HDAC6/aggresome processing pathway importance for inflammasome formation is context-dependent.

The inflammasome is a large multiprotein complex that assembles in the cell cytoplasm in response to stress or pathogenic infection. Its primary function is to defend the cell and promote the secretion of pro-inflammatory cytokines, including IL-1ß and IL-18. Previous research has shown that in immortalized bone marrow-derived macrophages (iBMDMs) inflammasome assembly is dependent on the deacetylase HDAC6 and the aggresome processing pathway (APP), a cellular pathway involved in the disposal of misfolded proteins. Here we used primary BMDMs from mice in which HDAC6 is ablated or impaired and found that inflammasome activation was largely normal. We also used human peripheral blood mononuclear cells and monocyte cell lines expressing a synthetic protein blocking the HDAC6-ubiquitin interaction and impairing the APP and found that inflammasome activation was moderately affected. Finally, we used a novel HDAC6 degrader and showed that inflammasome activation was partially impaired in human macrophage cell lines with depleted HDAC6. Our results therefore show that HDAC6 importance in inflammasome activation is context-dependent.

A quantitative model for virus uncoating predicts influenza A infectivity.

For virus infection of new host cells, the disassembly of the protective outer protein shell (capsid) is a critical step, but the mechanisms and host-virus interactions underlying the dynamic, active, and regulated uncoating process are largely unknown. Here, we develop an experimentally supported, multiscale kinetics model that elucidates mechanisms of influenza A virus (IAV) uncoating in cells. Biophysical modeling demonstrates that interactions between capsid M1 proteins, host histone deacetylase 6 (HDAC6), and molecular motors can physically break the capsid in a tug-of-war mechanism. Biochemical analysis and biochemical-biophysical modeling identify unanchored ubiquitin chains as essential and allow robust prediction of uncoating efficiency in cells. Remarkably, the different infectivity of two clinical strains can be ascribed to a single amino acid variation in M1 that affects binding to HDAC6. By identifying crucial modules of viral infection kinetics, the mechanisms and models presented here could help formulate novel strategies for broad-range antiviral treatment.

HDAC6/aggresome processing pathway importance for inflammasome formation is context dependent.

The inflammasome is a large multiprotein complex that assembles in the cell cytoplasm in response to stress or pathogenic infection. Its primary function is to defend the cell and promote the secretion of pro-inflammatory cytokines, including IL-1ß and IL-18. It was shown that in immortalized bone marrow derived macrophages (iBMDMs) inflammasome assembly is dependent on the deacetylase HDAC6 and the aggresome processing pathway (APP), a cellular pathway involved in the disposal of misfolded proteins. Here we used primary BMDMs from mice in which HDAC6 is ablated or impaired and found that inflammasome activation was largely normal. We also used human peripheral blood mononuclear cells and monocytes cell lines expressing a synthetic protein blocking HDAC6-ubiquitin interaction and impairing the APP and found that inflammasome activation was moderately affected. Finally, we used a novel HDAC6 degrader and showed that inflammasome activation was partially impaired in human macrophage cell lines with depleted HDAC6. Our results therefore show that HDAC6 importance in inflammasome activation is context dependent.

Chemoproteomic target deconvolution reveals Histone Deacetylases as targets of (R)-lipoic acid.

Lipoic acid is an essential enzyme cofactor in central metabolic pathways. Due to its claimed antioxidant properties, racemic (R/S)-lipoic acid is used as a food supplement but is also investigated as a pharmaceutical in over 180 clinical trials covering a broad range of diseases. Moreover, (R/S)-lipoic acid is an approved drug for the treatment of diabetic neuropathy. However, its mechanism of action remains elusive. Here, we performed chemoproteomics-aided target deconvolution of lipoic acid and its active close analog lipoamide. We find that histone deacetylases HDAC1, HDAC2, HDAC3, HDAC6, HDAC8, and HDAC10 are molecular targets of the reduced form of lipoic acid and lipoamide. Importantly, only the naturally occurring (R)-enantiomer inhibits HDACs at physiologically relevant concentrations and leads to hyperacetylation of HDAC substrates. The inhibition of HDACs by (R)-lipoic acid and lipoamide explain why both compounds prevent stress granule formation in cells and may also provide a molecular rationale for many other phenotypic effects elicited by lipoic acid.

Expression and Crystallization of HDAC6 Tandem Catalytic Domains.

Histone deacetylase 6 (HDAC6) is an atypical lysine deacetylase with tandem catalytic domains and an ubiquitin-binding zinc finger domain. HDAC6 is involved in various biological processes, such as cell motility or stress responses, and has been implicated in pathologies ranging from cancer to neurodegeneration. Due to this broad range of functions, there has been considerable interest in developing HDAC6-specific small molecule inhibitors, several of which are already available. The crystal structure of the tandem catalytic domains of zebrafish HDAC6 has revealed an arrangement with twofold symmetry and extensive surface interaction between the catalytic domains. Further dissection of the biochemical properties of HDAC6 and the development of novel inhibitors will benefit from being able to routinely express high-quality protein. We present here our optimized protocol for expression and crystallization of the zebrafish tandem catalytic domains.

Immunoprecipitation of HDAC6 and Interacting Proteins.

The lysine deacetylase HDAC6 has unique structural and functional properties: It contains tandem catalytic domains that can deacetylate a variety of proteins and a zinc finger domain that binds ubiquitin. HDAC6 has been implicated in a variety of biological processes, normal or pathological, such as cellular motility, stress response, cancer, neurodegeneration, or viral infection. Due to this, HDAC6 is considered an attractive therapeutic target, and there is a major interest to identify small molecule inhibitors. To gain a mechanistic understanding of how HDAC6 impacts these different biological processes, there is a continued need to discover additional substrates as well as interacting proteins in different paradigms. One approach to achieve this is to perform HDAC6 immunoprecipitations to identify partner proteins. We describe here our optimized protocols to immunoprecipitate HDAC6 with the goal to identify or validate interacting proteins.

Structure of the ciliogenesis-associated CPLANE complex.

Dysfunctional cilia cause pleiotropic human diseases termed ciliopathies. These hereditary maladies are often caused by defects in cilia assembly, a complex event that is regulated by the ciliogenesis and planar polarity effector (CPLANE) proteins Wdpcp, Inturned, and Fuzzy. CPLANE proteins are essential for building the cilium and are mutated in multiple ciliopathies, yet their structure and molecular functions remain elusive. Here, we show that mammalian CPLANE proteins comprise a bona fide complex and report the near-atomic resolution structures of the human Wdpcp-Inturned-Fuzzy complex and of the mouse Wdpcp-Inturned-Fuzzy complex bound to the small guanosine triphosphatase Rsg1. Notably, the crescent-shaped CPLANE complex binds phospholipids such as phosphatidylinositol 3-phosphate via multiple modules and a CPLANE ciliopathy mutant exhibits aberrant lipid binding. Our study provides critical structural and functional insights into an enigmatic ciliogenesis-associated complex as well as unexpected molecular rationales for ciliopathies.

Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways.

The deacetylase HDAC6 has tandem catalytic domains and a zinc finger domain (ZnF) binding ubiquitin (Ub). While the catalytic domain has an antiviral effect, the ZnF facilitates influenza A virus (IAV) infection and cellular stress responses. By recruiting Ub via the ZnF, HDAC6 promotes the formation of aggresomes and stress granules (SGs), dynamic structures associated with pathologies such as neurodegeneration. IAV subverts the aggresome/HDAC6 pathway to facilitate capsid uncoating during early infection. To target this pathway, we generate designed ankyrin repeat proteins (DARPins) binding the ZnF; one of these prevents interaction with Ub in vitro and in cells. Crystallographic analysis shows that it blocks the ZnF pocket where Ub engages. Conditional expression of this DARPin reversibly impairs infection by IAV and Zika virus; moreover, SGs and aggresomes are downregulated. These results validate the HDAC6 ZnF as an attractive target for drug discovery.

How Influenza Virus Uses Host Cell Pathways during Uncoating.

Influenza is a zoonotic respiratory disease of major public health interest due to its pandemic potential, and a threat to animals and the human population. The influenza A virus genome consists of eight single-stranded RNA segments sequestered within a protein capsid and a lipid bilayer envelope. During host cell entry, cellular cues contribute to viral conformational changes that promote critical events such as fusion with late endosomes, capsid uncoating and viral genome release into the cytosol. In this focused review, we concisely describe the virus infection cycle and highlight the recent findings of host cell pathways and cytosolic proteins that assist influenza uncoating during host cell entry.

OBF1 and Oct factors control the germinal center transcriptional program.

OBF1 is a specific coactivator of the POU family transcription factors OCT1 and OCT2. OBF1 and OCT2 are B cell-specific and indispensable for germinal center (GC) formation, but their mechanism of action is unclear. Here, we show by chromatin immunoprecipitation-sequencing that OBF1 extensively colocalizes with OCT1 and OCT2. We found that these factors also often colocalize with transcription factors of the ETS family. Furthermore, we showed that OBF1, OCT2, and OCT1 bind widely to the promoters or enhancers of genes involved in GC formation in mouse and human GC B cells. Short hairpin RNA knockdown experiments demonstrated that OCT1, OCT2, and OBF1 regulate each other and are essential for proliferation of GC-derived lymphoma cell lines. OBF1 downregulation disrupts the GC transcriptional program: genes involved in GC maintenance, such as BCL6, are downregulated, whereas genes related to exit from the GC program, such as IRF4, are upregulated. Ectopic expression of BCL6 does not restore the proliferation of GC-derived lymphoma cells depleted of OBF1 unless IRF4 is also depleted, indicating that OBF1 controls an essential regulatory node in GC differentiation.

Monitoring Acetylation of the RNA Helicase DDX3X, a Protein Critical for Formation of Stress Granules.

Stress granules are dynamic structures that assemble in response to various forms of stress, such as heat shock or oxidative stress, among others. We had previously shown that the lysine deacetylase HDAC6 is required for the formation of stress granules, but the substrate important for this function was not known. We recently found that the RNA helicase DDX3X is a novel HDAC6 substrate, which is critical for the formation of stress granules. Through a series of detailed experiments, we showed that, upon stress, DDX3X becomes acetylated in an intrinsically disordered region; this alters its propensity to undergo phase separation and inhibits growth of the stress granules. HDAC6, by deacetylating DDX3X, allows maturation of the stress granules. This work identified acetylation of an RNA helicase as a critical regulator of the stress response. Here, we present methods to analyze the acetylation of DDX3X; these methods can be easily adapted to study the acetylation of other helicases, or other proteins.

EEF1A1 deacetylation enables transcriptional activation of remyelination.

Remyelination of the peripheral and central nervous systems (PNS and CNS, respectively) is a prerequisite for functional recovery after lesion. However, this process is not always optimal and becomes inefficient in the course of multiple sclerosis. Here we show that, when acetylated, eukaryotic elongation factor 1A1 (eEF1A1) negatively regulates PNS and CNS remyelination. Acetylated eEF1A1 (Ac-eEF1A1) translocates into the nucleus of myelinating cells where it binds to Sox10, a key transcription factor for PNS and CNS myelination and remyelination, to drag Sox10 out of the nucleus. We show that the lysine acetyltransferase Tip60 acetylates eEF1A1, whereas the histone deacetylase HDAC2 deacetylates eEF1A1. Promoting eEF1A1 deacetylation maintains the activation of Sox10 target genes and increases PNS and CNS remyelination efficiency. Taken together, these data identify a major mechanism of Sox10 regulation, which appears promising for future translational studies on PNS and CNS remyelination.

The disordered N-terminus of HDAC6 is a microtubule-binding domain critical for efficient tubulin deacetylation.

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic enzyme having tubulin deacetylase activity that has been unequivocally assigned to the second of the tandem catalytic domains. However, virtually no information exists on the contribution of other HDAC6 domains on tubulin recognition. Here, using recombinant protein expression, site-directed mutagenesis, fluorimetric and biochemical assays, microscale thermophoresis, and total internal reflection fluorescence microscopy, we identified the N-terminal, disordered region of HDAC6 as a microtubule-binding domain and functionally characterized it to the single-molecule level. We show that the microtubule-binding motif spans two positively charged patches comprising residues Lys-32 to Lys-58. We found that HDAC6-microtubule interactions are entirely independent of the catalytic domains and are mediated by ionic interactions with the negatively charged microtubule surface. Importantly, a crosstalk between the microtubule-binding domain and the deacetylase domain was critical for recognition and efficient deacetylation of free tubulin dimers both in vitro and in vivo Overall, our results reveal that recognition of substrates by HDAC6 is more complex than previously appreciated and that domains outside the tandem catalytic core are essential for proficient substrate deacetylation.

Histone deacetylase 1 (HDAC1): A key player of T cell-mediated arthritis.

Rheumatoid Arthritis (RA) represents a chronic T cell-mediated inflammatory autoimmune disease. Studies have shown that epigenetic mechanisms contribute to the pathogenesis of RA. Histone deacetylases (HDACs) represent one important group of epigenetic regulators. However, the role of individual HDAC members for the pathogenesis of arthritis is still unknown. In this study we demonstrate that mice with a T cell-specific deletion of HDAC1 (HDAC1-cKO) are resistant to the development of Collagen-induced arthritis (CIA), whereas the antibody response to collagen type II was undisturbed, indicating an unaltered T cell-mediated B cell activation. The inflammatory cytokines IL-17 and IL-6 were significantly decreased in sera of HDAC1-cKO mice. IL-6 treated HDAC1-deficient CD4+ T cells showed an impaired upregulation of CCR6. Selective inhibition of class I HDACs with the HDAC inhibitor MS-275 under Th17-skewing conditions inhibited the upregulation of chemokine receptor 6 (CCR6) in mouse and human CD4+ T cells. Accordingly, analysis of human RNA-sequencing (RNA-seq) data and histological analysis of synovial tissue samples from human RA patients revealed the existence of CD4+CCR6+ cells with enhanced HDAC1 expression. Our data indicate a key role for HDAC1 for the pathogenesis of CIA and suggest that HDAC1 and other class I HDACs might be promising targets of selective HDAC inhibitors (HDACi) for the treatment of RA.

LATS1 but not LATS2 represses autophagy by a kinase-independent scaffold function.

Autophagy perturbation represents an emerging therapeutic strategy in cancer. Although LATS1 and LATS2 kinases, core components of the mammalian Hippo pathway, have been shown to exert tumor suppressive activities, here we report a pro-survival role of LATS1 but not LATS2 in hepatocellular carcinoma (HCC) cells. Specifically, LATS1 restricts lethal autophagy in HCC cells induced by sorafenib, the standard of care for advanced HCC patients. Notably, autophagy regulation by LATS1 is independent of its kinase activity. Instead, LATS1 stabilizes the autophagy core-machinery component Beclin-1 by promoting K27-linked ubiquitination at lysine residues K32 and K263 on Beclin-1. Consequently, ubiquitination of Beclin-1 negatively regulates autophagy by promoting inactive dimer formation of Beclin-1. Our study highlights a functional diversity between LATS1 and LATS2, and uncovers a scaffolding role of LATS1 in mediating a cross-talk between the Hippo signaling pathway and autophagy.

Cell-specific role of histone deacetylase 6 in chemotherapy-induced mechanical allodynia and loss of intraepidermal nerve fibers.

Chemotherapy-induced peripheral neuropathy (CIPN) is a serious adverse side effect of cancer treatment with no Food and Drug Administration-approved medication for its prevention or management. Using RNA sequencing analysis of dorsal root ganglia (DRG), we identify critical contributions of histone deacetylase 6 (HDAC6) and mitochondrial damage to the establishment of CIPN in a mouse model of cisplatin-induced neuropathy. We show that pharmacological inhibition of HDAC6 using ACY-1215 or global deletion of HDAC6 is sufficient to prevent cisplatin-induced mechanical allodynia, loss of intraepidermal nerve fibers (IENFs), and mitochondrial bioenergetic deficits in DRG neurons and peripheral nerves in male and female mice. The bioenergetic deficits in the neuronal cell bodies in the DRG are characterized by reduced oxidative phosphorylation, whereas the mitochondrial deficits in the nerves are due to a reduction in axonal mitochondrial content. Notably, deleting HDAC6 in sensory neurons protects against the cisplatin-induced loss of IENFs and the reduction in mitochondrial bioenergetics and content in the peripheral nerve. By contrast, deletion of HDAC6 in sensory neurons only partially and transiently prevents cisplatin-induced mechanical allodynia and does not protect against impairment of mitochondrial function in DRG neurons. We further reveal a critical role of T cells in the protective effects of HDAC6 inhibition on these signs of CIPN. In summary, we show that cisplatin-induced mechanical allodynia is associated with mitochondrial damage in DRG neurons, whereas the loss of IENFs is related to bioenergetic deficits in peripheral nerves. Moreover, our findings identify cell-specific contributions of HDAC6 to mechanical allodynia and loss of IENFs that characterize cisplatin-induced peripheral neuropathy.

HDAC1 and HDAC2 Regulate Intermediate Progenitor Positioning to Safeguard Neocortical Development.

Neural progenitors with distinct potential to generate progeny are associated with a spatially distinct microenvironment. Neocortical intermediate progenitors (IPs) in the subventricular zone (SVZ) of the developing brain generate neurons for all cortical layers and are essential for cortical expansion. Here, we show that spatial control of IP positioning is essential for neocortical development. We demonstrate that HDAC1 and HDAC2 regulate the spatial positioning of IPs to form the SVZ. Developmental stage-specific depletion of both HDAC1 and HDAC2 in radial glial progenitors results in mispositioning of IPs at the ventricular surface, where they divide and differentiate into neurons, thereby leading to the cortical malformation. We further identified the proneural gene Neurogenin2 as a key target of HDAC1 and HDAC2 for regulating IP positioning. Our results demonstrate the importance of the spatial positioning of neural progenitors in cortical development and reveal a mechanism underlying the establishment of the SVZ microenvironment.

Acetylation of intrinsically disordered regions regulates phase separation.

Liquid-liquid phase separation (LLPS) of proteins containing intrinsically disordered regions (IDRs) has been proposed as a mechanism underlying the formation of membrane-less organelles. Tight regulation of IDR behavior is essential to ensure that LLPS only takes place when necessary. Here, we report that IDR acetylation/deacetylation regulates LLPS and assembly of stress granules (SGs), membrane-less organelles forming in response to stress. Acetylome analysis revealed that the RNA helicase DDX3X, an important component of SGs, is a novel substrate of the deacetylase HDAC6. The N-terminal IDR of DDX3X (IDR1) can undergo LLPS in vitro, and its acetylation at multiple lysine residues impairs the formation of liquid droplets. We also demonstrated that enhanced LLPS propensity through deacetylation of DDX3X-IDR1 by HDAC6 is necessary for SG maturation, but not initiation. Our analysis provides a mechanistic framework to understand how acetylation and deacetylation of IDRs regulate LLPS spatiotemporally, and impact membrane-less organelle formation in vivo.

The Transcriptional Regulation of Germinal Center Formation.

Germinal centers (GCs) are essential structures of the humoral immune response, which form in the periphery in response to T cell dependent antigens. During the GC reaction, B cells undergo critical differentiation steps, which ultimately lead to the generation of antibodies with altered effector function and higher affinity for the selected antigen. Remarkably, many of the B cell tumors have their origin in the GCs; thus, understanding how the formation of these structures is regulated or deregulated is of high medical importance. This review gives an overview of the transcription factors that have been linked to the generation of GCs, and of their roles in the process.

Purification of Unanchored Polyubiquitin Chains from Influenza Virions.

Influenza A virus (IAV) is an enveloped virus with a segmented single-stranded negative-strand RNA genome. In general, the role of virally encapsidated host cell proteins in the viral life cycle is unclear. The virion contains abundant ubiquitin molecules some of which have been identified as unanchored polyubiquitin chains. These ubiquitin chains have been postulated to play a role in recruiting histone deacetylase 6 (HDAC6) to the cytosolic surface of late endosomes (LEs), promoting IAV uncoating via aggresome processing-a cellular machinery that disposes of protein waste. HDAC6, a class II HDAC, is unusual because it resides mostly in the cytosol instead of the nucleus. It is a unique protein consisting of two catalytic domains (CDs) and a zinc-finger ubiquitin-binding domain (ZnF-UBP) close to its C-terminus. This ZnF-UBP recognizes the unconjugated ubiquitin C-terminus (di-Gly motif) with very high affinity. Biochemical analyses showed that free di-Gly motifs are present in the form of unanchored ubiquitin inside IAV virions. These motifs are exposed following low pH-triggered viral fusion at the LEs and attract HDAC6 transiently to the cytosolic surface of vesicles. The binding of the two components promotes viral uncoating via HDAC6 interaction with cellular motor proteins dynein and myosin II and the viral M1 capsid. The cellular mechanism involved is related to aggresome processing, a pathway that promotes degradation of misfolded protein aggregates. K63-linked ubiquitin chains are thought to be the trigger for aggresome processing, though it is still not clear whether such types of chains are prevalent within the IAV capsid. Here, we present methods using purified ZnF-UBP domain of HDAC6 to immunoprecipitate viral unanchored ubiquitin chains, which can then be used for further biochemical analyses of ubiquitin chain linkage.