Effect of divalent cation on S203C microtubule gliding velocity.

<p>A) Microtubule gliding velocity of the S203C mutant in buffers with the divalent cations shown. The asterisk indicates that the velocity in Mn²⁺ is significantly greater than the velocity in Mg²⁺ (p<0.01), although still significantly lower than wild type in Mg²⁺ (p<0.01). Note the log scale of the Y-axis. B) Microtubule gliding velocity of the S203C mutant that was purified in the presence of Mg²⁺ (Mg-S203C) and Mn²⁺ (Mn-S203C). The single asterisk indicates that the velocity of Mg-S203C in Mn²⁺ is significantly greater than in Mg²⁺ (p<0.01), while the double asterisk indicates that the velocity of Mn-S203C in Mn²⁺ is significantly greater than in Mg²⁺ (p<0.01), and also significantly greater than the velocity of Mg-S203C in Mn²⁺ (p<0.05). C) Histograms of the data for S203C mutants in the conditions indicated. A similar number of microtubules (n = 90–110) were analyzed in each condition.</p

Molecular dynamics simulations of predicted altered kinesin structure.

<p>Images shown are the end point of a 10 ns simulation for wild type or mutant kinesin using the 4HNA structure as a starting point. Amino acids of interest are labeled in white. Intramolecular distances between potential bonding partners are shown in purple, with units of angstroms.</p

Microtubule gliding velocity.

<p>Microtubule gliding velocity.</p

Characterization of kinesin switch I mutations that cause hereditary spastic paraplegia

<div><p>Kif5A is a neuronally-enriched isoform of the Kinesin-1 family of cellular transport motors. 23 separate mutations in the motor domain of Kif5A have been identified in patients with the complicated form of hereditary spastic paraplegia (HSP). We performed in vitro assays on dimeric recombinant Kif5A with HSP-causing mutations in the Switch I domain, which participates in the coordination and hydrolysis of ATP by kinesin. We observed a variety of significantly reduced catalytic and mechanical activities as a result of each mutation, with the shared phenotype from each that motility was significantly reduced. Substitution of Mn²⁺ for Mg²⁺ in our reaction buffers provides a dose-dependent rescue in both the catalytic and ensemble mechanical properties of the S203C mutant. This work provides mechanistic insight into the cause of HSP in patients with these mutations and points to future experiments to further dissect the root cause of this disease.</p></div

Effects of HSP mutations on the basal and MT-Stimulated ATPase rate.

<p>Effects of HSP mutations on the basal and MT-Stimulated ATPase rate.</p

MT affinity in the presence of AMPPNP.

<p>MT pelleting assays were performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180353#pone.0180353.g001" target="_blank">Fig 1</a>, except 5 mM AMPPNP was substituted in the reaction buffer. Experiments were repeated in a range of tubulin concentrations from 0 to 20 μM and the results of the densitometry analysis of the western blots produced are plotted for each Kif5A mutant. The curves shown are the best fit to the data using the exponential decay function in Origin 8 in a user free computational method. A) Curves of fraction Kif5A bound as a function of tubulin concentration for the wild type and mutant proteins. B-H) Individual Kif5A curves (WT or mutant as indicated) with mean +/- SD for each concentration of tubulin analyzed.</p

New Triazole-Based Potent Inhibitors of Human Factor XIIa as Anticoagulants

Factor XIIa (FXIIa) functions as a plasma serine protease within the contact activation pathway. Various animal models have indicated a substantial role for FXIIa in thromboembolic diseases. Interestingly, individuals and animals with FXII deficiency seem to maintain normal hemostasis. Consequently, inhibiting FXIIa could potentially offer a viable therapeutic approach for achieving effective and safer anticoagulation without the bleeding risks associated with the existing anticoagulants. Despite the potential, only a limited number of small molecule inhibitors targeting human FXIIa have been documented. Thus, we combined a small library of 32 triazole and triazole-like molecules to be evaluated for FXIIa inhibition by using a chromogenic substrate hydrolysis assay under physiological conditions. Initial screening at 200 μM involved 18 small molecules, revealing that 4 molecules inhibited FXIIa more than 20%. In addition to being the most potent inhibitor identified in the first round, inhibitor 8 also exhibited a substantial margin of selectivity against related serine proteases, including factors XIa, Xa, and IXa. However, the molecule also inhibited thrombin with a similar potency. It also prolonged the clotting time of human plasma, as was determined in the activated partial thromboplastin time and prothrombin time assays. Subsequent structure–activity relationship studies led to the identification of several inhibitors with submicromolar activity, among which inhibitor 22 appears to demonstrate significant selectivity not only over factors IXa, Xa, and XIa, but also over thrombin. In summary, this study introduces novel triazole-based small molecules, specifically compounds 8 and 22, identified as potent and selective inhibitors of human FXIIa. The aim is to advance these inhibitors for further development as anticoagulants to provide a more effective and safer approach to preventing and/or treating thromboembolic diseases

Discovery of a Series of Thiazole Derivatives as Novel Inhibitors of Metastatic Cancer Cell Migration and Invasion

Effective inhibitors of cancer cell migration and invasion can potentially lead to clinical applications as a therapy to block tumor metastasis, the primary cause of death in cancer patients. To this end, we have designed and synthesized a series of thiazole derivatives that showed potent efficacy against cell migration and invasion in metastatic cancer cells. The most effective compound, <b>5k</b>, was found to have an IC₅₀ value of 176 nM in the dose-dependent transwell migration assays in MDA-MB-231cells. At a dose of 10 μM, <b>5k</b> also blocked about 80% of migration in HeLa and A549 cells and 60% of invasion of MDA-MB-231 cells. Importantly, the majority of the derivatives exhibited no apparent cytotoxicity in the clonogenic assays. The low to negligible inhibition of cell proliferation is a desirable property of these antimigration derivatives because they hold promise of low toxicity to healthy cells as potential therapeutic agents. Mechanistic studies analyzing the actin cytoskeleton by microscopy demonstrate that compound <b>5k</b> substantially reduced cellular f-actin and prevented localization of fascin to actin-rich membrane protrusions. These results suggest that the antimigration activity may result from impaired actin structures in protrusions that are necessary to drive migration

Increase in Lass5 expression Bypasses B7-1/CD28 requirement for T effectors’ response.

<p>Lass5 specific LnB5 T-cell clone (A) and T-cell bulk culture (B) were used to determine B7-1/CD28 requirement. (A): Lass5 high expressing RMA-S/pUB.Trh4 and RMA-S/B7-1.Trh4 cells were used as targets that were recognized by LnB5 T-cell clone. The antibodies against CD80 (B7-1) or CD28 molecules were used to block B7-1/CD28 axis. The isotype Ig was used as a control. (B): Lass5-peptide (50 micromole) pulsed RMA-S/pUB and RMA-S/B7-1 cells were used as targets that were recognized by T-cell bulk culture for ⁵¹Cr-release assays. Pep means Lass5 peptide. One out of two experiments with similar results for each assay was shown. * * and *** indicated no statistical significance.</p

Importance of B7-1:CD28 axis in enhancing a Lass5 specific LnB5 T-cell clone activation.

<p>The RMA-S/pUB and RMA-S/B7-1 transfectants were used as targets recognized by a Lass5 specific LnB5 T-cell clone. Lass5 specific T-cell clone activation detected by the intracellular IFN-gamma release assays were conducted with stimulators RMA-S/pUB and RMA-S/B7-1 cells in (A) to (D). (A): 8×10³ T-cells were incubated with indicated amounts of RMA-S/pUB and RMA-S/B7-1 cells. (B): 8×10³ T-cells were incubated with 1×10⁵ stimulators that previously incubated with either anti-B7-1 mAb or isotype control (for RMA-S/B7-1). (C): 8×10³ T cells were incubated with either anti-CD28 mAb or isotype control before co-culture with 1×10⁵ stimulators (RMA-S/pUB or RMA-S/B7-1). (D): Before co-culture of the T-cells and stimulators, 8×10³ T-cells were incubated with either anti-CD28 mAb or Isotype control and 1×10⁵ RMA-S/B7-1 stimulator cells were incubated with either anti-B7-1 mAb or Isotype control. One out of at least two experiments with similar results was shown. <b>*</b> and ** indicated that P-values were less than 0.05.</p