* Structure Activity Relationships of Cytochrome P450
* Membrane-Cytochrome P450 Interactions
* Regulation of Cytochrome P450 Gene Expression
3D-Structure of cytochrome P450
What is Cytochrome
P450? Cytochromes P450 (P450) are a superfamily of
hemoproteins which are involved in the oxidative
metabolism of many endogenous and exogenous compounds.
There are more than 50 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.
Research Interests
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 the proper
folding and membrane interactions of the protein.
In the gene expression studies, we
are studying the molecular mechanisms by which
phenobarbital induces the activity of
P450s. A phenobarbital-responsive
enhancer mediates the induction. This
enhancer has binding motifs for several regulatory
proteins and has been designated a phenobarbital
responsive unit (PBRU). A key regulatory factor is
constitutive androstane receptor (CAR), which in
response to phenobarbital treatment is translocated to
the nucleus, binds to the PBRU, and activates gene
expression. We are studying complex of proteins,
including CAR, other DNA binding proteins, and
co-regulators, that bind to PBRU induce expression of
the gene and the changes in chromatin structure mediated
by these proteins. We are also studying the mechanisms
of cytoplasmic/nuclear shuttling of CAR. These studies
involve analysis of protein-DNA and protein-protein
interactions; the analysis of P450 promoter activity by
a variety of techniques, including transfection of
continuously cultured cell lines and liver cells in
situ; in vivo footprinting, analysis of protein binding
and transcription of P450 genes reassembled into
chromatin in vitro, and determination of cellular
location of CAR chimera with fluorescent proteins and
confocal microscopy.
We also study the signals involved in
the insertion and retention of P450 into the endoplasmic
reticulum (ER) membrane and the mechanism of the
retention process. Expression of chimeric proteins of
P450 and green fluorescent protein has demonstrated that
there are redundant complex ER retention signals in both
the N-terminal hydrophobic signal sequence of P450 and
the cytoplasmic domain. In addition to cellular
targeting, we are examining the regions of P450 that
interact with the membrane, which include the N-terminal
signal anchor as well as peptide sequences in the
catalytic domain of the protein. We have also been
examining the role of the "linker" sequence, which
connects the signal anchor sequence and the catalytic
domain, and appears to facilitate assembly of the
protein. Our major methodologies in these studies are
mutagenesis or construction of chimeric proteins
followed by expression of the modified proteins in
mammalian cells, insect cells, or bacteria in order to
examine the functional properties of the mutant or
chimeric proteins.
Cytochromes P450 (P450) are a superfamily of
hemoproteins which are involved in the oxidative
metabolism of many endogenous and exogenous compounds.
There are more than 50 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. 
In the gene expression studies, we
are studying the molecular mechanisms by wh
We also study the signals involved in
the insertion and retention of P450 into the endoplasmic
reticulum (ER) membrane and the mechanism of the
retention process. Expression of chimeric proteins of
P450 and green fluorescent protein has demonstrated that
there are redundant complex ER retention signals in both
the N-terminal hydrophobic signal sequence of P450 and
the cytoplasmic domain. In addition to cellular
targeting, we are examining the regions of P450 that
interact with the membrane, which include the N-terminal
signal anchor as well as peptide sequences in the
catalytic domain of the protein. We have also been
examining the role of the "linker" sequence, which
connects the signal anchor sequence and the catalytic
domain, and appears to facilitate assembly of the
protein. Our major methodologies in these studies are
mutagenesis or construction of chimeric proteins
followed by expression of the modified proteins in
mammalian cells, insect cells, or bacteria in order to
examine the functional properties of the mutant or
chimeric proteins.