58 research outputs found
Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior
Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects
Structural determinants of microtubule minus end preference in CAMSAP CKK domains
CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which lacks minus-end specificity. Here we report near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes, which show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit different footprints on microtubules. NMR experiments show that both HsCKK and NgCKK are remarkably rigid. However, whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. Thus, in contrast to many MAPs, the HsCKK domain can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition
Transverse relaxation of multiple quantum coherence in nuclear magnetic resonance as a probe of molecular motion
Two-dimensional Fourier-transform techniques were used to observe the deuteron double quantum transition for DC.tbd.CCN and CDCl3 partially oriented in a nematic solvent. Obsd. linewidths agree well with those calcd. from appropriate spin-lattice relaxation measurements, demonstrating the potential of multiple quantum linewidths for studies of relaxation and mol. dynamics. [on SciFinder (R)
Effects of fasting, intermittent feeding or continuous parenteral nutrition on rat liver and brain energy metabolism as assessed by 31P-NMR.
We wanted to determine what happens to brain PCr and ATP relative to that in liver during a series of dietary manipulation consisting of a severe fast, during eating (when nutrients are intermittently supplied), and during and after PN-100, when an excess amount of nutrients are continuously supplied, using 31P-NMR spectroscopy, in rats randomized to a Fast or Fed group in which energy was provided either as chow or as PN-100. Liver ATP concentration, and brain and liver 31P-nuclear magnetic resonance (NMR) spectras were measured serially. Brain energy metabolism was not different between groups and among days. In contrast, Fasted group showed increased liver ATP/Pi ratio and decreased ATP concentration and ATP/phosphomonoester ratio, there being no difference between Fed and PN-100 groups. Data suggest that brain energy metabolism is maintained regardless of whether energy is supplied intermittently or continuously, and during a negative caloric intake period, brain energy metabolism is quantitatively preserved, suggesting that ATP production by liver is subservient to brain ATP state
Proton assisted recoupling and protein structure determination
We introduce a homonuclear version of third spin assisted recoupling, a second-order mechanism that can be used for polarization transfer between (13)C or (15)N spins in magic angle spinning (MAS) NMR experiments, particularly at high spinning frequencies employed in contemporary high field MAS experiments. The resulting sequence, which we refer to as proton assisted recoupling (PAR), relies on a cross-term between (1)H-(13)C (or (1)H-(15)N) couplings to mediate zero quantum (13)C-(13)C (or (15)N-(15)N recoupling). In particular, using average Hamiltonian theory we derive an effective Hamiltonian for PAR and show that the transfer is mediated by trilinear terms of the form C(1)(+/-)C(2)(-/+)H(Z) for (13)C-(13)C recoupling experiments (or N(1)(+/-)N(2)(-/+)H(Z) for (15)N-(15)N). We use analytical and numerical simulations to explain the structure of the PAR optimization maps and to delineate the PAR matching conditions. We also detail the PAR polarization transfer dependence with respect to the local molecular geometry and explain the observed reduction in dipolar truncation. Finally, we demonstrate the utility of PAR in structural studies of proteins with (13)C-(13)C spectra of uniformly (13)C, (15)N labeled microcrystalline Crh, a 85 amino acid model protein that forms a domain swapped dimer (MW=2x10.4 kDa). The spectra, which were acquired at high MAS frequencies (omega(r)2 pi>20 kHz) and magnetic fields (750-900 MHz (1)H frequencies) using moderate rf fields, exhibit numerous cross peaks corresponding to long (up to 6-7 A) (13)C-(13)C distances which are particularly useful in protein structure determination. Using results from PAR spectra we calculate the structure of the Crh protein
Novel sporadic and recurrent mutations in KRT5 and KRT14 genes in Polish epidermolysis bullosa simplex patients: further insights into epidemiology and genotype–phenotype correlation
Magic Angle Spinning NMR Investigation of Influenza A M2(18-60): Support for an Allosteric Mechanism of Inhibition
The tetrameric M2 proton channel from influenza A virus conducts protons at low pH and is inhibited by aminoadamantyl drugs such as amantadine and rimantadine (Rmt). We report magic angle spinning NMR spectra of POPC and DPhPC membrane-embedded M2[subscript 18−60], both apo and in the presence of Rmt. Similar line widths in the spectra of apo and bound M2 indicate that Rmt does not have a significant impact on the dynamics or conformational heterogeneity of this construct. Substantial chemical shift changes for many residues in the transmembrane region support an allosteric mechanism of inhibition. An Rmt titration supports a binding stoichiometry of >1 Rmt molecule per channel and shows that nonspecific binding or changes in membrane composition are unlikely sources of the chemical shift changes. In addition, doubling of spectral lines in all of the observed samples provides evidence that the channel assembles with twofold symmetry.National Institutes of Health (U.S.) (EB001960)National Institutes of Health (U.S.) (EB002026)National Institutes of Health (U.S.) (Al067438)National Science Foundation (U.S.). Graduate Research Fellowship Progra
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