Ubiquitin Signaling Research Articles

Multi-tiered actions of Legionella effectors to modulate host Rab10 dynamics.

Rab GTPases are representative targets of manipulation by intracellular bacterial pathogens for hijacking membrane trafficking. Legionella pneumophila recruits many Rab GTPases to its vacuole and exploits their activities. Here, we found that infection-associated regulation of Rab10 dynamics involves ubiquitin signaling cascades mediated by the SidE and SidC families of Legionella ubiquitin ligases. Phosphoribosyl-ubiquitination of Rab10 catalyzed by the SidE ligases is crucial for its recruitment to the bacterial vacuole. SdcB, the previously uncharacterized SidC-family effector, resides on the vacuole and contributes to retention of Rab10 at the late stages of infection. We further identified MavC as a negative regulator of SdcB. By the transglutaminase activity, MavC crosslinks ubiquitin to SdcB and suppresses its function, resulting in elimination of Rab10 from the vacuole. These results demonstrate that the orchestrated actions of many L. pneumophila effectors fine-tune the dynamics of Rab10 during infection.

Genome-Wide Identification and Characterization of the PPPDE Gene Family in Rice

Protein ubiquitination is common and crucial in cellular functions, however, little is known about how deubiquitinating enzymes (DUBs) reverse regulate the ubiquitination signaling process. PPPDE family proteins are a novel class of deubiquitinating peptidases with demonstrated deubiquitination/deSUMOylating activities. In this study, we identified 10 PPPDE genes from the rice (Oryza sativa L.) genome unevenly distributed on five chromosomes, where most of these members have not been reported to date. Based on the gene structure, the OsPPPDE family consists of three distinct subgroups within the phylogenetic tree. Cis-element analysis identified light/phytohormone-responsive, development, and abiotic stress-related elements in the promoters of OsPPPDE. Furthermore, we conducted and analyzed the transcript abundance of OsPPPDE under various tissues and stresses using the transcriptome data of 352 samples from the Rice Expression Database and GEO datasets. Moreover, OsPPPDE5 showed differential regulation of its transcript abundance during Cd and drought stress. Collinearity and syntenic analysis of 101 PPPDEs and PPPDE-like proteins in 10 plant genomes indicated that this family is evolutionarily conserved. Domestication analysis suggests that OsPPPDEs may contribute to indica–japonica divergence using the data from the 3K Rice Genome Project. Our study provides a foundation for further study on the function and molecular mechanism of the OsPPPDE gene family.

The Shigella kinase effector OspG modulates host ubiquitin signaling to escape septin-cage entrapment

Shigella flexneri is a Gram-negative bacterium causing severe bloody dysentery. Its pathogenesis is largely dictated by a plasmid-encoded type III secretion system (T3SS) and its associated effectors. Among these, the effector OspG has been shown to bind to the ubiquitin conjugation machinery (E2~Ub) to activate its kinase activity. However, the cellular targets of OspG remain elusive despite years of extensive efforts. Here we show by unbiased phosphoproteomics that a major target of OspG is CAND1, a regulatory protein controlling the assembly of cullin-RING ubiquitin ligases (CRLs). CAND1 phosphorylation weakens its interaction with cullins, which is expected to impact a large panel of CRL E3s. Indeed, global ubiquitome profiling reveals marked changes in the ubiquitination landscape when OspG is introduced. Notably, OspG promotes ubiquitination of a class of cytoskeletal proteins called septins, thereby inhibiting formation of cage-like structures encircling cytosolic bacteria. Overall, we demonstrate that pathogens have evolved an elaborate strategy to modulate host ubiquitin signaling to evade septin-cage entrapment.

Abstract LB015: Differential expression of deubiquitinating enzymes and its related DNA damage response genes in neuroblastoma heterogeneity

Abstract SK-N-AS and SK-N-DZ are two cell lines of pediatric solid tumors neuroblastomas well characterized to evaluate heterogeneity between different neuroblastomas and are presumed as models for differences between different neuroblastoma. Using this model system, we recently evaluated if radiation could improve efficacy of radiation therapy and to follow these studies later with animal models to determine decrease of tumor burden. Differences between the N-type and S-type neuroblastoma cell lines SK-N-DZ and SK-N-AS, respectively were observed to be high and their responses to radiation stress were equally heterogeneous. Ubiquitin signaling plays a key role during the DNA damage response in protein recruitment and protein interactions. Deubiquitinating enzymes (DUBs) deconjugate ubiquitin from ubiquitylated substrate proteins to control their activity and stability and are shown to regulate various biological processes. DUBs may play a role in regulating the radiosensitivity of cancer cells by modulating DNA damage response and repair pathways. Determination of specific DUBs that are involved in regulating radiosensitivity in cancer cells will provide opportunity for drug targeting and visa-vis enhance the efficacy of radiation therapy in neuroblastoma cancer treatment. Here, using bioinformatic tools, the differentially regulated DUBs were identified. The relationship between DEGs-DUBs with the genes involved in various DNA repair pathways (NHEJ, HR and Fanconi anemia pathway; KEGG pathway database) are described. We identified 32 DEGs (14 upregulated and 18 downregulated) with these neuroblastoma cell lines. USP22 and USP41 DUBs and its differential expression in these cell lines denotes the importance of such analysis. Especially considering USP22 a potentially biomarker for predicting the outcome of patients with neuroblastoma. These DEG-DUBs were likewise correlated with 91 DNA damage response (DDR) genes belonging to three different pathways (NHEJ, HR and Fanconi anemia pathway). Out of which 44 of the DDR genes showed significant correlation with DEG-DUBs. Further studies are needed to determine the mechanisms underlying the complex relationship of these DUBs and to determine the critical DUBs for drug targeting and to enhance the efficacy of radiation therapy in cancer treatment. Citation Format: Vaibhav Vaibhav, Fahika Nazeerulla, Tatjana Paunesku, Ekta Tripathi, Gayle Woloschak, Prashanthi Karyala, Rao V. Papineni. Differential expression of deubiquitinating enzymes and its related DNA damage response genes in neuroblastoma heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB015.

Deciphering functional roles of protein succinylation and glutarylation using genetic code expansion

Post-translational modifications (PTMs) dynamically regulate cellular processes. Lysine undergoes a range of acylations, including malonylation, succinylation (SucK) and glutarylation (GluK). These PTMs increase the size of the lysine side chain and reverse its charge from +1 to −1 under physiological conditions, probably impacting protein structure and function. To understand the functional roles of these PTMs, homogeneously modified proteins are required for biochemical studies. While the site-specific encoding of PTMs and their mimics via genetic code expansion has facilitated the characterization of the functional roles of many PTMs, negatively charged lysine acylations have defied this approach. Here we describe site-specific incorporation of SucK and GluK into proteins via temporarily masking their negative charge through thioester derivatives. We prepare succinylated and glutarylated bacterial and mammalian target proteins, including non-refoldable multidomain proteins. This allows us to study how succinylation and glutarylation impact enzymatic activity of metabolic enzymes and regulate protein–DNA and protein–protein interactions in biological processes from replication to ubiquitin signalling.

Neuroprotective mushrooms

Abstract Objectives Alternative medicines commonly supplement or, at times, replace standard medical treatment. One area of increasing attention is disease-modifying medicines for neurodegenerative diseases. However, few such alternatives have been investigated thoroughly with an eye towards understanding mechanisms of action for clinical use. Medicinal mushrooms have important health benefits and pharmacological activities with anti-inflammatory, antioxidant, antibacterial, antiviral, immunomodulatory, digestive, cytoprotective, homeostatic, and neuroprotective activities. Edible mushrooms are known to play roles in preventing age-related diseases. Several studies have revealed that polysaccharides, terpenes, and phenolic compounds are chemical components derived from mushrooms with pharmacological activities. Due to limited effective protocols for mushroom protein extraction for proteomic studies, information about these medicinally related proteins and their biological functions remains enigmatic. Methods Herein, we have performed proteomic studies of two mushroom species Laricifomes officinalis (agarikon) and Grifola frondosa (maitake). Results These studies serve to uncover a foundation for putative proteome-associated neuroprotective processes. The recovered proteins from both species show multiple cell-specific signaling pathways including unfolded protein response, and mitochondrial protein import as well as those linked to BAG2, ubiquitination, apoptosis, microautophagy, glycolysis, SNARE, and immunogenic cell signaling pathways. Conclusions This study uncovered mushroom proteome-associated proteins which serve to better understand the structural and functional properties of mushrooms used as alternative medicines for broad potential health benefits.

Role of Autophagy and Mitophagy in Neurodegenerative Disorders.

Autophagy is a self-destructive cellular process that removes essential metabolites and waste from inside the cell to maintain cellular health. Mitophagy is the process by which autophagy causes disruption inside mitochondria and the total removal of damaged or stressed mitochondria, hence enhancing cellular health. The mitochondria are the powerhouses of the cell, performing essential functions such as ATP (adenosine triphosphate) generation, metabolism, Ca2+ buffering, and signal transduction. Many different mechanisms, including endosomal and autophagosomal transport, bring these substrates to lysosomes for processing. Autophagy and endocytic processes each have distinct compartments, and they interact dynamically with one another to complete digestion. Since mitophagy is essential for maintaining cellular health and using genetics, cell biology, and proteomics techniques, it is necessary to understand its beginning, particularly in ubiquitin and receptor-dependent signalling in injured mitochondria. Despite their similar symptoms and emerging genetic foundations, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS) have all been linked to abnormalities in autophagy and endolysosomal pathways associated with neuronal dysfunction. Mitophagy is responsible for normal mitochondrial turnover and, under certain physiological or pathological situations, may drive the elimination of faulty mitochondria. Due to their high energy requirements and post-mitotic origin, neurons are especially susceptible to autophagic and mitochondrial malfunction. This article focused on the importance of autophagy and mitophagy in neurodegenerative illnesses and how they might be used to create novel therapeutic approaches for treating a wide range of neurological disorders.

Shared and distinct mechanisms of UBA1 inactivation across different diseases

Most cellular ubiquitin signaling is initiated by UBA1, which activates and transfers ubiquitin to tens of E2 enzymes. Clonally acquired UBA1 missense mutations cause an inflammatory-hematologic overlap disease called VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic) syndrome. Despite extensive clinical investigation into this lethal disease, little is known about the underlying molecular mechanisms. Here, by dissecting VEXAS-causing UBA1 mutations, we discovered that p.Met41 mutations alter cytoplasmic isoform expression, whereas other mutations reduce catalytic activity of nuclear and cytoplasmic isoforms by diverse mechanisms, including aberrant oxyester formation. Strikingly, non-p.Met41 mutations most prominently affect transthioesterification, revealing ubiquitin transfer to cytoplasmic E2 enzymes as a shared property of pathogenesis amongst different VEXAS syndrome genotypes. A similar E2 charging bottleneck exists in some lung cancer-associated UBA1 mutations, but not in spinal muscular atrophy-causing UBA1 mutations, which instead, render UBA1 thermolabile. Collectively, our results highlight the precision of conformational changes required for faithful ubiquitin transfer, define distinct and shared mechanisms of UBA1 inactivation in diverse diseases, and suggest that specific E1-E2 modules control different aspects of tissue differentiation and maintenance.

Deciphering non-canonical ubiquitin signaling: biology and methodology.

Ubiquitination is a dynamic post-translational modification that regulates virtually all cellular processes by modulating function, localization, interactions and turnover of thousands of substrates. Canonical ubiquitination involves the enzymatic cascade of E1, E2 and E3 enzymes that conjugate ubiquitin to lysine residues giving rise to monomeric ubiquitination and polymeric ubiquitination. Emerging research has established expansion of the ubiquitin code by non-canonical ubiquitination of N-termini and cysteine, serine and threonine residues. Generic methods for identifying ubiquitin substrates using mass spectrometry based proteomics often overlook non-canonical ubiquitinated substrates, suggesting that numerous undiscovered substrates of this modification exist. Moreover, there is a knowledge gap between in vitro studies and comprehensive understanding of the functional consequence of non-canonical ubiquitination in vivo. Here, we discuss the current knowledge about non-lysine ubiquitination, strategies to map the ubiquitinome and their applicability for studying non-canonical ubiquitination substrates and sites. Furthermore, we elucidate the available chemical biology toolbox and elaborate on missing links required to further unravel this less explored subsection of the ubiquitin system.

Attenuation of HOIL-1L ligase activity promotes systemic autoimmune disorders by augmenting linear ubiquitin signaling.

Linear ubiquitin chains, which are generated specifically by the linear ubiquitin assembly complex (LUBAC) ubiquitin ligase, play crucial roles in immune signaling, including NF-κB activation. LUBAC comprises catalytic large isoform of heme-oxidized iron regulatory protein 2 ubiquitin ligase 1 (HOIL-1L) interacting protein (HOIP), accessory HOIL-1L, and SHANK-associated RH domain-interacting protein (SHARPIN). Deletion of the ubiquitin ligase activity of HOIL-1L, an accessory ligase of LUBAC, augments LUBAC functions by enhancing LUBAC-mediated linear ubiquitination, which is catalyzed by HOIP. Here, we show that HOIL-1L ΔRING1 mice, which exhibit augmented LUBAC functions upon loss of the HOIL-1L ligase, developed systemic lupus erythematosus (SLE) and Sjögren's syndrome in a female-dominant fashion. Augmented LUBAC activity led to hyperactivation of both lymphoid and myeloid cells. In line with the findings in mice, we sought to identify missense single nucleotide polymorphisms/variations of the RBCK1/HOIL-1L gene in humans that attenuate HOIL-1L ligase activity. We found that the R464H variant, which is encoded by rs774507518 within the RBCK1/HOIL-1L gene, attenuated HOIL-1L ligase activity and augmented LUBAC-mediated immune signaling, including that mediated by Toll-like receptors. We also found that rs774507518 was enriched significantly in patients with SLE, strongly suggesting that RBCK1/HOIL-1L is an SLE susceptibility gene and that augmented linear ubiquitin signaling generated specifically by LUBAC underlies the pathogenesis of this prototype systemic autoimmune disease.

Effects of multi-walled carbon nanotubes on message and Micro-RNA in human lung BEAS-2B cells.

Multi-walled Carbon nanotubes (MWCNTs) lack sufficient quality cytotoxicity, toxicity, genotoxicity and genomic data on which to make environmental and regulatory decisions. Therefore, we did a multidisciplinary in vitro study of 3 MWCNTs in human lung cells (BEAS-2B) with the following endpoints: cytotoxicity, DNA damage, reactive oxygen and nitrogen species, lipid peroxidation and mRNA and microRNA expression analyses. The MWCNTs were either unfunctionalized or functionalized with either -OH or -COOH. Doses studied ranged from 0.3 to 100 ug/ml and were exposed to a human lung cell line in vitro for 72 h., with genomic studies being done from 30 ug/ml downward. Some of the genomic pathways that were altered by MWCNT exposure were NRF2 mediated oxidative stress response, DNA damage repair, nuclear excision repair, base excision repair, mitochondrial dysfunction, oxidative phosphorylation, HIF1α signaling, unfolded protein response, protein ubiquitination, ferroptosis and sirtuin signaling pathways. The data suggested that OH functionalized MWCNT caused more and larger gene/microRNA changes, followed by COOH functionalized MWCNT and unfunctionalized MWCNT being the least biologically active. From microRNA target filter analysis, there were altered signaling hubs. MYC is the only hub that altered by all 3 MWCNTs. Signaling hubs that are common to OH and COOH functionalized MWCNTs are GRB2, AR, TP63 and AGO2. The signaling hubs that were only present in OH functionalized MWCNTs are TP53, STAT3 and BRCA1. These signaling pathways and hubs we found in vitro correlated well with the published in vivo pathological effects like oxidative stress DNA damage, inflammation and cancer in MWCNTs treated mice.

Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1.

Ubiquitin ligases (E3s) are pivotal specificity determinants in the ubiquitin system by selecting substrates and decorating them with distinct ubiquitin signals. However, structure determination of the underlying, specific E3-substrate complexes has proven challenging owing to their transient nature. In particular, it is incompletely understood how members of the catalytic cysteine-driven class of HECT-type ligases (HECTs) position substrate proteins for modification. Here, we report a cryogenic electron microscopy (cryo-EM) structure of the full-length human HECT HACE1, along with solution-based conformational analyses by small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry. Structure-based functional analyses in vitro and in cells reveal that the activity of HACE1 is stringently regulated by dimerization-induced autoinhibition. The inhibition occurs at the first step of the catalytic cycle and is thus substrate-independent. We use mechanism-based chemical crosslinking to reconstitute a complex of activated, monomeric HACE1 with its major substrate, RAC1, determine its structure by cryo-EM and validate the binding mode by solution-based analyses. Our findings explain how HACE1 achieves selectivity in ubiquitinating the active, GTP-loaded state of RAC1 and establish a framework for interpreting mutational alterations of the HACE1-RAC1 interplay in disease. More broadly, this work illuminates central unexplored aspects in the architecture, conformational dynamics, regulation and specificity of full-length HECTs.

Breaking the K48-chain: linking ubiquitin beyond protein degradation.

The discovery of ubiquitin conjugation to lysines and the role of K48-linked polyubiquitin in targeting substrates for proteasomal degradation was followed by revelation of non-degradative roles of ubiquitination and, more recently, of non-canonical covalent ubiquitin linkages. Here we summarize findings of the ever-expanding array of ubiquitin signals and their biological roles.

P012 Intestinal epithelial cells as targets of thiopurines in Inflammatory Bowel Disease paediatric patients-derived organoids

Abstract Background Thiopurines, azathioprine (AZA) and mercaptopurine (MP), are immunomodulators used to maintain remission in patients with inflammatory bowel disease (IBD). Intestinal epithelial cells play a key role in the pathogenesis of IBD, however, their role as target cells for thiopurines has not been elucidated. The aim of the project is to establish an in vitro IBD model using organoids starting from intestinal biopsies of IBD paediatric patients to investigate thiopurine effects on intestinal epithelium. Methods Forty-four IBD patients (mean age 14.4 years, 21 males) were enrolled. Intestinal biopsies were used to isolate crypts and generate adult stem cell-derived organoids. The cytotoxicity of thiopurines (0.2-200 µM for 72 hours) was evaluated by CellTiter-Glo 3D assay. Gene expression was evaluated using TaqMan assays. Thiopurine metabolites were quantified by LC-MS/MS analysis. Proteomic profiles were analyzed by Q-Exactive Plus mass spectrometer, western blot and REACTOME pathway software. Statistical analysis was performed using GraphPad and R software. Results IBD patients-derived organoids were sensitive to thiopurine treatment in a dose-dependent manner with a high interpatient variability (Two-way ANOVA p-value<0.0001; IC50 2.24 µM for AZA (95% CI: 1.28–5.53 µM) and 2.65 µM for MP (95% CI: 1.43–8.27 µM)).A significant negative correlation was observed between the viability after thiopurine exposure and mRNA expression of candidate genes involved in thiopurine pharmacokinetics (ITPA, TPMT, PACSIN2 and NUDT15). The most abundant thiopurine metabolite measured on organoids treated with IC50 of MP was MeTIMP, with a concentration correlated with cell viability after MP exposure (p=0.0056, ρ=-0.99). Proteomic analysis on organoids treated with IC20 of thiopurines (0.2 μM) showed 45 differentially expressed proteins in common between AZA and MP (p<0.05). REACTOME enrichment analysis revealed as the most altered pathways: vesicular traffic, autophagy, protein ubiquitination and interferon signalling, while no proteins related to apoptosis were induced. Among these proteins, TRIM32, a ubiquitin ligase involved in STING activity, a key regulator of both interferon I expression and NF-kB activation, resulted downregulated by the treatment (t-test AZA: p=0.0006; MP: p=0.014) and its basal levels negatively correlated with viability rates after thiopurine exposure (AZA: p=0.006, ρ=-0.85; MP: p=0.002, ρ=-0.88). Organoid exposure to thiopurines also decreased p-STING/STING ratio, particularly after MP treatment (t-test p=0.025), and increased autophagy, assessed as LC3-II levels (t-test AZA: p=0.0006; MP: p=0.014). Conclusion Organoid cultures provide new insights on the mechanism of action of thiopurines in the intestinal epithelium.

Rad6, a ubiquitin conjugator required for insect-pathogenic lifestyle, UV damage repair, and genomic expression of Beauveria bassiana

The E2 ubiquitin conjugator Rad6 is required for DNA damage bypass in budding yeast but remain functionally unknown in filamentous fungi. Here, we report pleiotropic effect of Rad6 ortholog in Beauveria bassiana, a wide-spectrum fungal insecticide. Global ubiquitination signal was greatly attenuated in the absence of rad6. The blocked ubiquitination led to severe growth defect, blocked asexual development, and abolished infectivity/insect pathogenicity, which correlated with compromised conidial quality (including viability, hydrophobicity, adherence to insect cuticle, and thermotolerance) and blocked secretion of cuticle-degrading enzymes including Pr1 family proteases. Importantly, Rad6 played much greater role in photoreactivation of UVB-impaired conidia by a 3- or 5-h light plus 9- or 7-h dark incubation than in dark reactivation of those impaired conidia by a 12-h dark incubation. The high activity of Rad6 in photoreactivation in vivo was derived from its link to a protein complex cored by the photolyase regulators WC1 and WC2 via the strong interactions of Rad6 with the E3 partner Rad18 and Rad18 with WC2 revealed in yeast two-hybrid assays. Transcriptomic analysis resulted in identification of 2700 differentially regulated genes involved in various function categories and metabolism pathways, indicating a regulatory role of Rad6-mediated ubiquitination in gene expression networks and genomic stability. Conclusively, Rad6 is required for asexual and insect-pathogenic lifecycles, solar UV damage repair, and genomic expression of B. bassiana. The primary dependence of its strong anti-UV role on photoreactivation in vivo unveils a scenario distinct from the core role of its yeast ortholog in DNA damage bypass.

USP8 prevents aberrant NF-κB and Nrf2 activation by counteracting ubiquitin signals from endosomes.

K63-linked ubiquitin chains attached to plasma membrane proteins serve as tags for endocytosis and endosome-to-lysosome sorting. USP8 is an essential deubiquitinase for the maintenance of endosomal functions. Prolonged depletion of USP8 leads to cell death, but the major effects on cellular signaling pathways are poorly understood. Here, we show that USP8 depletion causes aberrant accumulation of K63-linked ubiquitin chains on endosomes and induces immune and stress responses. Upon USP8 depletion, two different decoders for K63-linked ubiquitin chains, TAB2/3 and p62, were recruited to endosomes and activated the TAK1-NF-κB and Keap1-Nrf2 pathways, respectively. Oxidative stress, an environmental stimulus that potentially suppresses USP8 activity, induced accumulation of K63-linked ubiquitin chains on endosomes, recruitment of TAB2, and expression of the inflammatory cytokine. The results demonstrate that USP8 is a gatekeeper of misdirected ubiquitin signals and inhibits immune and stress response pathways by removing K63-linked ubiquitin chains from endosomes.

Role of Ubiquitin Signaling in Modulating Dendritic Cell Function.

As a professional antigen-presenting cell, dendritic cell (DC) plays an essential role in the connection of innate and adaptive immune responses. Ubiquitination is a post-translational mechanism of protein modification that plays a pivotal role in regulating DC maturation and function. To date, considerable progress has been made in understanding the underlying mechanisms of ubiquitination in modulating the function of DC in various diseases. Recent studies have emphasized that ubiquitin signaling in DCs plays crucial roles in regulating immune tolerance and functions, which can be promising targets for DC-based immunotherapy. In this chapter, we will focus on discussing the recent progress regarding the molecular mechanisms and functions of ubiquitination in DC-mediated immune homeostasis and responses.

An insight into the roles of ubiquitin-specific proteases in plants: development and growth, morphogenesis, and stress response.

Ubiquitination is a highly conserved and dynamic post-translational modification in which protein substrates are modified by ubiquitin to influence their activity, localization, or stability. Deubiquitination enzymes (DUBs) counter ubiquitin signaling by removing ubiquitin from the substrates. Ubiquitin-specific proteases (UBPs), the largest subfamily of DUBs, are conserved in plants, serving diverse functions across various cellular processes, although members within the same group often exhibit functional redundancy. Here, we briefly review recent advances in understanding the biological roles of UBPs, particularly the molecular mechanism by which UBPs regulate plant development and growth, morphogenesis, and stress response, which sheds light on the mechanistic roles of deubiquitination in plants.

Crystal structure of bacterial ubiquitin ADP-ribosyltransferase CteC reveals a substrate-recruiting insertion

ADP-ribosylation is a post-translational modification involved in regulation of diverse cellular pathways. Interestingly, many pathogens have been identified to utilize ADP-ribosylation as a way for host manipulation. A recent study found that CteC, an effector from the bacterial pathogen Chromobacterium violaceum, hinders host ubiquitin signaling pathways via installing mono-ADP-ribosylation on threonine 66 of ubiquitin. However, the molecular basis of substrate recognition by CteC is not well-understood. In this paper, we probed the substrate specificity of this effector at protein and residue levels. We also determined the crystal structure of CteC in complex with NAD+, which revealed a canonical mono-ADP-ribosyltransferase fold with an additional insertion domain. The AlphaFold predicted model differed significantly from the experimentally determined structure, even in regions not used in crystal packing. Biochemical and biophysical studies indicated unique features of the NAD+ binding pocket, while showing selectivity distinction between ubiquitin and structurally close ubiquitin-like modifiers and the role of the insertion domain in substrate recognition. Together, this study provides insights into the enzymatic specificities and the key structural features of a novel bacterial ADP-ribosyltransferase involved in host-pathogen interaction.

Cross-family small GTPase ubiquitination by the intracellular pathogen Legionella pneumophila.

The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s ∼330 secreted effector proteins are ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p. hijacks host cell ubiquitin signaling, we generated a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection increases ubiquitination of host regulators of subcellular trafficking and membrane dynamics, most notably ∼40% of mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo nondegradative ubiquitination at the Legionella-containing vacuole (LCV) membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE family ligases in the polyubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Our findings position L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.