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ABSTRACT: It is an accepted paradigm that extended stress predisposes an individual to pathophysiology. However, the biological adaptations to minimize this risk are poorly understood. Using a computational model based upon realistic kinetic parameters we are able to reproduce the interaction of the stress hormone cortisol with its two nuclear receptors, the high-affinity glucocorticoid receptor and the low-affinity pregnane X-receptor. We demonstrate that regulatory signals between these two nuclear receptors are necessary to optimize the body's response to stress episodes, attenuating both the magnitude and duration of the biological response. In addition, we predict that the activation of pregnane X-receptor by multiple, low-affinity endobiotic ligands is necessary for the significant pregnane X-receptor-mediated transcriptional response observed following stress episodes. This integration allows responses mediated through both the high and low-affinity nuclear receptors, which we predict is an important strategy to minimize the risk of disease from chronic stress.
Nature Communications 01/2013; 4:1792. · 7.40 Impact Factor
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ABSTRACT: The complement system is a fundamental component of innate immunity that orchestrates complex immunological and inflammatory processes. Complement comprises over 30 proteins that eliminate invading microorganisms while maintaining host cell integrity. Protein-carbohydrate interactions play critical roles in both the activation and regulation of complement. Mannose-binding lectin (MBL) activates the lectin pathway of complement via the recognition of sugar arrays on pathogenic surfaces. To determine the solution structure of MBL, synchrotron x-ray scattering and analytical ultracentrifugation experiments showed that the carbohydrate-recognition domains in the MBL dimer, trimer, and tetramer are positioned close to each other in near-planar fan-like structures. These data were subjected to constrained modeling fits. A bent structure for the MBL monomer was identified starting from two crystal structures for its carbohydrate-recognition domain and its triple helical region. The MBL monomer structure was used to identify 10-12 near-planar solution structures for each of the MBL dimers, trimers, and tetramers starting from 900 to 6,859 randomized structures for each. These near-planar fan-like solution structures joined at an N-terminal hub clarified how the carbohydrate-recognition domain of MBL binds to pathogenic surfaces. They also provided insight on how MBL presents a structural template for the binding and auto-activation of the MBL-associated serine proteases to initiate the lectin pathway of complement activation.
Journal of Biological Chemistry 12/2011; 287(6):3930-45. · 4.77 Impact Factor
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Anna Phillips,
Amina Teunisse,
Suzanne Lam,
Kirsten Lodder,
Matthew Darley,
Muhammad Emaduddin,
Anja Wolf,
Julia Richter,
Job de Lange,
Matty Verlaan-de Vries,
Kristiaan Lenos,
Anja Böhnke,
Frank Bartel,
Jeremy P Blaydes,
Aart G Jochemsen
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ABSTRACT: The p53 regulatory network is critically involved in preventing the initiation of cancer. In unstressed cells, p53 is maintained at low levels and is largely inactive, mainly through the action of its two essential negative regulators, HDM2 and HDMX. p53 abundance and activity are up-regulated in response to various stresses, including DNA damage and oncogene activation. Active p53 initiates transcriptional and transcription-independent programs that result in cell cycle arrest, cellular senescence, or apoptosis. p53 also activates transcription of HDM2, which initially leads to the degradation of HDMX, creating a positive feedback loop to obtain maximal activation of p53. Subsequently, when stress-induced post-translational modifications start to decline, HDM2 becomes effective in targeting p53 for degradation, thus attenuating the p53 response. To date, no clear function for HDMX in this critical attenuation phase has been demonstrated experimentally. Like HDM2, the HDMX gene contains a promoter (P2) in its first intron that is potentially inducible by p53. We show that p53 activation in response to a plethora of p53-activating agents induces the transcription of a novel HDMX mRNA transcript from the HDMX-P2 promoter. This mRNA is more efficiently translated than that expressed from the constitutive HDMX-P1 promoter, and it encodes a long form of HDMX protein, HDMX-L. Importantly, we demonstrate that HDMX-L cooperates with HDM2 to promote the ubiquitination of p53 and that p53-induced HDMX transcription from the P2 promoter can play a key role in the attenuation phase of the p53 response, to effectively diminish p53 abundance as cells recover from stress.
Journal of Biological Chemistry 09/2010; 285(38):29111-27. · 4.77 Impact Factor
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Anna Phillips,
Amina Teunisse,
Suzanne Lam,
Kirsten Lodder,
Matthew Darley,
Muhammad Emaduddin,
Anja Wolf,
Julia Richter,
Job de Lange,
Matty Verlaan-de Vries,
Kristiaan Lenos,
Anja Böhnke,
Frank Bartel,
Jeremy P. Blaydes,
Aart G. Jochemsen
[show abstract]
[hide abstract]
ABSTRACT: The p53 regulatory network is critically involved in preventing the initiation of cancer. In unstressed cells, p53 is maintained
at low levels and is largely inactive, mainly through the action of its two essential negative regulators, HDM2 and HDMX.
p53 abundance and activity are up-regulated in response to various stresses, including DNA damage and oncogene activation.
Active p53 initiates transcriptional and transcription-independent programs that result in cell cycle arrest, cellular senescence,
or apoptosis. p53 also activates transcription of HDM2, which initially leads to the degradation of HDMX, creating a positive feedback loop to obtain maximal activation of p53.
Subsequently, when stress-induced post-translational modifications start to decline, HDM2 becomes effective in targeting p53
for degradation, thus attenuating the p53 response. To date, no clear function for HDMX in this critical attenuation phase
has been demonstrated experimentally. Like HDM2, the HDMX gene contains a promoter (P2) in its first intron that is potentially inducible by p53. We show that p53 activation in response
to a plethora of p53-activating agents induces the transcription of a novel HDMX mRNA transcript from the HDMX-P2 promoter. This mRNA is more efficiently translated than that expressed from the constitutive HDMX-P1 promoter, and it encodes a long form of HDMX protein, HDMX-L. Importantly, we demonstrate that HDMX-L cooperates with HDM2
to promote the ubiquitination of p53 and that p53-induced HDMX transcription from the P2 promoter can play a key role in the attenuation phase of the p53 response, to effectively diminish
p53 abundance as cells recover from stress.
Journal of Biological Chemistry 09/2010; 285(38):29111-29127. · 4.77 Impact Factor
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ABSTRACT: This review addresses the general structure and function of nuclear receptors and places specific emphasis on their role in xenosensing, resulting in the activation of a battery of genes mediating drug metabolism, conjugation, and transport. The pregnane-X receptor is a nuclear receptor that functions to control a battery of genes predominantly involved in drug metabolism and we place emphasis on how this important cellular mediator is transcriptionally activated. We have identified both positive and negative regulatory elements in the PXR promoter, the balance of which dictates the steady state expression of the PXR gene.
Drug Metabolism Reviews 02/2006; 38(1-2):31-49. · 6.40 Impact Factor
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ABSTRACT: Cell cycle progression in response to serum growth factors is dependent on the expression of functional Hdm2 (Mdm2), which inhibits p53-dependent transcription of anti-proliferative genes. In a well characterised non-transformed human fibroblast model, growth factors induce the expression of Hdm2 with rapid kinetics. Here we dissect the mechanistic basis for this critical response. In contrast to previous studies in which components of the growth factor signalling pathways were overexpressed, hdm2 mRNA expression is not induced with immediate-early kinetics in these cells. Rather, the elevated Hdm2 protein levels which follow growth factor stimulation are primarily a consequence of phosphatidylinositol-3 kinase-dependent stabilisation of the Hdm2 protein combined with a global increase in protein synthesis.
FEBS Letters 02/2006; 580(1):300-4. · 3.54 Impact Factor
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ABSTRACT: The physical and functional interaction between the transcription factor p53 and its negative regulatory partner protein Hdm2 (Mdm2 in mouse) is a key point of convergence of multiple signaling pathways that regulates cell proliferation and survival. hdm2 mRNA transcription is induced by p53, forming the basis of an auto-regulatory feedback loop. Growth and survival factor-activated Ras-Raf-MEK-ERK signaling can also regulate Hdm2 expression independently of p53, contributing to the pro-survival effect of these factors. In murine fibroblasts, this occurs through the regulation of mdm2 mRNA transcription. Here we show that, in human breast cancer epithelial cells, MEK-dependent regulation of Hdm2 expression also occurs at a post-transcriptional level. Pharmacological blockade of MEK activity in T47D cells inhibits Hdm2 protein synthesis by 80-90%. This occurs in the absence of changes in the expression of the major hdm2-P1 mRNA transcript and only an approximately 40% reduction in hdm2-P2 transcript levels. The amounts of both transcripts that are associated with polyribosomes and are, hence, being actively translated are reduced by >80% by the MEK inhibitor, U0126. We show here that this is due to the inhibition of hdm2 mRNA export from the nucleus when MEK activity is inhibited. In MCF-7 breast cancer cells that express wild-type p53, Hdm2 is required to suppress p53-dependent transcription when MEK kinase is active. Regulation of the nuclear export of hdm2 mRNA provides, therefore, a mechanism whereby mitogen-stimulated cells avoid p53-dependent cell cycle arrest or apoptosis by maintaining the dynamic equilibrium of the Hdm2-p53 feedback loop.
Journal of Biological Chemistry 05/2005; 280(17):16651-8. · 4.77 Impact Factor
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ABSTRACT: Recent data have made it increasingly clear that the gene expression profile of a cell system, and its alteration in response to external stimuli, is highly dependent on both the higher order chromatin structure of the genome and the interaction of gene products in interpreting stimuli. To further explore this phenomenon, we have examined the role of both of these factors in controlling xenobiotic-mediated gene expression changes in primary and transformed human hepatocytes (HuH7). Using quantitative polymerase chain reaction, expression levels of several transcription factors implicated in the liver-specific regulation of the CYP3A gene family were examined in human adult and fetal liver RNA samples. These expression profiles were then compared with those obtained from both primary and transformed human hepatocytes, showing that, in general, cultured cells exhibit a distinct profile compared with either the fetal or adult samples. Transcriptome profiles before and after exposure to the CYP3A transcriptional activators rifampicin, dexamethasone, pregnane-16alpha-carbonitrile, and phenobarbital were subsequently examined. Whereas exposure to these compounds elicited a dose-dependent increase in CYP3A transcription in primary hepatocytes, no alteration in expression levels was observed for the hepatoma cell line HuH7. Alteration in the expression levels of pregnane X receptor and chicken ovalbumin upstream promoter transcription factor I, and the disruption of higher order chromatin within HuH7 cells altered CYP3A expression and/or activation by xenobiotics toward that observed in primary hepatocytes. These data provide potential roles for these two processes in regulating CYP3A expression in vivo.
Drug Metabolism and Disposition 03/2005; 33(2):233-42. · 3.73 Impact Factor
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ABSTRACT: Sodium valproate (VPA) is clinically employed as an anti-convulsant and, to a lesser extent, mood stabilizer. While the incidence of toxicity associated with the clinical use of valproate is low, serious hepatotoxicity makes up a significant percentage. Rats treated with high doses of sodium valproate are subject to hepatotoxicity, and the study of the molecular mechanisms underlying this phenomenon may shed further light on the human situation. Exposure to sodium valproate results in the down regulation in rat liver of several transcripts whose products are involved in cellular energy homeostasis, resulting in time-dependent fluctuations in cellular ATP, possibly resulting in cell death. To further examine this, classical markers of apoptosis were examined in the rat hepatoma cell line FaO following sodium valproate exposure. Concentrations greater than 300 microM sodium valproate resulted in a transient wave of apoptosis, as assessed by chromatin condensation and DNA fragmentation assay. Analysis indicated that Fas-ligand and caspase-11 expression were increased at the transcriptome level, while caspase-3 was activated at the proteome level during the exposure period. These data demonstrates that sodium valproate causes cell death through apoptosis in a rat liver cell line, and provides information on the possible molecular mechanisms underlying this phenomenon in vivo.
Toxicology 12/2003; 192(2-3):219-27. · 3.68 Impact Factor
