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-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.     

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.     

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.     

组蛋白去乙酰化酶6:异常蛋白降解的关键调控因子

组蛋白去乙酰化酶6(HDAC6)是位于胞浆中的一种去乙酰化酶,参与调控细胞内多种重要的生物活性,可使α-微管蛋白(α-tubulin)、热休克蛋白90(Hsp90)和皮肌动蛋白(cortactin)去乙酰化,并与多种蛋白质缔结形成复合物。在细胞培养中,当产生的错误折叠蛋白超过了分子伴侣再折叠及泛素蛋白酶体系统(UPS)处理能力时,HDAC6可将其特异转运到细胞核周结构——异常蛋白包涵体(aggresome)中,从而使之被自噬有效降解,因此认为HDAC6在异常蛋白降解中发挥了关键的调控功能,是"蛋白构象病"的潜在治疗靶点。     

Development of the first small molecule histone deacetylase 6 (HDAC6) degraders

Histone deacetylases (HDACs) decrease the acetylation level of histones and other non-histone proteins. Over expression of HDACs have been observed in cancers and other diseases. Targeted protein degradation by “hijacking” the natural ubiquitin-proteasome-system (UPS) recently emerged as a novel technology to “knock-out” endogenous disease-causing proteins. We applied this strategy to the development of the first small molecule degraders for zinc-dependent HDACs by conjugating non-selective HDAC inhibitors with E3 ubiquitin ligase ligands. Through cell-based assays, we discovered novel bifunctional molecules (dHDAC6) that could selectively degrade HDAC6. Further mechanistic studies indicated that HDAC6 was selectively removed by the UPS.     

The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress

The efficient clearance of cytotoxic misfolded protein aggregates is critical for cell survival. Misfolded protein aggregates are transported and removed from the cytoplasm by dynein motors via the microtubule network to a novel organelle termed the aggresome where they are processed. However, the means by which dynein motors recognize misfolded protein cargo, and the cellular factors that regulate aggresome formation, remain unknown. We have discovered that HDAC6, a microtubule-associated deacetylase, is a component of the aggresome. We demonstrate that HDAC6 has the capacity to bind both polyubiquitinated misfolded proteins and dynein motors, thereby acting to recruit misfolded protein cargo to dynein motors for transport to aggresomes. Indeed, cells deficient in HDAC6 fail to clear misfolded protein aggregates from the cytoplasm, cannot form aggresomes properly, and are hypersensitive to the accumulation of misfolded proteins. These findings identify HDAC6 as a crucial player in the cellular management of misfolded protein-induced stress.     

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.     

Targeting the ‘garbage-bin’ to fight cancer: HDAC6 inhibitor WT161 has an anti-tumor effect on osteosarcoma and synergistically interacts with 5-FU

An imbalance between protein aggregation and protein degradation may induce ‘stress’ in the functionality of the endoplasmic reticulum (ER). There are quality control (QC) mechanisms to minimize misfolding and to eliminate misfolded proteins before aggregation becomes lethal for the cell. Proper protein folding and maturation is one of the crucial functions of the ER. Chaperones of the ER and folding enzymes guarantee correct conformational maturation of emerging secretory proteins. Histone deacetylase (HDAC) 6 (HDAC6) is a masterpiece coordinating the cell response to protein aggregate formation. The balance between HDAC6 and its partner Valosin-containing protein/p97 determines the fate of polyubiquitinated misfolded proteins. WT161 is a terrific, selective, and bioavailable HDAC6 inhibitor. WT161 selectively inhibits HDAC6 and adequately increases levels of acetylated α-tubulin. This compound induces accumulation of acetylated tubulin and cytotoxicity in multiple myeloma (MM) cells. In this journal, Sun et al. (Biosci. Rep. 41, DOI: 10.1042/BSR20203905) identified that WT161 suppresses the cell growth of osteosarcoma cells. This discovery opens the door to future chemotherapeutic regimens of this bone neoplasm.