Ubiquitin-binding Domain Research Articles

Seneca Valley virus 3C protease cleaves OPTN (optineurin) to Impair selective autophagy and type I interferon signaling

ABSTRACT Seneca Valley virus (SVV) causes vesicular disease in pigs, posing a threat to global pork production. OPTN (optineurin) is a macroautophagy/autophagy receptor that restricts microbial propagation by targeting specific viral or bacterial proteins for degradation. OPTN is degraded and cleaved at glutamine 513 following SVV infection via the activity of viral 3C protease (3C[pro]), resulting in N-terminal and a C-terminal OPTN fragments. Moreover, OPTN interacts with VP1 and targets VP1 for degradation to inhibit viral replication. The N-terminal cleaved OPTN sustained its interaction with VP1, whereas the degradation capacity targeting VP1 decreased. The inhibitory effect of N-terminal OPTN against SVV infection was significantly reduced, C-terminal OPTN failed to inhibit viral replication, and degradation of VP1 was blocked. The knockdown of OPTN resulted in reduced TBK1 activation and phosphorylation of IRF3, whereas overexpression of OPTN led to increased TBK1-IRF3 signaling. Additionally, the N-terminal OPTN diminished the activation of the type I IFN (interferon) pathway. These results show that SVV 3C[pro] targets OPTN because its cleavage impairs its function in selective autophagy and type I IFN production, revealing a novel model in which the virus develops diverse strategies for evading host autophagic machinery and type I IFN response for survival. Abbreviations: Co-IP: co-immunoprecipitation; GFP-green fluorescent protein; hpi: hours post-infection; HRP: horseradish peroxidase; IFN: interferon; IFNB/IFN-β: interferon beta; IRF3: interferon regulatory factor 3; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; OPTN: optineurin; PBS: phosphate-buffered saline; SVV: Seneca Valley virus; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious doses; UBAN: ubiquitin binding in TNIP/ABIN (TNFAIP3/A20 and inhibitor of NFKB/NF-kB) and IKBKG/NEMO; UBD: ubiquitin-binding domain; ZnF: zinc finger.

A Novel Interaction Between RAD23A/B and Y-family DNA Polymerases

The Y-family DNA polymerases – Pol ι, Pol η, Pol κ and Rev1 – are most well-known for their roles in the DNA damage tolerance pathway of translesion synthesis (TLS). They function to overcome replication barriers by bypassing DNA damage lesions that cannot be normally replicated, allowing replication forks to continue without stalling. In this work, we demonstrate a novel interaction between each Y-family polymerase and the nucleotide excision repair (NER) proteins, RAD23A and RAD23B. We initially focus on the interaction between RAD23A and Pol ι, and through a series of biochemical, cell-based, and structural assays, find that the RAD23A ubiquitin-binding domains (UBA1 and UBA2) interact with separate sites within the Pol ι catalytic domain. While this interaction involves the ubiquitin-binding cleft of UBA2, Pol ι interacts with a distinct surface on UBA1. We further find that mutating or deleting either UBA domain disrupts the RAD23A-Pol ι interaction, demonstrating that both interactions are necessary for stable binding. We also provide evidence that both RAD23 proteins interact with Pol ι in a similar manner, as well as with each of the Y-family polymerases. These results shed light on the interplay between the different functions of the RAD23 proteins and reveal novel binding partners for the Y-family TLS polymerases.

Fish Rhbdd3 positively regulates IFN response through RIG-I signaling pathway

Rhomboid domain-containing protein 3 (Rhbdd3) is a member of the rhomboid family, which can modulate the innate immune response in mammals. Nonetheless, the function and regulatory mechanism of fish Rhbdd3 during viral infection have not been characterized. In this study, Rhbdd3 was firstly cloned from common carp (Cyprinus carpio) and nominated as CcRhbdd3. Phylogenetically characterization showed that CcRhbdd3 shared a relatively long evolutionary distance with its mammalian homologs. In vivo experiment demonstrated that spring viraemia of carp virus (SVCV) infection promoted the expression of CcRhbdd3 in the liver, spleen, kidney and muscle tissues. Furthermore, overexpression of CcRhbdd3 significantly inhibited SVCV propagation, whereas knockdown of CcRhbdd3 markedly promoted SVCV replication in susceptible cells. RNA-seq and following validation showed that CcRhbdd3 overexpression upregulated the expression of several RIG-I signaling related genes, including TRIM25, TRAF2, MDA5, LGP2, IFN1, IFN3, RIG-I, IRF3 and ISG15. Moreover, CcRhbdd3 promoted the expression of NF-κB, a central immune regulator. Subcellular localization experiments showed that CcRhbdd3 was primarily distributed in the cytoplasm and co-localized with Rab5 in the early endosomes. Truncation experiments further demonstrated that the C-terminus containing the ubiquitin-binding associated domain, was crucial for both the subcellular localization and antiviral activity of CcRhbdd3. The findings in this study provide new insight into the host antiviral mechanism against aquatic RNA virus infection, and will facilitate the development of therapeutic strategies for the infection of SVCV.

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.

The Importance of the "Time Factor" for the Evaluation of Inhibition Mechanisms: The Case of Selected HDAC6 Inhibitors.

Histone deacetylases (HDACs) participate with histone acetyltransferases in the modulation of the biological activity of a broad array of proteins, besides histones. Histone deacetylase 6 is unique among HDAC as it contains two catalytic domains, an N-terminal microtubule binding region and a C-terminal ubiquitin binding domain. Most of its known biological roles are related to its protein lysine deacetylase activity in the cytoplasm. The design of specific inhibitors is the focus of a large number of medicinal chemistry programs in the academy and industry because lowering HDAC6 activity has been demonstrated to be beneficial for the treatment of several diseases, including cancer, and neurological and immunological disorders. Here, we show how re-evaluation of the mechanism of action of selected HDAC6 inhibitors, by monitoring the time-dependence of the onset and relief of the inhibition, revealed instances of slow-binding/slow-release inhibition. The same approach, in conjunction with X-ray crystallography, in silico modeling and mass spectrometry, helped to propose a model of inhibition of HDAC6 by a novel difluoromethyloxadiazole-based compound that was found to be a slow-binding substrate analog of HDAC6, giving rise to a tightly bound, long-lived inhibitory derivative.

Bioinformatics analysis and single-cell RNA sequencing: elucidating the ubiquitination pathways and key enzymes in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a prevalent subtype of lung cancer associated with high mortality rates. We aimed to utilize single-cell multiomics analysis to identify the key molecules involved in ubiquitination modification, which plays a role in LUAD development and progression. We use a systematic approach to analyze LUAD-related single-cell and bulk transcriptome datasets from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Single-cell RNA sequencing (scRNA-seq) data were normalized, clustered, and annotated with the Seurat package in R. InferCNV was used to distinguish malignant from epithelial cells, and AUCell evaluated the area under the curve (AUC) score of ubiquitination-related enzymes. Survival and differential analyses identified significant molecular markers associated with ubiquitination. PSMD14 expression was confirmed using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot assays, and its knockdown cell lines were assessed for effects on cellular processes and tumor formation in mice. PSMD14's interacting proteins were predicted, and its impact on AGR2 protein half-life and ubiquitination was evaluated. Rescue experiments involving PSMD14 overexpression and AGR2 silencing assessed their impact on malignant behaviors. By means of single-cell sequencing analysis, we probed the ubiquitination modification landscape in the LUAD microenvironment. Malignant cells had elevated scores for enzymes and ubiquitin-binding domains compared to normal epithelial cells, with 53 ubiquitination-related molecules showing prognostic disparities. FGR, PSMD14, and ZBTB16 were identified as genes with prognostic significance, with PSMD14 showing higher expression in epithelial and malignant cells. Two missense mutation sites were identified in PSMD14, which had a high copy number amplification ratio and positive correlation with messenger RNA (mRNA) expression. PSMD14 expression and tumor stage were found to be independent prognostic factors, and interfering with PSMD14 expression reduced the malignant behavior of LUAD cells. PSMD14 was found to bind to AGR2 protein and reduce its ubiquitination, leading to increased AGR2 stability. Knockdown of AGR2 inhibited the enhancement of cell viability, invasion, and migration resulting from PSMD14 overexpression. This study examined ubiquitination modifications in LUAD using sequencing data, identifying PSMD14's critical role in malignancy regulation and its potential as a prognostic and therapeutic biomarker. These insights enhance understanding of LUAD mechanisms and treatment.

Targeting of biomolecular condensates to the autophagy pathway.

Biomolecular condensates are membraneless compartments formed by liquid-liquid phase separation. They can phase transit into gel-like and solid states. The amount and state of biomolecular condensates must be tightly regulated to maintain normal cellular function. Autophagy targets biomolecular condensates to the lysosome for degradation or other purposes, which we term biocondensophagy. In biocondensophagy, autophagy receptors recognize biomolecular condensates and target them to the autophagosome, the vesicle carrier of autophagy. Multiple types of autophagy receptors have been identified and they are specifically involved in targeting biomolecular condensates with different phase transition states. The receptors also organize the phase transition of biomolecular condensate to facilitate biocondensophagy. Here, we briefly discuss the latest discoveries regarding how biomolecular condensates are recognized by autophagy receptors.

Repurposing the inhibitors of MMP-9 and SGLT-2 against ubiquitin specific protease 30 in Parkinson’s disease: computational modelling studies

Ubiquitin specific protease 30 (USP30) has been attributed to mitochondrial dysfunction and impediment of mitophagy in Parkinson’s disease (PD). This happens once ubiquitin that supposed to bind with deformed mitochondria at the insistence of Parkin, it’s been recruited by USP30 via the distal ubiquitin binding domain. This is a challenge when PINK1 and Parkin loss their functions due to mutation. Although, there are reports on USP30s’ inhibitors but no study on the repurposing of inhibitors approved against MMP-9 and SGLT-2 as potential inhibitors of USP30 in PD. Thus, the highlight therein, is to repurpose approved inhibitors of MMP-9 and SGLT-2 against USP30 in PD using extensive computational modelling framework. 3D structures of Ligands and USP30 were obtained from PubChem and protein database (PDB) servers respectively, and were subjected to molecular docking, ADMET evaluation, DFT calculation, molecular dynamics simulation (MDS) and free energy calculations. Out of the 18 drugs, 2 drugs showed good binding affinity to the distal ubiquitin binding domain, moderate pharmacokinetic properties and good stability. The findings showed canagliflozin and empagliflozin as potential inhibitors of USP30. Thus, we present these drugs as repurposing candidates for the treatment of PD. However, the findings in this current study needs to be validated experimentally. Communicated by Ramaswamy H. Sarma

The autophagy receptor NBR1 directs the clearance of photodamaged chloroplasts.

The ubiquitin-binding NBR1 autophagy receptor plays a prominent role in recognizing ubiquitylated protein aggregates for vacuolar degradation by macroautophagy. Here, we show that upon exposing Arabidopsis plants to intense light, NBR1 associates with photodamaged chloroplasts independently of ATG7, a core component of the canonical autophagy machinery. NBR1 coats both the surface and interior of chloroplasts, which is then followed by direct engulfment of the organelles into the central vacuole via a microautophagy-type process. The relocalization of NBR1 into chloroplasts does not require the chloroplast translocon complexes embedded in the envelope but is instead greatly enhanced by removing the self-oligomerization mPB1 domain of NBR1. The delivery of NBR1-decorated chloroplasts into vacuoles depends on the ubiquitin-binding UBA2 domain of NBR1 but is independent of the ubiquitin E3 ligases SP1 and PUB4, known to direct the ubiquitylation of chloroplast surface proteins. Compared to wild-type plants, nbr1 mutants have altered levels of a subset of chloroplast proteins and display abnormal chloroplast density and sizes upon high light exposure. We postulate that, as photodamaged chloroplasts lose envelope integrity, cytosolic ligases reach the chloroplast interior to ubiquitylate thylakoid and stroma proteins which are then recognized by NBR1 for autophagic clearance. This study uncovers a new function of NBR1 in the degradation of damaged chloroplasts by microautophagy.

Abstract LB335: Establishment of the effective dose of the STAT3 inhibitor WP1066 used in combination with STING activation for reprograming the preclinical glioma microenvironment

Abstract Purpose: Trials of immunotherapy in diffuse glioma patients have been mostly unsuccessful. We therefore sought to determine the combined effects of the signal transducer and activator of transcription 3 (STAT3) inhibitor WP1066 and the STING agonist IACS-8803 in a preclinical model of glioma. Experimental Design: C57BL/6 mice (n=9-10/group) with orthotopically engrafted GL261 cells were treated orally with WP1066, a blood-brain-barrier penetrant inhibitor of STAT3, in combination with the STING agonist IACS-8803 administered directly into the tumor. Analysis of treatment effects included immunoblots, ubiquitination, multiplex immunohistochemistry, and NanoString immune phenotyping of glioma-infiltrating immune cells. Results: The STING agonist 8803 at 2.5µg/mouse, in combination with WP1066 dosed at 30mg/kg with a 12-hour delay, increased animal subject median survival (MS) to 58 days, in contrast to monotherapy (WP1066=25 days, 8803=29 days) or control (25 days) (p=0.002 of combo relative to all other groups). Dose escalation of WP1066 to 60 mg/kg, administered simultaneously with 8803, completely abrogated the therapeutic combinatorial effect. Five ubiquitin-binding domains were predicted on STING using the UbPred program; concentrations of 5µM or higher of WP1066 induced polyubiquitination of phosphorylated STING. Immune profiling during the therapeutic window demonstrated a multi-pathway induction of anti-tumor immunity. Conclusions: Co-administration of a reduced effective dose of WP1066 is necessary with STING agonist 8803 to effectively enhance the anti-glioma immune reactivity in the tumor microenvironment. Citation Format: Hinda Najem, Shashwat Tripathi, Moloud Sooreshjani, Lisa Hurley, Corey Dussold, Victor Arrieta, Martina Ott, Jun Wei, Michael Curran, Anantha Marisetty, Waldemar Priebe, Leondias C. Platanias, Maciej S. Lesniak, Charles D. James, Craig Horbinski, David M. Ashley, Amy B. Heimberger. Establishment of the effective dose of the STAT3 inhibitor WP1066 used in combination with STING activation for reprograming the preclinical glioma microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB335.

Abstract 3959: Novel spliced isoform of the proteasome subunit ADRM1/Rpn13 promotes hepatocellular carcinoma (HCC) development through selective degradation of tumor suppressor p53

Abstract The Ubiquitin-Proteasome System (UPS) is the major pathway in eukaryotic cells that regulates intracellular protein degradation and turnover. Cumulative evidence has suggested that defects in the UPS machinery are involved in the pathogenesis of human diseases, including cancer. The proteasome-associated polyubiquitin receptor ADRM1 (also known as Rpn13) orchestrates the process of substrate recruitment with deubiquitination in the proteasome through its N-terminal ubiquitin-binding domain and C-terminal activation of deubiquitinating enzyme. Using long-read SMRT-seq to profile the RNA splicing landscape in hepatocellular carcinoma (HCC), we previously reported on an unannotated alternative spliced variant of ADRM1 in human hepatocellular carcinoma (HCC) (Hepatology 2019). This novel isoform exhibits an exon 9 skipping (ADRM1-ΔEx9) that resulted in an altered C-terminus of the protein. In this study, we undertook further investigations to characterize the detailed functional effects of ADRM1-ΔEx9 in HCC biology and the underlying mechanism. Initial study by junction-specific Taqman PCR assay showed that ADRM1-ΔEx9 is more frequently upregulated in HCC tumors than the canonical full-length counterpart (ADRM1-FL). There is an interchange between the two isoforms as tumor develops which underscores an isoform-switch in favor of HCC development. Indeed, our result showed that ADRM1-ΔEx9, but not ADRM1-FL, correlated significantly with inferior patient survival. Functionally, ADRM1-ΔEx9 knockdown profoundly suppressed proliferation by inducing spontaneous apoptosis in HCC cell lines and patient-derived HCC organoids. The corresponding effects were less apparent from ADRM1-FL knockdown. Consistently, overexpression of ADRM1-ΔEx9 in normal human liver organoids promoted pre-malignant features alongside increased in-vitro propagation time. Ubiquitin Proteome profiling revealed that ADRM1-ΔEx9 led to a reduced expression of the pivotal tumor suppressor p53. A parallel transcriptome also substantiated that ADRM1-ΔEx9 modulated genes involved in the p53 and apoptosis pathways. Collectively, our study demonstrated that the non-canonical spliced isoform ADRM1-ΔEx9 confers growth and pro-survival advantages in HCC through mediating selective degradation of tumor suppressor p53 protein. Citation Format: Yanmei Sun, Mingjing Xu, Ho Lee Wan, Alissa M. Wong, Kelvin K. Ng, Nathalie Wong. Novel spliced isoform of the proteasome subunit ADRM1/Rpn13 promotes hepatocellular carcinoma (HCC) development through selective degradation of tumor suppressor p53. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3959.

Characterisation of the OTU domain deubiquitinase complement of Toxoplasma gondii.

The phylum Apicomplexa contains several parasitic species of medical and agricultural importance. The ubiquitination machinery remains, for the most part, uncharacterised in apicomplexan parasites, despite the important roles that it plays in eukaryotic biology. Bioinformatic analysis of the ubiquitination machinery in apicomplexan parasites revealed an expanded ovarian tumour domain-containing (OTU) deubiquitinase (DUB) family in Toxoplasma, potentially reflecting functional importance in apicomplexan parasites. This study presents comprehensive characterisation of Toxoplasma OTU DUBs. AlphaFold-guided structural analysis not only confirmed functional orthologues found across eukaryotes, but also identified apicomplexan-specific enzymes, subsequently enabling discovery of a cryptic OTU DUB in Plasmodium species. Comprehensive biochemical characterisation of 11 Toxoplasma OTU DUBs revealed activity against ubiquitin- and NEDD8-based substrates and revealed ubiquitin linkage preferences for Lys6-, Lys11-, Lys48-, and Lys63-linked chain types. We show that accessory domains in Toxoplasma OTU DUBs impose linkage preferences, and in case of apicomplexan-specific TgOTU9, we discover a cryptic ubiquitin-binding domain that is essential for TgOTU9 activity. Using the auxin-inducible degron (AID) to generate knockdown parasite lines, TgOTUD6B was found to be important for Toxoplasma growth.

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

Coarse-grained model for simulating protein complexes: Folded vs. disordered proteins.

We develop a residue-level coarse-grained model for simulating multi-protein complexes. Contact potentials and distances between all pairs of residues are constructed via Boltzmann inversion of inter-molecular contacts of more than 22000 non-redundant unique interfaces of protein complexes. We show that the contact potentials between hydrophobic pairs are less sensitive to the residue identities when compared to the Miyazawa-Jernigan contact potentials derived from intra-molecular contacts. On the other hand, the contact potentials between hydrophilic pairs depend more strongly on the residue identities. These potentials and contact distances are adopted to the modified Wang-Frenkel potential for the short-range van der Waals (vdW) interactions and Deybe-Hückel electrostatics with a distance-dependent dielectric constant to simulate thermodynamic, kinetic, and structural properties of protein complexes of folded and disordered proteins. Upon parameterizing to fit the second virial coefficient of lysozyme and the binding affinities of the ubiquitin and ubiquitin-binding domains, we show that the potential form with a shorter cutoff distance performs best in predicting the binding affinities and structures for folded proteins. On the other hand, the structural properties of disordered proteins measured by the radius of gyration are in better agreement with experimental data at longer cutoff distances. Comparison between pairwise contact distributions for folded and disordered proteins shows that contacts between charged and polar residues are more prevalent in disordered proteins. The potentials are modified to account for this disparity. We show that the modified potentials perform well in predicting the binding affinities and structures for folded proteins as well as the radius of gyration for disordered proteins. The model can easily be implemented to many existing simulation packages to simulate multi-protein complexes containing disordered regions or proteins in order to gain insights into understanding many biological processes inside a cell.

METTL5 stabilizes c-Myc by facilitating USP5 translation to reprogram glucose metabolism and promote hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world, with a high likelihood of metastasis and a dismal prognosis. The reprogramming of glucose metabolism is critical in the development of HCC. The Warburg effect has recently been confirmed to occur in a variety of cancers, including HCC. However, little is known about the molecular biological mechanisms underlying the Warburg effect in HCC cells. In this study, we sought to better understand how methyltransferase 5, N6-adenosine (METTL5) controls the development of HCC and the Warburg effect. In the current study, quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of METTL5 in HCC tissues and cell lines. Several different cell models and animal models were established to determine the role of METTL5 in glucose metabolism reprogramming and the underlying molecular mechanism of HCC. Glutathione-S-transferase pulldown, coimmunoprecipitation, RNA sequencing, non-targeted metabolomics, polysome profiling, and luciferase reporter assays were performed to investigate the molecular mechanisms of METTL5 in HCC cells. We discovered that METTL5 drove glucose metabolic reprogramming to promote the proliferation and metastasis of HCC. Mechanistically, upregulation of METTL5 promoted c-Myc stability and thus activated its downstream glycolytic genes lactate dehydrogenase A (LDHA), enolase 1 (ENO1), triosephosphate isomerase 1 (TPI1), solute carrier family 2 member 1 (SLC2A1), and pyruvate kinase M2 (PKM2). The c-Box and ubiquitin binding domain (UBA) regions of ubiquitin specific peptidase 5 (USP5) binded to c-Myc protein and inhibited K48-linked polyubiquitination of c-Myc. Further study revealed that METTL5 controled the USP5 translation process, which in turn regulated the ubiquitination of c-Myc. Furthermore, we identified cAMP responsive element binding protein 1 (CREB1)/P300 as a critical transcriptional regulator of METTL5 that promoted the transcription of METTL5 in HCC. In patient-derived tumor xenograft (PDX) models, adenovirus-mediated knockout of METTL5 had a good antitumor effect and prolonged the survival of PDX-bearing mice. These findings point to a novel mechanism by which CREB1/P300-METTL5-USP5-c-Myc controls abnormal glucose metabolism and promotes tumor growth, suggesting that METTL5 is a potential therapeutic target and prognostic biomarker for HCC.

Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA

The versatility of ubiquitination to control vast domains of eukaryotic biology is due, in part, to diversification through differently linked poly-ubiquitin chains. Deciphering signaling roles for some chain types, including those linked via K6, has been stymied by a lack of specificity among the implicated regulatory proteins. Forged through strong evolutionary pressures, pathogenic bacteria have evolved intricate mechanisms to regulate host ubiquitin during infection. Herein, we identify and characterize a deubiquitinase domain of the secreted effector LotA from Legionella pneumophila that specifically regulates K6-linked poly-ubiquitin. We demonstrate the utility of LotA for studying K6 poly-ubiquitin signals. We identify the structural basis of LotA activation and poly-ubiquitin specificity and describe an essential "adaptive" ubiquitin-binding domain. Without LotA activity during infection, the Legionella-containing vacuole becomes decorated with K6 poly-ubiquitin as well as the AAA ATPase VCP/p97/Cdc48. We propose that LotA's deubiquitinase activity guards Legionella-containing vacuole components from ubiquitin-dependent extraction.

The Lambda Display Technology: A Useful Tool for the Identification of Ubiquitin-and Ubiquitin-Like-Binding Domains.

Ubiquitin-binding domains (UBDs) are modular units that mediate non-covalent recognition of ubiquitin modifications. They are found in ubiquitin (Ub)-binding proteins and recognize defined surface patches of a single Ub through typically weak interactions. Although more than 200 Ub-binding proteins have been identified to date, only 29 UBD types have been reported in the human proteome, suggesting that much remains to be learned about Ub recognition. Several methods, from bioinformatics to experimental, have successfully identified Ub-binding properties in several proteins. We here report the protocol to identify Ub-binding domains by panning a human brain cDNA library whose products are displayed on the surface of lambda capsid. In parallel, we carried out a panning experiment aimed at identifying domains interacting with the Ub-like NEDD8 (neural precursor cell-expressed developmentally downregulated), which is the Ub-like protein showing the closest sequence identity (58%) to Ub. This approach proved to be very effective for the discovery of the previously unidentified UBDs CUBAN and CoCUN, and it is in principle applicable to investigate the interaction network of any other Ub-like protein.

High-Content RNAi Phenotypic Screening Unveils the Involvement of Human Ubiquitin-Related Enzymes in Late Cytokinesis

CEP55 is a central regulator of late cytokinesis and is overexpressed in numerous cancers. Its post-translationally controlled recruitment to the midbody is crucial to the structural coordination of the abscission sequence. Our recent evidence that CEP55 contains two ubiquitin-binding domains was the first structural and functional link between ubiquitin signaling and ESCRT-mediated severing of the intercellular bridge. So far, high-content screens focusing on cytokinesis have used multinucleation as the endpoint readout. Here, we report an automated image-based detection method of intercellular bridges, which we applied to further our understanding of late cytokinetic signaling by performing an RNAi screen of ubiquitin ligases and deubiquitinases. A secondary validation confirmed four candidate genes, i.e., LNX2, NEURL, UCHL1 and RNF157, whose downregulation variably affects interconnected phenotypes related to CEP55 and its UBDs, as follows: decreased recruitment of CEP55 to the midbody, increased number of midbody remnants per cell, and increased frequency of intercellular bridges or multinucleation events. This brings into question the Notch-dependent or independent contributions of LNX2 and NEURL proteins to late cytokinesis. Similarly, the role of UCHL1 in autophagy could link its function with the fate of midbody remnants. Beyond the biological interest, this high-content screening approach could also be used to isolate anticancer drugs that act by impairing cytokinesis and CEP55 functions.

An expanded lexicon for the ubiquitin code.

Our understanding of the ubiquitin code has greatly evolved from conventional E1, E2 and E3 enzymes that modify Lys residues on specific substrates with a single type of ubiquitin chain to more complex processes that regulate and mediate ubiquitylation. In this Review, we discuss recently discovered endogenous mechanisms and unprecedented pathways by which pathogens rewrite the ubiquitin code to promote infection. These processes include unconventional ubiquitin modifications involving ester linkages with proteins, lipids and sugars, or ubiquitylation through a phosphoribosyl bridge involving Arg42 of ubiquitin. We also introduce the enzymatic pathways that write and reverse these modifications, such as the papain-like proteases of severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Furthermore, structural studies have revealed that the ultimate functions of ubiquitin are mediated not simply by straightforward recognition by ubiquitin-binding domains. Instead, elaborate multivalent interactions between ubiquitylated targets or ubiquitin chains and their readers (for example, the proteasome, the MLL1 complex or DOT1L) can elicit conformational changes that regulate protein degradation or transcription. The newly discovered mechanisms provide opportunities for innovative therapeutic interventions for diseases such as cancer and infectious diseases.

The AAA-ATPase ATAD1 and its partners promote degradation of desmin intermediate filaments in muscle.

Maintenance of desmin intermediate filaments (IF) is vital for muscle plasticity and function, and their perturbed integrity due to accelerated loss or aggregation causes atrophy and myopathies. Calpain-1-mediated disassembly of ubiquitinated desmin IF is a prerequisite for desmin loss, myofibril breakdown, and atrophy. Because calpain-1 does not harbor a bona fide ubiquitin-binding domain, the precise mechanism for desmin IF disassembly remains unknown. Here, we demonstrate that the AAA-ATPase, ATAD1, is required to facilitate disassembly and turnover of ubiquitinated desmin IF. We identified PLAA and UBXN4 as ATAD1's interacting partners, and their downregulation attenuated desmin loss upon denervation. The ATAD1-PLAA-UBXN4 complex binds desmin filaments and promotes a release of phosphorylated and ubiquitinated species into the cytosol, presenting ATAD1 as the only known AAA-ATPase that preferentially acts on phosphorylated substrates. Desmin filaments disassembly was accelerated by the coordinated functions of Atad1 and calpain-1, which interact in muscle. Thus, by extracting ubiquitinated desmin from the insoluble filament, ATAD1 may expose calpain-1 cleavage sites on desmin, consequently enhancing desmin solubilization and degradation in the cytosol.