Tsuneo Omura

Gene regulation of steroidogenesis

The biosynthesis of various steroid hormones in animal tissues are catalyzed by six forms of cytochrome P450 and two hydroxysteroid dehydrogenases. The tissue-specific expression of these enzymes, which are under the control of the pituitary gland and mainly regulated at the transcriptional level, determines the steroidogenesis of animal tissues. Analysis of the promoter regions of the steroidogenic P450 genes revealed various cis-acting elements, including cAMP-responsive sequences (CRS), Ad4, and GC-rich sequences, which were needed for the tissue-specific and cAMP-dependent expression of the genes. Some of the nuclear protein factors binding to the cis-acting elements were isolated and characterized. A zinc-finger protein binding to Ad4, which was termed Ad4BP or SF-1, seems to be of particular importance in steroidogenesis. Ad4BP was expressed in the cells of the steroidogenic tissues, adrenal gland and gonadal tissues, in the rat fetus prior to the expression of steroidogenic P450s, and remained expressed only in steroidogenic cells in adult animals. Close investigation of the temporal and spacial expression of Ad4BP in the fetal tissues suggested its role in the differentiation of the steroidogenic tissues and the sex determination of the gonadal tissues.

A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins.

Rat liver cytosol contained an activity that stimulated the import of wheat germ lysate‐synthesized precursor proteins into mitochondria. The activity was purified 10,000‐fold from the cytosol as a homogeneous heterodimeric protein. This protein (termed mitochondrial import stimulation factor or MSF) stimulated the binding and import of mitochondrial precursor proteins. MSF was also found to recognize the presequence portion of mitochondrial precursors and catalyze the depolymerization and unfolding of in vitro synthesized mitochondrial precursor proteins in an ATP‐dependent manner; in this connection, MSF exhibited ATPase activity depending on the important‐incompetent mitochondrial precursor protein. The mitochondrial binding and import‐stimulating activities were strongly inhibited by the pretreatment of MSF with NEM, whereas the ATP‐dependent depolymerization activity was insensitive to the NEM treatment, suggesting that the process subsequent to the unfolding was inhibited with the NEM treatment. We conclude that MSF is a multifunctional cytoplasmic chaperone specific for mitochondrial protein import.

Purification and characterization of a processing protease from rat liver mitochondria.

A processing protease has been purified from the matrix fraction of rat liver mitochondria. The purified protease contained two protein subunits of 55 kd (P‐55) and 52 kd (P‐52) as determined by SDS‐PAGE. The processing protease was estimated to be 105 kd in gel filtration, indicating that the two protein subunits form a heterodimeric complex. At high ionic conditions, the two subunits dissociated. The purified processing protease cleaved several mitochondrial protein precursors destined to different mitochondrial compartments, including adrenodoxin, malate dehydrogenase, P‐450(SCC) and P‐450(11 beta), but the processing efficiencies were different each other. The endoprotease nature of the processing protease was confirmed with the purified enzyme using adrenodoxin precursor as the substrate; both the mature form and the extension peptide were detected after the processing. The processing activity of the protease was inhibited by metal chelators, and reactivated by Mn2+, indicating that the protease is a metalloprotease.

Cytoplasmic chaperones in precursor targeting to mitochondria: the role of MSF and hsp 70.

Despite extensive study since the early 1980s, the mechanism by which newly synthesized protein precursors are unfolded in the cytoplasm and targeted correctly to the mitochondrial surface prior to translocation through the mitochondrial membranes is understood poorly. Recently, an N-ethylmaleimide (NEM)-sensitive cytoplasmic factor called mitochondrial import stimulation factor (MSF), which catalyses the ATP-dependent unfolding of precursor proteins, was described. Unlike the more general chaperone proteins of the hsp70 families, MSF not only unfolds proteins but also targets the unfolded precursor proteins to the mitochondria. Here, Mihara and Omura summarize what is known about MSF and speculate on how it, and other cytoplasmic factors, may be involved in mitochondrial import.

Molecular cloning and sequence analysis of cDNA encoding rat adrenal cytochrome P-45011β

A cDNA clone encoding cytochrome P‐45011β of rat adrenal has been cloned and sequenced using a bovine P‐45011β cDNA insert (pcP‐450(11β)‐2; (1987) J. Biochem. 102, 559–568) as a probe. The nucleotide sequence contains an open reading frame sufficient to encode the entire amino acid sequence of a P‐45011β precursor protein consisting of 499 amino acids including an extension peptide of 24 amino acids at the NH2‐terminus. The cDNA contains 1247 nucleotides at the 3′‐noncoding region including 51 nucleotides of poly A, but lacks the 5′‐noncoding region. The deduced amino acid sequence shows 61% similarity to that of bovine P‐45011β. Putative binding sites for heme and steroid are highly conserved among steroidogenic P‐450s of known structure.

The amino-terminal structures that determine topological orientation of cytochrome P-450 in microsomal membrane.

We previously showed that the amino‐terminal region of P‐450 is responsible not only for targeting to endoplasmic reticulum (ER) membrane but also for stable anchoring to the membrane. In the present study, we introduced several mutations or deletions into the signal‐anchor region of the chimeric proteins in which the amino‐terminal regions of two forms of cytochrome P‐450 were fused to the mature portion of interleukin 2. The amino‐terminal acidic amino acid residues were replaced with basic amino acid residues or the hydrophobic core sequences were partially deleted, and these mutant proteins were assayed in vitro for their capacity to be inserted into or translocated across the ER membrane. The proteins that received the former manipulations were processed and the IL‐2 portion was translocated across the membrane. In one case, the processing did not occur, thereby enabling the chimeric protein to anchor on the luminal side of the ER. Those that received the latter manipulation were also processed and the IL‐2 portion translocated across the ER. These results strongly suggest that the signal‐anchor function is determined both by the amino‐terminal charged amino acid residues and by the length of the hydrophobic stretch.

[90] Isolation of cytochromes P-450 and P-420

Publisher Summary This chapter describes the isolation of cytochromes P-450 and P-420. P-450 is identified by the formation of a yellow-colored CO compound of the reduced pigment with a broad absorption peak at 450 mμ. As P-450 has a high affinity for CO, bubbling the dithionitereduced sample with CO for 10-20 seconds is sufficient to give a fully developed spectrum of the CO compound of P-450. From the CO difference spectrum of the reduced sample, the concentration of P-450 can be calculated using the extinction coefficient-difference between 450 mμ (peak) and 490 mμ. Presence of P-420 or hemoglobin does not interfere with the determination of P-450 by this method. If P-450 is not present, the concentration of P-420 can be obtained by measuring the difference of the optical density between 420 mμ (peak) and 490 mμ of the CO-difference spectrum of dithionitereduced samples. As the spectrum of the CO compound of P-420 is almost identical with that of hemoglobin, the sample for the determination of P-420 should be free from the blood pigment. The chapter also describes the purification procedure and properties of cytochromes P-450 and P-420.

Immunochemical evidence for the participation of cytochrome b5 in the NADH synergism of the NADPH-dependent mono-oxidase system of hepatic microsomes.

Abstract Anti-cytochrome b5 immunoglobulin (AIg) from a rabbit was used to establish the role of cytochrome b5 in the transfer of electrons from NADH or NADPH to the hepatic microsomal mono-oxidase system of the rat. AIg inhibited ethylmorphine (EM) N-demethylase when both NADH and NADPH were present, but had little effect when NADPH was the only source of electrons. Inhibition was reversed when AIg was preincubated with pure cytochrome b5. Specificity of AIg was shown by its inhibitory effect on NADH cytochrome c reductase activity; it was without effect on NADPH-cytochrome P-450 reductase or aniline hydroxylase activities. It is concluded that the second electron required for EM N-demethylation can be donated by NADH via cytochrome b5.

The Transmembrane Region of Microsomal Cytochrome P450 Identified as the Endoplasmic Reticulum Retention Signal

Abstract Microsomal-type cytochrome P450s are integral membrane proteins bound to the membrane through their N-terminal transmembrane hydrophobic segment, the signal anchor sequence. To elucidate the determinants that enable the P450s to be located in the ER, we constructed cDNAs encoding chimeric proteins in which a secretory form of carboxyesterase, carboxyesterase Sec, was connected to the N-terminus of the full-length or truncated forms of a microsomal-type P450, P45O(M1), and the constructed plasmids were expressed in COS cells. Since carboxyesterase Sec is an iV-glycosylated secretory protein, endo H treatment could be used to determine whether these chimeric proteins were located in the ER or not. Carboxyesterase Sec with the N-terminal 20 amino acids, containing the transmembrane region, of P46O(M1), was located in the ER, as determined from the endo H sensitivity of the expressed protein and immunofluorescence staining of the cells. As the expressed protein exhibited carboxyesterase activity, it was not retained in the ER through the BiP-dependent quality control system recognizing unfolded proteins. Another chimeric protein construct in which carboxyesterase Sec was connected to the C-terminal region of rat UDP-glucuronosyltransferase (UDP-GT), that contained a double-lysin ER retention motif, was also located in the ER, as determined from the endo H sensitivity and immunofluorescence staining. On the other hand, the sugar moiety of the carboxyesterase Sec connected to the transmembrane segment of UDP-GT, Sec/GTd, was partially resistant to the endo H treatment. From the results of immunofluorescent staining and cell fractiona-tion, it was concluded that the Sec/GTd product was located in the Golgi apparatus. These observations indicated that the N-terminal hydrophobic segment of P45O(M1) is sufficient for the ER membrane retention, whereas the transmembrane segment of UDP-GT is not. To determine whether microsomal P450s are recycled between the ER and Golgi compartments or not, a DNA construct encoding cathepsin D connected to the N-terminus of P45O(M1) was prepared and expressed in COS cells. The fusion protein was phosphorylated, but the phosphorylation was sensitive to alkaline phosphatase. As a control, authentic cathepsin D was subjected to phosphorylation of its oligosaccharide chain that was resistant to the alkaline phosphatase treatment. Since GlcNAc-P-transferase, which forms the alkaline phosphatase-resistant phosphodiester in the sugar chains of lysosome-targeting proteins, is located in the Golgi apparatus, it was concluded that the oligosaccharide chain of the cathepsin D portion of the fusion protein was not phosphorylated, and that the chimeric protein did not go to the Golgi apparatus. These results indicate that P45O(M1) is not recycled from the Golgi compartments to the ER in cells.

Different requirement for cytochrome b5 in NADPH-supported O-deethylation of p-nitrophenetole catalyzed by two types of microsomal cytochrome P-450

Abstract The role of cytochrome b 5 in the NADPH-supported O-deethylation of p-nitrophenetole catalyzed by cytochrome P-450 was studied with reconstituted systems using two types of cytochrome P-450 (P-450 PB and P-450 MC ) purified from rat liver microsomes. The O-deethylation by P-450 PB absolutely required the presence of cytochrome b 5 , whereas the same reaction catalyzed by P-450 MC did not require cytochrome b 5 . These effects of cytochrome b 5 on the activities of reconstituted systems were confirmed by the use of antibodies to cytochrome b 5 . On the other hand, the oxidations of ethylmorphine and aniline by these two types of cytochrome P-450 did not show significant dependence on cytochrome b 5 . These observations suggest that the requirement for cytochrome b 5 in NADPH-supported drug oxidations depends not only on the species of cytochrome P-450 catalyzing the reactions, but also on the substrates oxidized.