The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

The cytochromes P450 (P450) are a superfamily of hemoproteins which are involved in the oxidative metabolism of many endogenous and exogenous compounds. There are, perhaps, 100 different P450s in a single mammalian species which are located in either the mitochondria or the microsomes. The microsomal forms are part of the liver microsomal detoxification system, which is responsible for the inactivation of many drugs, insecticides, carcinogens, and other lipophilic compounds and can metabolize endogenous compounds such as steroids and fatty acids. In some cases, the P450s are responsible for the activation of compounds, carcinogenic agents being prime examples. Our primary interests are the mechanism by which the expression of these genes are regulated, particularly by phenobarbital, the mechanisms of targeting and retention in the microsomes and the amino acids of P450 important for determining substrate specificity and activity of the enzyme in its membrane environment.

In the gene expression studies, we have analyzed the promoter activity of P450 genes by a variety of techniques, including transfection into continuously cultured cell lines and primary cultures of hepatocytes and construction of transgenic mice. These studies led to the identification of regulatory factors involved in the liver-specific expression of the genes but were not useful models for phenobarbital induction. Recently, we have discovered that P450 gene DNA is taken by hepatocytes and expressed after direct injection of DNA into rat liver in situ. A fragment of DNA from the gene for CYP2B2 has been identified which mediates phenobarbital induction of P450 genes injected this way. The sequence contains several regulatory elements which have the properties of a phenobarbital-dependent enhancer. We are presently dissecting the regulatory elements in this enhancer and examining protein binding to the enhancer and chromatin structure in vivo.

We have been studying the signals involved in the insertion and retention of P450 into the endoplasmic reticulum (ER) membrane. Expression of chimeric proteins of P450 and other proteins not normally targeted to the ER has demonstrated that there are redundant complex ER retention signals in both the N-terminal hydrophobic signal sequence of P450 and the cytoplasmic domain. We had shown previously that the N-terminal sequence functioned as a combination membrane insertion, halt transfer, and membrane anchor sequence. We are presently utilizing mutagenesis to define the regions of P450 that are involved in retention in the ER and expression and photobleaching of P450-green fluorescent protein chimeras to examine the mechanism of ER retention.

We are also studying structural features of P450 that are important for substrate specificity and required for a functional protein when inserted in the membrane. Substrate specificity has been approached by cassette mutagenesis of regions expected to contain substrate contacting residues and construction of chimeric proteins of P450s with different substrate specificities. The proteins are expressed in mammalian cells and in E. coli and assayed for enzymatic activity. These studies have identified a small number of amino acids scattered along the P450 molecule which are critical for substrate interactions. We are also focusing on the region between the membrane anchor and the cytoplasmic catalytic domain of P450. Mutagenesis studies indicate that a glycine rich segment in this region may serve as a linker between the membrane and cytoplasmic domains. In contrast, within a nearby proline-rich region, structural requirements for activity at the proline positions vary dramatically and may be consistent with intra- or inter-protein interactions by this region. Both regions appear to be important for the proper assembly of newly synthesized P450.