CYP3A4, an integral endoplasmic reticulum (ER)-anchored protein, is the major human liver cytochrome P450 enzyme responsible for the disposition of over 50% of clinically relevant drugs. Alterations of its protein turnover can influence drug metabolism, drug-drug interactions, and the bioavailability of chemotherapeutic drugs. Such CYP3A4 turnover occurs via a classical ER-associated degradation (ERAD) process involving ubiquitination by both UBC7/gp78 and UbcH5a/CHIP E2-E3 complexes for 26 S proteasomal targeting. These E3 ligases act sequentially and cooperatively in CYP3A4 ERAD because RNA interference knockdown of each in cultured hepatocytes results in the stabilization of a functionally active enzyme. We have documented that UBC7/gp78-mediated CYP3A4 ubiquitination requires protein phosphorylation by protein kinase (PK) A and PKC and identified three residues (Ser-478, Thr-264, and Ser-420) whose phosphorylation is required for intracellular CYP3A4 ERAD. We document herein that of these, Ser-478 plays a pivotal role in UBC7/ gp78-mediated CYP3A4 ubiquitination, which is accelerated and enhanced on its mutation to the phosphomimetic Asp residue but attenuated on its Ala mutation. Intriguingly, CYP3A5, a polymorphically expressed human liver CYP3A4 isoform (containing Asp-478) is ubiquitinated but not degraded to a greater extent than CYP3A4 in HepG2 cells. This suggests that although Ser-478 phosphorylation is essential for UBC7/gp78-mediated CYP3A4 ubiquitination, it is not sufficient for its ERAD. Additionally, we now report that CYP3A4 protein phosphorylation by PKA and/or PKC at sites other than Ser-478, Thr-264, and Ser-420 also enhances UbcH5a/CHIP-mediated ubiquitination. Through proteomic analyses, we identify (i) 12 additional phosphorylation sites that may be involved in CHIP-CYP3A4 interactions and (ii) 8 previously unidentified CYP3A4 ubiquitination sites within spatially associated clusters of Asp/Glu and phosphorylatable Ser/Thr residues that may serve to engage each E2-E3 complex. Collectively, our findings underscore the interplay between protein phosphorylation and ubiquitination in ERAD and, to our knowledge, provide the very first example of gp78 substrate recognition via protein phosphorylation. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.010132, 1-17, 2012. Hepatic cytochromes P450 (P450s)1 are endoplasmic reticulum (ER)-anchored hemoproteins involved in the metabolism of numerous endo-and xenobiotics. Of these, CYP3A4 is particularly noteworthy because it comprises 30% of the human liver microsomal P450 complement and is responsible for the metabolism of Ͼ50% clinically relevant drugs, as well as hepatotoxins such as aflatoxin B1 (1). In common with other ER-integral P450s, it is a monotopic protein, N-terminally anchored to the ER membrane, with the bulk of its catalytic domain in the cytosol. We have documented that CYP3A4, in common with its CYP3A orthologs, incurs ubiquitin (Ub)-dependent proteasomal degradation (UPD) in a classical ER-associated degradation (ERAD) process...

Peptides derived from protein kinase C (PKC) modulate its activity by interfering with critical protein-protein interactions within PKC and between PKC and PKC-binding proteins (Souroujon, M. C., and Mochly-Rosen, D. (1998) Nat. Biotechnol. 16, 919 -924). We previously demonstrated that the C2 domain of PKC plays a critical role in these interactions. By focusing on ⑀PKC and using a rational approach, we then identified one C2-derived peptide that acts as an isozyme-selective activator and another that acts as a selective inhibitor of ⑀PKC. These peptides were used to identify the role of ⑀PKC in protection from cardiac and brain ischemic damage, in prevention of complications from diabetes, in reducing pain, and in protecting transplanted hearts. The efficacy of these two peptides led us to search for additional C2-derived peptides with PKC-modulating activities. Here we report on the activity of a series of 5-9-residue peptides that are derived from regions that span the length of the C2 domain of ⑀PKC. These peptides were tested for their effect on PKC activity in cells in vivo and in an ex vivo model of acute ischemic heart disease. Most of the peptides acted as activators of PKC, and a few peptides acted as inhibitors. PKC-dependent myristoylated alanine-rich C kinase substrate phosphorylation in ⑀PKC knock-out cells revealed that only a subset of the peptides were selective for ⑀PKC over other PKC isozymes. These ⑀PKC-selective peptides were also protective of the myocardium from ischemic injury, an ⑀PKC-dependent function (Liu, G. S., Cohen, M. V., Mochly-Rosen, D., and Downey, J. M. (1999) J. Mol. Cell. Cardiol. 31, 1937-1948), and caused selective translocation of ⑀PKC over other isozymes when injected systemically into mice. Examination of the structure of the C2 domain from ⑀PKC revealed that peptides with similar activities clustered into discrete regions within the domain. We propose that these regions represent surfaces of protein-protein interactions within ⑀PKC and/or between ⑀PKC and other partner proteins; some of these interactions are unique to ⑀PKC, and others are common to other PKC isozymes.The protein kinase C (PKC) 3 family of serine/threonine protein kinases is involved in normal cell functions such as apoptosis (3, 4), cell proliferation (5-7), and secretion (8), as well as in disease states such as ischemic heart disease (9 -12) and stroke (13,14). PKC activation is associated with binding to the negatively charged phospholipids, phosphatidylserine, and different PKC isozymes have varying sensitivities to Ca 2ϩ and lipid-derived second messengers such as diacylglycerol (15). Upon activation, PKC isozymes translocate from the soluble to the particulate cell fraction (16), including cell membrane, nucleus (17), and mitochondria (18).PKC primary sequence can be broadly separated into two domains as follows: the N-terminal regulatory domain and the conserved C-terminal catalytic domain. The regulatory domain of PKC is composed of the C1 and C2 domains that mediate PKC interactions with se...

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, ...

A simple model of spike generation is described that gives rise to negative correlations in the interspike interval (ISI) sequence and leads to long-term spike train regularization. This regularization can be seen by examining the variance of the kth-order interval distribution for large k (the times between spike i and spike i + k). The variance is much smaller than would be expected if successive ISIs were uncorrelated. Such regularizing effects have been observed in the spike trains of electrosensory afferent nerve fibers and can lead to dramatic improvement in the detectability of weak signals encoded in the spike train data (Ratnam & Nelson, 2000). Here, we present a simple neural model in which negative ISI correlations and long-term spike train regularization arise from refractory effects associated with a dynamic spike threshold. Our model is derived from a more detailed model of electrosensory afferent dynamics developed recently by other investigators (Chacron, Longtin, St.-Hilaire, & Maler, 2000;Chacron, Longtin, & Maler, 2001). The core of this model is a dynamic spike threshold that is transiently elevated following a spike and subsequently decays until the next spike is generated. Here, we present a simplified version-the linear adaptive threshold model-that contains a single state variable and three free parameters that control the mean and coefficient of variation of the spontaneous ISI distribution and the frequency characteristics of the driven response. We show that refractory effects associated with the dynamic threshold lead to regularization of the spike train on long timescales. Furthermore, we show that this regularization enhances the detectability of weak signals encoded by the linear adaptive threshold model. Although inspired by properties of electrosensory afferent nerve fibers, such regularizing effects may play an important role in other neural systems where weak signals must be reliably detected in noisy spike trains. When modeling a neuronal system that exhibits this type of ISI correlation structure, the linear adaptive threshold model may provide a more appropriate starting point than conventional renewal process models that lack long-term regularizing effects.

The ribosome is a large macromolecular machine, and correlated motion between residues is necessary for coordinating function across multiple protein and RNA chains. We ran two all-atom, explicit solvent molecular dynamics simulations of the bacterial ribosome and calculated correlated motion between residue pairs by using mutual information. Because of the short timescales of our simulation (ns), we expect that dynamics are largely local fluctuations around the crystal structure. We hypothesize that residues that show coupled dynamics are functionally related, even on longer timescales. We validate our model by showing that crystallographic B-factors correlate well with the entropy calculated as part of our mutual information calculations. We reveal that A-site residues move relatively independently from P-site residues, effectively insulating A-site functions from P-site functions during translation.