivid 7-ethoxymethoxy-3-cyanocoumarin.…”

Expression and Function of Eicosanoid-Producing Cytochrome P450 Enzymes in Solid Tumors

“…Lung cancer Unknown (Leclerc et al, 2010;Alexanian et al, 2012;Alexanian and Sorokin, 2013) CYP4Z1 Epoxygenase Breast cancer -Pro-angiogenic (Yu et al, 2012;McDonald et al, 2017) includes surgical resection followed by adjuvant therapy whereas the more aggressive SCLC treatments rely exclusively on chemotherapy and targeted immunotherapy (Zhao et al, 2018;Duma et al, 2019). Despite best efforts, mortality rates remain among the highest compared to other cancers.…”

“…The other EETs (11,12-8,9-and 5,6-EETs) were not investigated, and thus it is unclear if they also promote similar resistance to chemotherapeutic intervention. Unfortunately, Luo et al specifically examined 14,15-EET and did not identify any specific epoxygenase responsible for its biosynthesis although CYP4Z1 is a possible candidate (McDonald et al, 2017). Studies by Luo et al and Wei et al both focused primarily on 14,15-EET and while the four isoforms of EETs are typically thought to elicit similar responses, there are no studies that identify definitive and distinct pathways for each regioisomer, if they exist.…”

Screening of cytochrome 4Z1 expression in human non-neoplastic, pre-neoplastic and neoplastic tissues

“…Moreover, high levels of 20-hydroxyeicosatetraenoic acid (20-HETE) metabolites were possibly generated through the metabolism of arachidonic acid by CYP4Z1 [ 19 ]. Conversely, a recent functional analysis reported the absence of 20-HETE metabolites in the CYP4Z1 reaction with arachidonic acid in a recombinant enzyme system [ 20 ]. This discrepancy needs further study with purified reconstituted CYP4Z1 to examine the enzyme’s substrate and product specificities.…”