Fig 1.
Alternative Splicing of CYP4B1.
(A) Human CYP4B1. The human CYP4B1 intron 5–6 harbors two splice acceptors (SAs) in frame allowing the generation of two alternative CYP4B1 transcripts, with (isoform 2: NM_001099772) or without (isoform 1: NM_000779) insSer207. (B) Rabbit CYP4B1. Intron 5–6 of rabbit CYP4B1 carries only a single SA and therefore generates a single transcript without insSer202 (isoform 1: NM_001082103). (C) Alignment of CYP4B1 sequences from other species. Alignment of the genomic DNA of CYP4B1 from other species shows that the insertion of the CAG triplet, which generates the additional splice acceptor site, exists only in humans and great apes, and not in rhesus, macaque, marmoset or other mammals.
Fig 2.
Frequency of insSer207 in human CYP4B1 transcripts.
(A) Schematic outline of CYP4B1. The forward (fw) primer binds in exon 4 and the reverse (rv) primer in exon 9, thus resulting in PCR products of 240 bp and 243 bp, respectively. (B) Frequency of human CYP4B1 without (w/o) and with insSer207. The plasmid DNA from 220 bacterial colonies was isolated and sequenced. The percentages of CYP4B1 transcripts with and without (w/o) insSer207 are shown.
Fig 3.
Functional analysis of CYP4B1.
(A) Schematic outline of the lentiviral (LV) vectors used for expression of CYP4B1. For functional testing, the different CYP4B1 forms were cloned into a LV vector with an IRES-EGFP site. CMV: CMV promoter; SD: splice donor; LTR: long terminal repeat; SA: splice acceptor; RRE: Rev responsive element, cPPT: central polypurine binding tract; SFFV U3: U3 promoter of the spleen focus-forming virus; mcs: multicloning site; IRES: internal ribosomal entry site; EGFP: enhanced green fluorescent protein. (B) Toxicity of HepG2 cells after 4-IPO exposure. Survival of EGFP+ HepG2 cells (%) after 24 and 48 h of incubation with 0, 2.9, 29, 290 or 1450 μM 4-IPO as measured by flow cytometry. The cells stably expressed the different CYP4B1 enzymes: h-S427: human wild-type (isoform 1); h-S427 insSer207: human wild-type with insSer207 (isoform 2), h-P427: human mutated p.S427P (artificial); h-P427 insSer207: human mutated p.S427P with insSer207 (artificial); r-P422: rabbit wild-type (isoform 1); r-P422 insSer202: rabbit with insSer202 (artificial). For each construct, the mean ± standard error of the mean (SEM) values are shown from at least three independent experiments. The transduction efficiency of HepG2 cells (not shown) was ≥90%, as determined by EGFP expression in flow cytometry. (C) Toxicity of T cells after 4-IPO exposure. Survival of EGFP+ primary human T cells (%) expressing the different CYP4B1 proteins [listed for (B)] after 24 and 48 h of incubation with increasing doses of 4-IPO as assessed by flow cytometry. For each construct, the mean ± SEM values are shown from at least three experiments.
Fig 4.
Bioactivation of 4-IPO to a stable NAC/NAL adduct.
The NADPH-dependent formation of NAC/NAL adducts by membranes isolated from HepG2 cells stably transduced with the different CYP4B1 cDNAs r-P422: rabbit wild-type (isoform 1); h-S427: human wild-type (isoform 1); h-P427: human mutated p.S427P (artificial); h-S427 insSer207: human wild-type with insSer207 (isoform 2) after incubation in 50 nM 4-IPO for 20 minutes is shown. The adduct formation rate is shown as the mean ± standard deviation (SD) of triplicate incubations.
Fig 5.
(A) Schematic outline of the lentiviral vector used for expression of CYP4B1. CMV: CMV promoter; SD: splice donor; LTR: long terminal repeat; SA: splice acceptor; RRE: Rev responsive element, cPPT: central polypurine binding tract; SFFV U3: U3 promoter of the spleen focus-forming virus; mcs: multicloning site; IRES: internal ribosomal entry site; neoR: neomycin phosphotransferase (nptII) resistance cDNA; EGFP: enhanced green fluorescent protein. (B) Protein half-life analysis of CYP4B1 isoforms in HepG2 cells. Protein half-life analysis was performed by adding 50 μg/ml cycloheximide (CHX) to the transduced and G418-selected HepG2 cells. The mean fluorescence intensity (MFI) of CYP4B1/EGFP fusion proteins was assessed at various time points by flow cytometry. For each construct, the mean ± SEM values are shown from at least three experiments.
Fig 6.
Structural model of human CYP4B1 with insSer207 located in a loop region from G198 to S210.
Cartoon representation of the structure of human CYP4B1. The side chain of insSer207 and the heme prosthetic group are shown as spacefilling and ball and stick representations, respectively. Carbon atoms are colored gray, oxygen atoms are colored red, nitrogen atoms are colored blue, and hydrogen atoms are colored white. The figure was generated using UCSF Chimera [43].