Douglas A. Gray

Explaining oscillations and variability in the p53-Mdm2 system.

BackgroundIn individual living cells p53 has been found to be expressed in a series of discrete pulses after DNA damage. Its negative regulator Mdm2 also demonstrates oscillatory behaviour. Attempts have been made recently to explain this behaviour by mathematical models but these have not addressed explicit molecular mechanisms. We describe two stochastic mechanistic models of the p53/Mdm2 circuit and show that sustained oscillations result directly from the key biological features, without assuming complicated mathematical functions or requiring more than one feedback loop. Each model examines a different mechanism for providing a negative feedback loop which results in p53 activation after DNA damage. The first model (ARF model) looks at the mechanism of p14ARF which sequesters Mdm2 and leads to stabilisation of p53. The second model (ATM model) examines the mechanism of ATM activation which leads to phosphorylation of both p53 and Mdm2 and increased degradation of Mdm2, which again results in p53 stabilisation. The models can readily be modified as further information becomes available, and linked to other models of cellular ageing.ResultsThe ARF model is robust to changes in its parameters and predicts undamped oscillations after DNA damage so long as the signal persists. It also predicts that if there is a gradual accumulation of DNA damage, such as may occur in ageing, oscillations break out once a threshold level of damage is acquired. The ATM model requires an additional step for p53 synthesis for sustained oscillations to develop. The ATM model shows much more variability in the oscillatory behaviour and this variability is observed over a wide range of parameter values. This may account for the large variability seen in the experimental data which so far has examined ARF negative cells.ConclusionThe models predict more regular oscillations if ARF is present and suggest the need for further experiments in ARF positive cells to test these predictions. Our work illustrates the importance of systems biology approaches to understanding the complex role of p53 in both ageing and cancer.

Akt promotes chemoresistance in human ovarian cancer cells by modulating cisplatin-induced, p53-dependent ubiquitination of FLICE-like inhibitory protein.

Although Akt is a determinant of cisplatin (cis-diaminedichloroplatinum (CDDP)) resistance in ovarian cancer cells, which is related in part to its inhibitory action on p53 activation, precisely how Akt confers CDDP resistance is unclear. In this study, we show that CDDP induced p53-dependent Fas-associated death domain-like interleukin-1β-converting enzyme (FLICE)-like inhibitory protein (FLIP) degradation in chemosensitive ovarian cancer cells but not their resistant counterparts. CDDP induced FLIP–p53–Itch interaction, colocalization and FLIP ubiquitination in chemosensitive but not chemoresistant ovarian cancer cells. Moreover, although activated Akt inhibited CDDP-induced FLIP degradation and apoptosis in sensitive cells, these responses were facilitated by dominant-negative Akt expression in chemoresistant cells. Inhibition of Akt function also facilitated p53–FLIP interaction and FLIP ubiquitination, which were attenuated by p53 silencing. These results suggest that Akt confers resistance, in part, by modulating CDDP-induced, p53-dependent FLIP ubiquitination. Understanding the precise etiology of chemoresistance may improve treatment for ovarian cancer.

FUS-immunoreactive intranuclear inclusions in neurodegenerative disease

Neuronal intranuclear inclusions (NIIs) are a histopathological hallmark of several neurodegenerative disorders. However, the role played by NIIs in neurodegenerative pathogenesis remains enigmatic. Defining their molecular composition represents an important step in understanding the pathophysiology of these disorders. Recently, a nuclear protein, “fused‐in‐sarcoma” (FUS) was identified as the pathological protein in two forms of frontotemporal lobar degeneration (FTLD‐IF, formerly known as neuronal intermediate filament inclusion disease, and FTLD‐UPS, formerly known as atypical FTLD‐U), both of which are characterized by the presence of NII. The objective of the present study was to determine the range of neurodegenerative disorders characterized by FUS‐positive NIIs. Immunostaining for FUS revealed intense reactivity of NIIs in FTLD‐IF and FTLD‐UPS as well as in Huntingtons disease, spinocerebellar ataxias 1 and 3, and neuronal intranuclear inclusion body disease. In contrast, there was no FUS staining of NIIs in inherited forms of FTLD‐TDP caused by GRN and VCP mutations, fragile‐X‐associated tremor ataxia syndrome, or oculopharyngeal muscular dystrophy. In a cell culture model of Huntingtons disease, NIIs were intensely FUS‐positive. NII‐bearing cells displayed loss of the normal diffuse nuclear pattern of FUS staining. This suggests that sequestration of nuclear FUS by NIIs may interfere with its normal nuclear localization.

Cisplatin Induces p53-Dependent FLICE-Like Inhibitory Protein Ubiquitination in Ovarian Cancer Cells

Understanding the mechanism of cisplatin (CDDP) action may improve therapeutic strategy for ovarian cancer. Although p53 and FLICE-like inhibitory protein (FLIP) are determinants of CDDP sensitivity in ovarian cancer, the interaction between p53 and FLIP remains poorly understood. Here, using two chemosensitive ovarian cancer cell lines and various molecular and cellular approaches, we show that CDDP induces p53-dependent FLIP ubiquitination and degradation, and apoptosis in vitro. Moreover, we showed that Itch (an E3 ligase) forms a complex with FLIP and p53 upon CDDP treatment. These results suggest that p53 facilitates FLIP down-regulation by CDDP-induced FLIP ubiquitination and proteasomal degradation.

Alpha‐synuclein in the appendiceal mucosa of neurologically intact subjects

Parkinsons disease is characterized by the pathological aggregation of Alpha‐synuclein. The dual‐hit hypothesis proposed by Braak implicates the enteric nervous system as an initial site of α‐synuclein aggregation with subsequent spread to the central nervous system. Regional variations in the spatial pattern or levels of α‐synuclein along the enteric nervous system could have implications for identifying sites of onset of this pathogenic cascade. We performed immunohistochemical staining for α‐synuclein on gastrointestinal tissue from patients with no history of neurological disease using the established LB509 antibody and a new clone, MJFR1, characterized for immunohistochemistry here. We demonstrate that the vermiform appendix is particularly enriched in α‐synuclein–containing axonal varicosities, concentrated in its mucosal plexus rather than the classical submucosal and myenteric plexuses. Unexpectedly, intralysosomal accumulations of α‐synuclein were detected within mucosal macrophages of the appendix. The abundance and accumulation of α‐synuclein in the vermiform appendix implicate it as a candidate anatomical locus for the initiation of enteric α‐synuclein aggregation and permits the generation of testable hypotheses for Parkinsons disease pathogenesis.

GSK3 and p53 - is there a link in Alzheimer's disease?

BackgroundRecent evidence suggests that glycogen synthase kinase-3β (GSK3β) is implicated in both sporadic and familial forms of Alzheimers disease. The transcription factor, p53 also plays a role and has been linked to an increase in tau hyperphosphorylation although the effect is indirect. There is also evidence that GSK3β and p53 interact and that the activity of both proteins is increased as a result of this interaction. Under normal cellular conditions, p53 is kept at low levels by Mdm2 but when cells are stressed, p53 is stabilised and may then interact with GSK3β. We propose that this interaction has an important contribution to cellular outcomes and to test this hypothesis we developed a stochastic simulation model.ResultsThe model predicts that high levels of DNA damage leads to increased activity of p53 and GSK3β and low levels of aggregation but if DNA damage is repaired, the aggregates are eventually cleared. The model also shows that over long periods of time, aggregates may start to form due to stochastic events leading to increased levels of ROS and damaged DNA. This is followed by increased activity of p53 and GSK3β and a vicious cycle ensues.ConclusionsSince p53 and GSK3β are both involved in the apoptotic pathway, and GSK3β overactivity leads to increased levels of plaques and tangles, our model might explain the link between protein aggregation and neuronal loss in neurodegeneration.

An in silico model of the ubiquitin-proteasome system that incorporates normal homeostasis and age-related decline

BackgroundThe ubiquitin-proteasome system is responsible for homeostatic degradation of intact protein substrates as well as the elimination of damaged or misfolded proteins that might otherwise aggregate. During ageing there is a decline in proteasome activity and an increase in aggregated proteins. Many neurodegenerative diseases are characterised by the presence of distinctive ubiquitin-positive inclusion bodies in affected regions of the brain. These inclusions consist of insoluble, unfolded, ubiquitinated polypeptides that fail to be targeted and degraded by the proteasome. We are using a systems biology approach to try and determine the primary event in the decline in proteolytic capacity with age and whether there is in fact a vicious cycle of inhibition, with accumulating aggregates further inhibiting proteolysis, prompting accumulation of aggregates and so on. A stochastic model of the ubiquitin-proteasome system has been developed using the Systems Biology Mark-up Language (SBML). Simulations are carried out on the BASIS (Biology of Ageing e-Science Integration and Simulation) system and the model output is compared to experimental data wherein levels of ubiquitin and ubiquitinated substrates are monitored in cultured cells under various conditions. The model can be used to predict the effects of different experimental procedures such as inhibition of the proteasome or shutting down the enzyme cascade responsible for ubiquitin conjugation.ResultsThe model output shows good agreement with experimental data under a number of different conditions. However, our model predicts that monomeric ubiquitin pools are always depleted under conditions of proteasome inhibition, whereas experimental data show that monomeric pools were depleted in IMR-90 cells but not in ts20 cells, suggesting that cell lines vary in their ability to replenish ubiquitin pools and there is the need to incorporate ubiquitin turnover into the model. Sensitivity analysis of the model revealed which parameters have an important effect on protein turnover and aggregation kinetics.ConclusionWe have developed a model of the ubiquitin-proteasome system using an iterative approach of model building and validation against experimental data. Using SBML to encode the model ensures that it can be easily modified and extended as more data become available. Important aspects to be included in subsequent models are details of ubiquitin turnover, models of autophagy, the inclusion of a pool of short-lived proteins and further details of the aggregation process.

SIRT1 is a Highly Networked Protein That Mediates the Adaptation to Chronic Physiological Stress

SIRT1 is a NAD(+)-dependent protein deacetylase that has a very large number of established protein substrates and an equally impressive list of biological functions thought to be regulated by its activity. Perhaps as notable is the remarkable number of points of conflict concerning the role of SIRT1 in biological processes. For example, evidence exists suggesting that SIRT1 is a tumor suppressor, is an oncogene, or has no effect on oncogenesis. Similarly, SIRT1 is variably reported to induce, inhibit, or have no effect on autophagy. We believe that the resolution of many conflicting results is possible by considering recent reports indicating that SIRT1 is an important hub interacting with a complex network of proteins that collectively regulate a wide variety of biological processes including cancer and autophagy. A number of the interacting proteins are themselves hubs that, like SIRT1, utilize intrinsically disordered regions for their promiscuous interactions. Many studies investigating SIRT1 function have been carried out on cell lines carrying undetermined numbers of alterations to the proteins comprising the SIRT1 network or on inbred mouse strains carrying fixed mutations affecting some of these proteins. Thus, the effects of modulating SIRT1 amount and/or activity are importantly determined by the genetic background of the cell (or the inbred strain of mice), and the effects attributed to SIRT1 are synthetic with the background of mutations and epigenetic differences between cells and organisms. Work on mice carrying alterations to the Sirt1 gene suggests that the network in which SIRT1 functions plays an important role in mediating physiological adaptation to various sources of chronic stress such as calorie restriction and calorie overload. Whether the catalytic activity of SIRT1 and the nuclear concentration of the co-factor, NAD(+), are responsible for modulating this activity remains to be determined. However, the effect of modulating SIRT1 activity must be interpreted in the context of the cell or tissue under investigation. Indeed, for SIRT1, we argue that context is everything.

Association of UNP, a ubiquitin-specific protease, with the pocket proteins pRb, p107 and p130.

The murine Unp gene encodes a widely expressed ubiquitin-specific protease. The predicted sequence of the UNP protein features motifs common to viral oncoproteins through which these proteins interact with the retinoblastoma gene product pRb, as well as the related ‘pocket proteins’ p107 and p130. We have explored the possibility that UNP interacts with pocket proteins, and report here that such associations can be detected in vitro and in cells. Associations of UNP and pocket proteins are sensitive to site-directed mutations in a manner directly analogous to those documented in viral oncoproteins. We conclude that within cells UNP does physically associate with pRb, and can also associate with p107 and p130.

Proviral inactivation of the Npat gene of Mpv 20 mice results in early embryonic arrest.

The Mpv 20 transgenic mouse strain was created by infection of embryos with a defective retrovirus. When Mpv 20 heterozygous animals were crossed, no homozygous neonatal mice or midgestation embryos were identified. When embryos from heterozygous crosses were cultured in vitro, approximately one quarter arrested as uncompacted eight-cell embryos, indicating that proviral insertion resulted in a recessive lethal defect whose phenotype was manifest very early in development. Molecular cloning of the Mpv 20 insertion site revealed that the provirus had disrupted the Npat gene, a gene of unknown function, resulting in the production of a truncated Npat mRNA. Expression of the closely linked Atm gene was found to be unaffected by the provirus.