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1
Alonso, Victoria Lucia, Gabriela Vanina Villanova, Carla Ritagliati, María Cristina Machado Motta, Pamela Cribb, and Esteban Carlos Serra. "Trypanosoma cruzi Bromodomain Factor 3 Binds Acetylated α-Tubulin and Concentrates in the Flagellum during Metacyclogenesis." Eukaryotic Cell 13, no. 6 (April 18, 2014): 822–31. http://dx.doi.org/10.1128/ec.00341-13.
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ABSTRACTBromodomains are highly conserved acetyl-lysine binding domains found mainly in proteins associated with chromatin and nuclear acetyltransferases. TheTrypanosoma cruzigenome encodes at least four bromodomain factors (TcBDFs). We describe here bromodomain factor 3 (TcBDF3), a bromodomain-containing protein localized in the cytoplasm.TcBDF3 cytolocalization was determined, using purified antibodies, by Western blot and immunofluorescence analyses in all life cycle stages ofT. cruzi. In epimastigotes and amastigotes, it was detected in the cytoplasm, the flagellum, and the flagellar pocket, and in trypomastigotes only in the flagellum. Subcellular localization ofTcBDF3 was also determined by digitonin extraction, ultrastructural immunocytochemistry, and expression ofTcBDF3 fused to cyan fluorescent protein (CFP). Tubulin can acquire different posttranslational modifications, which modulate microtubule functions. Acetylated α-tubulin has been found in the axonemes of flagella and cilia, as well as in the subpellicular microtubules of trypanosomatids.TcBDF3 and acetylated α-tubulin partially colocalized in isolated cytoskeletons and flagella fromT. cruziepimastigotes and trypomastigotes. Interaction between the two proteins was confirmed by coimmunoprecipitation and far-Western blot assays with synthetic acetylated α-tubulin peptides and recombinantTcBDF3.
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HARRISON, ALISTAIR, HOWARD STEBBINGS, and JEREMY S. HYAMS. "Different Patterns of α-Tubulin Post-Translational Modification in Ovarian Nutritive Tubes of Two Hemipteran Insects." Journal of Cell Science 100, no. 3 (November 1, 1991): 501–7. http://dx.doi.org/10.1242/jcs.100.3.501.
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Usage of the tyrosinated, detyrosinated and acetylated forms of α-tubulin in ovarian nutritive tube microtubules of the hemipterans Oncopeltus fasciatus and Notonecta glauca glauca was investigated by immunofluorescence microscopy of frozen sections of ovarioles with isotype-specific antibodies. In Oncopeltus, nutritive tubes at all stages of development contained tyrosinated α-tubulin and showed only a weak reaction to antibodies to the detyrosinated and acetylated forms. In Notonecta, tyrosinated α-tubulin was confined to a zone around the periphery of functional nutritive tubes; the body of these tubes, and the older, redundant, nutritive tubes stained strongly for both the detyrosinated and acetylated isotypes. The difference in isotype usage between the two species was confirmed by immunoblotting of 2-D gels of ovariole extracts. The results are consistent with the different time-course of oogenesis, and hence the longevity of the nutritive tube microtubules, in the two insects. A model for the insertion of new microtubules into nutritive tubes as they grow is proposed.
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Dorsch, Schuldt, Remedios, Schinkel, Jong, Michels, Kuster, Brundel, and Velden. "Protein Quality Control Activation and Microtubule Remodeling in Hypertrophic Cardiomyopathy." Cells 8, no. 7 (July 18, 2019): 741. http://dx.doi.org/10.3390/cells8070741.
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Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disorder. It is mainly caused by mutations in genes encoding sarcomere proteins. Mutant forms of these highly abundant proteins likely stress the protein quality control (PQC) system of cardiomyocytes. The PQC system, together with a functional microtubule network, maintains proteostasis. We compared left ventricular (LV) tissue of nine donors (controls) with 38 sarcomere mutation-positive (HCMSMP) and 14 sarcomere mutation-negative (HCMSMN) patients to define HCM and mutation-specific changes in PQC. Mutations in HCMSMP result in poison polypeptides or reduced protein levels (haploinsufficiency, HI). The main findings were 1) several key PQC players were more abundant in HCM compared to controls, 2) after correction for sex and age, stabilizing heat shock protein (HSP)B1, and refolding, HSPD1 and HSPA2 were increased in HCMSMP compared to controls, 3) α-tubulin and acetylated α-tubulin levels were higher in HCM compared to controls, especially in HCMHI, 4) myosin-binding protein-C (cMyBP-C) levels were inversely correlated with α-tubulin, and 5) α-tubulin levels correlated with acetylated α-tubulin and HSPs. Overall, carrying a mutation affects PQC and α-tubulin acetylation. The haploinsufficiency of cMyBP-C may trigger HSPs and α-tubulin acetylation. Our study indicates that proliferation of the microtubular network may represent a novel pathomechanism in cMyBP-C haploinsufficiency-mediated HCM.
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Labisso, Wajana, Ana-Caroline Raulin, Lucky Nwidu, Artur Kocon, Declan Wayne, Amaia Erdozain, Benito Morentin, et al. "The Loss of α- and β-Tubulin Proteins Are a Pathological Hallmark of Chronic Alcohol Consumption and Natural Brain Ageing." Brain Sciences 8, no. 9 (September 11, 2018): 175. http://dx.doi.org/10.3390/brainsci8090175.
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Repetitive excessive alcohol intoxication leads to neuronal damage and brain shrinkage. We examined cytoskeletal protein expression in human post-mortem tissue from Brodmann’s area 9 of the prefrontal cortex (PFC). Brain samples from 44 individuals were divided into equal groups of 11 control, 11 alcoholic, 11 non-alcoholic suicides, and 11 suicide alcoholics matched for age, sex, and post-mortem delay. Tissue from alcoholic cohorts displayed significantly reduced expression of α- and β-tubulins, and increased levels of acetylated α-tubulin. Protein levels of histone deacetylase-6 (HDAC6), and the microtubule-associated proteins MAP-2 and MAP-tau were reduced in alcoholic cohorts, although for MAPs this was not significant. Tubulin gene expressions increased in alcoholic cohorts but not significantly. Brains from rats administered alcohol for 4 weeks also displayed significantly reduced tubulin protein levels and increased α-tubulin acetylation. PFC tissue from control subjects had reduced tubulin protein expression that was most notable from the sixth to the eighth decade of life. Collectively, loss of neuronal tubulin proteins are a hallmark of both chronic alcohol consumption and natural brain ageing. The reduction of cytosolic tubulin proteins could contribute to the brain volumetric losses reported for alcoholic patients and the elderly.
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Luo, Jinping, Jose Rafael Rodriguez-Sosa, Lin Tang, Alla Bondareva, Susan Megee, and Ina Dobrinski. "Expression pattern of acetylated α-tubulin in porcine spermatogonia." Molecular Reproduction and Development 77, no. 4 (December 30, 2009): 348–52. http://dx.doi.org/10.1002/mrd.21153.
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Othman, Ahmad, Marcus Winogradzki, Shreya Patel, Waddell Holmes, Alan Blank, and Jitesh Pratap. "The Role of Runx2 in Microtubule Acetylation in Bone Metastatic Breast Cancer Cells." Cancers 14, no. 14 (July 15, 2022): 3436. http://dx.doi.org/10.3390/cancers14143436.
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Bone metastasis of breast cancer results in severe bone loss, fractures, and death. Crosstalk between breast cancer cells and bone resident cells promotes osteoclast activity and the release of growth factors from the bone matrix resulting in aggressive tumor growth and bone loss. We and others have shown that Runt-related transcription factor-2 (Runx2) promotes metastatic tumor growth-associated bone loss. Breast cancer cells also induce autophagy to survive metabolic stress at the metastatic site. Recently, we reported a Runx2-dependent increase in autophagy. In this study, to examine the underlying mechanisms of metastasis and tumor resistance to stress, we used a bone metastatic isogenic variant of breast cancer MDA-MB-231 cells isolated from a xenograft tumor mouse model of metastasis. Our results with immunofluorescence and biochemical approaches revealed that Runx2 promotes microtubule (MT) stability to facilitate autophagy. Stable MTs are critical for autophagosome trafficking and display increased acetylation at Lysine 40 of α-tubulin. Runx2 silencing decreases acetylated α-tubulin levels. The expression levels of HDAC6 and αTAT1, which serve to regulate the acetylation of α-tubulin, were not altered with Runx2 silencing. We found that HDAC6 interaction with α-tubulin is inhibited by Runt-related factor-2 (Runx2). We show that the expression of wild-type Runx2 can restore the acetylated polymer of MTs in Runx2 knockdown cells, while the C-terminal deletion mutant fails to rescue the polymer of MTs. Importantly, cellular stress, such as glucose starvation also increases the acetylation of α-tubulin. We found that the loss of Runx2 increases the sensitivity of breast cancer cells to MT-targeting agents. Overall, our results indicate a novel regulatory mechanism of microtubule acetylation and suggest that Runx2 and acetylated microtubules may serve as therapeutic targets for bone metastatic tumors.
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Denduluri, N., J. J. Lee, J. M. Walshe, S. X. Yang, U. Vatas, C. K. Chow, S. M. Steinberg, M. C. Cox, J. A. Low, and S. M. Swain. "Phase II clinical trial of ixabepilone in metastatic breast cancer (MBC) patients previously untreated with taxanes." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 651. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.651.
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651 Background: Ixabepilone, an epothilone B analog, stabilizes microtubules by binding to tubulin. The response rate (RR) in taxane-pretreated patients at our institution was 22%. Methods: Patients (pts) were eligible if they had MBC previously untreated with taxanes and measurable disease by RECIST criteria. Ixabepilone was given at 6mg/m2/d intravenously days 1–5 every 3 weeks until unacceptable toxicity or disease progression. Primary objectives included RR and toxicity. Pts underwent pre and/or post treatment tumor biopsies for correlative studies. Acetylated α-tubulin, Tau-1, and p53 were stained with anti-acetylated α-tubulin, anti-Tau-1, and anti-p53 antibodies in samples from 13 pts. Staining was scored quantitatively using the Automated Cellular Imaging System. Results: Twenty-three pts received 197 cycles (C). Median of 7C (range 2–22) per pt were administered. Median age was 55 (range 22–79). Seven pts received 1 prior metastatic chemotherapy regimen. Ten of 23 or 43% (exact 95% confidence interval: 23.2% to 65.5%) pts had partial responses (PR), 9 (39%) stable disease (SD) (2 unconfirmed PRs), and 4 (17%) progressive disease (PD). Median time to progression was 5.3 months; median duration of response was 5.4 months from date of best response. Four pts required dose reductions for neutropenia, neuropathy or fatigue. Grade 3/4 toxicities included neutropenia (22%), fatigue (13%), anorexia (9%), infection without neutropenia (9%), motor neuropathy (4%), and muscle weakness (4%). No grade 3/4 sensory neuropathy was seen, but 35% and 13% of pts had grades 1 and 2 neuropathy respectively. Median acetylated α-tubulin at baseline was 0.2 in responders and 17.6 in non-responders (p=0.069). There were no differences in response according to Tau-1 or p53 expression at baseline. Conclusion: Ixabepilone is an effective treatment for MBC with a 43% RR in 23 pts previously untreated with taxanes. There was minimal hematologic toxicity and no grade 3 sensory neuropathy. The use of baseline level of acetylated α-tubulin to predict response may warrant further study. No significant financial relationships to disclose.
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Yang, Wulin, Xiangxiang Guo, Shermaine Thein, Feng Xu, Shigeki Sugii, Peter W. Baas, George K. Radda, and Weiping Han. "Regulation of adipogenesis by cytoskeleton remodelling is facilitated by acetyltransferase MEC-17-dependent acetylation of α-tubulin." Biochemical Journal 449, no. 3 (January 9, 2013): 605–12. http://dx.doi.org/10.1042/bj20121121.
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Cytoskeleton remodelling is a prerequisite step for the morphological transition from preadipocytes to mature adipocytes. Although microtubules play a pivotal role in organizing cellular structure, regulation of microtubule dynamics during adipogenesis remains unclear. In the present paper we show that acetylation of α-tubulin is up-regulated during adipogenesis, and adipocyte development is dependent on α-tubulin acetylation, as expression of an acetylation-resistant α-tubulin mutant significantly inhibits adipogenesis. Moreover, acetylation of α-tubulin is under the control of the acetyltransferase MEC-17 and deacetylases SIRT2 (Sirtuin 2) and HDAC6 (histone deacetylase 6). Adipocyte development is inhibited in MEC-17-knockdown cells, but enhanced in MEC-17-overexpressing cells. Finally, we show that katanin, a microtubule-severing protein with enhanced activity on acetylated α-tubulin, is actively involved in adipogenesis. We propose that co-ordinated up-regulation of α-tubulin acetylation initiates cytoskeleton remodelling by promoting α-tubulin severing by katanin which, in turn, allows expansion of lipid droplets and accelerates the morphological transition toward mature adipocytes.
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Adamakis, Ioannis-Dimosthenis S., Emmanuel Panteris, and Eleftherios P. Eleftheriou. "Tubulin Acetylation Mediates Bisphenol A Effects on the Microtubule Arrays of Allium cepa and Triticum turgidum." Biomolecules 9, no. 5 (May 11, 2019): 185. http://dx.doi.org/10.3390/biom9050185.
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The effects of bisphenol A (BPA), a prevalent endocrine disruptor, on both interphase and mitotic microtubule array organization was examined by immunofluorescence microscopy in meristematic root cells of Triticum turgidum (durum wheat) and Allium cepa (onion). In interphase cells of A. cepa, BPA treatment resulted in substitution of cortical microtubules by annular/spiral tubulin structures, while in T. turgidum BPA induced cortical microtubule fragmentation. Immunolocalization of acetylated α-tubulin revealed that cortical microtubules of T. turgidum were highly acetylated, unlike those of A. cepa. In addition, elevation of tubulin acetylation by trichostatin A in A. cepa resulted in microtubule disruption similar to that observed in T. turgidum. BPA also disrupted all mitotic microtubule arrays in both species. It is also worth noting that mitotic microtubule arrays were acetylated in both plants. As assessed by BPA removal, its effects are reversible. Furthermore, taxol-stabilized microtubules were resistant to BPA, while recovery from oryzalin treatment in BPA solution resulted in the formation of ring-like tubulin conformations. Overall, these findings indicate the following: (1) BPA affects plant mitosis/cytokinesis by disrupting microtubule organization. (2) Microtubule disassembly probably results from impairment of free tubulin subunit polymerization. (3) The differences in cortical microtubule responses to BPA among the species studied are correlated to the degree of tubulin acetylation.
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Carbajal, Agustín, María E. Chesta, C. Gastón Bisig, and Carlos A. Arce. "A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype." Biochemical Journal 449, no. 3 (January 9, 2013): 643–48. http://dx.doi.org/10.1042/bj20121439.
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Tubulin can be acetylated/deacetylated on Lys40 of the α-subunit. Studies of the post-translational acetylation/deacetylation of tubulin using biochemical techniques require tubulin preparations that are enriched in AcTubulin (acetylated tubulin) and (for comparison) preparations lacking AcTubulin. Assembly–disassembly cycling of microtubules gives tubulin preparations that contain little or no AcTubulin. In the present study we demonstrated that this result is owing to the presence of high deacetylating activity in the extracts. This deacetylating activity in rat brain homogenates was inhibited by TSA (Trichostatin A) and tubacin, but not by nicotinamide, indicating that HDAC6 (histone deacetylase 6) is involved. TSA showed no effect on microtubule polymerization or depolymerization. We utilized these properties of TSA to prevent deacetylation during the assembly–disassembly procedure. The effective inhibitory concentration of TSA was 3 μM in the homogenate and 1 μM in the subsequent cycling steps. By comparison with immunopurified AcTubulin, we estimated that ~64% of the tubulin molecules in the three cycled preparations were acetylated. The protein profiles of these tubulin preparations, as assessed by SDS/PAGE and Coomassie Blue staining, were identical to that of a preparation completely lacking AcTubulin obtained by assembly–disassembly cycles in the absence of TSA. The tyrosination state and in vitro assembly–disassembly kinetics were the same regardless of the degree of acetylation.
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Eshun-Wilson, Lisa, Rui Zhang, Didier Portran, Maxence V. Nachury, Daniel B. Toso, Thomas Löhr, Michele Vendruscolo, Massimiliano Bonomi, James S. Fraser, and Eva Nogales. "Effects of α-tubulin acetylation on microtubule structure and stability." Proceedings of the National Academy of Sciences 116, no. 21 (May 9, 2019): 10366–71. http://dx.doi.org/10.1073/pnas.1900441116.
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Acetylation of K40 in α-tubulin is the sole posttranslational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule stabilization is correlative or causative. We have obtained high-resolution cryo-electron microscopy (cryo-EM) reconstructions of pure samples of αTAT1-acetylated and SIRT2-deacetylated microtubules to visualize the structural consequences of this modification and reveal its potential for influencing the larger assembly properties of microtubules. We modeled the conformational ensembles of the unmodified and acetylated states by using the experimental cryo-EM density as a structural restraint in molecular dynamics simulations. We found that acetylation alters the conformational landscape of the flexible loop that contains αK40. Modification of αK40 reduces the disorder of the loop and restricts the states that it samples. We propose that the change in conformational sampling that we describe, at a location very close to the lateral contacts site, is likely to affect microtubule stability and function.
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Luo, J., S. Megee, and I. Dobrinski. "288 THE EXPRESSION PATTERN OF ACETYLATED ALPHA-TUBULIN IS CONSERVED IN PORCINE AND MURINE SPERMATOGONIAL STEM CELLS." Reproduction, Fertility and Development 20, no. 1 (2008): 223. http://dx.doi.org/10.1071/rdv20n1ab288.
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During mammalian spermatogenesis, spermatogonial stem cells (SSCs) reside in the stem cell niche on the basement membrane where they undergo self-renewing divisions. Differentiating daughter cells are located progressively more toward the tubular lumen where they ultimately form spermatozoa. The mechanisms responsible for maintenance of SSCs at the basement membrane are unclear. Microtubules consisting of α/β-tubulin heterodimers are associated with many cellular functions. Reversible acetylation of α-tubulin at Lys40 has been implicated in regulating microtubule stability and function. Acetylation of α-tubulin is abundant in stable microtubules but absent from dynamic cellular structures. Deacetylation of α-tubulin is controlled by histone deacetylase 6 which is predominantly expressed in mouse testis. Here, we tested the hypothesis that differential acetylation of α-tubulin might be involved in maintenance of SSCs. Immunohistochemistry for acetylated α-tubulin (Ac-α-Tu) and the spermatogonia specific proteins PGP 9.5, DAZL, and PLZF were used to characterize the expression pattern of Ac-α-Tu in porcine and murine germ cells at different stages of testis development. In immature boar testes, Ac-α-Tu was present exclusively in gonocytes but not in other testicular cells at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating to the basement membrane of the seminiferous tubules, and Ac-α-Tu appeared to be polarized toward the basement membrane. In immature mouse testes, Ac-α-Tu was present in germ cells and Sertoli cells at 6 days of age, whereas at 2 weeks of age, Ac-α-Tu expression was stronger in spermatogonia co-expressing PGP 9.5 and in spermatocytes than in Sertoli cells or PGP 9.5-negative spermatogonia. In adult boar and mouse testes, Ac-α-Tu was detected in a few single or paired spermatogonia expressing PGP 9.5 localized on the basement membrane as well as in spermatocytes, spermatids, and spermatozoa. Spermatogonia with high levels of Ac-α-Tu expressed PLZF but did not express DAZL, suggesting that only undifferentiated spermatogonia maintain a high level of Ac-α-Tu. When seminiferous tubules from 1-week-old and adult boar testes were maintained in vitro for 1–2 days, high levels of Ac-α-Tu were detected in single or paired round spermatogonia with a large nucleus, compared to low levels in elongated paired and aligned spermatogonia. The unique expression pattern of Ac-α-Tu in undifferentiated germ cells during postnatal development appears to be conserved in mammalian testes. Since Ac-α-Tu is a component of long-lived stable microtubules and reducing acetylation of α-tubulin enhances cell motility, these results suggest that stabilization of microtubules might contribute to the maintenance of spermatogonial stem cells. This work was supported by 1R01 RR 17359-05.
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Pongrakhananon, Varisa, Hiroko Saito, Sylvain Hiver, Takaya Abe, Go Shioi, Wenxiang Meng, and Masatoshi Takeichi. "CAMSAP3 maintains neuronal polarity through regulation of microtubule stability." Proceedings of the National Academy of Sciences 115, no. 39 (September 6, 2018): 9750–55. http://dx.doi.org/10.1073/pnas.1803875115.
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The molecular mechanisms that guide each neuron to become polarized, forming a single axon and multiple dendrites, remain unknown. Here we show that CAMSAP3 (calmodulin-regulated spectrin-associated protein 3), a protein that regulates the minus-end dynamics of microtubules, plays a key role in maintaining neuronal polarity. In mouse hippocampal neurons, CAMSAP3 was enriched in axons. Although axonal microtubules were generally acetylated, CAMSAP3 was preferentially localized along a less-acetylated fraction of the microtubules. CAMSAP3-mutated neurons often exhibited supernumerary axons, along with an increased number of neurites having nocodazole-resistant/acetylated microtubules compared with wild-type neurons. Analysis using cell lines showed that CAMSAP3 depletion promoted tubulin acetylation, and conversely, mild overexpression of CAMSAP3 inhibited it, suggesting that CAMSAP3 works to retain nonacetylated microtubules. In contrast, CAMSAP2, a protein related to CAMSAP3, was detected along all neurites, and its loss did not affect neuronal polarity, nor did it cause increased tubulin acetylation. Depletion of α-tubulin acetyltransferase-1 (αTAT1), the key enzyme for tubulin acetylation, abolished CAMSAP3 loss-dependent multiple-axon formation. These observations suggest that CAMSAP3 sustains a nonacetylated pool of microtubules in axons, interfering with the action of αTAT1, and this process is important to maintain neuronal polarity.
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Chen, Ling, Qiguo Hu, Huaicun Liu, Yan Zhao, Sun-On Chan, and Jun Wang. "Nogo-A Induced Polymerization of Microtubule Is Involved in the Inflammatory Heat Hyperalgesia in Rat Dorsal Root Ganglion Neurons." International Journal of Molecular Sciences 22, no. 19 (September 26, 2021): 10360. http://dx.doi.org/10.3390/ijms221910360.
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The microtubule, a major constituent of cytoskeletons, was shown to bind and interact with transient receptor potential vanilloid subfamily member 1 (TRPV1), and serves a pivotal role to produce thermal hyperalgesia in inflammatory pain. Nogo-A is a modulator of microtubule assembly and plays a key role in maintaining the function of TRPV1 in inflammatory heat pain. However, whether the microtubule dynamics modulated by Nogo-A in dorsal root ganglion (DRG) neurons participate in the inflammatory pain is not elucidated. Here we reported that the polymerization of microtubules in the DRG neurons, as indicated by the acetylated α-tubulin, tubulin polymerization-promoting protein 3 (TPPP3), and microtubule numbers, was significantly elevated in the complete Freund’s adjuvant (CFA) induced inflammatory pain. Consistent with our previous results, knock-out (KO) of Nogo-A protein significantly attenuated the heat hyperalgesia 72 h after CFA injection and decreased the microtubule polymerization via up-regulation of phosphorylation of collapsin response mediator protein 2 (CRMP2) in DRG. The colocalization of acetylated α-tubulin and TRPV1 in DRG neurons was also reduced dramatically in Nogo-A KO rats under inflammatory pain. Moreover, the down-regulation of TRPV1 in DRG of Nogo-A KO rats after injection of CFA was reversed by intrathecal injection of paclitaxel, a microtubule stabilizer. Furthermore, intrathecal injection of nocodazole (a microtubule disruptor) attenuated significantly the CFA-induced inflammatory heat hyperalgesia and the mechanical pain in a rat model of spared nerve injury (SNI). In these SNI cases, the Nogo-A and acetylated α-tubulin in DRG were also significantly up-regulated. We conclude that the polymerization of microtubules promoted by Nogo-A in DRG contributes to the development of inflammatory heat hyperalgesia mediated by TRPV1.
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Schatten, Gerald, Calvin Simerly, David J. Asai, Ester Szöke, Peter Cooke, and Heide Schatten. "Acetylated α-tubulin in microtubules during mouse fertilization and early development." Developmental Biology 130, no. 1 (November 1988): 74–86. http://dx.doi.org/10.1016/0012-1606(88)90415-0.
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Bhagwat, Shweta, Veena Dalvi, Darshan Chandrasekhar, Tinsu Matthew, Kshitish Acharya, Rahul Gajbhiye, Vijay Kulkarni, Shobha Sonawane, Manish Ghosalkar, and Priyanka Parte. "Acetylated α-tubulin is reduced in individuals with poor sperm motility." Fertility and Sterility 101, no. 1 (January 2014): 95–104. http://dx.doi.org/10.1016/j.fertnstert.2013.09.016.
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Chiticariu, Elena, Alexandre Regamey, Marcel Huber, and Daniel Hohl. "CENPV Is a CYLD-Interacting Molecule Regulating Ciliary Acetylated α-Tubulin." Journal of Investigative Dermatology 140, no. 1 (January 2020): 66–74. http://dx.doi.org/10.1016/j.jid.2019.04.028.
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Liu, Pengfei, Shengwei Zhang, Jingyi Ma, Dongning Jin, Yali Qin, and Mingzhou Chen. "Vimentin inhibits α-tubulin acetylation via enhancing α-TAT1 degradation to suppress the replication of human parainfluenza virus type 3." PLOS Pathogens 18, no. 9 (September 15, 2022): e1010856. http://dx.doi.org/10.1371/journal.ppat.1010856.
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We previously found that, among human parainfluenza virus type 3 (HPIV3) proteins, the interaction of nucleoprotein (N) and phosphoprotein (P) provides the minimal requirement for the formation of cytoplasmic inclusion bodies (IBs), which are sites of RNA synthesis, and that acetylated α-tubulin enhances IB fusion and viral replication. In this study, using immunoprecipitation and mass spectrometry assays, we determined that vimentin (VIM) specifically interacted with the N–P complex of HPIV3, and that the head domain of VIM was responsible for this interaction, contributing to the inhibition of IB fusion and viral replication. Furthermore, we found that VIM promoted the degradation of α-tubulin acetyltransferase 1 (α-TAT1), through its head region, thereby inhibiting the acetylation of α-tubulin, IB fusion, and viral replication. In addition, we identified a 20-amino-acid peptide derived from the head region of VIM that participated in the interaction with the N–P complex and inhibited viral replication. Our findings suggest that VIM inhibits the formation of HPIV3 IBs by downregulating α-tubulin acetylation via enhancing the degradation of α-TAT1. Our work sheds light on a new mechanism by which VIM suppresses HPIV3 replication.
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Valenzuela-Fernández, Agustín, Susana Álvarez, Mónica Gordon-Alonso, Marta Barrero, Ángeles Ursa, J. Román Cabrero, Gerónimo Fernández, et al. "Histone Deacetylase 6 Regulates Human Immunodeficiency Virus Type 1 Infection." Molecular Biology of the Cell 16, no. 11 (November 2005): 5445–54. http://dx.doi.org/10.1091/mbc.e05-04-0354.
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Efficient human immunodeficiency virus (HIV)-1 infection depends on multiple interactions between the viral gp41/gp120 envelope (Env) proteins and cell surface receptors. However, cytoskeleton-associated proteins that modify membrane dynamics may also regulate the formation of the HIV-mediated fusion pore and hence viral infection. Because the effects of HDAC6-tubulin deacetylase on cortical α-tubulin regulate cell migration and immune synapse organization, we explored the possible role of HDAC6 in HIV-1-envelope-mediated cell fusion and infection. The binding of the gp120 protein to CD4+-permissive cells increased the level of acetylated α-tubulin in a CD4-dependent manner. Furthermore, overexpression of active HDAC6 inhibited the acetylation of α-tubulin, and remarkably, prevented HIV-1 envelope-dependent cell fusion and infection without affecting the expression and codistribution of HIV-1 receptors. In contrast, knockdown of HDAC6 expression or inhibition of its tubulin deacetylase activity strongly enhanced HIV-1 infection and syncytia formation. These results demonstrate that HDAC6 plays a significant role in regulating HIV-1 infection and Env-mediated syncytia formation.
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Aguilar, Andrea, Lars Becker, Thomas Tedeschi, Stefan Heller, Carlo Iomini, and Maxence V. Nachury. "α-Tubulin K40 acetylation is required for contact inhibition of proliferation and cell–substrate adhesion." Molecular Biology of the Cell 25, no. 12 (June 15, 2014): 1854–66. http://dx.doi.org/10.1091/mbc.e13-10-0609.
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Acetylation of α-tubulin on lysine 40 marks long-lived microtubules in structures such as axons and cilia, and yet the physiological role of α-tubulin K40 acetylation is elusive. Although genetic ablation of the α-tubulin K40 acetyltransferase αTat1 in mice did not lead to detectable phenotypes in the developing animals, contact inhibition of proliferation and cell–substrate adhesion were significantly compromised in cultured αTat1−/− fibroblasts. First, αTat1−/− fibroblasts kept proliferating beyond the confluent monolayer stage. Congruently, αTat1−/− cells failed to activate Hippo signaling in response to increased cell density, and the microtubule association of the Hippo regulator Merlin was disrupted. Second, αTat1−/− cells contained very few focal adhesions, and their ability to adhere to growth surfaces was greatly impaired. Whereas the catalytic activity of αTAT1 was dispensable for monolayer formation, it was necessary for cell adhesion and restrained cell proliferation and activation of the Hippo pathway at elevated cell density. Because α-tubulin K40 acetylation is largely eliminated by deletion of αTAT1, we propose that acetylated microtubules regulate contact inhibition of proliferation through the Hippo pathway.
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Marettová, E., and M. Maretta. "Localisation of S100 Protein and Acetylated Tubulin in Sheep Pancreas." Folia Veterinaria 65, no. 2 (June 1, 2021): 9–16. http://dx.doi.org/10.2478/fv-2021-0012.
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Abstract The pancreas plays a critical role in the control of nutritional homeostasis. It consists of two major parts, the exocrine pancreas, and the endocrine pancreas. In the present study S100 protein and acetylated α-tubulin were used to identify positive structures in both the exocrine and endocrine part of the ovine pancreas. In the exocrine part of the pancreas, a positive reaction to S100 protein was confined to centroacinar cells, intercalated, and intralobular ducts cells. In addition, the S100 protein was localized in the Schwann cells of nerve fibres. On the pancreatic islets, the S100 protein has been observed in Schwann cells of nerve axons, where they form a fine envelope that invests the islet surface. Inside the pancreatic islets, the Schwann cells positive for S100 protein envelope the endocrine cells of the islets. The difference in positivity of the S100 protein was found in relation to the endocrine cells. The relationship between endocrine cell positivity and positive exocrine duct cells was discussed. Acetylated α-tubulin (AT) was restricted to axons of the nerve fibres and was located within the connective tissue accompanying intralobular and inter-lobular ducts, and between secretory acini in close contact with secretory cells.
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Misawa, Takuma, Tatsuya Saitoh, Tatsuya Kozaki, Sehoon Park, Michihiro Takahama, and Shizuo Akira. "Resveratrol inhibits the acetylated α-tubulin-mediated assembly of the NLRP3-inflammasome." International Immunology 27, no. 9 (April 7, 2015): 425–34. http://dx.doi.org/10.1093/intimm/dxv018.
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Aslan, Joseph E., Kevin G. Phillips, Laura D. Healy, Asako Itakura, Jiaqing Pang, and Owen J. T. McCarty. "Histone deacetylase 6-mediated deacetylation of α-tubulin coordinates cytoskeletal and signaling events during platelet activation." American Journal of Physiology-Cell Physiology 305, no. 12 (December 15, 2013): C1230—C1239. http://dx.doi.org/10.1152/ajpcell.00053.2013.
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The tubulin cytoskeleton plays a key role in maintaining the characteristic quiescent discoid shape of resting platelets. Upon activation, platelets undergo a dramatic change in shape; however, little is known of how the microtubule system contributes to regulating platelet shape and function. Here we investigated the role of the covalent modification of α-tubulin by acetylation in the regulation of platelet physiology during activation. Superresolution microscopy analysis of the platelet tubulin cytoskeleton showed that the marginal band together with an interconnected web of finer tubulin structures collapsed upon platelet activation with the glycoprotein VI (GPVI)-agonist collagen-related peptide (CRP). Western blot analysis revealed that α-tubulin was acetylated in resting platelets and deacetylated during platelet activation. Tubacin, a specific inhibitor of the tubulin deacetylase HDAC6, prevented tubulin deacetylation upon platelet activation with CRP. Inhibition of HDAC6 upregulated tubulin acetylation and disrupted the organization of the platelet microtubule marginal band without significantly affecting platelet volume changes in response to CRP stimulation. HDAC6 inhibitors also inhibited platelet aggregation in response to CRP and blocked platelet signaling events upstream of platelet Rho GTPase activation. Together, these findings support a role for acetylation signaling in controlling the resting structure of the platelet tubulin marginal band as well as in the coordination of signaling systems that drive platelet cytoskeletal changes and aggregation.
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Rahimi, Amir Mohammad, Mingfang Cai, Irem Kılıҫ, Zahra Basir Kazerouni, Constanza Tapia Contreras, and Sigrid Hoyer-Fender. "Expression of α-Tubulin Acetyltransferase 1 and Tubulin Acetylation as Selective Forces in Cell Competition." Cells 10, no. 2 (February 14, 2021): 390. http://dx.doi.org/10.3390/cells10020390.
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The wound healing response of fibroblasts critically depends on the primary cilium, a sensory organelle protruding into the environment and comprising a stable axonemal structure. A characteristic marker for primary cilia is acetylation of axonemal tubulin. Although formation of primary cilia is under cell cycle control, the environmental cues affecting ciliation are not fully understood. Our purpose was, therefore, to study the impact of culture conditions on cilia formation in NIH3T3 fibroblasts. We quantified ciliation in different NIH3T3 sub-cell lines and culture conditions by immunodetection of primary cilia and counting. Quantitative Western blotting, qRT-PCR, and proliferation assays completed our investigation. We observed large differences between NIH3T3 sub-cell lines in their ability to generate acetylated primary cilia that correlated with cytoplasmic tubulin acetylation. We found no increased activity of the major tubulin deacetylase, HDAC6, but instead reduced expression of the α-tubulin acetyltransferase 1 (Atat1) as being causative. Our observations demonstrate that cells with reduced expression of Atat1 and tubulin acetylation proliferate faster, eventually displacing all other cells in the population. Expression of Atat1 and tubulin acetylation are therefore selective forces in cell competition.
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Shimazu, Tadahiro, Sueharu Horinouchi, and Minoru Yoshida. "Multiple Histone Deacetylases and the CREB-binding Protein Regulate Pre-mRNA 3′-End Processing." Journal of Biological Chemistry 282, no. 7 (December 17, 2006): 4470–78. http://dx.doi.org/10.1074/jbc.m609745200.
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Trichostatin A (TSA), a specific inhibitor of histone deacetylases (HDACs), induces acetylation of various non-histone proteins such as p53 and α-tubulin. We purified several acetylated proteins by the affinity to an anti-acetylated lysine (AcLys) antibody from cells treated with TSA and identified them by mass spectrometry. Here we report on acetylation of CFIm25, a component of mammalian cleavage factor Im (CF Im), and poly(A) polymerase (PAP), a polyadenylating enzyme for the pre-mRNA 3′-end. The residues acetylated in these proteins were mapped onto the regions required for interaction with each other. Whereas CBP acetylated these proteins, HDAC1, HDAC3, HDAC10, SIRT1, and SIRT2 were involved in in vivo deacetylation. Acetylation of the CFIm25 occurred depending on the cleavage factor complex formation. Importantly, the interaction between PAP and CF Im complex was decreased by acetylation. We also demonstrated that acetylation of PAP inhibited the nuclear localization of PAP by inhibiting the binding to the importin α/β complex. These results suggest that CBP and HDACs regulate the 3′-end processing machinery and modulate the localization of PAP through the acetylation and deacetylation cycle.
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Tomas-Roig, Jordi, Shyam Ramasamy, Diana Zbarsky, Ursula Havemann-Reinecke, and Sigrid Hoyer-Fender. "Psychosocial stress and cannabinoid drugs affect acetylation of α-tubulin (K40) and gene expression in the prefrontal cortex of adult mice." PLOS ONE 17, no. 9 (September 21, 2022): e0274352. http://dx.doi.org/10.1371/journal.pone.0274352.
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The dynamics of neuronal microtubules are essential for brain plasticity. Vesicular transport and synaptic transmission, additionally, requires acetylation of α-tubulin, and aberrant tubulin acetylation and neurobiological deficits are associated. Prolonged exposure to a stressor or consumption of drugs of abuse, like marihuana, lead to neurological changes and psychotic disorders. Here, we studied the effect of psychosocial stress and the administration of cannabinoid receptor type 1 drugs on α-tubulin acetylation in different brain regions of mice. We found significantly decreased tubulin acetylation in the prefrontal cortex in stressed mice. The impact of cannabinoid drugs on stress-induced microtubule disturbance was investigated by administration of the cannabinoid receptor agonist WIN55,212–2 and/or antagonist rimonabant. In both, control and stressed mice, the administration of WIN55,212–2 slightly increased the tubulin acetylation in the prefrontal cortex whereas administration of rimonabant acted antagonistically indicating a cannabinoid receptor type 1 mediated effect. The analysis of gene expression in the prefrontal cortex showed a consistent expression of ApoE attributable to either psychosocial stress or administration of the cannabinoid agonist. Additionally, ApoE expression inversely correlated with acetylated tubulin levels when comparing controls and stressed mice treated with WIN55,212–2 whereas rimonabant treatment showed the opposite.
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Denduluri, Neelima, Jennifer A. Low, James J. Lee, Arlene W. Berman, Janice M. Walshe, Ujala Vatas, Catherine K. Chow, Seth M. Steinberg, Sherry X. Yang, and Sandra M. Swain. "Phase II Trial of Ixabepilone, an Epothilone B Analog, in Patients With Metastatic Breast Cancer Previously Untreated With Taxanes." Journal of Clinical Oncology 25, no. 23 (August 10, 2007): 3421–27. http://dx.doi.org/10.1200/jco.2006.10.0784.
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Purpose Ixabepilone is an epothilone B analog that binds to microtubules and results in microtubule stabilization and mitotic arrest. Ixabepilone was evaluated for efficacy and safety in a phase II clinical trial for women with metastatic breast cancer. Patients and Methods Patients were eligible if they had not previously received treatment with a taxane and had measurable metastatic breast cancer. Ixabepilone was administered at 6 mg/m2/d intravenously days 1 through 5 every 3 weeks until unacceptable toxicity or disease progression. Patients underwent pretreatment and post-treatment tumor biopsies, and tissues were analyzed for acetylated α-tubulin, tau-1, and p53 expression when possible. Results Twenty-three patients received 210 cycles with a median of eight cycles (range, two to 22 cycles) per patient. Thirteen patients (57%; exact 95% CI, 34.5% to 76.8%) had partial responses, six patients (26%) had stable disease, and four patients (17%) had progressive disease. Median time to progression and duration of response were 5.5 and 5.6 months, respectively. Four patients required dose reductions for neutropenia, neuropathy, or fatigue. Grade 3 or 4 toxicities included neutropenia (22%), fatigue (13%), anorexia (9%), and motor neuropathy (4%). Thirty-nine percent of patients experienced grade 1, 13% experienced grade 2, and none experienced grade 3/4 sensory neuropathy. The six patients with paired biopsies all had increases in tumor α-tubulin acetylation after treatment. Baseline or cycle 2 acetylated α-tubulin, tau-1, or p53 expression did not correlate with clinical response. Conclusion Women with metastatic breast cancer previously untreated with taxanes have a meaningful durable response to single-agent ixabepilone therapy. Minimal hematologic toxicity and no grade 3 sensory neuropathy were noted.
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Santo, Loredana, Teru Hideshima, Andrew L. Kung, Jen-Chieh Tseng, David Tamang, Min Yang, Matthew Jarpe, et al. "Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma." Blood 119, no. 11 (March 15, 2012): 2579–89. http://dx.doi.org/10.1182/blood-2011-10-387365.
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Abstract Histone deacetylase (HDAC) enzymatic activity has been linked to the transcription of DNA in cancers including multiple myeloma (MM). Therefore, HDAC inhibitors used alone and in combination are being actively studied as novel therapies in MM. In the present study, we investigated the preclinical activity of ACY-1215, an HDAC6-selective inhibitor, alone and in combination with bortezomib in MM. Low doses of ACY-1215 combined with bortezomib triggered synergistic anti-MM activity, resulting in protracted endoplasmic reticulum stress and apoptosis via activation of caspase-3, caspase-8, and caspase-9 and poly (ADP) ribosome polymerase. In vivo, the anti-MM activity of ACY-1215 in combination with bortezomib was confirmed using 2 different xenograft SCID mouse models: human MM injected subcutaneously (the plasmacytoma model) and luciferase-expressing human MM injected intravenously (the disseminated MM model). Tumor growth was significantly delayed and overall survival was significantly prolonged in animals treated with the combination therapy. Pharmacokinetic data showed peak plasma levels of ACY-1215 at 4 hours after treatment coincident with an increase in acetylated α-tubulin, a marker of HDAC6 inhibition, by immunohistochemistry and Western blot analysis. These studies provide preclinical rationale for acetylated α-tubulin use as a pharmacodynamic biomarker in future clinical trials.
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Tonami, Kazuo, Yukiko Kurihara, Hiroyuki Aburatani, Yasunobu Uchijima, Tomoichiro Asano, and Hiroki Kurihara. "Calpain 6 Is Involved in Microtubule Stabilization and Cytoskeletal Organization." Molecular and Cellular Biology 27, no. 7 (April 1, 2007): 2548–61. http://dx.doi.org/10.1128/mcb.00992-06.
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ABSTRACT The calpains are a family of Ca2+-dependent cysteine proteases implicated in various biological processes. In this family, calpain 6 (Capn6) is unique in that it lacks the active-site cysteine residues requisite for protease activity. During the search for genes downstream of the endothelin 1 (ET-1) signaling in pharyngeal-arch development, we identified Capn6. After confirming that the expression of Capn6 in pharyngeal arches is downregulated in ET-1-null embryos by in situ hybridization, we investigated its function. In Capn6-transfected cells, cytokinesis was retarded and was often aborted to yield multinucleated cells. Capn6 overexpression also caused the formation of microtubule bundles rich in acetylated α-tubulin and resistant to the depolymerizing activity of nocodazole. Green fluorescent protein-Capn6 overexpression, immunostaining for endogenous Capn6, and biochemical analysis demonstrated interaction between Capn6 and microtubules, which appeared to be mainly mediated by domain III. Furthermore, RNA interference-mediated Capn6 inactivation caused microtubule instability with a loss of acetylated α-tubulin and induced actin reorganization, resulting in lamellipodium formation with membrane ruffling. Taken together, these results indicate that Capn6 is a microtubule-stabilizing protein expressed in embryonic tissues that may be involved in the regulation of microtubule dynamics and cytoskeletal organization.
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Santo, Loredana, Teru Hideshima, Andrew L. Kung, Jen-Chieh Tseng, David Tamang, Min Yang, Matthew Jarpe, et al. "Pharmacodynamic and Pharmacokinetic Properties of a Novel and Selective HDAC6 Inhibitor, ACY-1215, in Combination with Bortezomib in Multiple Myeloma." Blood 118, no. 21 (November 18, 2011): 2912. http://dx.doi.org/10.1182/blood.v118.21.2912.2912.
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Abstract Abstract 2912 HDAC enzymes are being studied as novel therapeutic targets in several cancers including multiple myeloma (MM). In particular, the combination of proteasome inhibitors (e.g. bortezomib (BZ)) with Histone Deacetylase (HDAC) inhibitors have shown very promising results in pre-clinical MM models. HDAC6, a Class II HDAC, has been linked to the activity of aggresomes that degrade unfolded and misfolded ubiquitinated proteins. Targeting both proteasomal and aggresomal protein degradation systems with proteasome inhibitors and HDAC inhibitors, respectively, induces accumulation of polyubiquitinated proteins, followed by activation of apoptotic cascades. Here we investigated the preclinical activity of an HDAC6 selective inhibitor ACY-1215 in MM, either alone or in combination with BZ. In vitro enzyme assays showed that ACY-1215 has potent inhibitory activity against HDAC6 (IC50 0.0054 mM) compared to the other HDACs, including Class I HDACs. Maximal cytotoxicity of ACY-1215 against MM cell lines was observed at 48h, with IC50 values ranging from 2–8 μM. To investigate the specific inhibitory effect of ACY-1215 on HDAC6 activity, we evaluated its effect on acetylation of a-tubulin. ACY-1215 induces potent acetylation of a-tubulin at low doses and triggers acetylation of lysine on histone H3 and histone H4 only at much higher doses, confirming its selective inhibitory effect on HDAC6 activity. Importantly, this selective inhibition was also observed in patient MM cells, where ACY-1215 increased acetylated a-tubulin after 4 h of treatment. We next combined low doses of ACY-1215 with BZ and showed synergistic anti MM activity, resulting in apoptosis via caspase-3, -8, -9 and poly (ADP) ribosome polymerase activation. Moreover, the combination of ACY-1215 plus BZ increased the accumulation of polyubiquitinated proteins compared to either agent alone. To investigate the effect of ACY-1215 on aggresome formation, MM.1S cells treated with ACY-1215 1 μM and/or BZ 2.5 nM for 12 h were stained with immunofluorescent anti-ubiquitin antibody. BZ-treated cells showed perinuclear structures consistent with aggresome formation, which was disrupted when BZ and ACY-1215 were combined. This result supports the synergistic anti MM activity of ACY-1215 with BZ. We also evaluated the in vivo anti-MM effect of combination therapy using two different xenograft models in SCID mice: plasmacytoma model and disseminated MM model. ACY-1215 in combination with BZ triggered more significant anti-MM activity than either agent alone in suppressing tumor growth and prolonging host survival in both models, without significant adverse effects. To optimize the design of future clinical trials, we conducted pharmacokinetic and pharmacodynamic studies in our plasmacytoma model. ACY-1215 peak plasma levels were observed at 4 h, which were unaffected by the addition of BZ. To further characterize the activity of ACY-1215 against HDAC6 in vivo, we evaluated the acetylation of α-tubulin in mouse blood cells by flow cytometry. The maximum levels of blood cell α-tubulin acetylation were observed at 4 h, providing an important biomarker for future clinical trials. Importantly, levels of acetylated α-tubulin were also detected in tumor tissue from treated mice in a similar time frame to peak blood levels, suggesting that ACY-1215 is readily absorbed by tumor tissue. Moreover, ACY-1215 did not accumulate in tumor tissue, as shown by the decline of acetylated α-tubulin in blood cells and tumor tissue by 24 h post-dose, which parallels the elimination of ACY-1215 from blood. We further confirmed the HDAC6 selectivity of ACY-1215 in our in vivo models by investigating the effect of the drug combination on histone acetylation in tumor tissue. WB analysis and IHC did not show a significant increase in acetylated histone H3 (lys 18), while demonstrating a robust acetylation of α-tubulin, the primary marker of HDAC6 inhibition by ACY-1215 at the cellular level. The results from our in vitro and in vivo studies therefore show significant and synergistic anti-MM activity of ACY-1215 in combination with BZ and provided the rationale for the ongoing phase I/II clinical trial in patients with relapsed or relapsed/refractory MM. Moreover, our pharmacodynamic helped inform the design of correlative studies, which will establish whether acetylated α-tubulin can be used as predictive biomarker of HDAC6 inhibition and disease response. Disclosures: Hideshima: Acetylon: Consultancy. Kung:Acetylon Pharmaceuticals, Inc.: Consultancy. Tamang:Acetylon Pharmaceuticals, Inc.: Employment. Yang:Acetylon Pharmaceuticals, Inc.: Employment. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment. Mazitschek:Acetylon Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Ogier:Acetylon Pharmaceuticals, Inc.: Employment. Bradner:Acetylon: Consultancy. Anderson:celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Acetylon Pharmaceuticals Inc: founder. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.
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Howes, Stuart C., Gregory M. Alushin, Toshinobu Shida, Maxence V. Nachury, and Eva Nogales. "Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure." Molecular Biology of the Cell 25, no. 2 (January 15, 2014): 257–66. http://dx.doi.org/10.1091/mbc.e13-07-0387.
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Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.
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Saunders, Harriet A. J., Dena M. Johnson-Schlitz, Brian V. Jenkins, Peter J. Volkert, Sihui Z. Yang, and Jill Wildonger. "Acetylated α-tubulin K394 regulates microtubule stability to shape the growth of axon terminals." Current Biology 32, no. 3 (February 2022): 614–30. http://dx.doi.org/10.1016/j.cub.2021.12.012.
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Protic, Isidora, Igor Golic, Marija Aleksic, Snezana Vidakovic, Bato Korac, and Aleksandra Korac. "Presence of acetylated α-tubulin in human sperm nuclei: A contributor to sperm heterogeneity." Medical Hypotheses 161 (April 2022): 110800. http://dx.doi.org/10.1016/j.mehy.2022.110800.
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Chu, Dan T. W., and Michael W. Klymkowsky. "The appearance of acetylated α-tubulin during early development and cellular differentiation in Xenopus." Developmental Biology 136, no. 1 (November 1989): 104–17. http://dx.doi.org/10.1016/0012-1606(89)90134-6.
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Hempen, Bart, and Jean-Pierre Brion. "Reduction of Acetylated α-Tubulin Immunoreactivity in Neurofibrillary Tangle-bearing Neurons in Alzheimerʼs Disease." Journal of Neuropathology and Experimental Neurology 55, no. 9 (September 1996): 964–72. http://dx.doi.org/10.1097/00005072-199609000-00003.
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García-Arrarás, JoséE, and Eduardo Viruet. "Enteric nerve fibers of holothurians are recognized by an antibody to acetylated α-tubulin." Neuroscience Letters 157, no. 2 (July 1993): 153–56. http://dx.doi.org/10.1016/0304-3940(93)90725-z.
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Yedowitz, Jamie C., Anna Kotsakis, Elisabeth F. M. Schlegel, and John A. Blaho. "Nuclear Localizations of the Herpes Simplex Virus Type 1 Tegument Proteins VP13/14, vhs, and VP16 Precede VP22-Dependent Microtubule Reorganization and VP22 Nuclear Import." Journal of Virology 79, no. 8 (April 15, 2005): 4730–43. http://dx.doi.org/10.1128/jvi.79.8.4730-4743.2005.
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ABSTRACT Herpes simplex virus type 1 (HSV-1) induces microtubule reorganization beginning at approximately 9 h postinfection (hpi), and this correlates with the nuclear localization of the tegument protein VP22. Thus, the active retention of this major virion component by cytoskeletal structures may function to regulate its subcellular localization (A. Kotsakis, L. E. Pomeranz, A. Blouin, and J. A. Blaho, J. Virol. 75:8697-8711, 2001). The goal of this study was to determine whether the subcellular localization patterns of other HSV-1 tegument proteins are similar to that observed with VP22. To address this, we performed a series of indirect immunofluorescence analyses using synchronously infected cells. We observed that tegument proteins VP13/14, vhs, and VP16 localized to the nucleus as early as 5 hpi and were concentrated in nuclei by 9 hpi, which differed from that seen with VP22. Microtubule reorganization was delayed during infection with HSV-1(RF177), a recombinant virus that does not produce full-length VP22. These infected cells did not begin to lose microtubule-organizing centers until 13 hpi. Repair of the unique long 49 (UL49) locus in HSV-1(RF177) yielded HSV-1(RF177R). Microtubule reorganization in HSV-1(RF177R)-infected cells occurred with the same kinetics as HSV-1(F). Acetylated tubulin remained unchanged during infection with either HSV-1(F) or HSV-1(RF177). Thus, while α-tubulin reorganized during infection, acetylated tubulin was stable, and the absence of full-length VP22 did not affect this stability. Our findings indicate that the nuclear localizations of tegument proteins VP13/14, VP16, and vhs do not appear to require HSV-1-induced microtubule reorganization. We conclude that full-length VP22 is needed for optimal microtubule reorganization during infection. This implies that VP22 mainly functions to reorganize microtubules later, rather than earlier, in infection. That acetylated tubulin does not undergo restructuring during VP22-dependent, virus-induced microtubule reorganization suggests that it plays a role in stabilizing the infected cells. Our results emphasize that VP22 likely plays a key role in cellular cytopathology during HSV-1 infection.
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VUGLER, ANTHONY A., MA'AYAN SEMO, ANNA JOSEPH, and GLEN JEFFERY. "Survival and remodeling of melanopsin cells during retinal dystrophy." Visual Neuroscience 25, no. 2 (March 2008): 125–38. http://dx.doi.org/10.1017/s0952523808080309.
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AbstractThe melanopsin positive, intrinsically photosensitive retinal ganglion cells (ipRGCs) of the inner retina have been shown to send wide-ranging projections throughout the brain. To investigate the response of this important cell type during retinal dystrophy, we use the Royal College of Surgeons (RCS) dystrophic rat, a major model of retinal degeneration. We find that ipRGCs exhibit a distinctive molecular profile that remains unaltered during early stages of outer retinal pathology (15 weeks of age). In particular, these cells express βIII tubulin, α-acetylated tubulin, and microtubule-associated proteins (MAPs), while remaining negative for other RGC markers such as neurofilaments, calretinin, and parvalbumin. By 14 months of age, melanopsin positive fibers invade ectopic locations in the dystrophic retina and ipRGC axons/dendrites become distorted (a process that may involve vascular remodeling). The morphological abnormalities in melanopsin processes are associated with elevated immunoreactivity for MAP1b and a reduction in α-acetylated tubulin. Quantification of ipRGCs in whole mounts reveals reduced melanopsin cell number with increasing age. Focusing on the retinal periphery, we find a significant decline in melanopsin cell density contrasted by a stability of melanopsin positive processes. In addition to these findings, we describe for the first time, a distinct plexus of melanopsin processes in the far peripheral retina, a structure that is coincident with a short wavelength opsin cone-enriched rim. We conclude that some ipRGCs are lost in RCS dystrophic rats as the disease progresses and that this loss may involve vascular remodeling. However, a significant number of melanopsin positive cells survive into advanced stages of retinal degeneration and show indications of remodeling in response to pathology. Our findings underline the importance of early intervention in human retinal disease in order to preserve integrity of the inner retinal photoreceptive network.
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Tamang, David, Simon S. Jones, Min Yang, and Jeff Supko. "Tubulin Hyper-Acetylation In Blood Lymphocytes: Pharmacodynamic (PD) Biomarker For The Selective Histone Deacetylase (HDAC) 6 Inhibitor ACY-1215 In Multiple Myeloma (MM) Patients." Blood 122, no. 21 (November 15, 2013): 3219. http://dx.doi.org/10.1182/blood.v122.21.3219.3219.
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Abstract Introduction HDACs are a family of enzymes that remove acetyl groups from proteins and are involved in many key cellular processes, including apoptosis, cell cycle arrest and angiogenesis. Nonselective HDAC inhibitors has shown promise in treating MM in combination with standard therapies such as proteasome inhibitors (PIs) [1]. However, nonselective HDAC inhibitors have been of limited utility due to adverse effects. HDAC6 is a Class IIb enzyme found in the cytoplasm that is intimately involved in cytoskeletal remodeling and vesicle transport associated with degradation of misfolded protein through the regulation of the acetylation status of α-tubulin. Recent work [2, 3] suggests that selectively inhibiting HDAC6 in MM is an important therapeutic modality as part of a combination therapy with PIs and immunomodulatory agents (IMiDs) and may minimize toxicity due to Class I HDAC inhibition. ACY-1215 is a novel HDAC6-selective small molecule inhibitor that has been shown to be effective in reducing tumor burden in preclinical animal models of MM, and the anti-tumor activities correlate with increased levels of acetyl-tubulin, a HDAC6 specific substrate, in blood and tumor cells. ACY-1215 has now advanced to phase I clinical trials as a combination agent for the treatment of relapsed and refractory MM. A clinical PD biomarker for HDAC6 inhibition is critical to demonstrate that therapeutic plasma levels of ACY-1215 also increase the HDAC6 specific biomarker, acetylated tubulin, versus the Class I HDAC biomarker, acetylated histones. In addition HDAC6 inhibition is expected to be well-tolerated and dose limiting toxicity may not be reached implying continued dose escalation in phase 1 may not be warranted if there is a suitable increase in the PD HDAC6 biomarker. We describe the development of a clinical PD biomarker of HDAC6 inhibition, acetylated α-tubulin, to determine when blood levels of ACY-1215 are reached that give a specific increase in the biomarker versus acetylated histones. Methods Assay sensitivity, dynamic range and reproducibility have been established using peripheral whole blood from healthy donors and MM patients incubated with ACY-1215 ex vivo. In the clinic, peripheral blood is collected at up to six different PK matched time points after drug administration.Lymphocytes were assessed for tubulin and histone hyper-acetylation by flow cytometry using specific antibodies that recognize acetyl groups in the context of the specific protein targets. ACY-1215 plasma concentration was also determined in the peripheral blood by an appropriate bioanalytical method. More than 50 MM patients have been enrolled in two separate clinical trials and received escalating oral ACY-1215 doses from 40 mg to 240 mg. The ACY-100 study is composed of two parts, ACY-1215 monotherapy and combination therapy with both bortezomib and dexamethasone. A second study, ACE-MM-101, examines ACY-1215 in combination with lenalidomide and dexamethasone. Results & Conclusion Patients in ACY-100 and ACE-MM-101 clinical trials have shown elevated tubulin hyper-acetylation in peripheral blood lymphocytes as ACY-1215 dose increases from 40 mg to 240 mg. ACY-1215 plasma concentration reaches a maximal level (Cmax) 1 hr after administration. An apparent exposure plateau was reached at dose levels >160 mg where Cmax reached 1.2 - 1.8 µM. Correlating to the PK levels, the PD biomarker acetyl-tubulin increases to a maximal level 1 hr after dosing compared to predose, and declines to basal level by >4 hr with an apparent lag period compared to plasma levels of ACY-1215. Acetyl-histone levels were not significantly changed at these doses indicating a predominant HDAC6 selective effect. As plasma levels increased with dose from 40 mg to 160 mg so do the number of patients that have a measurable increase in acetyl-tublin such that at dose levels ≥80 mg all patients have a measurable increase in acetylated tubulin. In conclusion the clinical PK and PD results, comparing to prior preclinical studies [4], suggest that ACY-1215 has reached a pharmacologically relevant level of HDAC6 inhibition at clinical doses ≥80 mg. These results will aid in the determination of the recommend phase 2 dose of ACY-1215 in combination with PIs and IMiDs. Disclosures: Tamang: Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Supko:Acetylon: Research Funding.
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Yi, Sang Ah, Ki Hong Nam, Sil Kim, Hae Min So, Rhim Ryoo, Jeung-Whan Han, Ki Hyun Kim, and Jaecheol Lee. "Vulpinic Acid Controls Stem Cell Fate toward Osteogenesis and Adipogenesis." Genes 11, no. 1 (December 23, 2019): 18. http://dx.doi.org/10.3390/genes11010018.
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Vulpinic acid, a naturally occurring methyl ester of pulvinic acid, has been reported to exert anti-fungal, anti-cancer, and anti-oxidative effects. However, its metabolic action has not been implicated yet. Here, we show that vulpinic acid derived from a mushroom, Pulveroboletus ravenelii controls the cell fate of mesenchymal stem cells and preadipocytes by inducing the acetylation of histone H3 and α-tubulin, respectively. The treatment of 10T1/2 mesenchymal stem cells with vulpinic acid increased the expression of Wnt6, Wnt10a, and Wnt10b, which led to osteogenesis inhibiting the adipogenic lineage commitment, through the upregulation of H3 acetylation. By contrast, treatment with vulpinic acid promoted the terminal differentiation of 3T3-L1 preadipocytes into mature adipocytes. In this process, the increase in acetylated tubulin was accompanied, while acetylated H3 was not altered. As excessive generation of adipocytes occurs, the accumulation of lipid drops was not concentrated, but dispersed into a number of adipocytes. Consistently, the expressions of lipolytic genes were upregulated and inflammatory factors were downregulated in adipocytes exposed to vulpinic acid during adipogenesis. These findings reveal the multiple actions of vulpinic acid in two stages of differentiation, promoting the osteogenesis of mesenchymal stem cells and decreasing hypertrophic adipocytes, which can provide experimental evidence for the novel metabolic advantages of vulpinic acid.
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Bolino, Matthew, Abigail Leff, Bradley Ferguson, and Steven Frese. "Bifidobacterium longum subsp. infantis Shifts Global Transcriptional and Post-translational Modification Signals in Preterm Infant Gut Epithelial Cells." Current Developments in Nutrition 6, Supplement_1 (June 2022): 624. http://dx.doi.org/10.1093/cdn/nzac061.008.
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Abstract Objectives Bifidobacterium longum subsp. infantis (B. infantis) is an important human infant gut symbiont that confers benefits to the infant by shaping the gut microbiome in vivo. Here, we investigated the transcriptomic and post-translational modification (PTM) impacts of B. infantis on host gut epithelial cells representative of preterm infants, in vitro. Methods RNA isolated from preterm infant intestinal epithelial cells (HIEC-6) exposed to sterile, cell-free B. infantis supernatants or controls (no supernatant) for 20 hours was subjected to RNA-sequencing to assess global transcriptional changes (n = 6/group). Using the same approach, HIEC-6 cells were incubated for 3 or 20 hours, with or without supernatant (10% v/v; n = 3/group), before harvest for cellular proteins and subsequent western blotting. Gels were probed for α-tubulin, acetylated α-tubulin, Extracellular signal-Regulated Kinase (ERK), and phosphorylated ERK as markers of PTM activity. Results Global transcriptomic profiles were significantly different between cells exposed to supernatant and those that were not (P = 0.006) and gene pathways involved in anti-inflammatory responses and chemotaxis were significantly upregulated (P < 0.01). Western blotting revealed significant increases in acetyl-α-tubulin and phospho-ERK in cells exposed to supernatant relative to the control group (P < 0.05), while total α-tubulin and ERK concentrations were not significantly different (P > 0.05). Conclusions These findings demonstrate the ability of B. infantis to alter global transcriptional and PTM responses affecting inflammation, intracellular trafficking of α-tubulin, and transcription factor recruitment by ERK phosphorylation. These pathways regulate key cellular processes such as cell proliferation, differentiation, adhesion, migration and survival, providing molecular insights into the ability of B. infantis to regulate transcriptional and PTM processes in vitro. Funding Sources This work was funded by the Department of Nutrition, the College of Agriculture, Biotechnology, & Natural Resources, the Nevada Agricultural Experiment Station, and the Vice President for Research and Innovation, as well as the NICHD, R03HD105881, and grants from the NIGMS (GM103440 and GM104944) from the NIH.
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Zhang, Xuelian, Yaru Qiao, Ronglin Han, Yingjie Gao, Xun Yang, Ying Zhang, Ying Wan, Wei Yu, Xianchao Pan, and Juan Xing. "A Charcot-Marie-Tooth-Causing Mutation in HSPB1 Decreases Cell Adaptation to Repeated Stress by Disrupting Autophagic Clearance of Misfolded Proteins." Cells 11, no. 18 (September 15, 2022): 2886. http://dx.doi.org/10.3390/cells11182886.
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Charcot-Marie-Tooth (CMT) disease is the most common inherited neurodegenerative disorder with selective degeneration of peripheral nerves. Despite advances in identifying CMT-causing genes, the underlying molecular mechanism, particularly of selective degeneration of peripheral neurons remains to be elucidated. Since peripheral neurons are sensitive to multiple stresses, we hypothesized that daily repeated stress might be an essential contributor to the selective degeneration of peripheral neurons induced by CMT-causing mutations. Here, we mainly focused on the biological effects of the dominant missense mutation (S135F) in the 27-kDa small heat-shock protein HSPB1 under repeated heat shock. HSPB1S135F presented hyperactive binding to both α-tubulin and acetylated α-tubulin during repeated heat shock when compared with the wild type. The aberrant interactions with tubulin prevented microtubule-based transport of heat shock-induced misfolded proteins for the formation of perinuclear aggresomes. Furthermore, the transport of autophagosomes along microtubules was also blocked. These results indicate that the autophagy pathway was disrupted, leading to an accumulation of ubiquitinated protein aggregates and a significant decrease in cell adaptation to repeated stress. Our findings provide novel insights into the molecular mechanisms of HSPB1S135F-induced selective degeneration of peripheral neurons and perspectives for targeting autophagy as a promising therapeutic strategy for CMT neuropathy.
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Wolf, Klaus Werner. "The spindle apparatus in early embryonic divisions of Ephestia Kuehniella Z. (Pyralidae, Lepidoptera) is formed by alignment of minispindles." Zygote 2, no. 1 (February 1994): 87–95. http://dx.doi.org/10.1017/s0967199400001805.
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SummarySpindles were isolated from deposited eggs of the Mediterranean mealmoth, Ephestia Kuehniella. Their structure and development were studied using anti-tubulin immunofluorescence. The microtubules were labelled with three different monoclonal antibodies. These were directed against β-tubulin, tyrosinated α-tubulin and acetylated α-tubulin. Significant differences in the staining behaviour were not detected with the three antibodies. An unusual mode of spindle formation was observed during the first mitotic division after fusion of the pronuclei. Several of the ensuing embryonic divisions may show the same phenomenon. Prophase of these divisions was characterised by an irregular arrangement of microtubules in the nuclear area. The microtubule mass in the nuclear area increased concomitantly with chromosome condensation. Microtubular foci, comparable to the forming asters of canonical spindles, were not detected. The formation of an orderly pattern in the microtubule mass was signalled by the appearance of minispindles apparently developing around individual chromosomes. Several minispindles subsequently aligned and formed metaphase-like entities within the nuclear area. The metaphase-like entities, in turn, aligned with one another and gave rise to a conventional bipolar metaphase spindle with small asters. The further development of the spindle was conventional. The chromosomes migrated towards the spindle poles and finally daughter nuclei formed. The anaphase and telophase spindles possessed both a prominent array of interzone microtubules and asters. The events in prophase of early embryonic mitosis of E. kuehniella may represent a rare case of chromosomeinduced spindle formation.
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Chang, Panpan, Yuzi Tian, Aaron M. Williams, Umar F. Bhatti, Baoling Liu, Yongqing Li, and Hasan B. Alam. "Inhibition of Histone Deacetylase 6 Protects Hippocampal Cells Against Mitochondria-mediated Apoptosis in a Model of Severe Oxygen-glucose Deprivation." Current Molecular Medicine 19, no. 9 (October 11, 2019): 673–82. http://dx.doi.org/10.2174/1566524019666190724102755.
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Background: Histone deacetylase (HDAC) 6 inhibitors have demonstrated significant protective effects in traumatic injuries. However, their roles in neuroprotection and underlying mechanisms are poorly understood. This study sought to investigate the neuroprotective effects of Tubastatin A (Tub-A), an HDAC6 inhibitor, during oxygenglucose deprivation (OGD) in HT22 hippocampal cells. Methods: HT22 hippocampal cells were exposed to OGD. Cell viability and cytotoxicity were assessed by cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assay. Cellular apoptosis was assessed by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Mitochondria membrane potential was detected using JC-1 dye. Expressions of acetylated α-tubulin, α-tubulin, cytochrome c, VDAC, Bax, Bcl- 2, cleaved caspase 3, phosphorylated Akt, Akt, phosphorylated GSK3β and GSK3β were analyzed by Western blot analysis. Results: Tub-A induced acetylation of α-tubulin, demonstrating appropriate efficacy. Tub-A significantly increased cell viability and attenuated LDH release after exposure to OGD. Furthermore, Tub-A treatment blunted the increase in TUNEL-positive cells following OGD and preserved the mitochondrial membrane potential. Tub-A also attenuated the release of cytochrome c from the mitochondria into the cytoplasm and suppressed the ratio of Bax/Bcl-2 and cleaved caspase 3. This was mediated, in part, by the increased phosphorylation of Akt and GSK3β signaling pathways. Conclusion: HDAC 6 inhibition, using Tub-A, protects against OGD-induced injury in HT22 cells by modulating Akt/GSK3β signaling and inhibiting mitochondria-mediated apoptosis.
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Bergmann, Laura, Alexander Lang, Christoph Bross, Simone Altinoluk-Hambüchen, Iris Fey, Nina Overbeck, Anja Stefanski, et al. "Subcellular Localization and Mitotic Interactome Analyses Identify SIRT4 as a Centrosomally Localized and Microtubule Associated Protein." Cells 9, no. 9 (August 24, 2020): 1950. http://dx.doi.org/10.3390/cells9091950.
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The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD+-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is present in the cytosol and predominantly localizes to centrosomes. Confocal spinning disk microscopy revealed that SIRT4 is found during the cell cycle dynamically at centrosomes with an intensity peak in G2 and early mitosis. Moreover, SIRT4 precipitates with microtubules and interacts with structural (α,β-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism and provides the first evidence that SIRT4 acts as a novel centrosomal/microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.
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Zhang, Yu, SoHee Kwon, Teppei Yamaguchi, Fabien Cubizolles, Sophie Rousseaux, Michaela Kneissel, Chun Cao, et al. "Mice Lacking Histone Deacetylase 6 Have Hyperacetylated Tubulin but Are Viable and Develop Normally." Molecular and Cellular Biology 28, no. 5 (January 7, 2008): 1688–701. http://dx.doi.org/10.1128/mcb.01154-06.
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ABSTRACT Posttranslational modifications play important roles in regulating protein structure and function. Histone deacetylase 6 (HDAC6) is a mostly cytoplasmic class II HDAC, which has a unique structure with two catalytic domains and a domain binding ubiquitin with high affinity. This enzyme was recently identified as a multisubstrate protein deacetylase that can act on acetylated histone tails, α-tubulin and Hsp90. To investigate the in vivo functions of HDAC6 and the relevance of tubulin acetylation/deacetylation, we targeted the HDAC6 gene by homologous recombination in embryonic stem cells and generated knockout mice. HDAC6-deficient mice are viable and fertile and show hyperacetylated tubulin in most tissues. The highest level of expression of HDAC6 is seen in the testis, yet development and function of this organ are normal in the absence of HDAC6. Likewise, lymphoid development is normal, but the immune response is moderately affected. Furthermore, the lack of HDAC6 results in a small increase in cancellous bone mineral density, indicating that this deacetylase plays a minor role in bone biology. HDAC6-deficient mouse embryonic fibroblasts show apparently normal microtubule organization and stability and also show increased Hsp90 acetylation correlating with impaired Hsp90 function. Collectively, these data demonstrate that mice survive well without HDAC6 and that tubulin hyperacetylation is not detrimental to normal mammalian development.
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Vo, Nguyen T. K., and Niels C. Bols. "Demonstration of primary cilia and acetylated α-tubulin in fish endothelial, epithelial and fibroblast cell lines." Fish Physiology and Biochemistry 42, no. 1 (August 7, 2015): 29–38. http://dx.doi.org/10.1007/s10695-015-0114-1.
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Low, Jennifer A., Suparna B. Wedam, James J. Lee, Arlene W. Berman, Adam Brufsky, Sherry X. Yang, Marianne S. Poruchynsky, et al. "Phase II Clinical Trial of Ixabepilone (BMS-247550), an Epothilone B Analog, in Metastatic and Locally Advanced Breast Cancer." Journal of Clinical Oncology 23, no. 12 (April 20, 2005): 2726–34. http://dx.doi.org/10.1200/jco.2005.10.024.
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Purpose Ixabepilone (BMS-247550) is an epothilone B analog that stabilizes microtubules and has antitumor activity in taxane-refractory patients in phase I studies. In a phase II trial, we evaluated the efficacy and safety of ixabepilone in women with metastatic and locally advanced breast cancer. Patients and Methods Breast cancer patients with measurable disease who had paclitaxel and/or docetaxel as prior neoadjuvant, adjuvant, or metastatic therapy were treated with ixabepilone at 6 mg/m2/d intravenously on days 1 through 5 every 3 weeks. Levels of glutamate (glu) -terminated and acetylated α-tubulin, markers of microtubule stabilization, were detected by Western blot and by immunohistochemistry in a subset of matched pre- and post-treatment tumor biopsies. Results Thirty-seven patients received 153 cycles of ixabepilone. The best responses were a complete response in one patient (3%), partial responses in seven patients (19%), and stable disease in 13 patients (35%). Grade 3 and 4 toxicities included neutropenia (35%), febrile neutropenia (14%), fatigue (14%), diarrhea (11%), nausea/vomiting (5%), myalgia/arthralgia (3%), and sensory neuropathy (3%). Two patients were removed from study because of prolonged grade 2 or 3 neurotoxicity, and three patients were removed from study for other grade 3 and 4 nonhematologic toxicities. Compared with baseline levels, levels of both glu-terminated and acetylated α-tubulin were increased in tumor biopsies performed after ixabepilone therapy. Conclusion An objective response was seen in 22% of the patients in a population who had been previously treated with a taxane. Sensory neuropathy was mild with grade 3 neurotoxicity rarely seen. Microtubule stabilization occurred in tumor biopsies after treatment with ixabepilone.
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Strassel, Catherine, Agnes Hovasse, Sylvie Moog, Magda Mageira, Morgane Batzenschlager, Christine Schaeffer-Reiss, Benjamin Kile, Christian Gachet, Carsten Janke, and Francois Lanza. "Selected Expression and Functional Importance of α4a-Tubulin in Platelet Biogenesis." Blood 128, no. 22 (December 2, 2016): 1360. http://dx.doi.org/10.1182/blood.v128.22.1360.1360.
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Abstract Platelets are produced from mature megakaryocytes (MK) following a profound cellular reorganization. This includes the assembly of microtubules (MT) into a unique submenbranous coiled structure, the marginal band (MB). This process is thought to depend on a specific αβ-tubulin isotype repertoire. The MK-restricted-β1-tubulin, the predominant isoform of the MB, is already known to be important for platelet biogenesis but the implication of other isotypes is currently unknown. Our goal was to establish the αβ-tubulin repertoire in platelets and during megakaryopoiesis and to evaluate the implication of selected isotypes in platelet formation. To establish an exhaustive list of the tubulin isotypes, we used combination of RT PCR and proteomic analyses to quantify the expression of each isotype in human platelets and in human MK differentiated in culture from CD34+ hematopoietic progenitors. Information gained on the hierarchical combination of tubulin isoforms in the course of platelet biogenesis has been extended at the functional level to investigate both their role in marginal band formation and platelet functions β6-, β5- and α1c-tubulin transcripts were already present in CD34+ cells and decreased during the final stages of megakaryopoiesis. On the other hand, β1-, α4A- and α8-tubulin transcripts were only observed later during MK differentiation and in platelets. Quantitative LC-SRM mass spectrometry confirmed the predominant expression of β1 and α4A-isotypes in platelets. A functional role of the newly identified α4a-tubulin was supported by the thrombocytopenia and enlarged platelets with a decreased number of MT coils (1-3) comprising less-acetylated tubulin in mice carrying a point mutation in tuba4a. Additionally, a tendency to increased responses to several agonists was observed in these platelets. This study reveals new information on the evolution of the tubulin isotype repertoire in platelet formation pointing to a role of less-widely expressed α-isotypes. Disclosures No relevant conflicts of interest to declare.
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Langella, Arianna, Sergio Domenico Gadau, Elisa Serra, Daniela Bebbere, and Sergio Ledda. "Microtubular Assessment of C6 Rat Glioma Cell Spheroids Developed in Transparent Liquid Marbles or Hanging Drops." Biology 11, no. 4 (March 23, 2022): 492. http://dx.doi.org/10.3390/biology11040492.
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Glioblastoma is a brain tumour frequently used as an experimental model to exploit innovative therapeutic approaches due to its high lethality and refractoriness to therapies. Part of these innovative anticancer therapies address cytoskeletal microtubules (MTs) since specific tubulin post-translational modifications (PTMs) are considered markers of tumour plasticity. In vitro studies, which traditionally employ two-dimensional (2D) culture systems, are now being replaced by three-dimensional (3D) systems that more closely mimic in vivo physiological conditions and allow a better understanding of the signalling between cells. In this work, we compared 2 liquid base 3D methods for the generation of spheroids from C6 rat glioma cells (RGCs) using 30 µL of liquid marble (LM) or the hanging drops (HDs), which contained 2 different cell numbers (5000 or 15,000). After 24 or 48 h of in vitro culture (IVC), the morphology of the spheroids was observed and the behaviour of the two main tubulin PTMs, tyrosinated α-tubulin (Tyr-T) and acetylated α-tubulin (Ac-T), was evaluated by fluorescence and Western blot (WB). RGCs spontaneously formed spherical agglomerates more rapidly in the LM than in the HD system. Cell density influenced the size of the spheroids, which reached a larger size (> of 300 µm Ø), with 15,000 cells compared to 5000 cells (150 µm Ø). Moreover, an increase in Tyr-T and Ac-T was observed in both the HD and LM system from 24 to 48 h, with the highest values shown in the 48 h/LM spheroids of 5000 cells (p < 0.05). In conclusion, by comparing the morphology and microtubular architecture of spheroids from C6 rat glioma cells developed by LM or HD methodology, our findings demonstrate that the use of a fumed silica microbioreactor boosts the induction and maintenance of a high plasticity state in glioma cells. RGCs cultured in LM express levels of tubulin PTMs that can be used to evaluate the efficacy of new anticancer therapies.