Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity

Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.     

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.     

Dysferlin interacts with histone deacetylase 6 and increases alpha-tubulin acetylation

Dysferlin is a multi-C2 domain transmembrane protein involved in a plethora of cellular functions, most notably in skeletal muscle membrane repair, but also in myogenesis, cellular adhesion and intercellular calcium signaling. We previously showed that dysferlin interacts with alpha-tubulin and microtubules in muscle cells. Microtubules are heavily reorganized during myogenesis to sustain growth and elongation of the nascent muscle fiber. Microtubule function is regulated by post-translational modifications, such as acetylation of its alpha-tubulin subunit, which is modulated by the histone deacetylase 6 (HDAC6) enzyme. In this study, we identified HDAC6 as a novel dysferlin-binding partner. Dysferlin prevents HDAC6 from deacetylating alpha-tubulin by physically binding to both the enzyme, via its C2D domain, and to the substrate, alpha-tubulin, via its C2A and C2B domains. We further show that dysferlin expression promotes alpha-tubulin acetylation, as well as increased microtubule resistance to, and recovery from, Nocodazole- and cold-induced depolymerization. By selectively inhibiting HDAC6 using Tubastatin A, we demonstrate that myotube formation was impaired when alpha-tubulin was hyperacetylated early in the myogenic process; however, myotube elongation occurred when alpha-tubulin was hyperacetylated in myotubes. This study suggests a novel role for dysferlin in myogenesis and identifies HDAC6 as a novel dysferlin-interacting protein.     

The histone deacetylase-6 inhibitor tubacin directly inhibits de novo sphingolipid biosynthesis as an off-target effect

Histone deacetylase 6 (HDAC6) controls acetylation of a number of cytosolic proteins, most prominently tubulin. Tubacin is a small molecule inhibitor of HDAC6 selected for its selective inhibition of HDAC6 relative to other histone deacetylases. For this reason it has become a useful pharmacological tool to discern the biological functions of HDAC6 in numerous cellular processes. The interest of this laboratory is in the function and regulation of sphingolipids, a family of lipids based on the sphingosine backbone. Sphingolipid biosynthesis is initiated by the rate limiting enzyme serine palmitoyltransferase (SPT). Sphingolipids have critical and diverse functions in cell survival, apoptosis, intra- and intercellular signaling, and in membrane structure. In the course of examining the role of HDAC6 in the regulation of sphingolipid biosynthesis we observed that tubacin strongly inhibited de novo synthesis whereas HDAC6 knockdown very moderately stimulated synthesis. We resolved these seemingly contradictory results by demonstrating that, surprisingly, tubacin is a direct inhibitor of SPT activity in permeabilized cells. Furthermore tubacin inhibits de novo sphingolipid synthesis in intact cells at doses commonly used to test HDAC6 function and does so in an HDAC6-independent manner. Niltubacin is a chemical analog of tubacin which lacks tubacin’s HDAC6 activity, and so is often used as a control for off-target effects of tubacin. We find that niltubacin is inactive in the inhibition of sphingolipid biosynthesis, and so does not serve to distinguish the inhibitory effects of tubacin on HDAC6 from those on sphingolipid biosynthesis. These results indicate that caution should be used in the use of tubacin to study the role of HDAC6.     

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.     

Requirement of a novel splicing variant of human histone deacetylase 6 for TGF-β1-mediated gene activation

Histone deacetylase 6 (HDAC6) belongs to the family of class IIb HDACs and predominantly deacetylates non-histone proteins in the cytoplasm via the C-terminal deacetylase domain of its two tandem deacetylase domains. HDAC6 modulates fundamental cellular processes via deacetylation of α-tubulin, cortactin, molecular chaperones, and other peptides. Our previous study indicates that HDAC6 mediates TGF-β1-induced epithelial-mesenchymal transition (EMT) in A549 cells. In the current study, we identify a novel splicing variant of human HDAC6, hHDAC6p114. The hHDAC6p114 mRNA arises from incomplete splicing and encodes a truncated isoform of the hHDAC6p114 protein of 114 kDa when compared to the major isoform hHDAC6p131. The hHDAC6p114 protein lacks the first 152 amino acids from N-terminus in the hHDAC6p131 protein, which harbors a nuclear export signal peptide and 76 amino acids of the N-terminal deacetylase domain. hHDAC6p114 is intact in its deacetylase activity against α-tubulin. The expression hHDAC6p114 is elevated in a MCF-7 derivative that exhibits an EMT-like phenotype. Moreover, hHDAC6p114 is required for TGF-β1-activated gene expression associated with EMT in A549 cells. Taken together, our results implicate that expression and function of hHDAC6p114 is differentially regulated when compared to hHDAC6p131.     

HDAC6-p97/VCP controlled polyubiquitin chain turnover

HDAC6 is a unique cytoplasmic deacetylase capable of interacting with ubiquitin. Using a combination of biophysical, biochemical and biological approaches, we have characterized the ubiquitin-binding domain of HDAC6, named ZnF-UBP, and investigated its biological functions. These studies show that the three Zn ion-containing HDAC6 ZnF-UBP domain presents the highest known affinity for ubiquitin monomers and mediates the ability of HDAC6 to negatively control the cellular polyubiquitin chain turnover. We further show that HDAC6-interacting chaperone, p97/VCP, dissociates the HDAC6-ubiquitin complexes and counteracts the ability of HDAC6 to promote the accumulation of polyubiquitinated proteins. We propose that a finely tuned balance of HDAC6 and p97/VCP concentrations determines the fate of ubiquitinated misfolded proteins: p97/VCP would promote protein degradation and ubiquitin turnover, whereas HDAC6 would favour the accumulation of ubiquitinated protein aggregates and inclusion body formation.     

Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally

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, alpha-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.     

Depression of mitochondrial metabolism by downregulation of cytoplasmic deacetylase, HDAC6

Mitochondria perform multiple functions critical to the maintenance of cellular homeostasis. Here we report that the downregulation of histone deacetylase 6 (HDAC6) causes a reduction in the net activity of mitochondrial enzymes, including respiratory complex II and citrate synthase. HDAC6 deacetylase and ubiquitin-binding activities were both required for recovery of reduced mitochondrial metabolic activity due to the loss of HDAC6. Hsp90, a substrate of HDAC6, localizes to mitochondria and partly mediates the regulation of mitochondrial metabolic activity by HDAC6. Our finding suggests that HDAC6 regulates mitochondrial metabolism and might serve as a cellular homeostasis surveillance factor.     

HDAC6 at the Intersection of Neuroprotection and Neurodegeneration

Histone deacetylase 6 (HDAC6) catalyzes multiple reactions. We summarize the current knowledge on HDAC6, its targets and functions. Among others, HDAC6 recognizes damaged proteins and assures that these proteins are destroyed by autophagy. On the other hand, HDAC6 also modifies the tracks used by the clearance mechanism so that axonal transport becomes less efficient. We hypothesize that a disturbance in the equilibrium between the different functions of HDAC6 could play an important role in neurodegeneration.     

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.     

aPKC Phosphorylation of HDAC6 Results in Increased Deacetylation Activity

The Class II histone deacetylase, HDAC6, has been shown to be involved in cell motility, aggresome formation and mitochondria transport. HDAC6 deacetylase activity regulates α-tubulin acetylation levels and thus plays a critical role in these processes. In turn, HDAC6 activity can be regulated by interaction with various proteins including multiple kinases. Kinase mediated phosphorylation of HDAC6 can lead to either increased or reduced activity. Our previous research has shown that sequestosome1/p62 (SQSTM1/p62) interacts with HDAC6 and regulates its activity. As SQSTM1/p62 is a scaffolding protein known to interact directly with the zeta isoform of Protein Kinase C (PKCζ), we sought to examine if HDAC6 could be a substrate for PKCζ phosphorylation and if so, how its activity might be regulated. Our data demonstrate that HDAC6 is not only present in a protein complex with PKCζ but can also be phosphorylated by PKCζ. We also show that specific phosphorylation of HDAC6 by PKCζ increases HDAC6 deacetylase activity resulting in reduced acetylated tubulin levels. Our findings provide novel insight into the molecular mechanism by which HDAC6, PKCζ and SQSTM1/p62 function together in protein aggregate clearance. These results also highlight a new research direction which may prove fruitful for understanding the underlying cause of several neurodegenerative diseases.     

Caspase-mediated specific cleavage of human histone deacetylase 4

Histone deacetylase 4 (HDAC4) is a class II HDAC implicated in controlling gene expression important for diverse cellular functions, but little is known about how its expression and stability are regulated. We report here that this deacetylase is unusually unstable, with a half-life of less than 8 h. Consistent with the instability of HDAC4 protein, its mRNA was also highly unstable (with a half-life of less than 4 h). The degradation of HDAC4 could be accelerated by exposure of cells to ultraviolet irradiation. HDAC4 degradation was not dependent on proteasome or CRM1-mediated export activity but instead was caspase-dependent and was detectable in diverse human cancer lines. Of two potential caspase consensus motifs in HDAC4, both lying within a region containing proline-, glutamic acid-, serine-, and threonine-rich (PEST) sequences, we identified, by site-directed mutagenesis, Asp-289 as the prime cleavage site. Notably, this residue is not conserved among other class IIa members, HDAC5, -7, and -9. Finally, the induced expression of caspase-cleavable HDAC4 led to markedly increased apoptosis. These results therefore unexpectedly link the regulation of HDAC4 protein stability to caspases, enzymes that are important for controlling cell death and differentiation.     

Interplay between histone deacetylases and autophagy--from cancer therapy to neurodegeneration

Histone deacetylases (HDACs) are chromatin modifiers that alter gene expression but also exert a broad range of functions outside the nucleus by deacetylating non-histone target proteins. They gained growing attention for their implications in disease treatment, mainly through research using HDAC-inhibiting compounds. Understanding the effects of HDAC function and deregulation has therefore become an important focus for both basic and applied research. One of the described effects of HDAC inhibition is induction of autophagy. Autophagy is a ubiquitous process of recycling cellular components in response to starvation or stress and therefore crucial for cell homeostasis. Because of its role in managing anomalous protein overloads, autophagy is of great interest for neurodegenerative disease research. However, autophagy can also promote cell death, which puts it in the focus of cancer research. This review provides an overview of what we know of the impact of HDACs on the autophagy pathway and describes the fields where promising progress has been achieved, although one has to state that the work to illuminate the connections has just begun. Therefore, one focus is the effect of HDAC inhibition on autophagy in several types and models of cancer, which aims to find combinations of treatments that circumvent the ability of cancer cells to escape from cell death. Another recently emerged aspect is the direct involvement of the cytosolic deacetylase HDAC6 in autophagy progression, which is of great potential for revealing disease mechanisms in neurodegeneration.