Structural and Biochemical Properties of the P450aro

The P450aro is a membrane-bound enzyme localized in the endoplasmic reticulum; it is present in the brain, in the gonads (both in the ovary and in the testis), as well as in several extragonadal tissues (e.g., the human placenta, the adipose tissue, the skin) (see ref. 41).

The enzyme is the product of the CYP19 gene, and is a member of the P450 cytochrome superfamily. In humans, it consists of a protein of 503 amino acids with an apparent molecular weight of about 58 kDa (42). Associated to the enzyme, is the flavoprotein NADPH-cytochrome P450 reductase, which is responsible for transferring the reducing equivalents from NADPH to the P450aro (43).

Studies on the amino-acid sequence of the P450aro, derived from the corresponding cDNA, revealed that the catalytic site is located towards the carboxy terminal of the protein, in a region rich in cysteine residues able to bind the heme iron. Near the heme-binding region, there is a portion (I-helix) which is believed to form the substrate-binding pocket. The putative membrane-spanning domain, characterized by a region of high hydrophobic amino acids, is located towards the amino-terminus of the protein. Site-directed mutagenesis experiments have revealed that the region between the amino acids 10 and 20 is critical for the conformational integrity of the enzyme (42,44).

The CYP19 gene is located on the long arm of the chromosome 15, spans at least 70 kb, and is composed of 10 exons, the first of which (exon I) is untranslated (45). The translation start site (ATG) is located in exon II, and the sequence encoding the open reading frame is identical in all tissues. Because of this, the aromatase proteins expressed in the various tissues have the same amino acid sequence. Owing to the high conservation of the protein during phylogenesis, there is a high degree of homology among the different species: for instance, 77, 81, and 73% between the human P450aro on one side and the rat, the mouse and the chicken enzymes on the other (46,47). The possible existence of two different P450aro isoforms has been described only in pigs (48).

The identity of the structure of the P450aro in the different tissues does not correspond to an analogous identity of the mRNAs coding for the protein. In some tissues (e.g., the human placenta, mouse and rat ovaries) the use of alternative splicing during the mRNA processing gives rise to P450aro transcripts of different length. In particular, three different mRNA species have been demonstrated in the rat ovary (49); among these, only the largest one seems to be able to produce a functional protein, because the two smaller species lack the heme-binding domain coding region, and contain an unspliced intron. However, these two transcripts have not been identified in brain structures (31). A high degree of heterogeneity has been demonstrated in the 5'-untranslated region of exon I. Exon I presents several subtypes (named exon I.a, I.b, and so forth, or, alternatively, I.1, I.2, and so forth) (47,50-52) which can be alternatively utilized for the synthesis of the different mRNA species in the various tissues. Moreover, the expression of tissue-specific P450aro transcripts occurs through the alternative utilization of multiple and distinct promoters located upstream to each exon subtype. Studies on the human, monkey, and rat brains have revealed that the major P450aro transcript of the hypothalamus and of the amygdala possesses an unique exon I (exon I-f), which is now considered the brain-specific one (50-52). In the human brain, it has been found that the promoter region of exon I.f contains putative TATA and CAAT boxes (which participate in the initiation of the transcription), as well as a consensus sequence for Ad4, a factor involved in the regulation of the genes of all steroidogenic enzymes. Moreover, the analysis of the DNA sequence of this promoter region revealed the presence of a potential androgen/glucocorticoid binding site at about 300 bp upstream from exon I.f (50). It is noteworthy that, both in humans and in rodents, the same promoter seems to be used for the transcription of the P450aro gene in the hypothalamus as well as in the amygdala. Because it is known that the expression of the enzyme is regulated in a different fashion by sex steroids in these two structures (see refs. 26 and 52; also next section), it might be hypothesized that these brain regions possess structural and functional peculiarities (e.g., neuronal inputs, cell-specific transacting factors, and so forth), which could explain this phenomenon. Yamada-Mouri and coworkers (52) have recently confirmed that, in the CNS, the amount of P450aro mRNA derived from the "brain-specific" exon I-f predominates over other types of P450aro mRNA carrying the information of other types of exons I (51). They have also shown that the ratio of the P450aro mRNA derived from exon I.f over the total P450aro mRNA is higher in the amygdala than in the hypothalamus. This observation further strengthens the region-specific regulation of the expression of this enzyme.

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