Biochemical and biophysical studies of MDM2-ligand interactions

MDM2, murine double minute 2, is a RING type-E3 ligase protein and also an oncogene. MDM2 plays a critical role in determining the steady levels and activity of p53 in cells using two mechanisms. The N-terminal domain of MDM2 binds to the transactivation domain of p53 and inhibits its transcriptional activity. The RING domain of MDM2 plays a role in the ubiquitination (and degradation) of p53. Several proteins are responsible for the ubiquitination mechanism including the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). Since the E2-E3 interaction is essential for ubiquitination, the protein-protein recognition site is a potential drug target. Two different MDM2 RING constructs were expressed and purified: MDM2RING (residues 386-491) and MDM2RING△C (residues 386-478). Both constructs were characterised using dynamic light scattering, size exclusion chromatography, mass spectrometry, NMR and electron microscopy. E3 ligase activity in vitro was also studied. Taken together these results showed that the MDM2RING construct formed a concentration-dependent oligomeric structure. In contrast, the MDM2RING△C construct formed a dimer at all concentrations. Both MDM2RING and MDM2RING △ C retain E3 ligase activity. However, the MDM2RING△C construct is less active. Full length E2 enzyme UbcH5a was also purified. Various biophysical techniques were used to study its interaction with MDM2 as well as with potential small molecule inhibitors as in principle, small molecules which disrupt the interaction between MDM2 and UbcH5a, could prevent/promote ubiquitination of p53. The dimerisation of MDM2 is important for its E3 activity and the C8-binding site potentially provides a second druggable site. In this work, peptide 9, which has the same sequence as the C-terminus of MDMX (an MDM2 homologue) was found to inhibit MDM2 E3 activity. Various biological techniques including NMR, fluorescence anisotropy, and electrospray mass spectrometry were used to investigate the interaction between two inhibitory peptides and MDM2. A major part of project involved virtual screening (VS) to search for small molecules which can affect MDM2-dependent ubiquitination. Three potential targets were considered: (1) the C8-binding site of MDM2; (2) the UbcH5a-binding site of MDM2; and (3) the MDM2-binding site of UbcH5a. Several small molecules were identified using our virtual screening database-mining and docking programs that were shown to affect MDM2-dependent ubiquitination of p53. In terms of understanding the complex biochemical mechanism of MDM2 this work provides two interesting and functionally relevant observations: (i) the MDM2 RING△C construct is a dimer as this would not be expected form the existing studies, and has less E3 ligase activity than MDM2RING; (ii) small molecules that bind MDM2 on the E2 binding site enhanced E3 ligase activity. One model to explain these observations is that binding of small molecule activators family to the RING induces a change in the conformation of the Cterminal tail residues which may enhance E2 binding

An Impulsive Three-Species Model with Square Root Functional Response and Mutual Interference of Predator

An impulsive two-prey and one-predator model with square root functional responses, mutual interference, and integrated pest management is constructed. By using techniques of impulsive perturbations, comparison theorem, and Floquet theory, the existence and global asymptotic stability of prey-eradication periodic solution are investigated. We use some methods and sufficient conditions to prove the permanence of the system which involve multiple Lyapunov functions and differential comparison theorem. Numerical simulations are given to portray the complex behaviors of this system. Finally, we analyze the biological meanings of these results and give some suggestions for feasible control strategies

Scaling Analysis of the Tensile Strength of Bamboo Fibers Using Weibull Statistics

This study demonstrates the effect of weak-link scaling on the tensile strength of bamboo fibers. The proposed model considers the random nature of fiber strength, which is reflected by using a two-parameter Weibull distribution function. Tension tests were performed on samples that could be scaled in length. The size effects in fiber length on the strength were analyzed based on Weibull statistics. The results verify the use of Weibull parameters from specimen testing for predicting the strength distributions of fibers of longer gauge lengths

Detection of gamma-ray emission from the Coma cluster with Fermi Large Area Telescope and tentative evidence for an extended spatial structure

Many galaxy clusters have giant halos of non-thermal radio emission, indicating the presence of relativistic electrons in the clusters. Relativistic protons may also be accelerated by merger and/or accretion shocks in galaxy clusters. These cosmic-ray (CR) electrons and/or protons are expected to produce gamma-rays through inverse-Compton scatterings or inelastic $pp$ collisions respectively. Despite of intense efforts in searching for high-energy gamma-ray emission from galaxy clusters, conclusive evidence is still missing so far. Here we report the discovery of $\ge 200$ MeV gamma-ray emission from the Coma cluster direction with an unbinned likelihood analysis of the 9 years of {\it Fermi}-LAT Pass 8 data. The gamma-ray emission shows a spatial morphology roughly coincident with the giant radio halo, with an apparent excess at the southwest of the cluster. Using the test statistic analysis, we further find tentative evidence that the gamma-ray emission at the Coma center is spatially extended. The extended component has an integral energy flux of $\sim 2\times 10^{-12}{\rm \ erg\ cm^{-2}\ s^{-1}}$ in the energy range of 0.2 - 300 GeV and the spectrum is soft with a photon index of $\simeq-2.7$. Interpreting the gamma-ray emission as arising from CR proton interaction, we find that the volume-averaged value of the CR to thermal pressure ratio in the Coma cluster is about $\sim 2\%$. Our results show that galaxy clusters are likely a new type of GeV gamma-ray sources, and they are probably also giant reservoirs of CR protons.Comment: 10 pages, 10 figures, Accepted by Physical Review D, more spatial models for the gamma-ray emission are used, systematic checks on the results are adde