The overall objective of this research is to understand the structural basis for the production of a functional cytochrome P450 (P450) molecule, which includes: a) proper targeting to the endoplasmic reticulum (ER), b) uptake of heme and assembly into an active hemoprotein, and c) achieving a membrane topology compatible with interactions with its substrates and P450 reductase. The N- terminal signal anchor sequence and following proline-rich region play an important role in each of these processes and the catalytic domain also contributes to membrane interaction.
The specific aims of the proposal are to: first, determine whether static retention of P450 2C2 involves targeting of P450 to ER subdomains different from those where transport vesicles are forming by using immunogold ultrastructural localization of P450 2C2 (statically retained), P450 2E1 (transported and retrieved to the ER), and sec13p, a marker of transport bud sites and by FRET analysis to determine whether P450 2E1 and P450 2C2 are localized together and form mixed oligomers; second, determine whether mutations in the signal anchor sequence that decrease ER retention alter the position of the signal anchor in the membrane by tryptophan fluorescence and its quenching by membrane depth-specific probes and by correlating oligomerization of P450 mutants with loss of static ER retention using FRET analysis; third, examine whether specific interactions of the signal anchor, particularly the linker region, with other proteins may be required for ER retention by substitution of the linker sequence into a heterologous membrane protein to test whether this specific sequence is sufficient to confer ER retention, by overexpression of the signal anchor sequence to determine whether the ER retention mechanism is saturable, and by yeast two-hybrid screening to identify any interacting proteins; fourth, identify the regions of the catalytic domain that interact with the membrane by tryptophan fluorescence scanning and the functional significance of the interactions by mutagenesis of the membrane interacting loops; and finally, to examine whether intramolecular interactions of the proline-rich region are important for assembly of P450 by examining reciprocal chimera of P450 2C2 and P450 3A4 which differ at one of the highly conserved prolines. These studies should provide significant insight into the structural determinants of P450s that are important for formation of a functional protein and the mechanisms of static ER retention. P450s mediate the detoxification of thousands of drugs and toxins, activation of carcinogens and pro-drugs, and biogenesis of many endogenous compounds. Mutations in human P450s have dramatic clinical effects on drug metabolism and steroid biogenesis. It is important to understand the genetic, or structural, basis for generating functional P450s, this proposal's objective, so that the role of these enzymes in normal and pathological human states can be better understood.
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