Α-tubulin Acetylation Research Articles

Drosophila CG17003/leaky (lky) is required for microtubule acetylation in early germ cells in Drosophila ovary.

Microtubule acetylation is found in populations of stable, long-lived microtubules, occurring on the conserved lysine 40 (K40) residue of α-tubulin by α-tubulin acetyltransferases (αTATs). α-tubulin K40 acetylation has been shown to stabilize microtubules via enhancing microtubule resilience against mechanical stress. Here we show that a previously uncharacterized αTAT, Drosophila CG17003/leaky (lky), is required for α-tubulin K40 acetylation in early germ cells in Drosophila ovary. We found that loss of lky resulted in a progressive egg chamber fusion phenotype accompanied with mislocalization of germline-specific Vasa protein in somatic follicle cells. The same phenotype was observed upon replacement of endogenous α-tubulin84B with non-acetylatable α-tubulin84BK40A, suggesting α-tubulin K40 acetylation is responsible for the phenotype. Chemical disturbance of microtubules by Colcemid treatment resulted in a mislocalization of Vasa in follicle cells within a short period of time (~30 min), suggesting that the observed mislocalization is likely caused by direct leakage of cellular contents between germline and follicle cells. Taken together, this study provides a new function of α-tubulin acetylation in maintaining the cellular identity possibly by preventing the leakage of tissue-specific gene products between juxtaposing distinct cell types.

Identification of novel aza-analogs of TN-16 as disrupters of microtubule dynamics through a multicomponent reaction

Despite novel biological targets emerging at an impressive rate for anticancer therapy, antitubulin drugs remain the backbone of numerous oncological protocols and their efficacy has been demonstrated in a wide variety of adult and pediatric cancers. In the present contribution, we set to develop analogs of a potent but neglected antitubulin agent, TN-16, originally discovered via modification of tenuazonic acid (3-acetyl-5-sec-butyltetramic acid).To this extent, we developed a novel multicomponent reaction to prepare TN-16, and then we applied the same reaction for the synthesis of aza-analogs. In brief, we prepared a library of 62 novel compounds, and three of these retained nanomolar potencies. TN-16 and the active analogs are cytotoxic on cancer cell lines and, as expected from antitubulin agents, induce G2/M cell cycle arrest. These agents lead to a disruption of the microtubules and an increase in α-tubulin acetylation and affect in vitro polymerization, although they have a lesser effect in cellular tubulin polymerization assays.

Panobinostat Synergistically Enhances the Cytotoxicity of Microtubule Destabilizing Drugs in Ovarian Cancer Cells.

Ovarian cancer (OC) is one of the most common gynecologic neoplasia and has the highest mortality rate, which is mainly due to late-stage diagnosis and chemotherapy resistance. There is an urgent need to explore new and better therapeutic strategies. We have previously described a family of Microtubule Destabilizing Sulfonamides (MDS) that does not trigger multidrug-mediated resistance in OC cell lines. MDS bind to the colchicine site of tubulin, disrupting the microtubule network and causing antiproliferative and cytotoxic effects. In this work, a novel microtubule-destabilizing agent (PILA9) was synthetized and characterized. This compound also inhibited OC cell proliferation and induced G2/M cell cycle arrest and apoptosis. Interestingly, PILA9 was significantly more cytotoxic than MDS. Here, we also analyzed the effect of these microtubule-destabilizing agents (MDA) in combination with Panobinostat, a pan-histone deacetylase inhibitor. We found that Panobinostat synergistically enhanced MDA-cytotoxicity. Mechanistically, we observed that Panobinostat and MDA induced α-tubulin acetylation and that the combination of both agents enhanced this effect, which could be related to the observed synergy. Altogether, our results suggest that MDA/Panobinostat combinations could represent new therapeutic strategies against OC.

Elongator promotes neuritogenesis via regulation of tau stability through acly activity.

The six subunits (Elp1 to Elp6) Elongator complex promotes specific uridine modifications in tRNA's wobble site. Moreover, this complex has been indirectly involved in the regulation of α-tubulin acetylation in microtubules (MTs) via the stabilization of ATP-Citrate Lyase (Acly), the main cytosolic source of acetyl-CoA production in cells, a key substrate used for global protein acetylation. Here, we report additional evidence that Elongator activity is important for proper cytoskeleton remodeling as cells lacking expression of Elp1 show morphology impairment; including distinct neurite process formation and disorganization and instability of MTs. Here, we show that loss of Elongator results in a reduction of expression of the microtubule associated protein Tau (MAPT). Tau, is a well-known key MT regulator in neurons whose lysines can be competitively acetylated or ubiquitylated. Therefore, we tested whether Tau is an indirect acetylation target of Elongator. We found that a reduction of Elongator activity leads to a decrease of lysine acetylation on Tau that favors its proteasomal degradation. This phenotype was prevented by using selective deacetylase or proteasomal inhibitors. Moreover, our data demonstrate that Acly's activity regulates the mechanism underlying Tau mediated neurite morphology defects found in Elp1 KD since both Tau levels and neurites morphology are restored due to Acly overexpression. This suggests a possible involvement of both Tau and Acly dysfunction in Familial Dysautonomia (FD), which is an autosomal recessive peripheral neuropathy caused by mutation in the ELP1 gene that severely affects Elp1 expression levels in the nervous system in FD patients in a similar way as found previously in Elp1 KD neuroblastoma cells.

Design, synthesis and biological evaluation of novel pyrazinone derivatives as PI3K/HDAC dual inhibitors

PI3Ks and HDACs play essential roles in the occurrence and progression of leukemia. Herein, a series of novel pyrazin-2(1H)-one derivatives were rationally designed and synthesized as novel dual PI3K and HDAC inhibitors based on scaffold replacement and heterozygous strategies. Most of the target compounds showed potent inhibitory potency to PI3Kα and HDAC6. Especially, compound 9q displayed PI3Kα and HDAC6 inhibitory with IC50 values of 372 nM and 4.5 nM, and anti-proliferative activity against MV4-11 cells with IC50 value of 0.093 ± 0.012 μM. Further mechanistic studies revealed that 9q induced apoptosis, arrested the cell cycle in the G2/M phase, promoted the acetylation of α-tubulin, and blocked the PI3K/AKT/mTOR signal way in MV4-11 cells. All the results demonstrated that 9q was a promising lead candidate for further development of novel PI3K/HDAC dual inhibitors for leukemia treatment.

Combination of Paclitaxel and PXR Antagonist SPA70 Reverses Paclitaxel-Resistant Non-Small Cell Lung Cancer.

Paclitaxel (PTX) is one of the most efficient drugs for late-stage non-small cell lung cancer (NSCLC) patients. However, most patients gradually develop resistance to PTX with long-term treatments. The identification of new strategies to reverse PTX resistance in NSCLC is crucially important for the treatment. PTX is an agonist for the pregnane X receptor (PXR) which regulates PTX metabolism. Antagonizing PXR, therefore, may render the NSCLC more sensitive to the PTX treatment. In this study, we investigated the PXR antagonist SPA70 and its role in PTX treatment of NSCLC. In vitro, SPA70 and PTX synergistically inhibited cell growth, migration and invasion in both paclitaxel-sensitive and paclitaxel-resistant A549 and H460 lung cancer cells. Mechanistically, we found PTX and SPA70 cotreatment disassociated PXR from ABCB1 (MDR1, P-gp) promoter, thus inhibiting P-gp expression. Furthermore, the combination regimen synergistically enhanced the interaction between PXR and Tip60, which abrogated Tip60-mediated α-tubulin acetylation, leading to mitosis defect, S-phase arrest and necroptosis/apoptosis. Combination of PXT and SPA70 dramatically inhibited tumor growth in a paclitaxel-resistant A549/TR xenograft tumor model. Taken together, we showed that SPA70 reduced the paclitaxel resistance of NSCLC. The combination regimen of PTX and SPA70 could be potential novel candidates for the treatment of taxane-resistant lung cancer.

Psychosocial stress and cannabinoid drugs affect acetylation of α-tubulin (K40) and gene expression in the prefrontal cortex of adult mice.

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.

Vimentin inhibits α-tubulin acetylation via enhancing α-TAT1 degradation to suppress the replication of human parainfluenza virus type 3.

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.

Cystathionine β-synthase is required for oocyte quality by ensuring proper meiotic spindle assembly.

Poor oocyte quality is detrimental to fertilization and embryo development, which causes infertility. Cystathionine β-synthase (CBS) is one of the key enzymes modulating the metabolism of homocysteine (Hcy). Studies have shown that CBS plays an important role in female reproduction. However, the role of CBS in regulating oocyte quality during meiotic maturation still needs further investigation. Immunohistochemistry, immunofluorescence, drug treatment, western blot, cRNA construct and in vitro transcription, microinjection of morpholino oligo and cRNA were performed for this study. We found that CBS was expressed both in human and mouse oocytes of follicles. In mouse oocytes, CBS was distributed in the nucleus at germinal vesicle (GV) stage and localized to spindle from germinal vesicle breakdown (GVBD) to metaphase II (MII). The expression of CBS was reduced in ovaries and oocytes of aged mice. CBS depletion resulted in meiotic arrest, spindle abnormality and chromosome misalignment, disrupted kinetochore-microtubule attachments and provoked spindle assembly checkpoint (SAC). CBS was disassembled when microtubules were disrupted with nocodazole, and co-localized with the stabilized microtubules after taxol treatment. Furthermore, CBS depletion decreased the acetylation of α-tubulin. These results reveal that CBS is required for the acetylation of α-tubulin to ensure proper spindle assembly in regulating oocyte quality during meiotic maturation.

Discovery of 2,5-diphenyl-1,3,4-thiadiazole derivatives as HDAC inhibitors with DNA binding affinity

Incorporating a DNA-binding fragment in HDAC inhibitors has been proved to be an effective strategy for the treatment of hematologic malignancies by our group. However, similar to other approved HDAC inhibitors, their effects on solid tumor were poor. For this issue, a series of 2,5-diphenyl-1,3,4-thiadiazole hydroxamate derivatives were designed and synthesized as the HDAC inhibitors with DNA binding affinity. Among the target compounds, 4j not only bound with DNA effectively but also exhibited the most potent inhibitory activity against HDAC1 with the IC50 of 15 nM. Compared to SAHA, compound 4j displayed stronger antiproliferative activity in tested tumor cell lines. Western blot analysis showed that 4j could enhance the acetylation of histone H3 and α-tubulin, as well as promote the activation of caspase 3 in HCT116 and MC38 cell lines. Furthermore, these responses resulted in significant suppression of tumor growth in the MC38 tumor model. This work validated that compound 4j was a promising lead compound for further structural optimization.

The Role of Runx2 in Microtubule Acetylation in Bone Metastatic Breast Cancer Cells.

Simple SummaryBreast cancer is the most commonly diagnosed cancer type, making up a quarter of all cases among women. While modern-day research has shown tremendous progress in the development of treatment options, complications related to bone metastasis remain an obstacle that demands further attention. Cancer cells that migrate into the bone microenvironment show significant alterations in their metabolism and gene expression profiles. In this study, we set out to better understand the changes induced by Runx2 in bone-derived breast cancer cells. We identified key changes in the stability of the microtubule cytoskeleton of bone-derived cells expressing Runx2. These changes have implications on a variety of processes related to metabolism, cellular stress response, and response to common chemotherapies. These studies help to shed light on the changes that take place as a tumor begins to metastasize and to better predict which therapies will benefit patients with bone metastasis.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.

Histone deacetylase inhibitor, panobinostat, exerts anti-proliferative effect with partial normalization from aberrant epigenetic states on granulosa cell tumor cell lines.

The prognosis of the patients with inoperable or advanced granulosa cell tumors (GCTs) is still poor, and therefore it is important to establish a novel treatment strategy. Here we investigated the in vitro effects of a histone deacetylase inhibitor, panobinostat (PS) on two GCT cell lines (KGN and COV434). GCT cell lines were found to be susceptible to PS treatment and it inhibited cell growth mainly by apoptosis. In cell cycle analysis, PS reduced only the ratio of S phase in GCT cell lines. Combined treatment of PS with a deubiquitinase inhibitor, VLX1570 enhanced the expression of p21, cleaved PARP, cleaved caspase-9, heme oxygenase-1, and the acetylation of histone H4 and α-tubulin, leading to an additive anti-proliferative effect on KGN and COV434. The gene set enrichment analysis revealed that PS treatment suppressed DNA replication- or cell cycle-related gene expression which led to chemotherapeutic cell death and in addition, this treatment induced activation of the gene set of adherens junction towards a normalized direction as well as activation of neuron-related gene sets that might imply unexpected differentiation potential due to epigenetic modification by a HDAC inhibitor in KGN cells. Exposure of KGN and COV434 cells to PS increased the expression of E-cadherin, one of the principal regulators associated with adherens junction in quantitative RT-PCR and immunoblotting analysis. In the present study, we indicate a basis of a novel therapeutic availability of a HDAC inhibitor for the treatment of GCTs and further investigations will be warranted.

Acetylation of α-tubulin on Lys40, a new way to modulate cardiac glucose transport

Cardiovascular disease remains the leading cause of death in the diabetic population. Diabetic patients develop a specific heart condition called diabetic cardiomyopathy that can evolve to heart failure and that is characterized by altered metabolism in the early stages of the disease. Our group previously demonstrated that overfueling the heart with branched-chain amino acids, ketone bodies or fatty acids, substrates that are upregulated in diabetes, inhibits glucose entry in cardiomyocytes through a global rise in protein acetylation that interferes with glucose transporter 4 (GLUT4) translocation to the plasma membrane. However, the acetylated proteins involved in this process were not yet identified. We investigated the role of α-tubulin, an important cytoskeleton element responsible for GLUT4 translocation, that is known to be acetylable on Lys40 (K40). Acetylation level of α-tubulin on K40 was evaluated in a mouse model of diabetes (high-fat diet, 4 months) and in primary cultured adult rat cardiomyocytes. Its acetylation level was modulated by using a pharmacological inhibitor of its deacetylase (tubacin 10 μM, 5 hrs) and by overexpressing a non-acetylable form of α-tubulin (mCherry α-tubulin K40A). GLUT4 translocation was assessed by fluorescent immunostaining in cells expressing the fusion protein HA-GLUT4-GFP. Glucose transport was measured by following the detritiation rate of 2-3H-glucose in insulin-sensitive and insulin-resistant cardiomyocytes. Acetylation of α-tubulin on K40 was significantly enhanced in the heart of diet-induced diabetic mice. Experimentally increasing its acetylation level in cardiomyocytes with tubacin impaired GLUT4 translocation to the plasma membrane and reduced both basal and insulin-dependent glucose uptake. On the other hand, decreasing α-tubulin K40 acetylation via the overexpression of the K40A form of α-tubulin stimulated glucose transport. Interestingly, reducing α-tubulin K40 acetylation similarly promoted glucose entry in insulin-resistant conditions. Cardiac glucose transport can be regulated by modulation of α-tubulin K40 acetylation, which may lead to new therapeutic strategies preventing the metabolic changes occurring during the development of diabetic cardiomyopathy.

소포체 스트레스에 의한 미세소관 중합 저해에 대한 국산 프로폴리스의 효과

Microtubules (MTs) are a major components of cytoskeletal structure. It is biopolymers formed through polymerization of heterodimers of α-tubulin and β-tubulin. MTs play a critical role for maintain of cell shape, cell division, migration and transport of materials in the cells. Endoplasmic reticulum (ER) stress disrupts posttranslational modification of protein and consequently inhibits the formation of MTs structure. Here, we investigated the improving effect of Korean propolis for microtubule polymerization during ER stress by tunicamycin. Tunicamycin and propolis concentration for PC12 cell treatment is 150 μg/mL and 10 μg/mL, respectively. Tunicamycin treatment decreases the expression level of acetylation of α-tubulin and it was inhibits MTs formation in tubulin polymerization assay. However, although the presence of tunicamycin, propolis extracts recovers acetylation of α-tubulin level and polymerized β-tubulin. Especially, pre-treatment of propolis group exhibits higher polymerization level more than tunicamycin pre-treatment group. Fluorescence microscopy analysis shows the MTs structure recovery by propolis treatment. Our results suggested that propolis can be prevents the apoptosis, allowing protects the cells.

α-Tubulin acetylation on lysine 40 controls cardiac glucose uptake.

Our group previously demonstrated that an excess of nutrients, as observed in diabetes, provokes an increase in cardiac protein acetylation responsible for a reduced insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The acetylated proteins involved in this event have yet not been identified. α-Tubulin is a promising candidate as a major cytoskeleton component involved, among other things, in the translocation of GLUT4-containing vesicles from their intracellular pools toward the plasma membrane. Moreover, α-tubulin is known to be acetylated, Lys40 (K40) being its best characterized acetylated residue. The present work sought to evaluate the impact of α-tubulin K40 acetylation on cardiac glucose entry, with a particular interest in GLUT4 translocation. First, we observed that a mouse model of high-fat diet-induced obesity presented an increase in cardiac α-tubulin K40 acetylation level. We next showed that treatment of insulin-sensitive primary cultured adult rat cardiomyocytes with tubacin, a specific tubulin acetylation inducer, reduced insulin-stimulated glucose uptake and GLUT4 translocation. Conversely, decreasing α-tubulin K40 acetylation by expressing a nonacetylable dominant form of α-tubulin (mCherry α-tubulin K40A mutant) remarkably intensified insulin-induced glucose transport. Finally, mCherry α-tubulin K40A expression similarly improved glucose transport in insulin-resistant cardiomyocytes or after AMP-activated protein kinase activation. Taken together, our study demonstrates that modulation of α-tubulin K40 acetylation level affects glucose transport in cardiomyocytes, offering new putative therapeutic insights regarding modulation of glucose metabolism in insulin-resistant and diabetic hearts.NEW & NOTEWORTHY Acetylation level of α-tubulin on K40 is increased in the heart of a diet-induced mouse model of type 2 diabetes. Pharmacological stimulation of α-tubulin K40 acetylation lowers insulin-mediated GLUT4 vesicles translocation to the plasma membrane, reducing glucose transport. Expressing a nonacetylable dominant form of α-tubulin boosts glucose uptake in both insulin-sensitive and insulin-resistant cardiomyocytes.

Microtubule depolymerization limits porcine betacoronavirus PHEV replication

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a typical neurotropic betacoronavirus causing digestive disease and/or neurological dysfunction in neonatal pigs. Actin filaments have been identified to implicate in PHEV invasion, but the effects of viral infection on microtubules (MTs) cytoskeleton are unknown. Here, we observed that PHEV infection induced MT depolymerization and was accompanied by the disappearance of microtubule organizing centers. Depolymerization of MTs induced by nocodazole significantly inhibited viral RNA replication, but over-polymerization of MTs induced by paclitaxel did not substantially affect PHEV infection. The expression of histone deacetylase 6 (HDAC6), an important regulator of MT acetylation, progressively increased during PHEV infection. Tramstatin A could alter HDAC6 deacetylase activity to enhance the acetylation of the substrate α-tubulin and MT polymerization, but does not increase PHEV proliferation. These findings suggest that PHEV could subvert host MT cytoskeleton to facilitate infection, and that MT depolymerization negatively affects viral replication independently of HDAC6 activity.

Rutin increases alpha-tubulin acetylation via histone deacetylase 6 inhibition.

Microtubules are dynamic cytoskeletal filaments composed of alpha- (α) and beta (β)-tubulin proteins. α-tubulin proteins are posttranslationally acetylated, and loss of acetylation is associated with axonal transport defects, a common alteration contributing to the pathomechanisms of several neurodegenerative diseases. Restoring α-tubulin acetylation by pharmacological inhibition of HDAC6, a primary α-tubulin deacetylase, can rescue impaired transport. Therefore, HDAC6 is considered a promising therapeutic target for neurodegenerative diseases, but currently, there is no clinically approved inhibitor for this purpose. In this study, using drug repurposing strategy, we aimed to identify compounds possessing HDAC6 inhibition activity and inducing α-tubulin acetylation. We systematically analyzed the FDA-approved library by utilizing virtual screening and consensus scoring approaches. Inhibition activities of promising compounds were tested using in vitro assays. Motor neuron-like NSC34 cells were treated with the candidate compounds, and α-tubulin acetylation levels were determined by Western blot. Our results demonstrated that rutin, a natural flavonoid, inhibits cellular HDAC6 activity without inducing any toxicity, and it significantly increases α-tubulin acetylation level in motor neuron-like cells.

Effect of ACY-1215 on cytoskeletal remodeling and histone acetylation in bovine somatic cell nuclear transfer embryos

The latest studies indicated that in addition to alterations in abnormal chromosome epigenetic modifications, the abnormal cytoskeletal changes are also an important cause for the developmental failure of somatic cell nuclear transfer (SCNT) embryos. In the present study, the effects of ACY-1215, a specific inhibitor of HDAC6, on the acetylation of α-tubulin, histone epigenetic modification, spindle formation and embryonic development of early bovine SCNT embryos were studied. The results showed that acetylation of α-tubulin, H3K9, and H4K16 was significantly lower in SCNT embryos than in vitro fertilization (IVF) embryos. After ACY-1215 treatment, the acetylation level of α-tubulin, H3K9, and H4K16 of SCNT embryos was closer to that of IVF embryos. ACY-1215 treatment reduced spindle abnormalities, delayed the time of first cleavage of embryos, increased the total cell number and trophectoderm cells numbers, and reduced apoptosis in SCNT blastocysts. ACY-1215 regulated the process of embryonic epigenetic modification and cytoskeletal protein acetylation, corrected abnormal development of SCNT embryos, and improved SCNT embryonic development potential.

The 19S proteasome subunit Rpt5 reversibly associates with cold-stable microtubules in glial cells at low temperatures.

The ubiquitin-proteasome system (UPS) degrades intracellular proteins through the 26S proteasome. We analysed how cold stress affects the UPS in glial cells. Together with a reduction in the 20S proteolytic activity and increased levels of polyubiquitinated proteins, exposure of glial cell cultures to cold induces a partial disassembly of the 26S proteasome. In particular, we found that Rpt5, a subunit of the 19S proteasome, relocates to cold-stable microtubules, although no apparent cytoskeletal redistribution was detected for other analysed subunits of the 19S or 20S complexes. Furthermore, we demonstrate that both the expression of the microtubule-associated protein MAP6 and the post-translational acetylation of α-tubulin modulate the association of Rpt5 with microtubules. This reversible association could be related to functional preservation of the proteolytic complex during cold stress.

N-Methylpropargylamine-Conjugated Hydroxamic Acids as Dual Inhibitors of Monoamine Oxidase A and Histone Deacetylase for Glioma Treatment.

Glioma treatment remains a challenge with a low survival rate due to the lack of effective therapeutics. Monoamine oxidase A (MAO A) plays a role in glioma development, and MAO A inhibitors reduce glioma growth. Histone deacetylase (HDAC) inhibition has emerged as a promising therapy for various malignancies including gliomas. We have synthesized and evaluated N-methylpropargylamine-conjugated hydroxamic acids as dual inhibitors of MAO A and HDAC. Compounds display potent MAO A inhibition with IC50 from 0.03 to <0.0001 μM and inhibit HDAC isoforms and cell growth in the micromolar to nanomolar IC50 range. These selective MAO A inhibitors increase histone H3 and α-tubulin acetylation and induce cell death via nonapoptotic mechanisms. Treatment with 15 reduced tumor size, reduced MAO A activity in brain and tumor tissues, and prolonged the survival. This first report on dual inhibitors of MAO A and HDAC establishes the basis of translational research for an improved treatment of glioma.