Acute hepatic heme deficiency is a biochemical hallmark of acute clinical attacks of hepatic porphyrias, genetic disorders stemming from inherited defects in heme synthesis. In genetically predisposed individuals, such acute attacks are commonly triggered by some prescription drugs, starvation, infections and/or inflammation. Inhibition of heme synthesis in rat or mouse hepatocytes also results in acute heme depletion and consequent autoactivation of hepatic heme-regulated inhibitor HRI eIF2? kinase, an exquisite heme-sensor. The ensuing increased eIF2? phosphorylation causes global translational arrest of de novo synthesis of a myriad of hepatic proteins including cytochromes P450 (P450s), hemoprotein enzymes engaged in the breakdown and clearance of endo- and xenobiotics such as drugs, carcinogens, toxins, natural and chemical products. Such hepatic heme depletion also results in the translationally arrested synthesis of hepatic I?B? (a NF?B-inhibitor) and consequent nuclear activation of NF?B, a proinflammatory factor involved in inflammation, cancer, autophagy apoptosis and immunity. Concurrently, autophagic-lysosomal degradation (ALD) of existing and otherwise longer-lived hepatic P450s is also rapidly accelerated, in a heme-reversible manner. Activation of the other three eIF2? kinases is associated with NF?B-mediated autophagy, thereby indicating that hepatic HRI activation could similarly induce P450 ALD. The specific role of hepatic HRI in NF?B activation and consequent P450 ALD remains to be established and will be examined in hepatocytes from mice with genetic HRI knockout (Hri-/-) relative to wild type (Hri+/+) controls. Relatively little is mechanistically known about P450 ALD other than its recognition as a slow bulk process. Studies are therefore proposed to characterize the molecular determinants and participants operating in normal P450 ALD pathway, and to elucidate those responsible for accelerating P450 degradation upon hepatic heme depletion. Such elucidation will rely on approaches such as site-directed mutagenesis, lentiviral shRNA interference, immunoaffinity capture and proteomic analyses. The liver is a major supplier of proteins, lipids and nutrients to the rest of the body, and thus translational arrest of de novo synthesis coupled with enhanced ALD of hepatic proteins is bound to have far-reaching physiological/ pathophysiological consequences. Our preliminary proteomic analyses reveal that within a few hours of hepatic heme depletion, several hepatic proteins including P450s are decreased >2-fold, while a few are actually increased >2-fold. Studies are proposed in Hri-/- and Hri+/+ hepatocytes using SILAC (stable isotope labeling of amino acids in culture) coupled with proteomics to identify and quantify the hepatic proteins whose synthesis and/or degradation are so dramatically altered. The specific hepatic proteins whose turnover is markedly altered upon acute heme depletion and HRI activation may provide insight into the symptomatology and manifestations of acute hepatic porphyria. Moreover, such global alterations of hepatic P450 turnover would also rationalize the impaired drug clearance clinically observed in porphyric patients.
Hepatic porphyrias are inherited disorders of heme biosynthesis, wherein one of the enzymes required for the production of heme for hemoglobin, myoglobin, mitochondrial cytochromes, microsomal drug-metabolizing cytochromes P450 (P450s) and other hemoproteins, is deficient or defective. Although heme production is essential for the survival of all cells, its production is most active in the liver and bone marrow. In genetically predisposed individuals, prescription drugs, infections and/or inflammation commonly precipitate acute attacks characterized by severe abdominal and neurological symptoms, that can be fatal if not treated with heme infusion. A hallmark of these attacks is acute heme deficiency. In the mouse and rat liver, we have shown that acute heme deficiency activates a heme-inhibitable 'heme-sensor' HRI eIF2? kinase, an enzyme also present in the human liver. We have found that such liver HRI activation not only shuts off the formation of new liver proteins but also results in the destruction of existing proteins. Such shut-off and destruction of liver P450s, (enzymes that are responsible for the breakdown and elimination of ingested drugs, carcinogens and other chemicals and dietary substances), we propose result in the markedly impaired drug clearance observed in porphyric patients. Furthermore, we find that arrested protein synthesis of I?B?, an inhibitor of the proinflammatory/proimmune NF-?B factor, leads to NF-?B activation, triggering a pathogenic cascade of events. This NF-?B activation in genetically susceptible individuals, we believe may account for the precipitation of an acute porphyric attack by infections and/or inflammation.
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