The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.
After completing the course, the reader will be able to:1. Educate community oncologists about the promise of anti-CTLA-4 monoclonal antibodies for the treatment of advanced cancer. 2. Suggest that CTLA-4 blockade overcomes barriers to effective immunotherapy for cancer. 3. Describe the rational design and clinical development strategy taken with the CTLA-4 antagonist tremelimumab.Access and take the CME test online and receive 1 AMA PRA Category 1 Credit ™ at CME.TheOncologist.com CME CME ABSTRACT
We propose a new model for the segmental flexibility of immunoglobulin G (IgG). The flexibility of native and mildly reduced anti-5-(dimethylamino)naphthalene-1-sulfonyl (anti-dansyl) antibody was reexamined by nanosecond fluorescence spectroscopy using deconvolution and lamp-shift corrections. The rabbit antibodies used for this study were purified of dimers and other aggregates. The original results indicated that the decay of fluorescence anisotropy involved two rotational correlation times. It was suggested that the short rotational correlation time, phi s, represented a flexible Fab arm motion over a restricted angle and that the long correlation time, phi L, represented global tumbling of the molecule [Yguerabide, J., Epstein, H. F., & Stryer, L. (1970) J. Mol. Biol. 51, 573--590]. Our new data indicate that the long correlation time primarily represents motions of the Fab segments and not global tumbling of the entire molecule. This interpretation implies a more flexible model for IgG. Thus, in solution the antibody arms appear to move over a wide angle and are not restricted to 33 degrees as was suggested in the earlier model. Simple diffusion calculations and other evidence suggest that phi s may represent V-module flexibility about the switch peptides or Fab twisting around its long axis, whereas phi L may represent wagging or wobbling motions of the Fab arms about the hinge region. The faster motions appear to occur over small angles whereas the slower wagging or wobbling motions responsible for most of the decay of anisotropy appear to be much less restricted. The biological function of IgG and anisotropy changes resulting from hinge disulfide cleavage are interpreted in terms of the proposed model. We also demonstrate a useful method for comparison of time-dependent and steady-state fluorescence polarization data.
The anti-tumor properties of Toll-like receptor (TLR) 9 agonist CpG oligodeoxynucleotides (ODN) are enhanced by combinations with several cytotoxic chemotherapy regimens. The mechanisms of this added benefit, however, remain unclear. We now report that, similar to the depletion of regulatory T cells (Treg) using anti-CD25, paclitaxel increased the anti-tumor effect of the TLR9 agonist PF-3512676 in a CD8(+) T cell-dependent fashion. Paclitaxel treatment decreased Treg numbers in a TLR4-independent fashion, and preferentially affected cycling Treg expressing high levels of FoxP3. The paclitaxel-induced reduction in Treg FoxP3 expression was associated with reduced inhibitory function. Adoptively transferred tumor-antigen specific CD8(+) T cells proliferated better in mice treated with paclitaxel and their recruitment in the tumor was increased. However, the systemic frequency of PF-3512676-induced tumor-antigen specific effector CD8(+) T cells decreased with paclitaxel, suggesting opposite effects of paclitaxel on the anti-tumor response. Finally, gene expression profiling and studies of tumor-associated immune cells revealed a complex modulation of the PF-3512676-induced immune response by paclitaxel, including a decrease of IL-10 expression and an increase in IL-17-secreting CD4(+) T cells. Collectively, these data suggest that paclitaxel combined with PF-3512676 may not only promote a better anti-tumor CD8(+) response though increased recruitment in the tumor, possibly through Treg depletion and suppression, but also exerts more complex immune modulatory effects.
1 UK-78,282, a novel piperidine blocker of the T lymphocyte voltage-gated K + channel, Kv1.3, was discovered by screening a large compound ®le using a high-throughput 86 Rb e ux assay. This compound blocks Kv1.3 with a IC 50 of *200 nM and 1 : 1 stoichiometry. A closely related compound, 325, containing a benzyl moiety in place of the benzhydryl in UK-78,282, is signi®cantly less potent. 2 Three lines of evidence indicate that UK-78,282 inhibits Kv1.3 in a use-dependent manner by preferentially blocking and binding to the C-type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at 750 mV enhances the channel's sensitivity to 282. Decreasing the number of inactivated channels by exposure to *160 mM external K + decreases the sensitivity to UK-78,282. Mutations that alter the rate of Ctype inactivation also change the channel's sensitivity to UK-78,282 and there is a direct correlation between t h and IC 50 values.3 Competition experiments suggest that UK-78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK-78,282's action on the channel. 4 UK-78,282 displays marked selectivity for Kv1.3 over several other closely related K + channels, the only exception being the rapidly inactivating voltage-gated K + channel, Kv1.4. 5 UK-78,282 e ectively suppresses human T-lymphocyte activation.
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