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Although the role of hydrophilic antioxidants in the development of hepatic insulin resistance and nonalcoholic fatty liver disease has been well studied, the role of lipophilic antioxidants remains poorly characterized. A known lipophilic hydrogen peroxide scavenger is bilirubin, which can be oxidized to biliverdin and then reduced back to bilirubin by cytosolic biliverdin reductase. Oxidation of bilirubin to biliverdin inside mitochondria must be followed by the export of biliverdin to the cytosol, where biliverdin is reduced back to bilirubin. Thus, the putative mitochondrial exporter of biliverdin is expected to be a major determinant of bilirubin regeneration and intracellular hydrogen peroxide scavenging. Here, we identified ABCB10 as a mitochondrial biliverdin exporter. ABCB10 reconstituted into liposomes transported biliverdin, and ABCB10 deletion caused accumulation of biliverdin inside mitochondria. Obesity with insulin resistance up-regulated hepatic ABCB10 expression in mice and elevated cytosolic and mitochondrial bilirubin content in an ABCB10-dependent manner. Revealing a maladaptive role of ABCB10-driven bilirubin synthesis, hepatic ABCB10 deletion protected diet-induced obese mice from steatosis and hyperglycemia, improving insulin-mediated suppression of glucose production and decreasing lipogenic SREBP-1c expression. Protection was concurrent with enhanced mitochondrial function and increased inactivation of PTP1B, a phosphatase disrupting insulin signaling and elevating SREBP-1c expression. Restoration of cellular bilirubin content in ABCB10 KO hepatocytes reversed the improvements in mitochondrial function and PTP1B inactivation, demonstrating that bilirubin was the maladaptive effector linked to ABCB10 function. Thus, we identified a fundamental transport process that amplifies intracellular bilirubin redox actions, which can exacerbate insulin resistance and steatosis in obesity.

Membrane proteins play important roles in health and disease. Despite their importance, the study of membrane proteins has been significantly limited by the difficulties inherent to their successful expression, purification, and stabilization once they have been extracted from the cell membrane. In addition, expression of human membrane proteins commonly requires the use of expensive and/or time-consuming eukaryotic systems, hence their successful expression in bacteria will be obviously beneficial for experimental research. Furthermore, since lipids can have critical effects on the activity of membrane proteins and given the composition similarities between the inner mitochondrial membrane and the bacterial plasma membrane, production of mitochondrial membrane proteins in E. coli represents a logical choice. Here, we present a novel protocol to produce a human mitochondrial ATP-Binding Cassette (ABC) transporter in E. coli. The function of the three known human mitochondrial ABC transporters is not fully understood, but X-ray crystallography models of ABCB10 produced in insect cells are available. We have successfully expressed and purified ABCB10 from E. coli. The yield is close to that of another bacterial ABC transporter routinely produced in our laboratory under similar conditions. In addition, we can efficiently reconstitute detergent purified ABCB10 into lipid nanodiscs. Measurements of ATPase activity of ABCB10 produced in E. coli show an ATP hydrolysis rate similar to other human ABC transporters. This novel protocol facilitates the production of this human mitochondrial transporter for biochemical, structural, and functional analysis, and can likely be adjusted for production of other mitochondrial transporters.

P-glycoprotein (Pgp) is an efflux pump important in multidrug resistance of cancer cells and in determining drug pharmacokinetics. Pgp is a prototype ATP-binding cassette transporter with two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Conformational changes at the NBDs (the Pgp engines) lead to changes across Pgp transmembrane domains that result in substrate translocation. According to current alternating access models (substrate-binding pocket accessible only to one side of the membrane at a time), binding of ATP promotes NBD dimerization, resulting in external accessibility of the drug-binding site (outward-facing, closed NBD conformation), and ATP hydrolysis leads to dissociation of the NBDs with the subsequent return of the accessibility of the binding site to the cytoplasmic side (inward-facing, open NBD conformation). However, previous work has not investigated these events under near-physiological conditions in a lipid bilayer and in the presence of transport substrate. Here, we used luminescence resonance energy transfer (LRET) to measure the distances between the two Pgp NBDs. Pgp was labeled with LRET probes, reconstituted in lipid nanodiscs, and the distance between the NBDs was measured at 37 °C. In the presence of verapamil, a substrate that activates ATP hydrolysis, the NBDs of Pgp reconstituted in nanodiscs were never far apart during the hydrolysis cycle, and we never observed the NBD-NBD distances of tens of Å that have previously been reported. However, we found two main conformations that coexist in a dynamic equilibrium under all conditions studied. Our observations highlight the importance of performing studies of efflux pumps under near-physiological conditions, in a lipid bilayer, at 37 °C, and during substrate-stimulated hydrolysis.

ATP-binding cassette (ABC) exporters transport substrates across biological membranes using ATP hydrolysis by a process that involves switching between inward- and outward-facing conformations. Most of the structural studies of ABC proteins have been performed with proteins in detergent micelles, locked in specific conformations and/or at low temperature. In this article, we present recent data from our laboratories where we studied the prototypical ABC exporter MsbA during ATP hydrolysis, at 37°C, reconstituted in a lipid bilayer. These studies were possible through the use of luminescence resonance energy transfer spectroscopy in MsbA reconstituted in nanodiscs. We found major differences between MsbA in these native-like conditions and in previous studies. These include a separation between the nucleotide-binding domains that was much smaller than previously thought, and a large fraction of molecules with associated nucleotide-binding domains in the nucleotide-free apo state. These studies stress the importance of studying membrane proteins in an environment that approaches physiological conditions.

Lipid nanodiscs have become a widely used approach for studying membrane proteins thanks to several advantages they offer. They have been especially useful for studying ABC transporters, despite the growing concern about the possible restriction of the conformational changes of the transporters due to the small size of the discs. Here, we performed a systematic study to determine the effect of the nanodisc size on the ATPase activity of model ABC transporters from human, plant, and bacteria. Our data confirm that the activity of the transporters and their response to regulatory molecules is affected by the nanodisc size. Our findings suggest the use of larger membrane scaffold proteins (MSPs), such as MSP2N2 nanodiscs, to minimize alterations caused by the commonly used small MSP1D1. [ABSTRACT FROM AUTHOR]