Interaction Of Tubulin Research Articles

Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length.

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAPF375A, with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAPEE343RR that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a ‘clutch-like' mechanism.

Isolation of Functional Tubulin Dimers and of Tubulin-Associated Proteins from Mammalian Cells

The microtubule (MT) cytoskeleton forms a dynamic filamentous network that is essential for many processes, including mitosis, cell polarity and shape, neurite outgrowth and migration, and ciliogenesis [1, 2]. MTs are built up of α/β-tubulin heterodimers, and their dynamic behavior is in part regulated bytubulin-associated proteins (TAPs). Here we describe a novel system to study mammalian tubulins and TAPs. We co-expressed equimolar amounts of triple-tagged α-tubulin and β-tubulin using a 2A "self-cleaving" peptide and isolated functional fluorescent tubulin dimers from transfected HEK293T cells with a rapid two-step approach. We also produced two mutant tubulins that cause brain malformations in tubulinopathy patients [3]. We then applied a paired mass-spectrometry-based method to identify tubulin-binding proteins in HEK293T cellsand describe both novel and known TAPs. We find that CKAP5 and the CLASPs, which are MT plus-end-tracking proteins with TOG(L)-domains[4], bind tubulin efficiently, as does the Golgi-associatedprotein GCC185, which interacts with the CLASPs [5]. The N-terminal TOGL domain of CLASP1 contributes to tubulin binding and allows CLASP1 to function as an autonomous MT-growth-promoting factor. Interestingly, mutant tubulins bind less well to a number of TAPs, including CLASPs and GCC185, and incorporate less efficiently into cellular MTs. Moreover, expression of these mutants in cells impairs several MT-growth-related processes involving TAPs. Thus, stable tubulin-TAP interactions regulate MT nucleation and growth in cells. Combined, our results provide a resource for investigating tubulin interactions and functions and widen the spectrum of tubulin-related disease mechanisms.

Characterizing Biophysical Features Driving the Self-Assembly of Microtubule Nano-Arrays

Microtubules (MTs) are biopolymeric filaments important in several cellular functions, including maintaining cell structure and providing platforms for intracellular transport. Biopolymers, including MTs, are being explored as nanostructure templates (i.e., for nanowire synthesis) due to their dimensions, self-assembly dynamics and versatile chemical functionalization. MTs have unique self-assembly processes scaling from polymerization of tubulin at the nanometer, to the head-to-tail micron-scale assembly of mature MTs, forming segmented nano-arrays. Controlling nano-array assembly requires characterization of the physicochemical properties driving self-assembly. Modelling studies have mapped the electrostatic potential of MTs and found that they are overall negatively charged with oppositely charged ends, suggesting electrostatically attractive interactions may drive assembly. Structural studies, however, suggest that end-to-end interactions of tubulin are driven by hydrophobic interactions. We demonstrate that biophysical features including electrostatics, hydrophobicity and temperature regulate the assembly of MT nano-arrays. Electrostatically shielding MTs, thus shielding the electrostatically polar ends, using salts resulted in increased rate of nano-array assembly. These results suggest that (1) decreasing the overall negative charge of MTs decreases electrostatic repulsion, increasing the probability of end-to-end interactions and (2) shielding electrostatically attractive ends of the MTs does not inhibit end-to-end interactions, suggesting that self-assembly is driven predominately by hydrophobic interactions. Finally, data exploring the role of temperature suggests that increasing temperature causes MT filaments to undergo a phase transition resulting in a discontinuous Arrhenius behavior. Together, these results provide insights into tunable parameters driving self-assembly of MT nano-arrays. Adjusting these parameters to optimize assembly will greatly aid in the application of biopolymeric templates for use in nanomaterials applications. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

Tubulin Nanorings

ABSTRACTBiological systems routinely produce nanoscopic molecular structures with considerably less dispersion in size and shape than encountered in most manufactured materials. Indeed, Biological structures are frequently and essentially monodisperse. An example of this uniformity, combined with an intriguing geometry, is the nanometer-scale protein nanorings produced by interaction of the protein tubulin with certain hydrophobic tri-, tetra- and pentapeptides originally extracted as natural products from marine biosystems. Different peptides produce different sized nanorings, but we focus on those produced by binding to tubulin of the cyclic depsipeptide cryptophycin. The nanorings that form upon binding of this ligand show a sharp mass distribution indicating that the nanorings are made of 8 tubulin dimers of 100 kDa.In this submission, we demonstrate how a combination of fluorescence correlation spectroscopy, dynamic light scattering, electron microscopy, analytical ultracentrifugation, small-angle neutron scattering, and modeling is applied to reveal interactions of tubulin and cryptophycin in solution and to characterize their structures. We find that the cryptophycin-tubulin nanorings (∼25 nm diameter) are single-walled, appear rigid, are composed of 8 tubulin dimers in a single closed ring, and are stable upon dilution to nanomolar concentrations.Similar studies with a different peptide, the linear pentapeptide dolastatin 10, demonstrated that binding of this peptide to tubulin produces larger nanorings (14 tubulin dimers, ∼45 nm diameter rings), with slightly different properties. The ability to adjust the ring size with different peptides, and produce uniform nanorings with properties that differ slightly between size classes, makes the tubulin-peptide ring structures an appealing structural system.

Tubulin Monomer-Monomer Association is Less Influenced by the Solvent than Dimer-Dimer Association: Structure and Function of Tubulin Interaction Interfaces

The ab-tubulin heterodimer is the basic unit of microtubules. Both a- and b-tubulins are members of the tubulin/FtsZ/CetZ superfamily of proteins, whose eukaryotic and prokaryotic members share the ability to bind guanine nucleotides and participate in the formation of linear polymers. Of all the members of this family only a- and b-tubulins form a dimer that is stable in dilute solutions. Other filament-forming members of this family, such as btubA/B and FtsZ, can form weak dimers but these species are not populated in dilute solutions. The association of the a- and b-tubulin monomers to form the heterodimer involves protein-protein interaction surfaces that are significantly different from those that form the dimer-dimer contact in protofilaments, so that heterodimers are stable in non-polymerizing conditions. We compared the properties of the intra-dimer interface (monomer-monomer) with the properties of the inter-dimer interface (dimer-dimer) in published models of tubulin protofilaments, including curved and straight filaments. Based on these analyzes we predict at least 10-fold enhanced stability of the monomer-monomer association over dimer-dimer association. We tested this prediction by measuring the dimer dissociation constant (Kd) and the critical concentration for polymerization (CC). We also measured the effects of changing the solution composition on dimer Kd (reflecting monomer-monomer association), and on CC (reflecting dimer-dimer association). We examined 4 changes to the solution: addition of ligands 1) GTP or GDP at 0.5 mM, addition of co-solvents 2) urea or 3) Trimethylamine N-oxide (TMAO), each at 0.3 M, and addition of 4) NaCl 0.5 M. Our experimental results demonstrate the enhanced stability and lower sensitivity to solution composition changes of tubulin monomer-monomer association compared to dimer-dimer association.

Kinesin-5 is a microtubule polymerase.

Kinesin-5 slides antiparallel microtubules during spindle assembly, and regulates the branching of growing axons. Besides the mechanical activities enabled by its tetrameric configuration, the specific motor properties of kinesin-5 that underlie its cellular function remain unclear. Here by engineering a stable kinesin-5 dimer and reconstituting microtubule dynamics in vitro, we demonstrate that kinesin-5 promotes microtubule polymerization by increasing the growth rate and decreasing the catastrophe frequency. Strikingly, microtubules growing in the presence of kinesin-5 have curved plus ends, suggesting that the motor stabilizes growing protofilaments. Single-molecule fluorescence experiments reveal that kinesin-5 remains bound to the plus ends of static microtubules for 7 s, and tracks growing microtubule plus ends in a manner dependent on its processivity. We propose that kinesin-5 pauses at microtubule plus ends and enhances polymerization by stabilizing longitudinal tubulin–tubulin interactions, and that these activities underlie the ability kinesin-5 to slide and stabilize microtubule bundles in cells.

Abstract 1667: Targeting acute myelogenous leukemia with novel combrestatin analogs and development of predictors of response

Abstract Introduction: Most adult acute myelogenous leukemia (AML) patients relapse and die of the disease. AML stem cells (AML-SCs) have been postulated to resist standard chemotherapeutic agents and thus give rise to relapse. Combretastatin A-4 (CA-4), a natural product isolated from the South African tree Combretum caffrum, has been reported to possess anti-leukemic activity in AML cell lines. To increase the repertoire of combretastatin analogs with potent anti-leukemic activity, about 100 CA-4 analogs were tested in AML cells, and studied the mechanism that AML cells resist to such microtubule (MT) inhibitors. Methods: Cell viability/apoptosis was determined by annexin V and 7-AAD staining and flow cytometry. Cellular ROS production was measured by CellROX. Cell cycle was determined by DAPI staining and flow cytometry. Stem cell function was evaluated by colony assay and mouse xenograft assays. Tubulin depolymerization was measured by immunofluorescence or immunoblotting for microtubule fraction. Interaction of tubulin with BTAN or p38 MAPK was determined by pull down assays. Results: One of the most potent analogs, BTAN, was further investigated in a panel of leukemic cell lines and primary AML samples. Similar to CA-4, BTAN inhibited tubulin polymerization in vitro and induced MT depolymerization and cell cycle arrest at G2/M in AML cells. BTAN induced cell death of AML blast, progenitor and stem cells via caspase activation regardless of their proliferating status and level of intracellular reactive oxygen species (ROS). In addition, BTAN impaired the physical contact of AML with stromal cells. To further identify the mechanism of resistance to BTAN, we investigated several signal transduction pathways including AKT, JAK-STAT, ERK1/2, JNK and p38. p38 MAPK inhibitor sensitized leukemia cells to BTAN treatment, and the sensitivity of AML cells to BTAN correlated with the basal level of active/phosphorylated p38 MAPK, suggesting p38 MAPK may interfere with the sensitivity to BTAN in AML. We demonstrated that tubulin interacted with p38 MAPK, especially its phosphorylated form, which was disrupted by treatment with a p38 MAPK inhibitor. In addition, in the presence of p38 inhibitor, the accumulation of BTAN was potentiated in AML cells, suggesting activated p38 may interfere with the interaction between BTAN and its tubulin targets. Conclusion: Our data demonstrated that BTAN is a newly identified anti-AML-SC agent that works by disrupting the MT cytoskeleton and inducing caspase activation. These data suggest that the MT network is involved in the regulation of pro-survival signaling pathways even in quiescent AML-SCs, and that p38 MAPK is a key modulator for the response of AML blasts to microtubule inhibitors and that co-treatment with p38 MAPK inhibitors could be beneficial to microtubule destabilizing agents in AML. Note: This abstract was not presented at the meeting. Citation Format: Hongliang Zong, Narsimha R. Penthala, Vijayakumar N. Sonar, Paraskevi Giannakakou, Gail J. Roboz, Peter A. Crooks, Monica L. Guzman. Targeting acute myelogenous leukemia with novel combrestatin analogs and development of predictors of response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1667. doi:10.1158/1538-7445.AM2015-1667

βI-tubulin mutations in the laulimalide/peloruside binding site mediate drug sensitivity by altering drug–tubulin interactions and microtubule stability

Peloruside A (PLA) and laulimalide (LAU) are potent microtubule-stabilizing natural products that are effective against a broad spectrum of cancer cells. The interactions of PLA and LAU with tubulin have attracted a great deal of attention, mainly because they bind to β-tubulin at a site that is different from the classical taxoid site. Multiple βI-tubulin amino acid residues have been predicted by computer modelling studies and more recently by protein crystallography to participate in the binding of PLA and LAU to tubulin. The relevance of these residues in determining cellular sensitivity to the compounds, however, remains largely uncertain. To determine the role of four binding site residues, Q291, D295, V333, and N337 on PLA and LAU activity, we introduced single mutations to these sites by site-directed mutagenesis and transfected each mutant tubulin separately into HEK and/or HeLa cells. We found that a Q291M βI-tubulin mutation increased sensitivity of the cells to PLA, but not to LAU, paclitaxel (PTX), or vinblastine (VBL). In contrast, V333W and N337L mutations led to less stable microtubules, with the V333W causing resistance to PLA and PTX, but not LAU, and the N337L causing resistance to PLA, LAU, and PTX. Moreover, cells expressing either W333 or L337 were hypersensitive to the microtubule-destabilizing agent, VBL. The D295I mutation conferred resistance to both PLA and LAU without affecting microtubule stability or sensitivity to PTX or ixabepilone (IXB). This study identifies the first mammalian βI-tubulin mutation that specifically increases sensitivity to PLA, and reports mutations at PLA and LAU binding site residues that can either reduce microtubule stability or impair drug–tubulin binding, conferring resistance to these microtubule-stabilizing agents. This information provides insights on β-tubulin residues important for maintaining microtubule structural integrity and for sensitivity to microtubule-targeting agents, and suggests novel directions for rational structure-based design of new and more potent agents for cancer treatment that target the LAU/PLA site.

Clinically acceptable colchicine concentrations have potential for the palliative treatment of human cholangiocarcinoma

Microtubules are an ideal target for anticancer drugs because of their essential role in mitosis. Colchicine is a microtubule destabilizer. Whether the clinically acceptable colchicine concentrations had anticancer effects on human cholangiocarcinoma cells was investigated. Two human cholangiocarcinoma cell lines (C14/KMUH, C51/KMUH) were investigated using clinically acceptable plasma colchicine concentrations (2 ng/mL and 6 ng/mL for the in vitro experiment, 0.07 mg colchicine/kg/d × 14 days for the nude mouse experiment). Our results showed that colchicine caused significantly dose-dependent antiproliferative effects on both cell lines (all p < 0.0001). Nude mouse (BALB/c-nu) experiments showed that the increased tumor volume ratios in colchicine-treated mice were significantly lower than control mice started from the 11th day of treatment (p = 0.0167). The tumor growth rates in colchicine-treated mice after 14 days of treatment were significantly lower than in control mice (0.147 ± 0.004/d vs. 0.274 ± 0.003/d, p = 0.0015). In addition to the well-known direct colchicine–tubulin interaction as a common anticancer mechanism of colchicine, microarray and quantitative reverse transcriptase-polymerase chain reaction showed that the antiproliferative effects of both 2 ng/mL and 6 ng/mL colchicine on C14/KMUH cells could be partially explained by downregulations of both HSD11B2 and MT-COI. There was no effect of colchicine on MT-COI expression in C51/KMUH cells, however, 6 ng/mL colchicine also downregulated HSD11B2 in this cell line. In conclusion, clinically acceptable colchicine concentrations can inhibit the proliferation of human cholangiocarcinoma cells. This drug has good potential for the palliative treatment of cholangiocarcinoma due to its low cost and our long-standing prescription experience.

High-affinity accumulation of a maytansinoid in cells via weak tubulin interaction.

The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4–6 × 107 copies). Efflux of 3 [H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.

The kinesin&amp;ndash;tubulin complex: considerations in structural and functional complexity

The kinesin&ndash;tubulin complex: considerations in structural and functional complexity Zachary T Olmsted, Andrew G Colliver, Janet L Paluh State University of New York Polytechnic Institute, Colleges of Nanoscale Science and Engineering, College of Nanoscale Science, Nanobioscience Constellation, Albany, NY, USA Abstract: The ability of cells to respond to external cues by appropriately manipulating their internal environment requires a dynamic microtubule cytoskeleton that is facilitated by associated kinesin motor interactions. The evolutionary adaptations of kinesins and tubulins when merged generate a highly adaptable communication and infrastructure cellular network that is important to understanding specialized cell functions, human disease, and disease therapies. Here, we review the state of the field in the complex relationship of kinesin&ndash;tubulin interactions. We propose 12 mechanistic specializations of kinesins. In one category, referred to as sortability, we describe how kinesin interactions with tubulin isoforms, isotypes, or posttranslationally modified tubulins contribute to diverse cellular roles. Fourteen kinesin families have previously been described. Here, we illustrate the great depth of functional complexity that is possible in members within a single kinesin family by mechanistic specialization through discussion of the well-studied Kinesin-14 family. This includes new roles of Kinesin-14 in regulating supramolecular structures such as the microtubule-organizing center &gamma;-tubulin ring complex of centrosomes. We next explore the value of an improved mechanistic understanding of kinesin&ndash;tubulin interactions in regard to human development, disease mechanisms, and improving treatments that target kinesin&ndash;tubulin complexes. The ability to combine the current kinesin nomenclature along with a more precisely defined kinesin and tubulin molecular toolbox is needed to support more detailed exploration of kinesin&ndash;tubulin interaction mechanisms including functional uniqueness, redundancy, or adaptations to new roles upon cell specialization, and to thereby accelerate applications in human health. Keywords: microtubule, cellular network, cell functions, sortability, transport, disease, signaling

Drug–tubulin interactions interrogated by transient absorption spectroscopy

The triplet excited state of complexed COL and MTC gives well defined transient spectra undetectable in the absence of TU.

The role of tubulin in the mitochondrial metabolism and arrangement in muscle cells.

Tubulin, a well-known component of the microtubule in the cytoskeleton, has an important role in the transport and positioning of mitochondria in a cell type dependent manner. This review describes different functional interactions of tubulin with cellular protein complexes and its functional interaction with the mitochondrial outer membrane. Tubulin is present in oxidative as well as glycolytic type muscle cells, but the kinetics of the in vivo regulation of mitochondrial respiration in these muscle types is drastically different. The interaction between VDAC and tubulin is probably influenced by such factors as isoformic patterns of VDAC and tubulin, post-translational modifications of tubulin and phosphorylation of VDAC. Important factor of the selective permeability of VDAC is the mitochondrial creatine kinase pathway which is present in oxidative cells, but is inactive or missing in glycolytic muscle and cancer cells. As the tubulin-VDAC interaction reduces the permeability of the channel by adenine nucleotides, energy transfer can then take place effectively only through the mitochondrial creatine kinase/phosphocreatine pathway. Therefore, closure of VDAC by tubulin may be one of the reasons of apoptosis in cells without the creatine kinase pathway. An important question in tubulin regulated interactions is whether other proteins are interacting with tubulin. The functional interaction may be direct, through other proteins like plectins, or influenced by simultaneous interaction of other complexes with VDAC.

Total synthesis and cytotoxicity of the marine natural product malevamide D and a photoreactive analog

The marine natural product malevamide D from the cyanobacterium Symploca hydnoides was synthesized for the first time. The final peptide coupling linked the dolaisoleuine and dolaproine subunits. The phenyl group of malevamide D was also functionalized with a photoreactive diazirine moiety, which was carried through seven reaction steps. Comprehensive assessment of the cytotoxicity in a panel of 42 human cancer cell lines revealed a geomean IC70 value of 1.5 nM (IC50 0.7 nM) for malevamide D, whereas the photoreactive derivative proved to be less active by a factor of at least 200. COMPARE analysis indicated tubulin interaction as likely mode of action of malevamide D.

Targeting cytotoxicity and tubulin polymerization by metal-carbene complexes on a purine tautomer platform.

This communication describes the synthesis, structural investigation and tubulin binding of purine rare imino-tautomer based Ag(i) and Hg(ii)-carbene complexes. These complexes exhibit cytotoxicity through tubulin interaction by binding to a site close to the GTP binding site.

An amyloid inhibitor octapeptide forms amyloid type fibrous aggregates and affects microtubule motility

An amyloid inhibitor octapeptide simultaneously forms amyloid type fibrous aggregates on its own and interacts with the microtubule lattice three times stronger than a Xenopus Microtubule Associated Protein (XMAP215).

Abstract LB-59: ERG induces taxane resistance in castration-resistant prostate cancer.

Abstract Taxanes are the only chemotherapies to prolong survival for patients with metastatic castration-resistant prostate cancer (CRPC), and both docetaxel (doc) and cabazitaxel (cab) are FDA approved for this indication. Despite the initial efficacy of taxanes in treating CRPC, all patients ultimately fail due to the development of drug resistance. In this study we show that ERG over-expression, which occurs in roughly 50% of prostate cancers, is associated with both doc and cab resistance using in vitro and in vivo models of CRPC. Using either tetracycline-inducible or constitutively active expression systems in multiple prostate cancer cell lines, we found that ERG over-expression leads to at least a 10 fold increase in IC50 doses of doc or cab based on cell viability assays and inhibition of taxane-induced apoptosis. Similarly, ERG over-expressing prostate cancer xenografts were reproducibly more resistant to cab compared to the cab-sensitive control xenografts. Based on a biochemical and cell biological characterization, we observed that ERG binds directly to tubulin in the cytoplasm of prostate cancer cells, as well as in circulating tumor cells from patients treated with doc, and prevents taxane-induced microtubule stabilization. ERG over-expression affects several parameters of microtubule dynamics and inhibits effective drug-target engagement of doc or cab with tubulin, either directly via its tubulin interaction or indirectly by affecting transcription of genes that modulate microtubule homeostasis. In addition, ERG induces and binds to clusterin, a molecular chaperone also associated with taxane resistance. Clusterin knock-down with siRNA abrogates ERG-induced taxane resistance. Altogether, this data suggests that ERG, an oncogenic transcription factor, plays a novel role beyond regulating gene expression and functions outside the nucleus to cooperate with clusterin and tubulin in promoting taxane resistance. Determining ERG status in patient tumors may aid in patient selection for doc or cab therapy and/or influence co-targeting approaches using the clusterin antisense inhibitor OGX-011. Citation Format: Giuseppe Galletti, Himisha Beltran, Alexandre Matov, Jacqueline Fontugne, Juan Miguel Mosquera, Ladan Fazli, Scott Tagawa, David Nanus, Martin Gleave, Mark Rubin, Paraskevi Giannakakou, David S. Rickman. ERG induces taxane resistance in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-59. doi:10.1158/1538-7445.AM2013-LB-59

Abstract 2961: The N-terminal projection domain of the microtubule associated protein tau inhibits TNFα signaling.

Abstract Tumor Necrosis Factor-alpha (TNFα) is a multifunctional pro-inflammatory cytokine implicated in inducing both tumor promotion and tumor inhibition. This paradoxical effect can be partly explained by the fact that TNFα acts as a regulator of both proliferation and apoptosis; however the mechanisms and factors associated with how or whether a tumor cell responds to a microenvironment rich in TNFα have not been fully resolved. Here we provide evidence that the microtubule associate protein Tau inhibits TNFα signaling through its N-terminal domain and implicate it as a novel regulator of the cellular response to this key cytokine. Our findings indicate that over-expression of Tau in the human breast cancer cell lines MCF7, SkBr3 and in the pheochromocytoma cell line PC12 suppresses TNFα induced apoptosis evident in the control cells. TNFα induced cell apoptosis in control cells is triggered as early as 6 hrs post-treatment with hallmark features of cell shrinkage, membrane blebbing, disruption of tubulin architecture and caspase-3 activation. While over-expression of wild type full length Tau isoform in these cell lines, completely abrogates all TNFα associated apoptotic phenotypes. Aside from inhibiting TNFα induced apoptosis, Tau over-expression also interferes with TNFα induced NFκB activation. This is evident by a significant decrease in NFκB nuclear translocation in Tau over-expressed cells compared to controls. Suppression of TNFα signaling by Tau is not associated with its tubulin interaction since a truncated N-terminal projection domain lacking all microtubule binding domains still provides resistance to TNFα induced apoptosis and NFκB activation. These findings lead us to conclude that the functional domain of Tau that regulates TNFα response lies within its N-terminal region and current mechanistic studies indicate this region inhibits TNFα receptor clustering, which is needed for signaling. Our data provide direct evidence that the N-terminal domain of the microtubule associated protein Tau can inhibit TNFα signaling in tumor cells. These findings indicate a novel function for Tau and add to our knowledge of how a tumor cell responds to TNFα signaling. Citation Format: Shawon Debnath, Peter Hannon, Kristina Toropova, Nelson Nunez Rodriguez, Alejandra Alonso, Jimmie Eugene Fata. The N-terminal projection domain of the microtubule associated protein tau inhibits TNFα signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2961. doi:10.1158/1538-7445.AM2013-2961 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Cytotoxic 3,4,5-trimethoxychalcones as mitotic arresters and cell migration inhibitors

Based on classical colchicine site ligands and a computational model of the colchicine binding site on beta tubulin, two classes of chalcone derivatives were designed, synthesized and evaluated for inhibition of tubulin assembly and toxicity in human cancer cell lines. Docking studies suggested that the chalcone scaffold could fit the colchicine site on tubulin in an orientation similar to that of the natural product. In particular, a 3,4,5-trimethoxyphenyl ring adjacent to the carbonyl group appeared to benefit the ligand–tubulin interaction, occupying the same subcavity as the corresponding moiety in colchicine. Consistent with modeling predictions, several 3,4,5-trimethoxychalcones showed improved cytotoxicity to murine acute lymphoblastic leukemia cells compared with a previously described parent compound, and inhibited tubulin assembly in vitro as potently as colchicine. The most potent chalcones inhibited the growth of human leukemia cell lines at nanomolar concentrations, caused microtubule destabilization and mitotic arrest in human cervical cancer cells, and inhibited human breast cancer cell migration in scratch wound and Boyden chamber assays.

Identification of a new CRMP5 isoform present in the nucleus of cancer cells and enhancing their proliferation

Collapsin Response Mediator Protein 5 (CRMP5) belongs to a family of five cytosolic proteins highly expressed in the developing nervous system but downregulated in the adult brain. When expressed at the adult stage, CRMP5 is involved in neurological disorders. Indeed, CRMP5 is found expressed in cancer cells of some brain tumors, such as glioblastoma, or in small cell lung cancer causing paraneoplastic neurological syndromes as a result of cancer-induced auto-immune processes. Nevertheless, its role in cancer pathology is still obscure. Here, we show a new short isoform, derived from C-terminal processing of CRMP5, presenting a nuclear localization both in human glioblastoma, and in cancer cell lines (H69, GL15). By mutational analysis, we demonstrate that nuclear translocation occurs via nuclear localization signal (NLS), where the essential residue for nuclear location is K391. Direct CRMP5/ tubulin interaction, previously shown during brain development, does not occur for cytosolic CRMP5 in pathological conditions, leading to the suggestion that in cancer cells CRMP5 is not sequestered in the cytosol; therefore it may undergo C-terminal truncation allowing the exposure of the NLS for active translocation. Moreover, we show that the function associated with the CRMP5 nuclear targeting is an increase of cell proliferation activity.