Heme Insertase machinery for cytochrome P450 NO synthase

University Of Michigan At Ann Arbor, Ann Arbor MI

Investigators

Abstract

Abstract The heme prosthetic moiety of P450 cytochromes is often damaged and destroyed during metabolism of drugs and other xenobiotics leaving dysfunctional apo-P450 protein. Although it was widely believed from studies with purified hemeproteins that heme is inserted without any factors, we discovered that Hsp90 plays a critical role in the cellular process. The long-term objective of the research is to elucidate the biological process of heme insertion into P450. The central hypothesis is that a heme insertase machinery comprising of Hsp90/Hsp70- based chaperones and novel protein factor(s) are responsible for heme reconstitution of P450. The overall objective is to define the role of Hsp70 and define the protein factor(s) that make up the heme insertase. The rationale is to take advantage of a recently developed subcellular system that faithfully recapitulates the Hsp90- dependent cellular heme insertion process with the use of apo-neuronal NO synthase (apo-nNOS), a P450-like enzyme. The nNOS has historically been a useful model for the study of chaperone regulation of liver P450 cytochromes and preliminary evidence with CYP2E1 indicates a similar Hsp90/Hsp70-dependent heme insertion process. MS studies also point to tubulin as a novel factor that works with Hsp90/Hsp70 chaperones. The hypothesis will be tested by pursuing three specific aims: 1) To characterize the role of tubulin in the heme insertase; 2) To define the role of Hsp70 in the heme insertion; 3) To characterize the heme insertase for CYP2E1. We take advantage of nNOS in the first two aims to define the minimal heme insertase machine and the roles of tubulin and Hsp70. The role of Hsp70 needs to be defined as Hsp90 does not always work with Hsp70 and no established precedence exists for the role of Hsp70 in heme insertion. We also aim to develop novel Hsp70 specific nanobodies. In Aim 3, we use cellular and subcellular systems to define the heme insertase for apo- CYP2E1. We will compare and contrast our findings to that for nNOS. Our studies will provide better tools to study heme insertion as standard techniques to knock down or overexpress chaperones often lead to variety of cellular effects that confound interpretation of the results. These studies are thought to be innovative as heme insertion was long thought to be independent of special protein factors and the identification of a heme insertase machine would be groundbreaking. These contributions are thought to be significant in understanding the fundamental process of heme insertion into P450 and maintenance of functional enzyme levels. Furthermore, effects on heme insertion should be considered in clinical testing of Hsp90 inhibitors and Hsp70-based therapies currently being developed.