Silybin regulates P450s activity by attenuating endoplasmic reticulum stress in mouse nonalcoholic fatty liver disease.

Cytochrome P450s are important phase I metabolic enzymes located on endoplasmic reticulum (ER) involved in the metabolism of endogenous and exogenous substances. Our previous study showed that a hepatoprotective agent silybin restored CYP3A expression in mouse nonalcoholic fatty liver disease (NAFLD). In this study we investigated how silybin regulated P450s activity during NAFLD. C57BL/6 mice were fed a high-fat-diet (HFD) for 8 weeks to induce NAFLD, and were administered silybin (50, 100 mg ·kg-1 ·d-1, i.g.) in the last 4 weeks. We showed that HFD intake induced hepatic steatosis and ER stress, leading to significant inhibition on the activity of five primary P450s including CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP3A in liver microsomes. These changes were dose-dependently reversed by silybin administration. The beneficial effects of silybin were also observed in TG-stimulated HepG2 cells in vitro. To clarify the underlying mechanism, we examined the components involved in the P450 catalytic system, membrane phospholipids and ER membrane fluidity, and found that cytochrome b5 (cyt b5) was significantly downregulated during ER stress, and ER membrane fluidity was also reduced evidenced by DPH polarization and lower polyunsaturated phospholipids levels. The increased ratios of NADP+/NADPH and PC/PE implied Ca2+ release and disruption of cellular Ca2+ homeostasis resulted from mitochondria dysfunction and cytochrome c (cyt c) release. The interaction between cyt c and cyt b5 under ER stress was an important reason for P450s activity inhibition. The effect of silybin throughout the whole course suggested that it regulated P450s activity through its anti-ER stress effect in NAFLD. Our results suggest that ER stress may be crucial for the inhibition of P450s activity in mouse NAFLD and silybin regulates P450s activity by attenuating ER stress.

Role of cytochrome b5 in the modulation of the enzymatic activities of cytochrome P450 17α-hydroxylase/17,20-lyase (P450 17A1).

Cytochrome b5 (cyt b5) is a small hemoprotein that plays a significant role in the modulation of activities of an important steroidogenic enzyme, cytochrome P450 17α-hydroxylase/17,20-lyase (P450 17A1, CYP17A1). Located in the zona fasciculata and zona reticularis of the adrenal cortex and in the gonads, P450 17A1 catalyzes two different reactions in the steroidogenic pathway; the 17α-hydroxylation and 17,20-lyase, in the endoplasmic reticulum of these respective tissues. The activities of P450 17A1 are regulated by cyt b5 that enhances the 17,20-lyase reaction by promoting the coupling of P450 17A1 and cytochrome P450 reductase (CPR), allosterically. Cyt b5 can also act as an electron donor to enhance the 16-ene-synthase activity of human P450 17A1. In this review, we discuss the many roles of cyt b5 and focus on the modulation of CYP17A1 activities by cyt b5 and the mechanisms involved.

Stabilization of cytochrome b5 by a conserved tyrosine in the secondary sphere of heme active site: A spectroscopic and computational study.

Heme proteins perform a large array of biological functions, with the heme group bound non-covalently or covalently. To probe the stabilization role of conserved tyrosine residue in the secondary sphere of heme site in heme proteins, we herein used cytochrome b5 (Cyt b5) as a model protein, and mutated Tyr30 to Phe or His by removal of Tyr30 associated H-bond network and hydrophobic interaction. We performed thermal-induced unfolding studies for the two mutants, Y30F Cyt b5 and Y30H Cyt b5, as monitored by both UV-Vis and CD spectroscopy, as well as heme transfer studies from these proteins to apo-myoglobin, with wild-type Cyt b5 under the same conditions for comparison. The reduced stability of both mutants indicates that both the H-bonding and hydrophobic interactions associated with Tyr30 contribute to the protein stability. Moreover, we performed molecular modeling studies, which revealed that the hydrophobic interaction in the local region of Y30F Cyt b5 was well-remained, whereas Y30H Cyt b5 formed an H-bond network. These observations suggest that the conserved Tyr30 in Cyt b5 is not replaceable due to the presence of both the H-bond network and hydrophobic interaction in the secondary sphere of the heme active site. As demonstrated here for Cyt b5, it may be of practical importance for design of artificial heme proteins by engineering a Tyr in the secondary sphere with improved properties and functions.

Dynamics of α-Hb chain binding to its chaperone AHSP depends on heme coordination and redox state.

BACKGROUND: AHSP is an erythroid molecular chaperone of the α-hemoglobin chains (α-Hb). Upon AHSP binding, native ferric α-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7). METHODS: Recombinant AHSP, WT α-Hb:AHSP and α-Hb(HE7Q):AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated α-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of α-Hb bound to AHSP as well as α-Hb release from the α-Hb:AHSP complex were measured by time-resolved absorption spectroscopy. RESULTS: AHSP binding to α-Hb is kinetically controlled to prevail over direct binding with ß-chains and is also thermodynamically controlled by the α-Hb redox state and not the liganded state of the ferrous α-Hb. The dramatic instability of isolated ferric α-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in α-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric α-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to α-Hb within the Hb-tetramer. CONCLUSION: α-Hb reduction could trigger its release from AHSP toward its final Hb ß-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis. GENERAL SIGNIFICANCE: The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia.

Active site proton delivery and the lyase activity of human CYP17A1.

Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon-carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional "Compound I" rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17's physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon-carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.