Chaperone-like activity of tubulin

Tubulin, a ubiquitous protein of eukaryotic cytoskeleton, is a building block unit of microtubule. Although several cellular processes are known to be mediated through the tubulin-microtubule system, the participation of tubulin or microtubule in protein folding pathway has not yet been reported. Here we show that goat brain tubulin has some functions and features similar to many known molecular chaperones. Substoichiometric amounts of tubulin can suppress the non-thermal and thermal aggregation of a number of unrelated proteins such as insulin, equine liver alcohol dehydrogenase, and soluble eye lens proteins containing β- and γ-crystallins. This chaperone-like activity of tubulin becomes more pronounced as temperature increases. Aging of tubulin solution at 37° C also enhances its chaperone-like activity. Tubulin loses its chaperone-like activity upon removal of its flexible hydrophilic C-terminal tail. These results suggest that both electrostatic and hydrophobic interactions are important in substrate binding by tubulin and that the negatively charged C-terminal tails play a crucial role for its chaperone-like activity

Chaperone-like activity of tubulin. Binding and reactivation of unfolded substrate enzymes

The eukaryotic cytoskeletal protein tubulin is a heterodimer of two subunits, α and β , and is a building block unit of microtubules. In a previous communication we demonstrated that tubulin possesses chaperonelike activities by preventing the stress-induced aggregation of various proteins (Guha, S., Manna, T. K., Das, K. P., and Bhattacharyya, B. (1998) J. Biol. Chem. 273, 30077-30080). As an extension of this observation, we explored whether tubulin, like other known chaperones, also protected biological activity of proteins against thermal stress or increased the yields of active proteins during refolding from a denatured state. We show here that tubulin not only prevents the thermal aggregation of alcohol dehydrogenase and malic dehydrogenase but also protects them from loss of activity. We also show that tubulin prevents the aggregation of substrates during their refolding from a denatured state and forms a stable complex with denatured substrate. The activity of malic dehydrogenase, α -glucosidase, and lactate dehydrogenase during their refolding from urea or guanidium hydrochloride denatured states increased significantly in presence of tubulin compared with that without tubulin. These results suggest that tubulin, in addition to its role in mitosis, cell motility, and other cellular events, might be implicated in protein folding and protection from stress

Sulfhydryls of tubulin

The 20 cysteine residues of tubulin are heterogeneously distributed throughout its three-dimensional structure. In the present work, we have used the reactivity of these cysteine residues with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe to detect the global conformational changes of tubulin under different experimental conditions. The 20 sulfhydryl groups can be classified into two categories: fast and slow reacting. Colchicine binding causes a dramatic decrease in the reactivity of the cysteine residues and causes complete protection of 1.4 cysteine residues. Similarly, other colchicine analogs that bind reversibly initially decrease the rate of reaction; but unlike colchicine they do not cause complete protection of any sulfhydryl groups. Interestingly, in all cases we find that all the slow reacting sulfhydryl groups are affected to the same extent, that is, have a single rate constant. Glycerol has a major inhibitory effect on all these slow reacting sulfhydryls, suggesting that the reaction of slow reacting cysteines takes place from an open state at equilibrium with the native. Ageing of tubulin at 37 ° C leads to loss of self-assembly and colchicine binding activity. Using DTNB kinetics, we have shown that ageing leads to complete protection of some of the sulfhydryl groups and increased reaction rate for other slow reacting sulfhydryl groups. Ageing at 37 ° C also causes aggregation of tubulin as indicated by HPLC analysis. The protection of some sulfhydryl groups may be a consequence of aggregation, whereas the increased rate of reaction of other slow reacting sulfhydryls may be a result of changes in global dynamics. CD spectra and acrylamide quenching support such a notion. Binding of 8-anilino-1-naphthalenesulfonate (ANS) and bis-ANS by tubulin cause complete protection of some cysteine residues as indicated by the DTNB reaction, but has little effect on the other slow reacting cysteines, suggesting local effects

Electronic structures of the electron donor-acceptor complexes of fullerenes C₆₀ and C₇₀ with azulene and some of its derivatives employing <i>ab initio</i> and DFT methods

1461-1467Quantum mechanical calculations have been carried out to determine the structural and electronic properties of the electron donor-acceptor complexes of C60 and C70 with azulene and some of its derivative, viz, 1,3-dichloroazulene, 2-hydroxyazulene and 4,6,8-trimetrhyl azulene. The molecules studied are optimized first with semi-empirical molecular orbital theory at the third parametric level, and, then further optimized by Hartree Fock method. The optimized geometries, calculated energies, spatial distribution, HOMO and LUMO energies along with the electrostatic potential maps of the individual molecules and the electron donor-acceptor complexes are obtained by ab initio and density functional theory methods. The results suggest that the LUMO state of these complexes is localized on the fullerene moiety whereas the HOMO state is positioned on the azulenes. The energy difference of localized LUMO levels strongly depend on the functional group attached to the azulene and the structure of the fullerene-azulene molecular complexes

Impact of Some Innovative Programmes for Improving Agricultural Productivity– A Study in Arid Zones of West Bengal, India

India at present faces a daunting challenge to provide food security to the burgeoning population. Rainfed areas falling mostly in arid zones accounts 60% of the total cultivated area in India. More than one-third share of the total population in the arid zone of India is below the poverty line. There is an urgent need to explore the possibilities of adopting innovative techniques of production in agriculture in the arid zones. The present paper provides some cases of innovative technologies to improve agricultural productivity in the arid zones in India. The innovative technologies that have been considered here are Happa, System of Rice Intensification (SRI), rain shelter, and mango orchard. The paper highlights the economics of these technologies for tribal households in the study area of Nayagram Block in Jungle Mahal in the state of West Bengal in India. All these technologies have been found to be economically beneficial for the farmer households in the arid zones under study

Stathmin Strongly Increases the Minus End Catastrophe Frequency and Induces Rapid Treadmilling of Bovine Brain Microtubules at Steady State in Vitro

International audienceStathmin is a ubiquitous microtubule destabilizing protein that isbelieved to play an important role linking cell signaling to the reg-ulation of microtubule dynamics. Here we show that stathminstrongly destabilizes microtubule minus endsin vitroat steadystate, conditions in which the soluble tubulin and microtubule lev-els remain constant. Stathmin increased the minus end catastrophefrequency13-fold at a stathmin:tubulin molar ratio of 1:5. Stath-min steady-state catastrophe-promoting activity was considerablystronger at the minus ends than at the plus ends. Consistent with itsability to destabilize minus ends, stathmin strongly increased thetreadmilling rate of bovine brain microtubules. By immunofluores-cence microscopy, we also found that stathmin binds to purifiedmicrotubules along their lengthsin vitro. Co-sedimentation of puri-fied microtubules polymerized in the presence of a 1:5 initial molarratio of stathmin to tubulin yielded a binding stoichiometry of 1 molof stathmin per14.7 mol of tubulin in the microtubules. Theresults firmly establish that stathmin can increase the steady-statecatastrophe frequency by a direct action on microtubules, and fur-thermore, they indicate that an important regulatory action ofstathmin in cells may be to destabilize microtubule minus ends

Conformational properties of α-tubulin tail peptide: implications for tail-body interaction

The carboxy-terminal segments of the α/β-tubulins are flexible regions rich in acidic amino acid residues. It is generally believed that these regions play crucial roles in tubulin polymerization and interaction with many ligands, including colchicine. Exactly how these effects are exerted are not known at present. One such interesting aspect is the pH dependence of colchicine-tubulin interaction and the influence of the α-tail on the binding interaction. We have investigated the location of the colchicine-binding site on tubulin by docking. It has been located on the α/β interface on the N-terminal side, which is also supported by much of the solution data. This location is too far from the tail regions, suggesting that influence of the tail region is transmitted by a pH-dependent conformational change. Two-dimensional NMR studies indicate that at pH 7 a 13-residue peptide corresponding to α-tubulin tail shows little NOE constraints, suggesting extended conformation. On the contrary, at pH 5, a relatively compact structure was deduced from the interproton NOE constraints. Pulsed field gradient measurement of diffusion constant indicates that the peptide at pH 5 is substantially faster diffusing than at pH 7. The Perrin factors calculated from diffusion data indicates that the peptide structure at pH is significantly more compact than at pH 7. Temperature coefficients of several amide protons at pH 5 fall below 5 ppb/°K, indicating a degree of protection. A difference is also seen in the CD spectra obtained at different pHs, consistent with the NMR data. We have investigated the probable spatial organization of the tail of the α-subunit of tubulin, in the high pH extended form and the low pH compact form. On the basis of correlation of pH dependence of many properties of tubulin and the conformation of the α-tail peptide, we propose that the intrinsic conformational preference of the tail-region modulate the tail-body interaction, which in turn has important bearing on colchicine binding properties