Tau – an inhibitor of deacetylase HDAC6 function

Analysis of brain microtubule protein from patients with Alzheimer's disease showed decreased alpha tubulin levels along with increased acetylation of the alpha tubulin subunit, mainly in those microtubules from neurons containing neurofibrillary tau pathology. To determine the relationship of tau protein and increased tubulin acetylation, we studied the effect of tau on the acetylation-deacetylation of tubulin. Our results indicate that tau binds to the tubulin-deacetylase, histone deacetylase 6 (HDAC6), decreasing its activity with a consequent increase in tubulin acetylation. As expected, increased acetylation was also found in tubulin from wild-type mice compared with tubulin from mice lacking tau because of the tau-mediated inhibition of the deacetylase. In addition, we found that an excess of tau protein, as a HDAC6 inhibitor, prevents induction of autophagy by inhibiting proteasome function.     

Carbamazepine promotes Her-2 protein degradation in breast cancer cells by modulating HDAC6 activity and acetylation of Hsp90

Histone deacetylase 6 (HDAC6) inhibition, recently, has been shown to promote the acetylation of heat-shock protein 90 (Hsp90) and disrupt its chaperone function. Her-2 oncoprotein is identified as a client protein of Hsp90. Therefore, in this study we examined the effect of carbamazepine, which could inhibit HDAC on Hsp90 acetylation and Her-2 stability. The results of this study demonstrate that while carbamazepine had no effect on the Her-2 mRNA level, it induced Her-2 protein degradation via the proteasome pathway by disrupting the chaperone function of Hsp90 in SK-BR-3 cells. Mechanistically, carbamazepine could enhance the acetylation of α-tubulin, indicating its inhibitory effect on HDAC6. Functionally, carbamazepine could synergize with trastuzumab or geldanamycin to promote Her-2 degradation and inhibit breast cancer cell proliferation. Thus, this study has potential clinical implications by providing a promising strategy to overcome the development of resistance against trastuzumab therapy for breast cancer.     

An acetylation site in the middle domain of Hsp90 regulates chaperone function

Heat-shock protein 90 (Hsp90) chaperones a key subset of signaling proteins and is necessary for malignant transformation. Hsp90 is subject to an array of posttranslational modifications that affect its function, including acetylation. Histone deacetylase (HDAC) inhibitors and knockdown of HDAC6 induce Hsp90 acetylation and inhibit its activity. However, direct determination of the functional consequences of Hsp90 acetylation has awaited mapping of specific sites. We now demonstrate that Hsp90 K294 is acetylated. Mutational analysis of K294 shows that its acetylation status is a strong determinant of client protein and cochaperone binding. In yeast, Hsp90 mutants that cannot be acetylated at K294 have reduced viability and chaperone function compared to WT or to mutants that mimic constitutive acetylation. These data suggest that acetylation/deacetylation of K294 plays an important role in regulating the Hsp90 chaperone cycle.     

Histone deacetylase 6 regulates growth factor-induced actin remodeling and endocytosis

Histone deacetylase 6 (HDAC6) is a cytoplasmic deacetylase that uniquely catalyzes α-tubulin deacetylation and promotes cell motility. However, the mechanism underlying HDAC6-dependent cell migration and the role for microtubule acetylation in motility are not known. Here we show that HDAC6-induced global microtubule deacetylation was not sufficient to stimulate cell migration. Unexpectedly, in response to growth factor stimulation, HDAC6 underwent rapid translocation to actin-enriched membrane ruffles and subsequently became associated with macropinosomes, the vesicles for fluid-phase endocytosis. Supporting the importance of these associations, membrane ruffle formation, macropinocytosis, and cell migration were all impaired in HDAC6-deficient cells. Conversely, elevated HDAC6 levels promoted membrane ruffle formation with a concomitant increase in macropinocytosis and motility. In search for an HDAC6 target, we found that heat shock protein 90 (Hsp90), another prominent substrate of HDAC6, was also recruited to membrane ruffles and macropinosomes. Significantly, inhibition of Hsp90 activity suppressed membrane ruffling and cell migration, while expression of an acetylation-resistant Hsp90 mutant promoted ruffle formation. Our results uncover a surprising role for HDAC6 in actin remodeling-dependent processes and identify the actin cytoskeleton as an important target of HDAC6-regulated protein deacetylation.     

FK228 inhibits Hsp90 chaperone function in K562 cells via hyperacetylation of Hsp70

Some pan-histone-deacetylase (HDAC) inhibitors have recently been reported to exert their anti-leukemia effect by inhibiting the activity of class IIB HDAC6, which is the deacetylase of Hsp90 and α-tubulin, thereby leading to hyperacetylation of Hsp90, disruption of its chaperone function and apoptosis. In this study, we compared the effect of a class I HDAC inhibitor FK228 with the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on the Hsp90 chaperone function of K562 cells. We demonstrated that, although having a weaker inhibitory effect on HDAC6, FK228 mediated a similar disruption of Hsp90 chaperone function compared to SAHA. Unlike SAHA, FK228 did not mediate hyperacetylation of Hsp90, instead the acetylation of Hsp70 was increased and Bcr-Abl was increasingly associated with Hsp70 rather than Hsp90, forming an unstable complex that promotes Bcr-Abl degradation. These results indicated that FK228 may disrupt the function of Hsp90 indirectly through acetylation of Hsp70 and inhibition of its function.     

HDAC6: a key regulator of cytoskeleton, cell migration and cell-cell interactions

Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that regulates many important biological processes, including cell migration, immune synapse formation, viral infection, and the degradation of misfolded proteins. HDAC6 deacetylates tubulin, Hsp90 and cortactin, and forms complexes with other partner proteins. Although HDAC6 enzymatic activity seems to be required for the regulation of cell morphology, the role of HDAC6 in lymphocyte chemotaxis is independent of its tubulin deacetylase activity. The diverse functions of HDAC6 suggest that it is a potential therapeutic target for the treatment of a range of diseases. This review examines the biological actions of HDAC6, focusing on its deacetylase activity and its potential scaffold functions in the regulation of cell migration and other key biological processes in which the cytoskeleton plays an important role.     

Regulation of ERα Signaling Pathway in Neuronal HN10 Cells: Role of Protein Acetylation and Hsp90

Estrogen has a variety of neuroprotective effects but the molecular basis of its function is still mainly unclear. Estrogen receptor (ER) signaling is highly dependent on posttranslational modifications and the assembly of coactivator and corepressor complexes. Several proteins involved in ERα signaling have recently been found to be acetylated, including ERα itself and Hsp90, a key chaperone in the functional regulation of ERα. ERα complexes also contain histone deacetylases (HDAC) which repress transactivation. Our purpose was to clarify the role of protein acetylation and Hsp90 function in the ERE-mediated ERα signaling in neuronal HN10 cells. We observed that increasing protein/histone acetylation status with trichostatin A, a potent HDAC inhibitor, increased the 17β-estradiol (E2)-induced transactivation of ERE-driven luciferase in non-transfected cells, and even more extensively in pERα-transfected cells. E2-induced ERE-driven transactivation was blocked by ICI 182.780. Several ER antagonists, such as raloxifene and tamoxifen, were unresponsive. Valproate, an antiepileptic drug which is recently characterized as a HDAC inhibitor, was also able to potentiate the E2-induced ERE-transactivation. Inhibition of the function of Hsp90 chaperone with geldanamycin strongly inhibited the E2-induced ERE-transactivation. Overexpression of SIRT2 protein deacetylase did not inhibit the acetylation-potentiated ERE-driven transactivation indicating that SIRT2 deacetylase is not involved in ERα signaling. Our results reveal that ERα signaling is dependent on protein acetylation and epigenetic regulation.     

HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo

Microtubules are cylindrical cytoskeletal structures found in almost all eukaryotic cell types which are involved in a great variety of cellular processes. Reversible acetylation on the epsilon-amino group of alpha-tubulin Lys40 marks stabilized microtubule structures and may contribute to regulating microtubule dynamics. Yet, the enzymes catalysing this acetylation/deacetylation have remained unidentified until recently. Here we report that beta-tubulin interacts with histone deacetylase-6 (HDAC-6) in a yeast two-hybrid assay and in vitro. We find that HDAC-6 is a micro tubule-associated protein capable of deacetylating alpha-tubulin in vivo and in vitro. HDAC-6's microtubule binding and deacetylation functions both depend on the hdac domains. Overexpression of HDAC-6 in mammalian cells leads to tubulin hypoacetylation. In contrast, inhibition of HDAC-6 function by two independent mechanisms--pharmacological (HDAC inhibitors) or genetic (targeted inactivation of HDAC-6 in embryonic stem cells)--leads to hyperacetylation of tubulin and microtubules. Taken together, our data provide evidence that HDAC-6 might act as a dual deacetylase for tubulin and histones, and suggest the possibility that acetylated non-histone proteins might represent novel targets for pharmacological therapy by HDAC inhibitors.     

Inhibition of HDAC6 Deacetylase Activity Increases Its Binding with Microtubules and Suppresses Microtubule Dynamic Instability in MCF-7 Cells

The post-translational modification of tubulin appears to be a highly controlled mechanism that regulates microtubule functioning. Acetylation of the ϵ-amino group of Lys-40 of α-tubulin marks stable microtubules, although the causal relationship between tubulin acetylation and microtubule stability has remained poorly understood. HDAC6, the tubulin deacetylase, plays a key role in maintaining typical distribution of acetylated microtubules in cells. Here, by using tubastatin A, an HDAC6-specific inhibitor, and siRNA-mediated depletion of HDAC6, we have explored whether tubulin acetylation has a role in regulating microtubule stability. We found that whereas both pharmacological inhibition of HDAC6 as well as its depletion enhance microtubule acetylation, only pharmacological inhibition of HDAC6 activity leads to an increase in microtubule stability against cold and nocodazole-induced depolymerizing conditions. Tubastatin A treatment suppressed the dynamics of individual microtubules in MCF-7 cells and delayed the reassembly of depolymerized microtubules. Interestingly, both the localization of HDAC6 on microtubules and the amount of HDAC6 associated with polymeric fraction of tubulin were found to increase in the tubastatin A-treated cells compared with the control cells, suggesting that the pharmacological inhibition of HDAC6 enhances the binding of HDAC6 to microtubules. The evidence presented in this study indicated that the increased binding of HDAC6, rather than the acetylation per se, causes microtubule stability. The results are in support of a hypothesis that in addition to its deacetylase function, HDAC6 might function as a MAP that regulates microtubule dynamics under certain conditions.     

Modulating molecular chaperone Hsp90 functions through reversible acetylation

The molecular chaperone protein Hsp90 is a key regulator of approximately 100 'client' proteins crucial for numerous cell signaling processes. Consequently, understanding the molecular underpinnings that regulate Hsp90 activity is an important biological endeavor. Exciting new results now suggest that, at least for nuclear receptor activity, Hsp90 function is directly regulated by histone deacetylase 6 (HDAC6). These observations have consequences for various biological processes and potentially important implications for the development of cancer therapeutics.     

Acetylation of heat shock protein 20 (Hsp20) regulates human myometrial activity

Phosphorylation of heat shock protein 20 (Hsp20) by protein kinase A (PKA) is now recognized as an important regulatory mechanism modulating contractile activity in the human myometrium. Thus agonists that stimulate cyclic AMP production may cause relaxation with resultant beneficial effects on pathologies that affect this tissue such as the onset of premature contractions prior to term. Here we describe for the first time that acetylation of Hsp20 is also a potent post-translational modification that can affect human myometrial activity. We show that histone deacetylase 8 (HDAC8) is a non-nuclear lysine deacetylase (KDAC) that can interact with Hsp20 to affect its acetylation. Importantly, use of a selective linkerless hydroxamic acid HDAC8 inhibitor increases Hsp20 acetylation with no elevation of nuclear-resident histone acetylation nor marked global gene expression changes. These effects are associated with significant inhibition of spontaneous and oxytocin-augmented contractions of ex vivo human myometrial tissue strips. A potential molecular mechanism by which Hsp20 acetylation can affect myometrial activity by liberating cofilin is described and further high-lights the use of specific effectors of KDACs as therapeutic agents in regulating contractility in this smooth muscle.     

Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis for antileukemia activity of histone deacetylase inhibitors

The hydroxamic acid (HAA) analogue pan-histone deacetylase (HDAC) inhibitors (HDIs) LAQ824 and LBH589 have been shown to induce acetylation and inhibit the ATP binding and chaperone function of heat shock protein (HSP) 90. This promotes the polyubiquitylation and degradation of the pro-growth and pro-survival client proteins Bcr-Abl, mutant FLT-3, c-Raf, and AKT in human leukemia cells. HDAC6 is a member of the class IIB HDACs. It is predominantly cytosolic, microtubule-associated alpha-tubulin deacetylase that is also known to promote aggresome inclusion of the misfolded polyubiquitylated proteins. Here we demonstrate that in the Bcr-abl oncogene expressing human leukemia K562 cells, HDAC6 can be co-immunoprecipitated with HSP90, and the knock-down of HDAC6 by its siRNA induced the acetylation of HSP90 and alpha-tubulin. Depletion of HDAC6 levels also inhibited the binding of HSP90 to ATP, reduced the chaperone association of HSP90 with its client proteins, e.g. Bcr-Abl, and induced polyubiquitylation and partial depletion of Bcr-Abl. Conversely, the ectopic overexpression of HDAC6 inhibited LAQ824-induced acetylation of HSP90 and alpha-tubulin and reduced LAQ824-mediated depletion of Bcr-Abl, AKT, and c-Raf. Collectively, these findings indicate that HDAC6 is also an HSP90 deacetylase. Targeted inhibition of HDAC6 leads to acetylation of HSP90 and disruption of its chaperone function, resulting in polyubiquitylation and depletion of pro-growth and pro-survival HSP90 client proteins including Bcr-Abl. Depletion of HDAC6 sensitized human leukemia cells to HAA-HDIs and proteasome inhibitors.     

HDAC6 Regulates Mutant SOD1 Aggregation through Two SMIR Motifs and Tubulin Acetylation

Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS.     

Anti-metastatic activity of MPT0G211, a novel HDAC6 inhibitor,in human breast cancer cells in vitro and in vivo

Triple-negative breast cancer (TNBC) is associated with an increased risk of metastasis and a poor prognosis. The invasive ability of TNBC relies on actin reorganization and is regulated by histone deacetylase 6 (HDAC6). The present study aimed to examine the effect of MPT0G211, a novel HDAC6 inhibitor, on cell migration and microtubule association in both in vitro and in vivo models of TNBC. Here MPT0G211 more selectively and potently targeted and inhibited HDAC6, compared with tubastatin A, another selective HDAC6 inhibitor. In vitro, MPT0G211 decreased the migration of the TNBC cell line MDA-MB-231, particularly when administered together with paclitaxel, and increased heat shock protein 90 (Hsp90) acetylation, leading to the dissociation of Hsp90 from aurora-A and proteasomal degradation. Furthermore, MPT0G211 significantly disrupted F-actin polymerization by increasing cortactin acetylation and downregulating slingshot protein phosphatase 1 (SSH1) and active cofilin expression. In vivo, MPT0G211 treatment significantly ameliorated TNBC metastasis. In conclusion, our results demonstrate that MPT0G211 reduces TNBC cell motility by promoting cortactin acetylation and aurora-A degradation, and inhibiting the cofilin–F-actin pathway via HDAC6 activity attenuation. MPT0G211 therefore demonstrates therapeutic potential for invasive TNBC.     

Deactylase inhibitors disrupt cellular complexes containing protein phosphatases and deacetylases

Affinity isolation of protein serine/threonine phosphatases on the immobilized phosphatase inhibitor microcystin-LR identified histone deacetylase 1(HDAC1), HDAC6, and HDAC10 as novel components of cellular phosphatase complexes. Other HDACs, specifically HDAC2, -3, -4, and -5, were excluded from such complexes. In vitro biochemical studies showed that recombinant HDAC6, but not HDAC4, bound directly to the protein phosphatase (PP)1 catalytic subunit. No association was observed between HDAC6 and PP2A, another major protein phosphatase. PP1 binding was mapped to the second catalytic domain and adjacent C-terminal sequences in HDAC6, and treatment of cells with trichostatin A (TSA) disrupted endogenous HDAC6.PP1 complexes. Consistent with the inhibition of tubulin deactylase activity of HDAC6, TSA enhanced cellular tubulin acetylation, and acetylated tubulin was present in the PP1 complexes from TSA-treated cells. Trapoxin B, a weak HDAC6 inhibitor, and calyculin A, a cell-permeable phosphatase inhibitor, had no effect on the stability of the HDAC6.PP1 complexes or on tubulin acetylation. Mutations that inactivated HDAC6 prevented its incorporation into cellular PP1 complexes and suggested that when bound together both enzymes were active. Interestingly, TSA disrupted all the cellular HDAC.phosphatase complexes analyzed. This study provided new insight into the mechanism by which HDAC inhibitors elicited coordinate changes in cellular protein phosphorylation and acetylation and suggested that changes in these protein modifications at multiple subcellular sites may contribute to the known ability of HDAC inhibitors to suppress cell growth and transformation.     

TPPP/p25 Promotes Tubulin Acetylation by Inhibiting Histone Deacetylase 6

TPPP/p25 (tubulin polymerization-promoting protein/p25) is an unstructured protein that induces microtubule polymerization in vitro and is aligned along the microtubule network in transfected mammalian cells. In normal human brain, TPPP/p25 is expressed predominantly in oligodendrocytes, where its expression is proved to be crucial for their differentiation process. Here we demonstrated that the expression of TPPP/p25 in HeLa cells, in doxycycline-inducible CHO10 cells, and in the oligodendrocyte CG-4 cells promoted the acetylation of α-tubulin at residue Lys-40, whereas its down-regulation by specific small interfering RNA in CG-4 cells or by the withdrawal of doxycycline from CHO10 cells decreased the acetylation level of α-tubulin. Our results indicate that TPPP/p25 binds to HDAC6 (histone deacetylase 6), an enzyme responsible for tubulin deacetylation. Moreover, we demonstrated that the direct interaction of these two proteins resulted in the inhibition of the deacetylase activity of HDAC6. The measurement of HDAC6 activity showed that TPPP/p25 is able to induce almost complete (90%) inhibition at 3 μm concentration. In addition, treatment of the cells with nocodazole, vinblastine, or cold exposure revealed that microtubule acetylation induced by trichostatin A, a well known HDAC6 inhibitor, does not cause microtubule stabilization. In contrast, the microtubule bundling activity of TPPP/p25 was able to protect the microtubules from depolymerization. Finally, we demonstrated that, similarly to other HDAC6 inhibitors, TPPP/p25 influences the microtubule dynamics by decreasing the growth velocity of the microtubule plus ends and also affects cell motility as demonstrated by time lapse video experiments. Thus, we suggest that TPPP/p25 is a multiple effector of the microtubule organization.