The xenobiotic metabolizing cytochromes P450 (P450s) are among the most versatile biological catalysts known, but knowledge of the structural basis for their broad substrate specificity has been limited. P450 2B4 has been frequently used as an experimental model for biochemical and biophysical studies of these membrane proteins. A 1.6-Å crystal structure of P450 2B4 reveals a large open cleft that extends from the protein surface directly to the heme iron between the ␣-helical and ␤-sheet domains without perturbing the overall P450 fold. This cleft is primarily formed by helices B to C and F to G. The conformation of these regions is dramatically different from that of the other structurally defined mammalian P450, 2C5/3LVdH, in which the F to G and B to C regions encapsulate one side of the active site to produce a closed form of the enzyme. The open conformation of 2B4 is trapped by reversible formation of a homodimer in which the residues between helices F and G of one molecule partially fill the open cleft of a symmetryrelated molecule, and an intermolecular coordinate bond occurs between H226 and the heme iron. This dimer is observed both in solution and in the crystal. Differences between the structures of 2C5 and 2B4 suggest that defined regions of xenobiotic metabolizing P450s may adopt a substantial range of energetically accessible conformations without perturbing the overall fold. This conformational flexibility is likely to facilitate substrate access, metabolic versatility, and product egress. T he cytochromes P450 (P450s) are a superfamily of hemecontaining monooxygenases. They are responsible for the metabolism of an unusually wide range of endogenous and exogenous substrates, including synthesis of steroid hormones, bile acids, and cholesterol, and the degradation of steroids, fatty acids, drugs, toxins, and procarcinogens (1). P450s from families 1, 2, and 3 have evolved to convert lipophilic xenobiotics to more polar metabolites readily conjugated by phase II enzymes, and thus targeted for elimination. The stereo-and regiospecificity of metabolite formation by individual xenobiotic metabolizing P450s suggest very specific substrate-enzyme interactions, whereas the range of substrates metabolized suggests an induced fit type of substrate recognition. Understanding the basis for this specific, yet versatile, metabolism by mammalian xenobiotic metabolizing P450s has been limited by the dearth of structural information for these membrane proteins.In 2000, the first mammalian P450 structure was published (2, 3), that of P450 2C5, which was engineered to delete the single N-terminal transmembrane domain and to mutate a peripheral membrane-binding site. Recent structures of 2C5 bound with the substrates, diclofenac (4) and 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (DMZ) (5), indicate that flexible regions of the protein adapt for substrate binding, and that ligands may bind in multiple orientations. The 2C5 structures generally reveal the enzyme closed around these substrates wi...

Prostate cancer is the most commonly diagnosed malignancy among men in industrialized countries, accounting for the second leading cause of cancer-related deaths. While we now know that the androgen receptor (AR) is important for progression to the deadly advanced stages of the disease, it is poorly understood what AR-regulated processes drive this pathology. Here, we demonstrate that AR regulates prostate cancer cell growth via the metabolic sensor 5′-AMP-activated protein kinase (AMPK), a kinase that classically regulates cellular energy homeostasis. In patients, activation of AMPK correlated with prostate cancer progression. Using a combination of radiolabeled assays and emerging metabolomic approaches, we also show that prostate cancer cells respond to androgen treatment by increasing not only rates of glycolysis, as is commonly seen in many cancers, but also glucose and fatty acid oxidation. Importantly, this effect was dependent on androgen-mediated AMPK activity. Our results further indicate that the AMPK-mediated metabolic changes increased intracellular ATP levels and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-mediated mitochondrial biogenesis, affording distinct growth advantages to the prostate cancer cells. Correspondingly, we used outlier analysis to determine that PGC-1α is overexpressed in a subpopulation of clinical cancer samples. This was in contrast to what was observed in immortalized benign human prostate cells and a testosterone-induced rat model of benign prostatic hyperplasia. Taken together, our findings converge to demonstrate that androgens can co-opt the AMPK-PGC-1α signaling cascade, a known homeostatic mechanism, to increase prostate cancer cell growth. The current study points to the potential utility of developing metabolic-targeted therapies directed towards the AMPK-PGC-1α signaling axis for the treatment of prostate cancer.

To better understand ligand-induced structural transitions in cytochrome P450 2B4, protein-ligand interactions were investigated using a bulky inhibitor. Bifonazole, a broad spectrum antifungal agent, inhibits monooxygenase activity and induces a type II binding spectrum in 2B4dH ( Cytochromes P4503 of the 1, 2, and 3 families play a central role in drug metabolism and detoxification (1). Unlike most classical enzymes with their strict substrate selectivity, these microsomal P450s can each bind and metabolize a number of substrates that are different in size, shape, and stereochemistry. A challenging problem is to understand how microsomal P450s accommodate different substrates and oxidize each in a regiospecific and stereo-specific manner. At the tertiary structural level, P450s exhibit a similar overall fold with a well conserved heme-binding core (2). However, recent results demonstrate that microsomal P450s exhibit striking conformational diversity and plasticity (3-5). Not only do different P450s exhibit different conformations, but an individual P450 can adopt multiple conformations in response to various ligands, making it difficult to model protein-ligand interactions even when one atomic structure of a P450 is known. Therefore, a good sampling of P450 "conformational space" is a prerequisite for understanding P450-ligand interactions and for performing modeling studies with high predictive power.P450s from the 2B subfamily are inducible by barbiturates and have been the object of numerous biochemical and biophysical investigations for several decades. P450 2B4, in particular, was the first microsomal P450 to be purified and is one of the best characterized xenobiotic metabolizing P450s (6). Recent structural investigations using an N-terminal modified form of P450 2B4 (2B4dH) have revealed that the enzyme can adopt strikingly different conformations (4, 5), making it one of the best sources for study of P450 conformational plasticity. In addition, a wealth of site-directed mutagenesis data on 2B4 and other 2B enzymes are available to correlate structural features with functional properties (7). When P450 2B4dH was crystallized without ligand, a tight dimer was formed by two symmetrical molecules that each adopted a widely open conformation (4). A large cleft is formed by helices F through G on one side and the BЈ/C loop and helix C on the other side (Fig. 1A). The cleft is ϳ15 Å wide and is filled by helices FЈ and GЈ of the symmetry-related molecule, whose His-226 forms an intermolecular coordinate bond to the heme iron of the other monomer. In order to investigate the structure of 2B4 with a ligand bound, the H226Y mutant was created to minimize dimer formation (5). When 2B4dH(H226Y) was crystallized with the inhibitor 4-(4-chlorophenyl)imidazole (CPI), a closed conformation was observed (Fig. 1B), which resembles those of P450s 2C5 (8 -10), 2C8 (11), 2C9 (12, 13), and 3A4 (14, 15). The BЈ/C loop and the N terminus of helix I move toward the active site, making contact with CPI. Helices F through G m...

A 1.9-Å molecular structure of the microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site was determined by x-ray crystallography. In contrast to the previous experimentally determined 2B4 structure, this complex adopted a closed conformation similar to that observed for the mammalian 2C enzymes. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B and C, the N terminus of helix I, and the ␤ 4 region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F and G helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding. Cytochromes P450 (P450)1 are involved in steroidogenesis, fatty acid metabolism, synthesis of bile and retinoid acids, and production of plant toxins, but it is their function in the elimination of xenobiotics that has received the most attention. In mammals, xenobiotic metabolizing P450s play the central role in detoxification of hydrophobic drugs, carcinogens, and toxins by decreasing the lipid solubility of these chemicals and, thus, promoting excretion. In contrast to the strict substrate selectivity of classical enzymes, xenobiotic-metabolizing P450s can each bind and oxidize a set of substrates with distinct sizes, shapes, and stereochemical features. Although the variety of substrates binding to a given P450 is often broad, the oxidation of each is usually remarkably regiospecific and stereospecific.Identification of the structural basis for the specific monooxygenation and binding of a diverse but select set of substrates has been a particularly challenging goal, which is an important prerequisite for understanding selective substrate oxidation. Although the diversity of substrates might suggest an easily accessible active site, initial structures of both soluble bacterial (1) and microsomal mammalian (2) P450s revealed active sites buried within the globular structure of the protein.A few recent bacterial structures, however, suggest a "lid" domain composed of helices F and G, the motion of which controls substrate entry (3-5). Protein ...

Retromer is a membrane coat complex that is recruited to endosomes by the small GTPase Rab7 and sorting nexin 3. The timing of this interaction and consequent endosomal dynamics are thought to be regulated by the guanine nucleotide cycle of Rab7. Here we demonstrate that TBC1d5, a GTPase-activating protein (GAP) for Rab7, is a high-affinity ligand of the retromer cargo selective complex VPS26/VPS29/VPS35. The crystal structure of the TBC1d5 GAP domain bound to VPS29 and complementary biochemical and cellular data show that a loop from TBC1d5 binds to a conserved hydrophobic pocket on VPS29 opposite the VPS29–VPS35 interface. Additional data suggest that a distinct loop of the GAP domain may contact VPS35. Loss of TBC1d5 causes defective retromer-dependent trafficking of receptors. Our findings illustrate how retromer recruits a GAP, which is likely to be involved in the timing of Rab7 inactivation leading to membrane uncoating, with important consequences for receptor trafficking.

Although checkpoint inhibitors that block CTLA-4 and PD-1 have improved cancer immunotherapies, targeting additional checkpoint receptors may be required to broaden patient response to immunotherapy. PVRIG is a coinhibitory receptor of the DNAM/TIGIT/CD96 nectin family that binds to PVRL2. We report that antagonism of PVRIG and TIGIT, but not CD96, increased CD8 þ T-cell cytokine production and cytotoxic activity. The inhibitory effect of PVRL2 was mediated by PVRIG and not TIGIT, demonstrating that the PVRIG-PVRL2 pathway is a nonredundant signaling node. A combination of PVRIG blockade with TIGIT or PD-1 blockade further increased T-cell activation. In human tumors, PVRIG expression on T cells was increased relative to normal tissue and trended with TIGIT and PD-1 expression. Tumor cells coexpressing PVR and PVRL2 were observed in multiple tumor types, with highest coexpression in endometrial cancers. Tumor cells expressing either PVR or PVRL2 were also present in numbers that varied with the cancer type, with ovarian cancers having the highest percentage of PVR À PVRL2 þ tumor cells and colorectal cancers having the highest percentage of PVR þ PVRL2 À cells. To demonstrate a role of PVRIG and TIGIT on tumor-derived T cells, we examined the effect of PVRIG and TIGIT blockade on human tumor-infiltrating lymphocytes. For some donors, blockade of PVRIG increased T-cell function, an effect enhanced by combination with TIGIT or PD-1 blockade. In summary, we demonstrate that PVRIG and PVRL2 are expressed in human cancers and the PVRIG-PVRL2 and TIGIT-PVR pathways are nonredundant inhibitory signaling pathways. See related article on p. 244

By converting cholesterol to 24S-hydroxycholesterol, cytochrome P450 46A1 (CYP46A1) initiates the major pathway for cholesterol removal from the brain. Two crystal structures of CYP46A1 were determined. First is the 1.9-Å structure of CYP46A1 complexed with a high-affinity substrate cholesterol 3-sulfate (CH-3S). The second structure is that of the substrate-free CYP46A1 at 2.4-Å resolution. CH-3S is bound in the productive orientation and occupies the entire length of the banana-shaped hydrophobic active-site cavity. A unique helix B -C loop insertion (residues 116 -120) contributes to positioning cholesterol for oxygenation catalyzed by CYP46A1. A comparison with the substrate-free structure reveals substantial substrate-induced conformational changes in CYP46A1 and suggests that structurally distinct compounds could bind in the enzyme active site. In vitro assays were performed to characterize the effect of different therapeutic agents on cholesterol hydroxylase activity of purified full-length recombinant CYP46A1, and several strong inhibitors and modest coactivators of CYP46A1 were identified. Structural and biochemical data provide evidence that CYP46A1 activity could be altered by exposure to some therapeutic drugs and potentially other xenobiotics.cholesterol metabolism ͉ monooxygenase ͉ drug interactions ͉ cholesterol 3-sulfate A ccumulating evidence indicates that neurodegeneration and development of neurological disorders such as Alzheimer's disease (AD) are associated with disturbances in cholesterol homeostasis in the brain (1-5). It is also becoming increasingly clear that the conversion of cholesterol to 24S-hydroxycholesterol (24OH-CH) is an important mechanism that controls cholesterol turnover in the central nervous system (6-8). Cholesterol 24-hydroxylation is carried out by cytochrome P450 46A1 (CYP46A1) and represents the first step in the major pathway for cholesterol elimination from the brain (9-11). Unlike cholesterol, 24OH-CH can cross the blood-brain barrier and be delivered to the liver for further degradation to bile acids.Studies of CYP46A1-knockout mice indicate that the continued synthesis and turnover of cerebral cholesterol via 24-hydroxylation are necessary for memory and learning (12). There appears to be a link between CYP46A1 and AD; the CYP46A1 expression pattern in the brain and levels of 24OH-CH in the serum and cerebrospinal f luid are different in healthy people and those affected by AD (13-17). The association between polymorphisms in the CYP46A1 gene and susceptibility to AD was also demonstrated in a number of studies (www.alzforum.org). However, this association has not yet been unambiguously proven and is still under investigation. Evidence has also been obtained in cultured cells and mice that supports a role for side-chain oxysterols, including 24OH-CH, as endogenous ligands for liver X receptors, that regulate the expression of genes involved in fatty acid and cholesterol metabolism (7). 24OH-CH was also shown to inhibit the formation of amyloid ␤-peptides, a ha...

Cancer cells display an increased demand for glucose. Therefore, identifying the specific aspects of glucose metabolism that are involved in the pathogenesis of cancer may uncover novel therapeutic nodes. Recently, there has been a renewed interest in the role of the pentose phosphate pathway in cancer. This metabolic pathway is advantageous for rapidly growing cells because it provides nucleotide precursors and helps regenerate the reducing agent NADPH, which can contribute to reactive oxygen species (ROS) scavenging. Correspondingly, clinical data suggest glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, is upregulated in prostate cancer. We hypothesized that androgen receptor (AR) signaling, which plays an essential role in the disease, mediated prostate cancer cell growth in part by increasing flux through the pentose phosphate pathway. Here, we determined that G6PD, NADPH and ribose synthesis were all increased by AR signaling. Further, this process was necessary to modulate ROS levels. Pharmacological or molecular inhibition of G6PD abolished these effects and blocked androgen-mediated cell growth. Mechanistically, regulation of G6PD via AR in both hormone-sensitive and castration-resistant models of prostate cancer was abolished following rapamycin treatment, indicating that AR increased flux through the pentose phosphate pathway by the mammalian target of rapamycin (mTOR)-mediated upregulation of G6PD. Accordingly, in two separate mouse models of Pten deletion/elevated mTOR signaling, Pb-Cre;Ptenf/f and K8-CreERT2;Ptenf/f, G6PD levels correlated with prostate cancer progression in vivo. Importantly, G6PD levels remained high during progression to castration-resistant prostate cancer. Taken together, our data suggest that AR signaling can promote prostate cancer through the upregulation of G6PD and therefore, the flux of sugars through the pentose phosphate pathway. Hence, these findings support a vital role for other metabolic pathways (that is, not glycolysis) in prostate cancer cell growth and maintenance.