Báo cáo khoa học: Expression and purification of orphan cytochrome P450 4X1 and oxidation of anandamide

Expression and purification of orphan cytochrome P450 4X1 and oxidation of anandamide

Tác giả: Katarina Stark, Miroslav Dostalek, F. P. Guengerich

Lĩnh vực: Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN, USA

Nội dung tài liệu: Nghiên cứu này tập trung vào việc biểu hiện và tinh chế protein P450 4X1, một loại enzyme “mồ côi” (orphan) chưa rõ chức năng sinh học. P450 4X1 đã được biểu hiện trong vi khuẩn E. coli và cho thấy khả năng chuyển hóa anandamide, một phân tử tín hiệu quan trọng, thành sản phẩm monooxygenated là 14,15-epoxyeicosatrienoic (EET) ethanolamide. Nghiên cứu cũng xác định sự biểu hiện mRNA của P450 4X1 ở nhiều vùng ngoài gan, bao gồm não, tim, tuyến tiền liệt và vú. Kết quả cho thấy vai trò tiềm năng của P450 4X1 trong việc điều hòa tín hiệu anandamide trong não.

Mục lục chi tiết:

• Keywords
• Correspondence
• Present address
• Cytochrome P450 (P450, EC 1.14.14.1, also termed ‘heme thiolate P450’) [1] monooxygenases are involved in tissue-specific conversions of many naturally occurring substances, for example, vitamins, hormones and signaling molecules, including the diverse group of the so-called eicosanoids [2]. P450 families 1-3 are primarily involved in the metabolism of therapeutic drugs and other xenobiotic chemicals, whereas families 4-51 consist of enzymes involved in the endogenous metabolism of important biological compounds, for example, steroids, fatty acids, vitamins and eicosanoids [3]. P450 subfamily 4F members are known to primarily oxidize endogenous compounds, for example, fatty acids and arachidonic acid derivatives [4]. The primary site of P450 metabolism is the liver, and the amount of P450 found in brain is relatively low, ranging from 1 to 10% of that found in liver [3]. P450 metabolism of fatty acids may be of importance in brain, as neurotransmitters and fatty acids are oxidized by P450s [4,5]. Arachidonic acid derivatives have been implicated in a large number of physiologically important processes. The arachidonic acid derivative anandamide (arachidonoyl ethanolamide) is a natural endocannabinoid
• Abbreviations
• Expression and purification of orphan cytochrome P450 4X1 and oxidation of anandamide
• Results
• Synthesis of codon-optimized P450 4X1 cDNA
• Expression of N-terminal variants
• Fig. 1. Optimizations introduced into the P450 4X1 cDNA for E. coli expression. Upper line, predicted amino acid sequence; middle line, nucleotide sequence predicted from genomic sequence; lower line, nucleotide sequence optimized for E. coli expression.
• Table 1. N-Terminal modifications used for heterologous expression of P450 4X1 membranes in E. coli [18] (supplementary Fig. S2). Amino acid changes are in italics and underlined.
• Fig. 2. Fe2+-CO versus Fe2+ difference spectra. (A) P450 4X1 construct 3 expression was performed in E. coli (with pGroES/EL12). The spectrum was recorded using 1/2 dilutions of whole-cell extracts and reducing with Na2S2O4. (B) Solubilized P450 4X1 (1.5 μμ). (C) Difference spectrum of purified P450 4X1 (0.14 μμ).
• Fig. 3. SDS/PAGE of purified recombinant P450 4X1. Lane 1, M, markers; lane 2, purified P450 4X1 (4 pmol).
• Fig. 4. Tissue distribution of P450 4X1 mRNA measured by real-time PCR. The relative levels of P450 4X1 mRNA were determined using real-time PCR in the tissues indicated, using GAPDH as a ref- erence standard. Different human cDNAs were used as templates and SYBR Green was used for detection. The mRNA levels are shown as the ratio of P450 4X1 to GAPDH and represent the mean of triplicate measurements from each sample. The relative expres- sion was calculated using the AC method (Livak). The graphs have standard deviations shown.
• Search for catalytic properties of P450 4X1
• Fig. 5. LC-MS analysis of the oxidized product formed from anandamide. The chromatogram shows selective ion monitoring of m/z 364 (MH+ of ananamide + 16). (A) Control reaction (no protein). (B) P450 4X1, NADPH-P450 reductase and NADPH. (C) P450 4X1 (and NADPH- P450 reductase) in the absence of NADPH. (D) Overlay of the product formation chromatograms from (B) and (C). Upper (-): P450 4X1 in the presence of NADPH; lower (——-): P450 4X1 in the absence of NADPH. (E) MS/MS spectra of 14,15-EET ethanolamide formed by P450 4X1, with the insert showing the x10 expansion of the indicated section of the spectrum.
• Discussion
• Experimental procedures
• Optimization of P450 4X1 and vector preparation
• Heterologous expression of P450 4X1
• Purification of recombinant P450 4X1
• Real-time PCR analysis of P450 4X1 expression
• LC-MS/MS analysis
• Assay of cholesterol oxidation
• Other assays and methods
• Data analysis
• Acknowledgements
• References
• Supplementary material