Peptide aptamers as new tools to modulate clathrin-mediated internalisation--inhibition of MT1-MMP internalisation.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Peptide aptamers are combinatorial protein reagents that bind to targets with a high specificity and a strong affinity thus providing a molecular tool kit for modulating the function of their targets in vivo. RESULTS: Here we report the isolation of a peptide aptamer named swiggle that interacts with the very short (21 amino acid long) intracellular domain of membrane type 1-metalloproteinase (MT1-MMP), a key cell surface protease involved in numerous and crucial physiological and pathological cellular events. Expression of swiggle in mammalian cells was found to increase the cell surface expression of MT1-MMP by impairing its internalisation. Swiggle interacts with the LLY573 internalisation motif of MT1-MMP intracellular domain, thus disrupting the interaction with the mu2 subunit of the AP-2 internalisation complex required for endocytosis of the protease. Interestingly, swiggle-mediated inhibition of MT1-MMP clathrin-mediated internalisation was also found to promote MT1-MMP-mediated cell migration. CONCLUSIONS: Taken together, our results provide further evidence that peptide aptamers can be used to dissect molecular events mediated by individual protein domains, in contrast to the pleiotropic effects of RNA interference techniques

Drosophila mutants in the 55 kDa regulatory subunit of protein phosphatase 2A show strongly reduced ability to dephosphorylate substrates of p34^(cdc2)

The 55 kDa regulatory subunit of Drosophila protein phosphatase 2A is located in the cytoplasm at all cell cycle stages, by the criterion of immunofluorescence. We are unable to detect significant change in protein phosphatase activity during the nuclear division cycle of syncytial embryos. However, cell cycle function of the enzyme is suggested by the mitotic defects exhibited by two Drosophila mutants, aar¹ and twins^P, defective in the gene encoding the 55 kDa subunit. The reduced levels of the 55 kDa subunit correlate with the loss of protein phosphatase 2A-like, okadaic acid-sensitive phosphatase activity of brain extracts against caldesmon and histone H1 phosphorylated by p34^(cdc2)/cyclin B kinase, but not against phosphorylase a. Thus the mitotic defects of aar¹ and twins^P are likely to result from the lack of dephosphorylation of specific substrates by protein phosphatase 2A

Discovery of a QPO in the X-ray pulsar 1A 1118-615: correlated spectral and aperiodic variability

Our goal is to investigate the X-ray timing and spectral variability of the high-mass X-ray binary 1A 1118-615 during a type-II outburst. We performed a detailed color, spectral and timing analysis of a giant outburst from 1A 1118-615, using RXTE data. Results. We report the discovery of a variable quasi-periodic oscillation (QPO) in the power spectral density of 1A 1118-615, with a centroid frequency of ~0.08 Hz. The centroid frequency of the QPO correlates with the X-ray flux, as expected according to the most accredited models for QPO production. For energies above ~4 keV, the QPO rms variability decreases as the energy increases. Pulse profiles display energy dependence, with a two-peak profile at lower energies, and a single peak at higher energies. From spectral analysis, we confirm the presence of a cyclotron absorption feature at ~60 keV, the highest value measured for an X-ray pulsar. We find that the spectral parameters (photon index, cutoff energy, iron fluorescence line strength) display a marked dependence with flux. We detect two different levels of neutral hydrogen column density, possibly due to the Be companion activity. We report for the first time a correlation between the timing and spectral parameters in an X-ray pulsar. All the correlations found between spectral/timing parameters and X-ray flux are present up to a flux of ~6x10^-9 erg cm^-2 s^-1, when a saturation level is reached. We propose that the saturation observed corresponds to the minimum extent of the neutron star magnetosphere. We estimate the magnetic field of the neutron star from two independent ways, using results from spectral (cyclotron line energy) and timing (QPO frequency) analysis, obtaining consistent values, of ~7-8x10^12 G. Results from the comprehensive spectral and timing analysis are discussed in comparison with other X-ray pulsars.Comment: 9 pages, 11 figures. Accepted for publication in Astronomy and Astrophysic

Probing stellar winds and accretion physics in high-mass X-ray binaries and ultra-luminous X-ray sources with LOFT

This is a White Paper in support of the mission concept of the Large Observatory for X-ray Timing (LOFT), proposed as a medium-sized ESA mission. We discuss the potential of LOFT for the study of high-mass X-ray binaries and ultra-luminous X-ray sources. For a summary, we refer to the paper.Comment: White Paper in Support of the Mission Concept of the Large Observatory for X-ray Timing. (v2 few typos corrected

Clumpy wind studies and the non-detection of cyclotron line in OAO 1657-415

Winds of massive stars are suspected to be inhomogeneous (or clumpy), which biases the measures of their mass loss rates. In High Mass X-ray Binaries (HMXBs), the compact object can be used as an orbiting X-ray point source to probe the wind and constrain its clumpiness. We perform spectro-timing analysis of the HMXB OAO 1657-415 with non-simultaneous NuSTAR and NICER observations. We compute the hardness ratio from the energy-resolved light curves, and using an adaptive rebinning technique, we thus select appropriate time segments to search for rapid spectral variations on timescales of a few hundreds to thousands of seconds. Column density and intensity of Iron K$\alpha$ line were strongly correlated, and the recorded spectral variations were consistent with accretion from a clumpy wind. We also illustrate a novel framework to measure clump sizes, masses in HMXBs more accurately based on absorption measurements and orbital parameters of the source. We then discuss the limitations posed by current X-ray spacecrafts in such measurements and present prospects with future X-ray missions. We find that the source pulse profiles show a moderate dependence on energy. We identify a previously undetected dip in the pulse profile visible throughout the NuSTAR observation near spin phase 0.15 possibly caused by intrinsic changes in accretion geometry close to the neutron star. We do not find any evidence for the debated cyclotron line at $\sim$ 36\,keV in the time-averaged or the phase-resolved spectra with NuSTAR.Comment: 32 pages, 11 figures in main text, 7 figures in Appendix, Accepted for publication in Ap

The ansamycin antibiotic, rifamycin SV, inhibits BCL6 transcriptional repression and forms a complex with the BCL6-BTB/POZ domain

BCL6 is a transcriptional repressor that is over-expressed due to chromosomal translocations, or other abnormalities, in ~40% of diffuse large B-cell lymphoma. BCL6 interacts with co-repressor, SMRT, and this is essential for its role in lymphomas. Peptide or small molecule inhibitors, which prevent the association of SMRT with BCL6, inhibit transcriptional repression and cause apoptosis of lymphoma cells in vitro and in vivo. In order to discover compounds, which have the potential to be developed into BCL6 inhibitors, we screened a natural product library. The ansamycin antibiotic, rifamycin SV, inhibited BCL6 transcriptional repression and NMR spectroscopy confirmed a direct interaction between rifamycin SV and BCL6. To further determine the characteristics of compounds binding to BCL6-POZ we analyzed four other members of this family and showed that rifabutin, bound most strongly. An X-ray crystal structure of the rifabutin-BCL6 complex revealed that rifabutin occupies a partly non-polar pocket making interactions with tyrosine58, asparagine21 and arginine24 of the BCL6-POZ domain. Importantly these residues are also important for the interaction of BLC6 with SMRT. This work demonstrates a unique approach to developing a structure activity relationship for a compound that will form the basis of a therapeutically useful BCL6 inhibitor