Zinc-finger Ubiquitin Binding Domain Research Articles

Structure-based virtual screening of novel USP5 inhibitors targeting the zinc finger ubiquitin-binding domain

The equilibrium of cellular protein levels is pivotal for maintaining normal physiological functions. USP5 belongs to the deubiquitination enzyme (DUBs) family, controlling protein degradation and preserving cellular protein homeostasis. Aberrant expression of USP5 is implicated in a variety of diseases, including cancer, neurodegenerative diseases, and inflammatory diseases. In this paper, a multi-level virtual screening (VS) approach was employed to target the zinc finger ubiquitin-binding domain (ZnF-UBD) of USP5, leading to the identification of a highly promising candidate compound 0456–0049. Molecular dynamics (MD) simulations were then employed to assess the stability of complex binding and predict hotspot residues in interactions. The results indicated that the candidate stably binds to the ZnF-UBD of USP5 through crucial interactions with residues ARG221, TRP209, GLY220, ASN207, TYR261, TYR259, and MET266. Binding free energy calculations, along with umbrella sampling (US) simulations, underscored a superior binding affinity of the candidate relative to known inhibitors. Moreover, US simulations revealed conformational changes of USP5 during ligand dissociation. These insights provide a valuable foundation for the development of novel inhibitors targeting USP5.

Design, synthesis, and biological evaluation of HDAC6 inhibitors targeting L1 loop and serine 531 residue

Histone deacetylases (HDACs) are a group of enzymes that remove acetyl groups from histones, leading to the silencing of genes. Targeting specific isoforms of HDACs has emerged as a promising approach for cancer therapy, as it can overcome drawbacks associated with pan-HDAC inhibitors. HDAC6 is a unique HDAC isoform that deacetylates non-histone proteins and is primarily located in the cytoplasm. It also has two catalytic domains and a zinc-finger ubiquitin binding domain (Zf-UBD) unlike other HDACs. HDAC6 plays a critical role in various cellular processes, including cell motility, protein degradation, cell proliferation, and transcription. Hence, the deregulation of HDAC6 is associated with various malignancies. In this study, we report the design and synthesis of a series of HDAC6 inhibitors. We evaluated the synthesized compounds by HDAC enzyme assay and identified that compound 8g exhibited an IC50 value of 21 nM and 40-fold selective activity towards HDAC6. We also assessed the effect of compound 8g on various cell lines and determined its ability to increase protein acetylation levels by Western blotting. Furthermore, the increased acetylation of α-tubulin resulted in microtubule polymerization and changes in cell morphology. Our molecular docking study supported these findings by demonstrating that compound 8g binds well to the catalytic pocket via L1 loop of HDAC6 enzyme. Altogether, compound 8g represents a preferential HDAC6 inhibitor that could serve as a lead for the development of more potent and specific inhibitors.

Discovery and Characterization of a Chemical Probe Targeting the Zinc-Finger Ubiquitin-Binding Domain of HDAC6.

Histone deacetylase 6 (HDAC6) inhibition is an attractive strategy for treating numerous cancers, and HDAC6 catalytic inhibitors are currently in clinical trials. The HDAC6 zinc-finger ubiquitin-binding domain (UBD) binds free C-terminal diglycine motifs of unanchored ubiquitin polymer chains and protein aggregates, playing an important role in autophagy and aggresome assembly. However, targeting this domain with small molecule antagonists remains an underdeveloped avenue of HDAC6-focused drug discovery. We report SGC-UBD253 (25), a chemical probe potently targeting HDAC6-UBD in vitro with selectivity over nine other UBDs, except for weak USP16 binding. In cells, 25 is an effective antagonist of HDAC6-UBD at 1 μM, with marked proteome-wide selectivity. We identified SGC-UBD253N (32), a methylated derivative of 25 that is 300-fold less active, serving as a negative control. Together, 25 and 32 could enable further exploration of the biological function of the HDAC6-UBD and investigation of the therapeutic potential of targeting this domain.

Discovery and characterization of a chemical probe targeting the zinc-finger ubiquitin-binding domain of HDAC6

Discovery and characterization of a chemical probe targeting the zinc-finger ubiquitin-binding domain of HDAC6

Structure-Activity Relationship of USP5 Inhibitors.

USP5 is a deubiquitinase that has been implicated in a range of diseases, including cancer, but no USP5-targeting chemical probe has been reported to date. Here, we present the progression of a chemical series that occupies the C-terminal ubiquitin-binding site of a poorly characterized zinc-finger ubiquitin binding domain (ZnF-UBD) of USP5 and competitively inhibits the catalytic activity of the enzyme. Exploration of the structure-activity relationship, complemented with crystallographic characterization of the ZnF-UBD bound to multiple ligands, led to the identification of 64, which binds to the USP5 ZnF-UBD with a KD of 2.8 μM and is selective over nine proteins containing structurally similar ZnF-UBD domains. 64 inhibits the USP5 catalytic cleavage of a di-ubiquitin substrate in an in vitro assay. This study provides a chemical and structural framework for the discovery of a chemical probe to delineate USP5 function in cells.

Expression, purification and characterization of the second DUSP domain of deubiquitinase USP20/VDU2

Deubiquitinase USP20/VDU2 (VHL-interacting Deubiquitinating Enzyme 2) has been proved to play vital roles in multiple cellular processes by controlling the life-span of substrate proteins including hypoxia-inducible factor HIF1α, β2-adrenergic receptor, and type 2 iodothyronine deiodinase etc. USP20 contains four distinct structural domains, which include the N-terminal zinc-finger ubiquitin binding domain (ZnF-UBP), the catalytic domain (USP domain), and two tandem DUSP domains (DUSP1 and DUSP2). Here in this study, we report the setting up of the production approach for USP20 DUSP2, and the NMR characterization of the produced target protein. With the assistance of GB1 tag and glycerol, both the solubility and stability of USP20 DUSP2 are significantly enhanced. And by using the optimized protein production procedure, monomeric and stable 15N, 13C-labeled USP20 DUSP2 sample for NMR data acquisition was obtained. The secondary structural elements of USP20 DUSP2 were then revealed by the analysis of recorded NMR spectra, and USP20 DUSP2 forms an AB3 fold in solution. The production protocol and NMR characterization results reported in this manuscript could be utilized in the extended structural and functional studies of USP20 DUSP2.

Discovery of Small Molecule Antagonists of the USP5 Zinc Finger Ubiquitin-Binding Domain.

USP5 disassembles unanchored polyubiquitin chains to recycle free monoubiquitin, and is one of the 12 ubiquitin specific proteases featuring a zinc finger ubiquitin-binding domain (ZnF-UBD). This distinct structural module has been associated with substrate positioning or allosteric modulation of catalytic activity, but its cellular function remains unclear. We screened a chemical library focused on the ZnF-UBD of USP5, crystallized hits in complex with the protein, and generated a preliminary structure-activity relationship, which enables the development of more potent and selective compounds. This work serves as a framework for the discovery of a chemical probe to delineate the function of USP5 ZnF-UBD in proteasomal degradation and other ubiquitin signaling pathways in health and disease.

Structural and functional studies of USP20 ZnF-UBP domain by NMR.

Deubiquitinase USP20/VDU2 has been demonstrated to play important roles in multiple cellular processes by controlling the life span of substrate proteins including hypoxia-inducible factor HIF1α, and so forth. USP20 contains four distinct structural domains including the N-terminal zinc-finger ubiquitin binding domain (ZnF-UBP), the catalytic domain (USP domain), and two tandem DUSP domains, and none of the structures for these four domains has been solved. Meanwhile, except for the ZnF-UBP domain, the biological functions for USP20's catalytic domain and tandem DUSP domains have been at least partially clarified. Here in this study, we determined the solution structure of USP20 ZnF-UBP domain and investigated its binding properties with mono-ubiquitin and poly-ubiquitin (K48-linked di-ubiquitin) by using NMR and molecular modeling techniques. USP20's ZnF-UBP domain forms a spherically shaped fold consisting of a central β-sheet with either one α-helix or two α-helices packed on each side of the sheet. However, although having formed a canonical core structure essential for ubiquitin recognition, USP20 ZnF-UBP presents weak ubiquitin binding capacity. The structural basis for understanding USP20 ZnF-UBP's ubiquitin binding capacity was revealed by NMR data-driven docking. Although the electrostatic interactions between D264 of USP5 (E87 in USP20 ZnF-UBP) and R74 of ubiquitin are kept, the loss of the extensive interactions formed between ubiquitin's di-glycine motif and the conserved and non-conserved residues of USP20 ZnF-UBP domain (W41, E55, and Y84) causes a significant decrease in its binding affinity to ubiquitin. Our findings indicate that USP20 ZnF-UBP domain might have a physiological role unrelated to its ubiquitin binding capacity.

Identification and Structure-Activity Relationship of HDAC6 Zinc-Finger Ubiquitin Binding Domain Inhibitors.

HDAC6 plays a central role in the recruitment of protein aggregates for lysosomal degradation and is a promising target for combination therapy with proteasome inhibitors in multiple myeloma. Pharmacologically displacing ubiquitin from the zinc-finger ubiquitin-binding domain (ZnF-UBD) of HDAC6 is an underexplored alternative to catalytic inhibition. Here, we present the discovery of an HDAC6 ZnF-UBD-focused chemical series and its progression from virtual screening hits to low micromolar inhibitors. A carboxylate mimicking the C-terminal extremity of ubiquitin, and an extended aromatic system stacking with W1182 and R1155, are necessary for activity. One of the compounds induced a conformational remodeling of the binding site where the primary binding pocket opens up onto a ligand-able secondary pocket that may be exploited to increase potency. The preliminary structure-activity relationship accompanied by nine crystal structures should enable further optimization into a chemical probe to investigate the merit of targeting the ZnF-UBD of HDAC6 in multiple myeloma and other diseases.

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.

Small Molecule Antagonists of the Interaction between the Histone Deacetylase 6 Zinc-Finger Domain and Ubiquitin.

Inhibitors of HDAC6 have attractive potential in numerous cancers. HDAC6 inhibitors to date target the catalytic domains, but targeting the unique zinc-finger ubiquitin-binding domain (Zf-UBD) of HDAC6 may be an attractive alternative strategy. We developed X-ray crystallography and biophysical assays to identify and characterize small molecules capable of binding to the Zf-UBD and competing with ubiquitin binding. Our results revealed two adjacent ligand-able pockets of HDAC6 Zf-UBD and the first functional ligands for this domain.

1H, 13C and 15N backbone and side-chain resonance assignments of the ZnF-UBP domain of USP20/VDU2.

Deubiquitinase USP20/VDU2 has been identified as a regulator of multiple proteins including hypoxia-inducible factor (HIF)-1α, β2-adrenergic receptor, and tumor necrosis factor receptor associated factor 6 etc. It contains four structural domains, including an N-terminal zinc-finger ubiquitin binding domain (ZnF-UBP) that potentially helps USP20 to recruit its ubiquitin substrates. Here we report the 1H, 13C and 15N backbone and side-chain resonance assignments of the ZnF-UBP domain of USP20/VDU2. The BMRB accession number is 26901. The secondary structural elements predicted from the NMR data reveal a global fold consisting of three α-helices and four β-strands. The complete assignments can be used to explore the protein dynamics of the USP20 ZnF-UBP and its interactions with monoubiquitin and ubiquitin chains.

Molecular dynamics of zinc-finger ubiquitin binding domains: a comparative study of histone deacetylase 6 and ubiquitin-specific protease 5

HDAC6 is a unique cytoplasmic histone deacetylase characterized by two deacetylase domains, and by a zinc-finger ubiquitin binding domain (ZnF-UBP) able to recognize ubiquitin (Ub). The latter has recently been demonstrated to be involved in the progression of neurodegenerative diseases and in mediating infection by the influenza A virus. Nowadays, understanding the dynamic and energetic features of HDAC6 ZnF-UBP-Ub recognition is considered as a crucial step for the conception of HDAC6 potential modulators. In this study, the atomic, solvent-related, and thermodynamic features behind HDAC6 ZnF-UBP-Ub recognition have been analyzed through molecular dynamics simulations. The behavior was then compared to the prototypical ZnF-UBP from ubiquitin-specific protease 5 (USP5) in order to spot relevant differences useful for selective drug design. Principal component analysis highlighted flapping motions of the L2A loop which were lowered down upon Ub binding in both systems. While polar and nonpolar interactions involving Ub G75 and G76 residues were also common features stabilizing both complexes, salt bridges showed a different pattern, more significant in HDAC6 ZnF-UBP-Ub, whose energetic contribution in USP5 ZnF-UBP-Ub was compensated by the presence of a more stable bridging water molecule. Whereas molecular mechanics/Poisson–Boltzmann surface area (MM-PBSA) free energies of binding were comparable for both systems, in agreement with experiments, computational alanine scanning and free energy decomposition data revealed that HDAC6 E1141 and D1178 are potential hotspots for the design of selective HDAC6 modulators.

The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing

Sad1 is an essential splicing factor initially identified in a genetic screen in Saccharomyces cerevisiae for snRNP assembly defects. Based on sequence homology, Sad1, or USP39 in humans, is predicted to comprise two domains: a zinc finger ubiquitin binding domain (ZnF-UBP) and an inactive ubiquitin-specific protease (iUSP) domain, both of which are well conserved. The role of these domains in splicing and their interaction with ubiquitin are unknown. We first used splicing microarrays to analyze Sad1 function in vivo and found that Sad1 is critical for the splicing of nearly all yeast intron-containing genes. By using in vitro assays, we then showed that it is required for the assembly of the active spliceosome. To gain structural insights into Sad1 function, we determined the crystal structure of the full-length protein at 1.8 Å resolution. In the structure, the iUSP domain forms the characteristic ubiquitin binding pocket, though with an amino acid substitution in the active site that results in complete inactivation of the enzymatic activity of the domain. The ZnF-UBP domain of Sad1 shares high structural similarly to other ZnF-UBPs; however, Sad1's ZnF-UBP does not possess the canonical ubiquitin binding motif. Given the precedents for ZnF-UBP domains to function as activators for their neighboring USP domains, we propose that Sad1's ZnF-UBP acts in a ubiquitin-independent capacity to recruit and/or activate Sad1's iUSP domain to interact with the spliceosome.

The Ubiquitin Binding Domain ZnF UBP Recognizes the C-Terminal Diglycine Motif of Unanchored Ubiquitin

Ubiquitin binding proteins regulate the stability, function, and/or localization of ubiquitinated proteins. Here we report the crystal structures of the zinc-finger ubiquitin binding domain (ZnF UBP) from the deubiquitinating enzyme isopeptidase T (IsoT, or USP5) alone and in complex with ubiquitin. Unlike other ubiquitin binding domains, this domain contains a deep binding pocket where the C-terminal diglycine motif of ubiquitin is inserted, thus explaining the specificity of IsoT for an unmodified C terminus on the proximal subunit of polyubiquitin. Mutations in the domain demonstrate that it is required for optimal catalytic activation of IsoT. This domain is present in several other protein families, and the ZnF UBP domain from an E3 ligase also requires the C terminus of ubiquitin for binding. These data suggest that binding the ubiquitin C terminus may be necessary for the function of other proteins.