Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), chemicals present in a multitude of consumer products, are persistent organic pollutants. Both compounds induce hepatotoxic effects in rodents, including steatosis, hepatomegaly and liver cancer. The mechanisms of PFOA- and PFOS-induced hepatic dysfunction are not completely understood. We present evidence that PFOA and PFOS induce their hepatic effects via targeting hepatocyte nuclear factor 4-alpha (HNF4α). Human hepatocytes treated with PFOA and PFOS at a concentration relevant to occupational exposure caused a decrease in HNF4α protein without affecting HNF4α mRNA or causing cell death. RNA sequencing analysis combined with Ingenuity Pathway Analysis of global gene expression changes in human hepatocytes treated with PFOA or PFOS indicated alterations in the expression of genes involved in lipid metabolism and tumorigenesis, several of which are regulated by HNF4α. Further investigation of specific HNF4α target gene expression revealed that PFOA and PFOS could promote cellular dedifferentiation and increase cell proliferation by down regulating positive targets (differentiation genes such as CYP7A1) and inducing negative targets of HNF4α (pro-mitogenic genes such as CCND1). Furthermore, in silico docking simulations indicated that PFOA and PFOS could directly interact with HNF4α in a similar manner to endogenous fatty acids. Collectively, these results highlight HNF4α degradation as novel mechanism of PFOA and PFOS-mediated steatosis and tumorigenesis in human livers.

Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein–protein interactions play a critical role in this process. Historically, the study of CYP–protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein–protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein–protein interactions with CYP enzymes.

The human organic cation transporter 1 (OCT1) is a polyspecific transporter involved in the uptake of positively charged and neutral small molecules in the liver. To date, few endogenous compounds have been identified as OCT1 substrates; more importantly, the effect of drugs on endogenous substrate transport has not been examined. In this study, we established monoamine neurotransmitters as substrates for OCT1, specifically characterizing serotonin transport in human embryonic kidney 293 cells. Kinetic analysis yielded a K m of 197 micomolar and a V max of 561 pmol/mg protein/ minute for serotonin. Furthermore, we demonstrated that serotonin uptake was inhibited by diphenhydramine, fluoxetine, imatinib, and verapamil, with IC 50 values in the low micromolar range. These results were recapitulated in primary human hepatocytes, suggesting that OCT1 plays a significant role in hepatic elimination of serotonin and that xenobiotics may alter the elimination of endogenous compounds as a result of interactions at the transporter level.

Cytochrome P450 enzymes are versatile catalysts involved in a wide variety of biological processes from hormonal regulation and antibiotic synthesis to drug metabolism. A hallmark of their versatility is their promiscuous nature, allowing them to recognize a wide variety of chemically diverse substrates. However, the molecular details of this promiscuity have remained elusive. Here, we have utilized two-dimensional heteronuclear single quantum coherence NMR spectroscopy to examine a series of mutants site-specific labeled with the unnatural amino acid, [ 13 C]p-methoxyphenylalanine, in conjunction with all-atom molecular dynamics simulations to examine substrate and inhibitor binding to CYP119, a P450 from Sulfolobus acidocaldarius. The results suggest that tight binding hydrophobic ligands tend to lock the enzyme into a single conformational substate, whereas weak binding low affinity ligands bind loosely in the active site, resulting in a distribution of localized conformers. Furthermore, the molecular dynamics simulations suggest that the ligand-free enzyme samples ligand-bound conformations of the enzyme and, therefore, that ligand binding may proceed largely through a process of conformational selection rather than induced fit.Recently, the dynamic nature of enzymes has drawn much attention (1-3). Protein dynamics are not only important for ligand recognition and binding, but also for bringing catalytic residues in close proximity to the bound substrate so that a reaction can occur (4, 5). It has long been known that conformational flexibility is critical for the recognition of a wide variety of substrates and inhibitors by the human liver drug-metabolizing cytochrome P450 enzymes (6 -9). These enzymes are members of a superfamily of hemoproteins that catalyze oxidative transformations of xenobiotic compounds (10). These "promiscuous" enzymes utilize a conserved mechanism of oxygen activation to oxidize a host of structurally diverse molecules (10). The crystal structures of several human P450 isoforms have recently been obtained, in many cases co-crystallized with known ligands (9,(11)(12)(13). In some cases, ligands have been found bound at some distance from the heme iron or even outside the active site (11,14). These particular structures imply that concerted conformational changes have to take place in the enzyme to position the ligand favorably for oxidation. However, it is not clear how this type of conformational change manifests itself in this important enzyme family. Two competing, albeit not mutually exclusive, theories have emerged to explain how P450s are able to adapt themselves to accommodate such a large number of chemically diverse compounds. The first, a derivative of Koshland's classic induced fit model, relies on substrate binding to induce conformational changes in the enzyme in a stepwise fashion that ultimately advance the ligand into the active site and place it in a productive orientation for oxidation (9, 15-17). The second model, derived from Monod-WymanChangeux allostery theory, ...