Molecular determinants for α-tubulin methylation by SETD2

Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. It was recently shown that methylation of tubulin by the histone methyltransferase SETD2 occurs on mitotic spindle microtubules during cell division, with its absence resulting in mitotic defects. However, the catalytic mechanism of methyl addition to tubulin is unclear. We used a truncated version of human wild type SETD2 (tSETD2) containing the catalytic SET and C-terminal Set2–Rpb1–interacting (SRI) domains to investigate the biochemical mechanism of tubulin methylation. We found that recombinant tSETD2 had a higher activity toward tubulin dimers than polymerized microtubules. Using recombinant single-isotype tubulin, we demonstrated that methylation was restricted to lysine 40 of α-tubulin. We then introduced pathogenic mutations into tSETD2 to probe the recognition of histone and tubulin substrates. A mutation in the catalytic domain (R1625C) allowed tSETD2 to bind to tubulin but not methylate it, whereas a mutation in the SRI domain (R2510H) caused loss of both tubulin binding and methylation. Further investigation of the role of the SRI domain in substrate binding found that mutations within this region had differential effects on the ability of tSETD2 to bind to tubulin versus the binding partner RNA polymerase II for methylating histones in vivo, suggesting distinct mechanisms for tubulin and histone methylation by SETD2. Finally, we found that substrate recognition also requires the negatively charged C-terminal tail of α-tubulin. Together, this study provides a framework for understanding how SETD2 serves as a dual methyltransferase for both histone and tubulin methylation.     

TRPV1-tubulin complex: involvement of membrane tubulin in the regulation of chemotherapy-induced peripheral neuropathy

Existence of microtubule cytoskeleton at the membrane and submembranous regions, referred as 'membrane tubulin' has remained controversial for a long time. Since we reported physical and functional interaction of Transient Receptor Potential Vanilloid Sub Type 1 (TRPV1) with microtubules and linked the importance of TRPV1-tubulin complex in the context of chemotherapy-induced peripheral neuropathy, a few more reports have characterized this interaction in in vitro and in in vivo condition. However, the cross-talk between TRPs with microtubule cytoskeleton, and the complex feedback regulations are not well understood. Sequence analysis suggests that other than TRPV1, few TRPs can potentially interact with microtubules. The microtubule interaction with TRPs has evolutionary origin and has a functional significance. Biochemical evidence, Fluorescence Resonance Energy Transfer analysis along with correlation spectroscopy and fluorescence anisotropy measurements have confirmed that TRPV1 interacts with microtubules in live cell and this interaction has regulatory roles. Apart from the transport of TRPs and maintaining the cellular structure, microtubules regulate signaling and functionality of TRPs at the single channel level. Thus, TRPV1-tubulin interaction sets a stage where concept and parameters of 'membrane tubulin' can be tested in more details. In this review, I critically analyze the advancements made in biochemical, pharmacological, behavioral as well as cell-biological observations and summarize the limitations that need to be overcome in the future.     

Interfacing protein lysine acetylation and protein phosphorylation: ?Ancient modifications meet on ancient proteins

Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature.     

微管蛋白酪氨酸连接酶类似物12通过干扰微管蛋白酪氨酸硝基化促进Hep-2细胞生长

硝基化酪氨酸与酪氨酸在结构上相似,它在病理情况下会出现,并在细胞内与微管蛋白结合,从而阻碍微管的正常功能. 硝基化酪氨酸在肿瘤中的作用,目前研究甚少.本文利用头颈鳞癌Hep-2细胞株,研究微管蛋白酪氨酸连接酶类似物12（tubulin tyrosine ligase like 12,TTLL12）和硝基化酪氨酸对头颈鳞癌Hep-2生长的影响,通过Western 印迹试验和MTT试验发现,随着硝基化酪氨酸的浓度升高,细胞内生成的硝基化酪氨酸微管蛋白含量也增高,同时细胞生长受抑制的程度显著增高; 对建立的TTLL12高表达细胞株加入硝基化酪氨酸培养,结果显示,TTLL12高表达细胞株内的硝基化酪氨酸微管蛋白含量明显低于对照组细胞;对照组细胞的生长明显受到抑制,而高表达细胞株的生长无明显改变,两者的细胞生长有显著性差异（P＜0.05）.本研究结果提示,TTLL12可通过阻碍硝基化酪氨酸与微管蛋白的结合,使头颈鳞癌Hep-2细胞逃避硝基化酪氨酸的打击. 对这一调控机制的进一步研究,必将有助于控制肿瘤细胞的生长,为治疗肿瘤寻找到新的治疗靶点.     

Reorganization of mitotic apparatus in the etoposide-treated CHO-K1 cells precedes apoptotic death

In a culture of CHO-K1 cells, etoposide (1 h, 25 μM) has been shown to produce interphase arrest, after which the cells resume mitotic division and, after some time, are submitted to apoptotic death. Accumulation of apoptotic cells in the culture follows a gradual increase in the number of multipolar mitoses. Our findings provide the first evidence for differences in the pattern of immunofluorescent staining of multipolar mitotic spindle microtubules with antibodies to α-tubulin, acetylated α-tubulin, and tyrosinated α-tubulin in mitotic cells dividing in the period preceding apoptosis. Moreover, some parts of the multipolar mitotic spindle can differ by the presence of antigenic determinants accessible to anti-tyrosinated α-tubulin antibodies. These abnormalities of the mitotic apparatus are aggravated immediately before the increase in the number of cells submitted to apoptosis. Our data have also shown that some cells pass through at least two mitotic cycles prior to a sharp increase in the number of apoptotic cells in the cell culture.     

Identification and characterization of a novel neural cell adhesion molecule (NCAM)-associated protein from quail myoblasts: relationship to myotube formation and induction of neurite-like protrusions

Abstract We identified a novel neural cell adhesion molecule (NCAM)-associated protein, myo genesis-related and N CAM- a ssociated p rotein (MYONAP), the expression of which increases during the formation of myotubes in quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells). MYONAP shares homology with PL48 in human cytotrophoblasts and KIAA0386 in human brain. Excess expression of MYONAP in presumptive QM-RSV myoblasts induced long protrusions like neurites in cooperation with microtubules. Suppression of MYONAP by antisense cDNA prevented myotubes from forming in spite of the expression of myogenin, creatine kinase, and myosin, and rendered myoblast membranes resistant to fusion. Yeast two-hybrid screening showed that MYONAP interacted with NCAM specifically. Deletion of the NCAM-associated domain resulted in a loss of the function that induces neurite-like protrusions to form and disturbed the elongation of microtubules. The results suggested that MYONAP influenced the functions of microtubules and was involved in the formation of myotubes via its interaction with NCAM.     

Lipase-catalysed deacetylation of androstane and pregnane derivatives: influence of ring D substitution

A series of acetoxy derivatives of androstane and pregnane was deacetylated in organic solvents by microbial lipases. The best results were obtained with lipase from Candida antarctica (CAL B), Candida rugosa (CRL) and Pseudomonas sp. (PSL). In some derivatives, CAL B and CRL showed a regioselective behaviour towards the removal of the 3β- or 16/16β-acetyl group. The results of the enzymatic deacetylation of pregnanes and androstanes substituted by various groups containing an sp²-hybridised C-atom in ring D could suggest that CAL B activity seems to be conditioned by the occurrence of a polar carbon double bond in this part of the steroid skeleton. Ten new steroid derivatives were obtained through this approach.     

Methyltransferase-inhibition interferes with neuronal differentiation of P19 embryonal carcinoma cells

We have analyzed the importance of substrate methylation by S-adenosylmethionine-dependent methyltransferases for neuronal differentiation of P19 embryonal carcinoma cells. We show that treatment of cells with methyltransferase inhibitor adenosine dialdehyde (AdOx) interferes with neuronal differentiation. Retinoic acid (RA) and AdOx co-treated cells had a decreased number of neurites and a flattened morphology compared with cells differentiated by RA. Also, the amount of neuronal class III tubulin (Tuj1) decreased from 76% to 9.6% with AdOx-treatment. Gene expression levels of wnt-1, brn-2, neuroD, and mash-1 were also down-regulated by AdOx-treatment. But AdOx-treatment did not up-regulate BMP-4 and GFAP genes. Treatment of RA decreased E-cadherin expression during neuronal differentiation. However, in AdOx/RA co-treated cells, E-cadherin expression was restored to the control level. Also, mRNA expression of N-cadherin decreased with AdOx-treatment. Taken together, these data show that methylation reactions might influence the cell-fate decision and neuronal differentiation of P19 cells.     

Virions and the Coat Protein of the Potato Virus X Interact with Microtubules and Induce Tubulin Polymerization In Vitro

A study was made of the in vitro interactions of virions and the coat protein (CP) of the potato virus X (PVX) with microtubules (MT). Both virions and CP cosedimented with taxol-stabilized MT. In the presence of PVX CP, tubulin polymerized to produce structures resistant to chilling. Electron microscopy revealed the aberrant character of the resulting tubulin polymers (protofilaments and their sheets), which differed from MT assembled in the presence of cell MAP2. In contrast, PVX virions induced the assembly of morphologically normal MT sensitive to chilling. Virions were shown to compete with MAP2 for MT binding, suggesting an overlap for the MT sites interacting with MAP2 and with PVX virions. It was assumed that PVX virions interact with MT in vivo and that, consequently, cytoskeleton elements participate in intracellular compartmentalization of the PVX genome.