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

Visible-near-infrared and fluorescent copper sensors based on julolidine conjugates: selective detection and fluorescence imaging in living cells

We present novel Schiff base ligands julolidine–carbonohydrazone 1 and julolidine–thiocarbonohydrazone 2 for selective detection of Cu<SUP>2+</SUP> in aqueous medium. The planar julolidine-based ligands can sense Cu<SUP>2+</SUP> colorimetrically with characteristic absorbance in the near-infrared (NIR, 700–1000 nm) region. Employing molecular probes 1 and 2 for detection of Cu<SUP>2+</SUP> not only allowed detection by the naked eye, but also detection of varying micromolar concentrations of Cu<SUP>2+</SUP> due to the appearance of distinct coloration. Moreover, Cu<SUP>2+</SUP> selectively quenches the fluorescence of julolidine–thiocarbonohydrazone 2 among all other metal ions, which increases the sensitivity of the probe. Furthermore, quenched fluorescence of the ligand 2 in the presence of Cu<SUP>2+</SUP> was restored by adjusting the complexation ability of the ligand. Hence, by treatment with ethylenediaminetetraacetic acid (EDTA), thus enabling reversibility and dual-check signaling, julolidine–thiocarbonohydrazone (2) can be used as a fluorescent molecular probe for the sensitive detection of Cu<SUP>2+</SUP> in biological systems. The ligands 1 and 2 can be utilized to monitor Cu<SUP>2+</SUP> in aqueous solution over a wide pH range. We have investigated the structural, electronic, and optical properties of the ligands using ab initio density functional theory (DFT) combined with time-dependent density functional theory (TDDFT) calculations. The observed absorption band in the NIR region is attributed to the formation of a charge-transfer complex between Cu<SUP>2+</SUP> and the ligand. The fluorescence-quenching behavior can be accounted for primarily due to the excited-state ligand 2 to metal (Cu<SUP>2+</SUP>) charge-transfer (LMCT) processes. Thus, experimentally observed characteristic NIR and fluorescence optical responses of the ligands upon binding to Cu<SUP>2+</SUP> are well supported by the theoretical calculations. Subsequently, we have employed julolidine–thiocarbonohydrazone 2 for reversible fluorescence sensing of intracellular Cu<SUP>2+</SUP> in cultured HEK293T cells

Discrete dinuclear complex to extended 2D compound in a Cu–azido system by controlling coligand stoichiometry: synthesis and magneto-structural correlations

This article describes syntheses, structural characterizations and magnetic studies of two different Cu(II)–azido compounds, a discrete dinuclear complex and an extended 2D network. The compounds, [Cu(&#x000B5;<SUB>1,1</SUB>-N<SUB>3</SUB>)(N<SUB>3</SUB>)(Me<SUB>2</SUB>en)]<SUB>2</SUB> (1) and [Cu<SUB>3</SUB>(&#x000B5;<SUB>1,1,1</SUB>-N<SUB>3</SUB>)<SUB>2</SUB>(&#x000B5;<SUB>1,1,3</SUB>-N<SUB>3</SUB>)(&#x000B5;<SUB>1,1,</SUB>-N<SUB>3</SUB>)2(&#x000B5;<SUB>1,3</SUB>-N<SUB>3</SUB>)(Me<SUB>2</SUB>en)]<SUB>n</SUB> (2), have been synthesized by controlling the relative concentration of the blocking ligand, N,N-dimethylethylenediamine (Me<SUB>2</SUB>en). Compound 1 is a dinuclear compound which is formed by a doubly asymmetric &#x000B5;<SUB>1,1</SUB>-N<SUB>3</SUB> bridging ligand, while 2 is a rare Cu–azido system where four different types of binding modes of azide ligands are present in a single compound. Compound 2 contains a hexanuclear core, where the Cu(II) centres are connected to each other by &#x000B5;<SUB>1,1,1</SUB>, &#x000B5;<SUB>1,1</SUB> and &#x000B5;<SUB>1,1,3</SUB> bridging azide ligands. The hexanuclear core acts as a secondary building block and further assembles via &#x000B5;<SUB>1,3</SUB> and &#x000B5;<SUB>1,1,3</SUB> azide groups, forming a 2D network in the crystallographic ac plane. Interestingly, temperature-dependent magnetic study suggests that the dinuclear compound 1 exhibits an antiferromagnetic interaction through the &#x000B5;<SUB>1,1</SUB>-N<SUB>3</SUB> bridge, which has also been supported by density functional theory (DFT) calculations. In the case of 2, an overall dominant ferromagnetic interaction is observed while antiferromagnetic interaction operates between the hexanuclear cores

Human SAS‑6 C‑Terminus Nucleates and Promotes Microtubule Assembly <i>in Vitro</i> by Binding to Microtubules

Centrioles are essential components of the animal centrosome and play crucial roles in the formation of cilia and flagella. They are cylindrical structures composed of nine triplet microtubules organized around a central cartwheel. Recent studies have identified spindle assembly abnormal protein SAS-6 as a critical component necessary for formation of the cartwheel. However, the molecular details of how the cartwheel participates in centriolar microtubule assembly have not been clearly understood. In this report, we show that the C-terminal tail (residues 470–657) of human SAS-6, HsSAS-6 C, the region that has been shown to extend toward the centriolar wall where the microtubule triplets are organized, nucleated and induced microtubule polymerization <i>in vitro</i>. The N-terminus (residues 1–166) of HsSAS-6, the domain known to be involved in formation of the central hub of the cartwheel, did not, however, exert any effect on microtubule polymerization. HsSAS-6 C bound to the microtubules and localized along the lengths of the microtubules <i>in vitro</i>. Microtubule pull-down and coimmunoprecipitation (Co-IP) experiments with S-phase synchronized HeLa cell lysates showed that the endogenous HsSAS-6 coprecipitated with the microtubules, and it mediated interaction with tubulin. Isothermal calorimetry titration and size exclusion chromatography showed that HsSAS-6 C bound to the αβ-tubulin dimer <i>in vitro</i>. The results demonstrate that HsSAS-6 possesses an intrinsic microtubule assembly promoting activity and further implicate that its outer exposed C-terminal tail may play critical roles in microtubule assembly and stabilizing microtubule attachment with the centriolar cartwheel