Analysis of human cytochrome P450 3A4 cooperativity: Construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics

Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure–activity study from another laboratory using various effector–substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6β-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by α-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6β-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.

13C NMR study of the effects of leptin treatment on kinetics of hepatic intermediary metabolism

The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin’s biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin’s effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive 13C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring flux of 13C-labeled substrate through several multistep pathways. In perfused liver from either ob/ob or lean mice, we found that acute treatment with leptin in vitro modulates pathways controlling carbohydrate flux into 13C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of ob/ob liver also caused a rapid stimulation of long-chain fatty acid synthesis from 13C-labeled acetyl-CoA by the de novo synthesis route. Chronic leptin treatment in vivo induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-13C]pyruvate in ob/ob mouse liver perfused in the absence of the hormone. Consistent with the 13C NMR results, leptin treatment of the ob/ob mouse in vivo resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin in vivo exerted the opposite effect of acute treatment in vitro and markedly decreased hepatic de novo synthesis of fatty acids in ob/ob mouse liver. In agreement with the 13C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the ob/ob mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.

Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm−1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s−1 under zero force up to 15 s−1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.

Kinetics of T cell receptor β, γ, and δ rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44+CD25+ Pro-T thymocytes

We performed a comprehensive analysis of T cell receptor (TCR) γ rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that TCRγ rearrangements are present in CD44+CD25+ Pro-T thymocytes much earlier than expected. TCRγ rearrangements increase significantly from the Pro-T to the CD44−CD25+ Pre-T cell transition, and follow different patterns depending on each Vγ gene segment, suggesting that ordered waves of TCRγ rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of TCRγ genes occur concurrently with TCRδ and D-Jβ rearrangements, but before Vβ gene assembly. Productive TCRγ rearrangements do not increase significantly before the Pre-T cell stage and are depleted in CD4+CD8+ double-positive cells from normal mice. In contrast, double-positive thymocytes from TCRδ−/− mice display random proportions of TCRγ rearranged alleles, supporting a role for functional TCRγ/δ rearrangements in the γδ divergence process.

In vivo kinetics of a redox-regulated transcriptional switch

SoxR is a transcription activator governing a cellular response to superoxide and nitric oxide in Escherichia coli. SoxR protein is a homodimer, and each monomer has a redox-active [2Fe–2S] cluster. Oxidation and reduction of the [2Fe–2S] clusters can reversibly activate and inactivate SoxR transcriptional activity. Here, we use electron paramagnetic resonance spectroscopy to follow the redox-switching process of SoxR protein in vivo. SoxR [2Fe–2S] clusters were in the fully reduced state during normal aerobic growth, but were completely oxidized after only 2-min aerobic exposure of the cells to superoxide-generating agents such as paraquat. The oxidized SoxR [2Fe–2S] clusters were rapidly re-reduced in vivo once the oxidative stress was removed. The in vivo kinetics of SoxR [2Fe–2S] cluster oxidation and reduction exactly paralleled the increase and decrease of transcription of soxS, the target gene for SoxR. The kinetic analysis also revealed that an oxidative stress-linked decrease in soxS mRNA stability contributes to the rapid attainment of a new steady state after SoxR activation. Such a redox stress-related change in soxS mRNA stability may represent a new level of biological control.

Structural kinetics of transcription activation at the malT promoter of Escherichia coli by UV laser footprinting

We have studied the kinetics of transcriptional initiation and activation at the malT and malTp1 promoters of Escherichia coli using UV laser footprinting. Contrary to previous studies and because of the very rapid signal acquisition by this technique, we can obtain structural information about true reaction intermediates of transcription initiation. The consequences of adding a transcriptional activator, the cAMP receptor protein/cAMP complex (CRP), are monitored in real time, permitting us to assign specific interactions to the activation of discrete steps in transcription initiation. Direct protein–protein contacts between CRP and the RNA polymerase appeared very rapidly, followed by DNA melting around the −10 hexamer. CRP slightly increased the rate of this isomerization reaction but, more importantly, favored the establishment of additional contacts between the DNA upstream of the CRP binding site and RNA polymerase subsequent to open complex formation. These contacts make a major contribution to transcriptional activation by stabilizing open forms of the promoter complex, thereby indirectly accelerating promoter escape. The ensemble of the kinetic, structural signals demonstrated directly that CRP exerts most of its activating effects on the late stages of transcriptional initiation at the malT promoter.

Neuronal nicotinic threonine-for-leucine 247 α7 mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine

Mutation of the highly conserved leucine residue (Leu-247) converts 5-hydroxytryptamine (5HT) from an antagonist into an agonist of neuronal homomeric α7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We show here that acetylcholine (AcCho) activates two classes of single channels with conductances of 44 pS and 58 pS, similar to those activated by 5HT. However, the mean open time of AcCho-gated ion channels (11 ms) is briefer than that of 5HT-gated ion channels (18 ms). Furthermore, whereas the open time of AcCho channels lengthens with hyperpolarization, that of 5HT channels is decreased. In voltage-clamped oocytes, the apparent affinity of the α7 mutant receptor for 5HT is not modified by the presence of dihydro-β-erythroidine, which acts on the AcCho binding site in a competitive manner. This indicates a noncompetitive action of 5HT on nicotinic acetylcholine receptors. Considered together, our findings show that AcCho gates α7 mutant channels with similar conductance but with different kinetic profile than the channels gated by 5HT, suggesting that the two agonists act on different docking sites. These results will help to understand the crosstalk between cholinergic and serotonergic systems in the central nervous system.

RNA folding kinetics regulates translation of phage MS2 maturation gene

The gene for the maturation protein of the single-stranded RNA coliphage MS2 is preceded by an untranslated leader of 130 nt, which folds into a cloverleaf, i.e., three stem–loop structures enclosed by a long distance interaction (LDI). This LDI prevents translation because its 3′ moiety contains the Shine–Dalgarno sequence of the maturation gene. Previously, several observations suggested that folding of the cloverleaf is kinetically delayed, providing a time window for ribosomes to access the RNA. Here we present direct evidence for this model. In vitro experiments show that ribosome binding to the maturation gene is faster than refolding of the denatured cloverleaf. This folding delay appears related to special properties of the leader sequence. We have replaced the three stem–loop structures by a single five nt loop. This change does not affect the equilibrium structure of the LDI. Nevertheless, in this construct, the folding delay has virtually disappeared, suggesting that now the RNA folds faster than ribosomes can bind. Perturbation of the cloverleaf by an insertion makes the maturation start permanently accessible. A pseudorevertant that evolved from an infectious clone carrying the insertion had overcome this defect. It showed a wild-type folding delay before closing down the maturation gene. This experiment reveals the biological significance of retarded cloverleaf formation.

RGS proteins reconstitute the rapid gating kinetics of Gβγ-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Gα(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20–40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of “regulators of G protein signaling” (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor → GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent “basal” GIRK current was suppressed by ≈40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of “slow” postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.

Protein folding kinetics exhibit an Arrhenius temperature dependence when corrected for the temperature dependence of protein stability

The anomalous temperature dependence of protein folding has received considerable attention. Here we show that the temperature dependence of the folding of protein L becomes extremely simple when the effects of temperature on protein stability are corrected for; the logarithm of the folding rate is a linear function of 1/T on constant stability contours in the temperature–denaturant plane. This convincingly demonstrates that the anomalous temperature dependence of folding derives from the temperature dependence of the interactions that stabilize proteins, rather than from the super Arrhenius temperature dependence predicted for the configurational diffusion constant on a rough energy landscape. However, because of the limited temperature range accessible to experiment, the results do not rule out models with higher order temperature dependences. The significance of the slope of the stability-corrected Arrhenius plots is discussed.

Slow dimer dissociation of the TATA binding protein dictates the kinetics of DNA binding

The association of the TATA binding protein (TBP) to eukaryotic promoters is a possible rate-limiting step in gene expression. Slow promoter binding might be related to TBP’s ability to occlude its DNA binding domain through dimerization. Using a “pull-down” based assay, we find that TBP dimers dissociate slowly (t½ = 6–10 min), and thus present a formidable kinetic barrier to TATA binding. At 10 nM, TBP appears to exist as a mixed population of monomers and dimers. In this state, TATA binding displays burst kinetics that appears to reflect rapid binding of monomers and slow dissociation of dimers. The kinetics of the slow phase is in excellent agreement with direct measurements of the kinetics of dimer dissociation.

Pressure-induced protein-folding/unfolding kinetics

We use an off-lattice minimalist model to describe the effects of pressure in slowing down the folding/unfolding kinetics of proteins when subjected to increasingly larger pressures. The potential energy function used to describe the interactions between beads in the model includes the effects of pressure on the pairwise interaction of hydrophobic groups in water. We show that pressure affects the participation of contacts in the transition state. More significantly, pressure exponentially decreases the chain reconfigurational diffusion coefficient. These results are consistent with experimental results on the kinetics of pressure-denaturation of staphylococcal nuclease.

The Kinetics of Mannose 6-Phosphate Receptor Trafficking in the Endocytic Pathway in HEp-2 Cells: The Receptor Enters and Rapidly Leaves Multivesicular Endosomes without Accumulating in a Prelysosomal Compartment

We have previously shown that in HEp-2 cells, multivesicular bodies (MVBs) processing internalized epidermal growth factor–epidermal growth factor receptor complexes mature and fuse directly with lysosomes in which the complexes are degraded. The MVBs do not fuse with a prelysosomal compartment enriched in mannose 6-phosphate receptor (M6PR) as has been described in other cell types. Here we show that the cation-independent M6PR does not become enriched in the endocytic pathway en route to the lysosome, but if a pulse of M6PR or an M6PR ligand, cathepsin D, is followed, a significant fraction of these proteins are routed from the trans-Golgi to MVBs. Accumulation of M6PR does not occur because when the ligand dissociates, the receptor rapidly leaves the MVB. At steady state, most M6PR are distributed within the trans-Golgi and trans-Golgi network and in vacuolar structures distributed in the peripheral cytoplasm. We suggest that these M6PR-rich vacuoles are on the return route from MVBs to the trans-Golgi network and that a separate stable M6PR-rich compartment equivalent to the late endosome/prelysosome stage does not exist on the endosome–lysosome pathway in these cells.

Long-term kinetics of T cell production in HIV-infected subjects treated with highly active antiretroviral therapy

The long-term kinetics of T cell production following highly active antiretroviral therapy (HAART) were investigated in blood and lymph node in a group of HIV-infected subjects at early stage of established infection and prospectively studied for 72 wk. Before HAART, CD4 and CD8 T cell turnover was increased. However, the total number of proliferating CD4+ T lymphocytes, i.e., CD4+Ki67+ T lymphocytes, was not significantly different in HIV-infected (n = 73) and HIV-negative (n = 15) subjects, whereas proliferating CD8+Ki67+ T lymphocytes were significantly higher in HIV-infected subjects. After HAART, the total body number of proliferating CD4+Ki67+ T lymphocytes increased over time and was associated with an increase of both naive and memory CD4+ T cells. The maximal increase (2-fold) was observed at week 36, whereas at week 72 the number of proliferating CD4+ T cells dropped to baseline levels, i.e., before HAART. The kinetics of the fraction of proliferating CD4 and CD8 T cells were significantly correlated with the changes in the total body number of these T cell subsets. These results demonstrate a direct relationship between ex vivo measures of T cell production and quantitative changes in total body T lymphocyte populations. This study provides advances in the delineation of the kinetics of T cell production in HIV infection in the presence and/or in the absence of HAART.

Zic2 regulates the kinetics of neurulation

Mutation in human ZIC2, a zinc finger protein homologous to Drosophila odd-paired, causes holoprosencephaly (HPE), which is a common, severe malformation of the brain in humans. However, the pathogenesis is largely unknown. Here we show that reduced expression (knockdown) of mouse Zic2 causes neurulation delay, resulting in HPE and spina bifida. Differentiation of the most dorsal neural plate, which gives rise to both roof plate and neural crest cells, also was delayed as indicated by the expression lag of a roof plate marker, Wnt3a. In addition the development of neural crest derivatives such as dorsal root ganglion was impaired. These results suggest that the Zic2 expression level is crucial for the timing of neurulation. Because the Zic2 knockdown mouse is the first mutant with HPE and spina bifida to survive to the perinatal period, the mouse will promote analyses of not only the neurulation but also the pathogenesis of human HPE.