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Different means of inducing tubulin hyperacetylation lead to the formation of distinct microtubule structures. (A) Schematic overview of the enzymes involved in tubulin deacetylation. Tubulin is deacetylated by HDAC6 and SIRT2. HDAC6-mediated deacetylation results in the release of acetate, and the activity can be inhibited by the specific HDAC6 inhibitor tubacin, or the general HDAC class I and II inhibitor Trichostatin A (TSA). SIRT2-mediated deacetylation requires NAD + as a co-substrate and results in the release of nicotinamide (Nam) and O-acetyl ADP-ribose. SIRT2 can be inhibited by the selective SIRT2 inhibitor AGK2, or inactivated through NAD + depletion. NAD + depletion can be achieved through inhibition of NamPRT by FK866. NamPRT is an NAD-biosynthetic enzyme that converts the NAD precursor nicotinamide into nicotinamide mononucleotide. FK866-induced NAD depletion can be bypassed through addition of the NamPRT-independent NAD precursor nicotinic acid (NA). (B) Different microtubule morphologies are detected in HeLaS3 cells cultured in either standard culture medium, or 6 h after treatment with 5 µM tubacin or 5 µM taxol, or 1 h after treatment with 0.25 M NaCl. MAP2c overexpression (30 h) was monitored by immunocytochemical detection of the C-terminal Myc tag. (C) SIRT2 inhibition leads to formation of hyperacetylated perinuclear microtubules. HeLaS3 cells were treated with 100 µM AGK2 or the solvent dimethyl sulfoxide (DMSO) for 6 h, or with 2 µM FK866 or the solvent dimethyl formamide (DMF) for 48 h. Ac α-tubulin, acetylated α-tubulin. Scale bars: 15 µm.
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Deacetylation of α-tubulin, lysine 40, is catalyzed by two enzymes, the NAD-dependent deacetylase SIRT2 and the NAD-independent deacetylase HDAC6, in apparently redundant reactions. In the present study, we tested whether these two enzymes might have distinguishable preferences for the deacetylation of different microtubule structures. Using variou...
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... deacetylases (HDACs, class I and II) perform deacetylation by hydrolytic cleavage resulting in the release of free acetate (de Ruijter et al., 2003). In the deacetylation reaction catalyzed by sirtuins (HDAC class III), nicotinamide adenine dinucleotide (NAD) is required as a co-substrate and is degraded in the process ( Sanders et al., 2010) (Fig. ...
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... deacetylating activities. Therefore, selective modulation of HDAC6 or SIRT2 activities could be used to reveal their contributions to the regulation of different microtubule structures. First, we made use of several pharmacological treatments that are known to induce the formation of distinct, hyperacetylated microtubule structures. As shown in Fig. 1B, the tubulin acetylation levels in HeLa cells cultivated under standard conditions were relatively low (Fig. 1B, medium). Addition of the specific HDAC6 inhibitor tubacin (Fig. 1A) increased the level of tubulin acetylation throughout the cell without affecting the morphology of the microtubule network (Fig. 1B). In contrast, exposure ...
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... their contributions to the regulation of different microtubule structures. First, we made use of several pharmacological treatments that are known to induce the formation of distinct, hyperacetylated microtubule structures. As shown in Fig. 1B, the tubulin acetylation levels in HeLa cells cultivated under standard conditions were relatively low (Fig. 1B, medium). Addition of the specific HDAC6 inhibitor tubacin (Fig. 1A) increased the level of tubulin acetylation throughout the cell without affecting the morphology of the microtubule network (Fig. 1B). In contrast, exposure to the cytostatic drug taxol led to the assembly of sharp wedge-like hyperacetylated microtubule structures (Fig. 1B), ...
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... First, we made use of several pharmacological treatments that are known to induce the formation of distinct, hyperacetylated microtubule structures. As shown in Fig. 1B, the tubulin acetylation levels in HeLa cells cultivated under standard conditions were relatively low (Fig. 1B, medium). Addition of the specific HDAC6 inhibitor tubacin (Fig. 1A) increased the level of tubulin acetylation throughout the cell without affecting the morphology of the microtubule network (Fig. 1B). In contrast, exposure to the cytostatic drug taxol led to the assembly of sharp wedge-like hyperacetylated microtubule structures (Fig. 1B), whereas overexpression of the neuronal microtubule-associated ...
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... microtubule structures. As shown in Fig. 1B, the tubulin acetylation levels in HeLa cells cultivated under standard conditions were relatively low (Fig. 1B, medium). Addition of the specific HDAC6 inhibitor tubacin (Fig. 1A) increased the level of tubulin acetylation throughout the cell without affecting the morphology of the microtubule network (Fig. 1B). In contrast, exposure to the cytostatic drug taxol led to the assembly of sharp wedge-like hyperacetylated microtubule structures (Fig. 1B), whereas overexpression of the neuronal microtubule-associated protein MAP2c (an isoform of MAP2) led to a drastic reorganization of the microtubules into long hyperacetylated bundles (Fig. 1B). ...
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... low (Fig. 1B, medium). Addition of the specific HDAC6 inhibitor tubacin (Fig. 1A) increased the level of tubulin acetylation throughout the cell without affecting the morphology of the microtubule network (Fig. 1B). In contrast, exposure to the cytostatic drug taxol led to the assembly of sharp wedge-like hyperacetylated microtubule structures (Fig. 1B), whereas overexpression of the neuronal microtubule-associated protein MAP2c (an isoform of MAP2) led to a drastic reorganization of the microtubules into long hyperacetylated bundles (Fig. 1B). Incubation of the cells with a hyperosmotic NaCl solution triggered only a moderate increase in tubulin acetylation. However, this treatment ...
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... network (Fig. 1B). In contrast, exposure to the cytostatic drug taxol led to the assembly of sharp wedge-like hyperacetylated microtubule structures (Fig. 1B), whereas overexpression of the neuronal microtubule-associated protein MAP2c (an isoform of MAP2) led to a drastic reorganization of the microtubules into long hyperacetylated bundles (Fig. 1B). Incubation of the cells with a hyperosmotic NaCl solution triggered only a moderate increase in tubulin acetylation. However, this treatment caused microtubule disintegration as indicated by the rather diffuse distribution of tubulin (Fig. 1B) (Croom et al., 1986). SIRT2 deacetylates the same lysine residue in α-tubulin as HDAC6. ...
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... isoform of MAP2) led to a drastic reorganization of the microtubules into long hyperacetylated bundles (Fig. 1B). Incubation of the cells with a hyperosmotic NaCl solution triggered only a moderate increase in tubulin acetylation. However, this treatment caused microtubule disintegration as indicated by the rather diffuse distribution of tubulin (Fig. 1B) (Croom et al., 1986). SIRT2 deacetylates the same lysine residue in α-tubulin as HDAC6. Therefore, inhibition of this NAD-dependent enzyme would be expected to result in a similar increase in tubulin acetylation as seen following tubacin treatment. However, treatment with the SIRT2 inhibitor AGK2 (Fig. 1A) only led to hyperacetylation ...
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... rather diffuse distribution of tubulin (Fig. 1B) (Croom et al., 1986). SIRT2 deacetylates the same lysine residue in α-tubulin as HDAC6. Therefore, inhibition of this NAD-dependent enzyme would be expected to result in a similar increase in tubulin acetylation as seen following tubacin treatment. However, treatment with the SIRT2 inhibitor AGK2 (Fig. 1A) only led to hyperacetylation of perinuclear microtubules (Fig. 1C). FK866, an inhibitor of the NAD biosynthetic enzyme NamPRT (also known as NAMPT), leads to NAD depletion in cells that rely on nicotinamide as NAD precursor. We have previously shown that FK866-induced NAD depletion results in indirect SIRT2 inactivation and gives rise ...
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... SIRT2 deacetylates the same lysine residue in α-tubulin as HDAC6. Therefore, inhibition of this NAD-dependent enzyme would be expected to result in a similar increase in tubulin acetylation as seen following tubacin treatment. However, treatment with the SIRT2 inhibitor AGK2 (Fig. 1A) only led to hyperacetylation of perinuclear microtubules (Fig. 1C). FK866, an inhibitor of the NAD biosynthetic enzyme NamPRT (also known as NAMPT), leads to NAD depletion in cells that rely on nicotinamide as NAD precursor. We have previously shown that FK866-induced NAD depletion results in indirect SIRT2 inactivation and gives rise to tubulin hyperacetylation ( Skoge et al., 2014) (Fig. 1A). In ...
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... microtubules (Fig. 1C). FK866, an inhibitor of the NAD biosynthetic enzyme NamPRT (also known as NAMPT), leads to NAD depletion in cells that rely on nicotinamide as NAD precursor. We have previously shown that FK866-induced NAD depletion results in indirect SIRT2 inactivation and gives rise to tubulin hyperacetylation ( Skoge et al., 2014) (Fig. 1A). In fact, FK866-induced indirect inactivation of SIRT2 led to the formation of hyperacetylated perinuclear microtubules similar to the hyperacetylated structures observed upon treatment of cells with AGK2 (Fig. ...
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... FK866-induced NAD depletion results in indirect SIRT2 inactivation and gives rise to tubulin hyperacetylation ( Skoge et al., 2014) (Fig. 1A). In fact, FK866-induced indirect inactivation of SIRT2 led to the formation of hyperacetylated perinuclear microtubules similar to the hyperacetylated structures observed upon treatment of cells with AGK2 (Fig. ...
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... with this hypothesis, we found that the microtubules in FK866- treated cells were only partially depolymerized and deacetylated when cells were incubated for 15 min on ice ( Fig. 3A; green and red channel, respectively). Addition of nicotinic acid restores the NAD level in FK866-treated cells and, in turn, reactivates SIRT2 ( Skoge et al., 2014) (Fig. 1A). Indeed, cells that were treated with nicotinic acid after pre-incubation with FK866 exhibited a normal arrangement of microtubules (Fig. 3A, green channel) and the microtubules were depolymerized and deacetylated when exposed to cold, similar to control-treated cells (Fig. 3A, green and red channel, respectively). Therefore, the ...
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... occurrence of hyperacetylated perinuclear microtubules, following incubation of cells with AGK2 or FK866 (Fig. 1C), strongly suggested that this phenomenon was brought about by diminished SIRT2 activity. However, it cannot be excluded that the pharmacological inhibitors used might have affected other NAD- dependent mechanisms. To establish whether the perinuclear microtubule hyperacetylation was indeed due to diminished SIRT2 activity, we treated ...
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INTRODUCTORY PARAGRAPH
Long-lived microtubules endow the eukaryotic cell with long-range transport abilities. While long-lived microtubules are acetylated on lysine 40 of α-tubulin (αK40), acetylation takes place after stabilization¹ and does not protect against depolymerization². Instead, αK40 acetylation has been proposed to mechanically stabiliz...
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... The histone deacetylase 6 (HDAC6) and sirtuin type 2 (SIRT2) can deacetylate tubulin. Although both enzymes can remove the acetyl group from α-tubulin interdependently, the activity of HDAC6 accounts for the majority of cytoplasmic microtubule deacetylation, whereas the activity of SIRT2 is more perinuclear and cell cycledependent [31][32][33][34] . Besides its regulatory function for cell motility through deacetylation of α-tubulin, the cytoplasmic HDAC6 plays an important role in regulating pro-apoptotic p53 acetylation and controlling chaperone Hsp90 required for cell signaling 35,36 . ...
... Furthermore, in vitro experiments demonstrated that microtubules can be deacetylated by HDAC6 stochastically along their entire length 37,38 . This is consistent with the idea that HDAC6 enters the microtubule lumen in a similar way to αTAT1, although HDAC6 is three-times larger than αTAT1 (140 versus 45 kDa) 34,38 . This difference in the size of their folded domains ( Supplementary Fig. 1) could affect their differential ability to enter through damage sites and, once inside, their differential diffusion along the~17 nm wide microtubule lumen. ...
... Upon inhibition of HDAC6 for 1 h, 80% of the microtubule network was acetylated, compared to only 36% of the network in control conditions (Fig. 5a-d and Supplementary Fig. 5e, f). This confirms previous findings that compared to other tubulin deacetylases like Sirt2, HDAC6 is a potent deacetylase of the interphase microtubule network 34 . ...
The properties of single microtubules within the microtubule network can be modulated through post-translational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could enter through the microtubule ends and at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen can be modulated by kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase αTAT1 is independent of kinesin-1-caused shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de)acetylation and scales with the size of the cells. The control of shaft damage represents a mechanism to regulate PTMs inside the microtubule by giving access to the lumen.
... Самыми главными результатами представляемого исследования можно считать: 1) Выявление этапов и корреляций с эмбриогенезом сердца в развитии, созревании и функциональности кардиомиоцитов пациента, полученных in vitro в процессе дифференцировки [4]. 2) Создание тестирования, выявляющего риск возникновения аритмий как под воздействием как внешних факторов, так и врожденных пациент-специфичных патологий [5,6] Вариабельные механические характеристики поли-ɛ-капролактона (ПКЛ) и простота манипуляций с полимером позволили адаптировать материал для широкого спектра задач регенеративной медицины. К тому же введение дополнительных функциональных групп в молекулу полимера в процессе модификации позволяет повысить биосовместимость ПКЛ и минимизировать негативный эффект таких его свойств, как гидрофобность, отсутствие функциональных групп и в целом синтетическую природу материала [1]. ...
... ВВ и КСБ добавляли одновременно с ЛПС, секрецию NO определяли через 24 часа с помощью реакции Грисса. Статистический анализ выполняли в SigmaPlot 12. 5 [1] кардиомиоцитах, которые получают из стволовых клеток человека. Эти клетки используют для тестирования лекарств и изучения механизмов возникновения аритмий. ...
... В дифференцированных клетках также значительно повышалась экспрессия гена Slc25a51, кодирующего митохондриальный переносчик, опосредующий импорт NAD из цитозоля в митохондриальный матрикс. Более того, с помощью иммуноблоттинга мы продемонстрировали, что уровень ацетилирования альфа-тубулина по лизину 40 -мишени NAD-зависимой цитозольной деацетилазы белков Sirt2 [5] -в дифференцированных клетках значительно превышает таковой в клетках Е14, находящихся в плюрипотентном состоянии. Это может быть связано с тем, что в процессе дифференцировки уровень NAD в цитозоле снижается, что, в свою очередь, приводит к подавлению активности Sirt2. ...
В процессе длительного культивирования мезенхимные стволовые клетки (МСК) человека подвержены репликативному старению (РС), которое может серьезно ухудшить их функции как in vitro, так и in vivo [1]. Поэтому понимание молекулярных процессов, сопровождающих РС МСК, имеет решающее значение для применения МСК в регенеративной медицине. Активные формы кислорода (АФК) в физиологических концентрациях регулируют пролиферацию и дифференцировку МСК, однако избыточное накопление АФК вызывает окислительный стресс и приводит к патологическим последствиям для клеток. Для защиты от избыточных АФК клетки синтезируют восстановленный глутатион (GSH), при этом в процессе РС в МСК происходит значительное снижение уровня GSH [2]. Малые ГТФазы Rho-семейства регулируют окислительно- восстановительное состояние клеток, контролируя ферменты, которые генерируют и преобразуют АФК [3]. Ранее нами были показано, что в процессе РС в клетках MSCWJ-1 происходит изменение субклеточной локализации RhoA [4].
Цель работы – анализ изменений уровня GSH в МСК человека под влиянием модуляторов RhoA-ассоциированных сигнальных путей (LPA – активатор RhoA; Y-27632 – ингибитор киназы ROCK1 на разных стадиях РС.
В работе использовали линию MSCWJ-1, выделенную из Вартонова студня пупочного канатика человека и полученную из ЦКП “Коллекция культур клеток позвоночных” ИНЦ РАН. Уровни GSH в живых клетках определяли с помощью флуоресцецнтного зонда FreSHtracer и системы цитометрии Image ExFluorer (LCI, Южная Корея). Клетки на 12 и 38 пассажах высевали на 24-луночные культуральные планшеты, инкубировали с FreSHtracer в течение 1,5 ч, затем измеряли соотношение флуоресценции (F510/F580) для каждой клетки (n>400). Результаты анализировали с использованием GraphPad Prism версии 8.4.1 для Windows (GraphPad Software, США).
Результаты исследования демонстрируют, что РС клеток линии MSCWJ-1 сопровождается значительным снижением уровня восстановленного GSH в старых клетках по сравнению с молодыми. LPA (1 μM) в молодых клетках вызывает снижение уровня GSH, а в старых клетках – его увеличение. Y-27632 (10 μM) вызывает значительное увеличение уровня GSH как на раннем, так и на позднем пассаже в активной стадии РС. Исходя из полученных результатов можно предположить, что РС МСК сопровождается изменением количества рецепторов LPA.
1. Childs B. G. et al. Cellular senescence in aging and age-related disease: from mechanisms to therapy // Nature medicine. – 2015. – Т. 21. – No. 12. – С. 1424-1435.
2. Benjamin D. I. et al. Multiomics reveals glutathione metabolism as a driver of bimodality during stem cell aging // Cell Metabolism. – 2023. – Т. 35. – No. 3. – С. 472-486. e6.
3. Hobbs G. A. et al. Rho GTPases, oxidation, and cell redox control // Small GTPases. – 2014. – Т. 5. – No. 2. – С. e28579.
4. Bobkov D. et al. Replicative senescence in MSCWJ-1 human umbilical cord mesenchymal stem cells is marked by characteristic changes in motility, cytoskeletal organization, and RhoA localization // Molecular Biology Reports. – 2020. – Т. 47. – No. 5. – С. 3867-3883.
... In human, seven sirtuins have been identified grouping them into four phylogentic branches, i.e., class 1 (sirtuins 1-3), class 2 (SIRT4), class 3 (SIRT5), and class 4 (sirtuins 6 and 7) [2,3]. These proteins function in epigenetic regulation and gene expression control in the nucleus (SIRT1, 2, 6, and 7; [4]), microtubule dynamics (SIRT2, SIRT4; [5][6][7]), proliferation/cell survival, senescence and aging (e.g. SIRT4 and SIRT6; [8,9]), life-span regulation (e.g. ...
SIRT4, together with SIRT3 and SIRT5, comprises the mitochondrially localized subgroup of sirtuins. SIRT4 regulates mitochondrial bioenergetics, dynamics (mitochondrial fusion), and quality control (mitophagy) via its NAD ⁺ ‐dependent enzymatic activities. Here, we address the regulation of SIRT4 itself by characterizing its protein stability and degradation upon CoCl 2 ‐induced pseudohypoxic stress that typically triggers mitophagy. Interestingly, we observed that of the mitochondrial sirtuins, only the protein levels of SIRT4 or ectopically expressed SIRT4‐eGFP decrease upon CoCl 2 treatment of HEK293 cells. Co‐treatment with BafA1, an inhibitor of autophagosome–lysosome fusion required for autophagy/mitophagy, or the use of the proteasome inhibitor MG132, prevented CoCl 2 ‐induced SIRT4 downregulation. Consistent with the proteasomal degradation of SIRT4, the lysine mutants SIRT4(K78R) and SIRT4(K299R) showed significantly reduced polyubiquitination upon CoCl 2 treatment and were more resistant to pseudohypoxia‐induced degradation as compared to SIRT4. Moreover, SIRT4(K78R) and SIRT4(K299R) displayed increased basal protein stability as compared to wild‐type SIRT4 when subjected to MG132 treatment or cycloheximide (CHX) chase assays. Thus, our data indicate that stress‐induced protein degradation of SIRT4 occurs through two mechanisms: (a) via mitochondrial autophagy/mitophagy, and (b) as a separate process via proteasomal degradation within the cytoplasm.
... Here, we focused, for the first time, on the earliest step of α-synuclein aggregation that gives rise to the formation of α-synuclein oligomers. Furthermore, we inhibited the acetylase activity of HDAC6, and not SIRT2, known to deacetylate different subsets of acetylated microtubules [56]. The accumulation of acetylated α-tubulin, caused by the inhibition of HDAC6 deacetylase activity, induced an increase in α-synuclein oligomers, indicating that the alteration in this post-translational modification of the microtubule cytoskeleton could lead to the aggregation of α-synuclein and thus be implicated in PD pathology. ...
Emerging evidence supports that altered α-tubulin acetylation occurs in Parkinson’s disease (PD), a neurodegenerative disorder characterized by the deposition of α-synuclein fibrillary aggregates within Lewy bodies and nigrostriatal neuron degeneration. Nevertheless, studies addressing the interplay between α-tubulin acetylation and α-synuclein are lacking. Here, we investigated the relationship between α-synuclein and microtubules in primary midbrain murine neurons and the substantia nigra of post-mortem human brains. Taking advantage of immunofluorescence and Proximity Ligation Assay (PLA), a method allowing us to visualize protein–protein interactions in situ, combined with confocal and super-resolution microscopy, we found that α-synuclein and acetylated α-tubulin colocalized and were in close proximity. Next, we employed an α-synuclein overexpressing cellular model and tested the role of α-tubulin acetylation in α-synuclein oligomer formation. We used the α-tubulin deacetylase HDAC6 inhibitor Tubacin to modulate α-tubulin acetylation, and we evaluated the presence of α-synuclein oligomers by PLA. We found that the increase in acetylated α-tubulin significantly induced α-synuclein oligomerization. In conclusion, we unraveled the link between acetylated α-tubulin and α-synuclein and demonstrated that α-tubulin acetylation could trigger the early step of α-synuclein aggregation. These data suggest that the proper regulation of α-tubulin acetylation might be considered a therapeutic strategy to take on PD.
... However, it is possible that the two enzymes catalyze the deacetylation of different subsets of MTs. In fact, Skoge and Ziegler [236] found that HDAC6 inhibition led to a general hyperacetylation of the MT network throughout the cell, whereas hyperacetylation induced by SIRT2 inactivation was limited to perinuclear MTs. Following HDAC6 overexpression, hyperacetylation of these perinuclear MTs was maintained, while reactivation of SIRT2 restored the basal acetylation level and a normal MT network. ...
Microtubules (MTs), dynamic polymers of α/β-tubulin heterodimers found in all eukaryotes, are involved in cytoplasm spatial organization, intracellular transport, cell polarity, migration and division, and in cilia biology. MTs functional diversity depends on the differential expression of distinct tubulin isotypes and is amplified by a vast number of different post-translational modifications (PTMs). The addition/removal of PTMs to α- or β-tubulins is catalyzed by specific enzymes and allows combinatory patterns largely enriching the distinct biochemical and biophysical properties of MTs, creating a code read by distinct proteins, including microtubule-associated proteins (MAPs), which allow cellular responses. This review is focused on tubulin-acetylation, whose cellular roles continue to generate debate. We travel through the experimental data pointing to α-tubulin Lys40 acetylation role as being a MT stabilizer and a typical PTM of long lived MTs, to the most recent data, suggesting that Lys40 acetylation enhances MT flexibility and alters the mechanical properties of MTs, preventing MTs from mechanical aging characterized by structural damage. Additionally, we discuss the regulation of tubulin acetyltransferases/desacetylases and their impacts on cell physiology. Finally, we analyze how changes in MT acetylation levels have been found to be a general response to stress and how they are associated with several human pathologies.
... SIRT2 and HDAC6 can deacetylate α-tubulin either together [94], or separately [93]. Interestingly, the inhibition of SIRT2 increased acetylation of the microtubular α-tubulin mainly in the perinuclear zone, whereas inhibition of HDAC6 caused the general hyperacetylation of microtubules throughout the cell [95]. Although some studies pointed to the pathological role of SIRT2 after cerebral ischemia [76,96], the functions of SIRT2 in the ischemic brain are possibly more complicated than only pathological or only neuroprotective. ...
Cerebral ischemia is the second leading cause of death in the world and multimodal stroke therapy is needed. The ischemic stroke generally reduces the gene expression due to suppression of acetylation of histones H3 and H4. Histone deacetylases inhibitors have been shown to be effective in protecting the brain from ischemic damage. Histone deacetylases inhibitors induce neurogenesis and angiogenesis in damaged brain areas promoting functional recovery after cerebral ischemia. However, the role of different histone deacetylases isoforms in the survival and death of brain cells after stroke is still controversial. This review aims to analyze the data on the neuroprotective activity of nonspecific and selective histone deacetylase inhibitors in ischemic stroke.
... We reasoned that disrupting tubulin deacetylating enzymes may affect cilia morphology and function, as inhibition of Hdac6 was shown to stabilize cilia from regulated resorption (Luxton and Gundersen, 2007;Pugacheva et al., 2007;de Diego et al., 2014;Ran et al., 2015). To evaluate acetylation of ciliary microtubules, we immunostained triple mutant embryos using anti-acetyl-α-tubulin (Lys40) (D20G3), a well-characterized and highly specific antibody that recognizes acetylated tubulin and is commonly used in cilia visualization (Skoge and Ziegler, 2016;Viau et al., 2018;Sheu et al., 2019;Zhang et al., 2019). This analysis revealed increased acetylation of cytoplasmic microtubules in triple mutants compared to wild type ( Figure 3A and Supplementary Figure 2). ...
Cilia are evolutionarily highly conserved organelles with important functions in many organs. The extracellular component of the cilium protruding from the plasma membrane comprises an axoneme composed of microtubule doublets, arranged in a 9 + 0 conformation in primary cilia or 9 + 2 in motile cilia. These microtubules facilitate transport of intraflagellar cargoes along the axoneme. They also provide structural stability to the cilium, which may play an important role in sensory cilia, where signals are received from the movement of extracellular fluid. Post-translational modification of microtubules in cilia is a well-studied phenomenon, and acetylation on lysine 40 (K40) of alpha tubulin is prominent in cilia. It is believed that this modification contributes to the stabilization of cilia. Two classes of enzymes, histone acetyltransferases and histone deacetylases, mediate regulation of tubulin acetylation. Here we use a genetic approach, immunocytochemistry and behavioral tests to investigate the function of tubulin deacetylases in cilia in a zebrafish model. By mutating three histone deacetylase genes (Sirt2, Hdac6, and Hdac10), we identify an unforeseen role for Hdac6 and Sirt2 in cilia. As expected, mutation of these genes leads to increased acetylation of cytoplasmic tubulin, however, surprisingly it caused decreased tubulin acetylation in cilia in the developing eye, ear, brain and kidney. Cilia in the ear and eye showed elevated levels of mono-glycylated tubulin suggesting a compensatory mechanism. These changes did not affect the length or morphology of cilia, however, functional defects in balance was observed, suggesting that the level of tubulin acetylation may affect function of the cilium.
... In accordance with previous observations, ⍺TAT1 was shown to have a higher affinity for and a higher catalytic rate on polymerized microtubules compared to unpolymerized tubulin heterodimers [25][26][27] . Lys40 acetylation can be removed, primarily by histone deacetylase 6 (HDAC6), but also by NAD-dependent deacetylase sirtuin 2 (SIRT2) (Fig. 2B) [28][29][30] . Recently, it has been shown that HDAC6 preferentially acts on free tubulin heterodimers, but can also target polymerized microtubules 31 . ...
The cytoskeleton plays important roles in many essential processes at the cellular and organismal levels, including cell migration and motility, cell division, and the establishment and maintenance of cell and tissue architecture. In order to facilitate these varied functions, the main cytoskeletal components – microtubules, actin filaments, and intermediate filaments – must form highly diverse intracellular arrays in different subcellular areas and cell types. The question of how this diversity is conferred has been the focus of research for decades. One key mechanism is the addition of post-translational modifications (PTMs) to the major cytoskeletal proteins. This post-translational addition of various chemical groups dramatically increases the complexity of the cytoskeletal proteome and helps facilitate major global and local cytoskeletal functions. Cytoskeletal proteins undergo many PTMs, most of which are not well understood. Recent technological advances in proteomics and cell biology have allowed for the in-depth study of individual PTMs and their functions in the cytoskeleton. Here, we provide an overview of the major PTMs that occur on the main structural components of the three cytoskeletal systems – tubulin, actin, and intermediate filament proteins – and highlight the cellular function of these modifications.
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... Acetylation and deacetylation of microtubules is a dynamic process and post translational modifications including acetylation balance at α-tubulin K40 have been suggested to affect microtubule stability and flexibility in turn minimizing microtubule aging and optimizing resilience [209][210][211][212][213]. Acetyl α-tubulin K40 is unique since it is the only lysine residue in the internal lumen of the microtubule. HDAC6 regulates the majority of microtubule deacetylation at K40 but SIRT2 also plays a significant K40 deacetylation role in the perinuclear region of the cell [214]. High intensity immunostaining for NQO1 in cells was reflective of an oxidized pyridine nucleotide environment in cells immunostaining and proximity ligation assays showed co-localization of NQO1 with acetylated microtubules and SIRT2 [177]. ...
In this review, we summarize the multiple functions of NQO1, its established roles in redox processes and potential roles in redox control that are currently emerging. NQO1 has attracted interest due to its roles in cell defense and marked inducibility during cellular stress. Exogenous substrates for NQO1 include many xenobiotic quinones. Since NQO1 is highly expressed in many solid tumors, including via upregulation of Nrf2, the design of compounds activated by NQO1 and NQO1-targeted drug delivery have been active areas of research. Endogenous substrates have also been proposed and of relevance to redox stress are ubiquinone and vitamin E quinone, components of the plasma membrane redox system. Established roles for NQO1 include a superoxide reductase activity, NAD⁺ generation, interaction with proteins and their stabilization against proteasomal degradation, binding and regulation of mRNA translation and binding to microtubules including the mitotic spindles. We also summarize potential roles for NQO1 in regulation of glucose and insulin metabolism with relevance to diabetes and the metabolic syndrome, in Alzheimer’s disease and in aging. The conformation and molecular interactions of NQO1 can be modulated by changes in the pyridine nucleotide redox balance suggesting that NQO1 may function as a redox-dependent molecular switch.
... Immunostaining for acetyl α-tubulin confirmed the immunoblot data and showed that following treatment with FK866 there was a gradual increase in immunostaining for acetyl α-tubulin in both cell lines (Fig. 1C). These data also showed that the increase in immunostaining for acetyl α-tubulin initially appeared in perinuclear regions, which is consistent with previous studies that have suggested that SIRT2 may be responsible for regulating acetylation of perinuclear microtubules [27]. We have confirmed the perinuclear localization of SIRT2 and NQO1 in 16HBE cells (Fig. 1D). ...
... Tubulin acetyltransferase aTAT1 and the deacetylase HDAC6 regulate a large majority of the microtubule acetylome including α-tubulin K 40 but smaller roles for other deacetylases have emerged [33]. A role for SIRT2 in regulating acetyl α-tubulin K 40 levels in a subset of perinuclear microtubules has been described [27]. ...
The localization of NQO1 near acetylated microtubules has led to the hypothesis that NQO1 may work in concert with the NAD⁺-dependent deacetylase SIRT2 to regulate acetyl α-tubulin (K⁴⁰) levels on microtubules. NQO1 catalyzes the oxidation of NADH to NAD⁺ and may supplement levels of NAD⁺ near microtubules to aid SIRT2 deacetylase activity. While HDAC6 has been shown to regulate the majority of microtubule acetylation at K⁴⁰, SIRT2 is also known to modulate microtubule acetylation (K⁴⁰) in the perinuclear region. In this study we examined the potential roles NQO1 may play in modulating acetyl α-tubulin levels. Knock-out or knock-down of NQO1 or SIRT2 did not change the levels of acetyl α-tubulin in 16HBE human bronchial epithelial cells and 3T3-L1 fibroblasts; however, treatment with a mechanism-based inhibitor of NQO1 (MI2321) led to a short-lived temporal increase in acetyl α-tubulin levels in both cell lines without impacting the intracellular pools of NADH or NAD⁺. Inactivation of NQO1 by MI2321 resulted in lower levels of NQO1 immunostaining on microtubules, consistent with redox-dependent changes in NQO1 conformation as evidenced by the use of redox-specific, anti-NQO1 antibodies in immunoprecipitation studies. Given the highly dynamic nature of acetylation-deacetylation reactions at α-tubulin K⁴⁰ and the crowded protein environment surrounding this site, disruption in the binding of NQO1 to microtubules may temporally disturb the physical interactions of enzymes responsible for maintaining the microtubule acetylome.
