Kamlesh Gupta

Dietary antioxidant curcumin inhibits microtubule assembly through tubulin binding

Curcumin, a component of turmeric, has potent antitumor activity against several tumor types. However, its molecular target and mechanism of antiproliferative activity are not clear. Here, we identified curcumin as a novel antimicrotubule agent. We have examined the effects of curcumin on cellular microtubules and on reconstituted microtubules in vitro. Curcumin inhibited HeLa and MCF‐7 cell proliferation in a concentration‐dependent manner with IC50 of 13.8 ± 0.7 µm and 12 ± 0.6 µm, respectively. At higher inhibitory concentrations (> 10 µm), curcumin induced significant depolymerization of interphase microtubules and mitotic spindle microtubules of HeLa and MCF‐7 cells. However, at low inhibitory concentrations there were minimal effects on cellular microtubules. It disrupted microtubule assembly in vitro, reduced GTPase activity, and induced tubulin aggregation. Curcumin bound to tubulin at a single site with a dissociation constant of 2.4 ± 0.4 µm and the binding of curcumin to tubulin induced conformational changes in tubulin. Colchicine and podophyllotoxin partly inhibited the binding of curcumin to tubulin, while vinblastine had no effect on the curcumin–tubulin interactions. The data together suggested that curcumin may inhibit cancer cells proliferation by perturbing microtubule assembly dynamics and may be used to develop efficacious curcumin analogues for cancer chemotherapy.

Paclitaxel-resistant Human Ovarian Cancer Cells Undergo c-Jun NH2-terminal Kinase-mediated Apoptosis in Response to Noscapine

We have previously discovered the opium alkaloid noscapine as a microtubule interacting agent that binds to tubulin, alters the dynamics of microtubule assembly, and arrests mammalian cells at mitosis (Ye, K., Ke, Y., Keshava, N., Shanks, J., Kapp, J. A., Tekmal, R. R., Petros, J., and Joshi, H. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1601–1606; Ye, K., Zhou, J., Landen, J. W., Bradbury, E. M., and Joshi, H. C. (2001) J. Biol. Chem. 276, 46697–46700; Zhou, J., Panda, D., Landen, J. W., Wilson, L., and Joshi, H. C. (2002) J. Biol. Chem. 277, 17200–17208). Here we show that noscapine does not compete with paclitaxel for tubulin binding and can efficiently inhibit the proliferation of both paclitaxel-sensitive and paclitaxel-resistant human ovarian carcinoma cells (i.e. the parental cell line 1A9 and two derivative cell lines, 1A9PTX10 and 1A9PTX22, which harbor β-tubulin mutations that impair paclitaxel-tubulin interaction (Giannakakou, P., Sackett, D. L., Kang, Y. K., Zhan, Z., Buters, J. T., Fojo, T., and Poruchynsky, M. S. (1997) J. Biol. Chem. 272, 17118–17125). Strikingly, these cells undergo apoptotic death upon noscapine treatment, accompanied by activation of the c-Jun NH2-terminal kinases (JNK). Furthermore, inhibition of JNK activity by treatment with antisense oligonucleotide or transfection with dominant-negative JNK blocks noscapine-induced apoptosis. These findings thus indicate a great potential for noscapine in the treatment of paclitaxel-resistant human cancers. In addition, our results suggest that the JNK pathway plays an essential role in microtubule inhibitor-induced apoptosis.

Glutamate-induced assembly of bacterial cell division protein FtsZ.

The polymerization of FtsZ is a finely regulated process that plays an essential role in the bacterial cell division process. However, only a few modulators of FtsZ polymerization are known. We identified monosodium glutamate as a potent inducer of FtsZ polymerization. In the presence of GTP, glutamate enhanced the rate and extent of polymerization of FtsZ in a concentration-dependent manner; ∼90% of the protein was sedimented as polymer in the presence of 1 mglutamate. Electron micrographs of glutamate-induced polymers showed large filamentous structures with extensive bundling. Furthermore, glutamate strongly stabilized the polymers against dilution-induced disassembly, and it decreased the GTPase activity of FtsZ. Calcium induced FtsZ polymerization and bundling of FtsZ polymers; interestingly, although 1 m glutamate produced a larger light-scattering signal than produced by 10 mm calcium, the amount of polymer sedimented in the presence of 1 mglutamate and 10 mm calcium was similar. Thus, the increased light scattering in the presence of glutamate must be due to its ability to induce more extensive bundling of FtsZ polymers than calcium. The data suggest that calcium and glutamate might induce FtsZ polymerization by different mechanisms.

Interactions between EB1 and Microtubules: DRAMATIC EFFECT OF AFFINITY TAGS AND EVIDENCE FOR COOPERATIVE BEHAVIOR*

Plus end tracking proteins (+TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved +TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip.

Mechanism for the catastrophe-promoting activity of the microtubule destabilizer Op18/stathmin

Significance The microtubule (MT) cytoskeleton is a dynamic polymer network that plays a crucial role in cell function and disease. MT assembly and dynamics are precisely controlled; a key regulator is the MT destabilizer known as stathmin. Stathmin’s mechanism of action remains controversial: one well-supported model is that it reduces polymer indirectly by sequestering MT subunits; the alternative is that it acts directly on MTs by an as yet unknown mechanism. We provide a resolution to this debate by presenting experimental evidence that stathmin can act directly on MTs and does so by binding and destabilizing exposed protofilaments. Computer simulations performed in parallel suggest that both the direct and sequestering activities are likely to be significant in a cellular context. Regulation of microtubule dynamic instability is crucial for cellular processes, ranging from mitosis to membrane transport. Stathmin (also known as oncoprotein 18/Op18) is a prominent microtubule destabilizer that acts preferentially on microtubule minus ends. Stathmin has been studied intensively because of its association with multiple types of cancer, but its mechanism of action remains controversial. Two models have been proposed. One model is that stathmin promotes microtubule catastrophe indirectly, and does so by sequestering tubulin; the other holds that stathmin alters microtubule dynamics by directly destabilizing growing microtubules. Stathmin’s sequestration activity is well established, but the mechanism of any direct action is mysterious because stathmin binds to microtubules very weakly. To address these issues, we have studied interactions between stathmin and varied tubulin polymers. We show that stathmin binds tightly to Dolastatin-10 tubulin rings, which mimic curved tubulin protofilaments, and that stathmin depolymerizes stabilized protofilament-rich polymers. These observations lead us to propose that stathmin promotes catastrophe by binding to and acting upon protofilaments exposed at the tips of growing microtubules. Moreover, we suggest that stathmins minus-end preference results from interactions between stathmins N terminus and the surface of α-tubulin that is exposed only at the minus end. Using computational modeling of microtubule dynamics, we show that these mechanisms could account for stathmins observed activities in vitro, but that both the direct and sequestering activities are likely to be relevant in a cellular context. Taken together, our results suggest that stathmin can promote catastrophe by direct action on protofilament structure and interactions.

Minimal Plus-end Tracking Unit of the Cytoplasmic Linker Protein CLIP-170

Cytoplasmic linker protein 170 (CLIP-170) is the prototype microtubule (MT) plus-end tracking protein (+TIP) and is involved in regulating MT dynamics. A comprehensive understanding of the process by which CLIP-170 tracks MT plus ends would provide insight into its function. However, the precise molecular mechanism of CLIP-170 +TIP behavior is unknown, and many potential models have been presented. Here, by separating the two CLIP-170 CAP-Gly domains and their adjacent serine-rich regions into fragments of varied size, we have characterized the minimal plus-end tracking unit of CLIP-170 in vivo. Each CLIP-170 fragment was also characterized for its tubulin polymerization activity in vitro. We found that the two CAP-Gly domains have different activities, whereas CAP-Gly-1 appears incompetent to mediate either +TIP behavior or MT nucleation, a CLIP-170 fragment consisting of the second CAP-Gly domain and its adjacent serine-rich region can both track MT plus ends in vivo and induce tubulin polymerization in vitro. These observations complement recent work on CLIP-170 fragments, demonstrate that CAP-Gly motifs do not require dimerization for +TIP and polymerization-promoting activities, and provide insight into CLIP-170 function and mechanism.

A novel multistage decision fusion for cognitive sensor networks using AND and OR rules

We propose a centralized radix-2 multistage decision fusion strategy comprising simple AND and OR rules for cooperative spectrum sensing in cognitive sensor networks. Earlier works on centralized decision fusion show the half-voting and majority rules to be optimum in many spectrum sensing scenarios in terms of minimizing the decision error (or equivalently maximizing the probability of correct decision). We consider a commonly occurring case in spectrum sensing in which the detection probability of a cognitive radio enabled sensor node is greater than its false-alarm probability. For this case, we consider five scenarios and demonstrate that the proposed method either performs better than half-voting and majority rules or exhibits a comparable performance. In this context, we also establish a criterion to make a choice between the AND and OR rules and compute the optimum number of nodes participating in cooperative spectrum sensing for these rules to maximize the correct decision probability.

A multistage approach to decision fusion using a distributed network of non-identical nodes

A distributed sensor network deployed to detect a binary event using a hard decision fusion scheme is considered. Earlier works show that the K-out-of-N counting rule is optimum in minimizing the total error rate when the sensor nodes participating in the event detection have identical performance indexes (detection and false-alarm probability pairs). In most scenarios, this optimum rule turns out to be the half-voting or majority rule. However, when the sensor nodes have non-identical performance indexes, the optimum value of K that gives the maximum correct decision probability may be anything from 1 to N and no general expression for it is available. Trying all possibilities before choosing the best value of K is computationally very demanding and the complexity grows rapidly as the number of sensor nodes increases, making it an impractical approach for a large sensor network. We propose a multistage decision fusion scheme for such networks using unanimity fusion rules and show that it gives an improved performance over unanimity, half-voting, majority, and some soft fusion rules and a comparable performance to the best K-out-of-N fusion rule in many cases with considerably reduced computational complexity in determining the global detection and false-alarm probabilities. HighlightsA new multistage approach for decision fusion for a network of non-identical nodes.Computation of multiplications required for the conventional and proposed schemes.Low computational complexity in determining the system level performance.Superior performance of the proposed scheme to the conventional fusion rules.Performance of the proposed strategy reaching the ideal one for larger networks.