Host Ubiquitin Research Articles

A host E3 ubiquitin ligase regulates Salmonella virulence by targeting an SPI-2 effector involved in SIF biogenesis.

Salmonella Typhimurium creates an intracellular niche for its replication by utilizing a large cohort of effectors, including several that function to interfere with host ubiquitin signaling. Although the mechanism of action of many such effectors has been elucidated, how the interplay between the host ubiquitin network and bacterial virulence factors dictates the outcome of infection largely remains undefined. In this study, we found that the SPI-2 effector SseK3 inhibits SNARE pairing to promote the formation of a Salmonella-induced filament by Arg-GlcNAcylation of SNARE proteins, including SNAP25, VAMP8, and Syntaxin. Further study reveals that host cells counteract the activity of SseK3 by inducing the expression of the E3 ubiquitin ligase TRIM32, which catalyzes K48-linked ubiquitination on SseK3 and targets its membrane-associated portion for degradation. Hence, TRIM32 antagonizes SNAP25 Arg-GlcNAcylation induced by SseK3 to restrict Salmonella-induced filamentbiogenesis and Salmonella replication. Our study reveals a mechanism by which host cells inhibit bacterial replication by eliminating specific virulence factors.

Structural insights into ubiquitin chain cleavage by Legionella ovarian tumor deubiquitinases.

Although ubiquitin is found only in eukaryotes, several pathogenic bacteria and viruses possess proteins that hinder the host ubiquitin system. Legionella, a gram-negative intracellular bacterium, possesses an ovarian tumor (OTU) family of deubiquitinases (Lot DUBs). Herein, we describe the molecular characteristics of Lot DUBs. We elucidated the structure of the LotA OTU1 domain and revealed that entire Lot DUBs possess a characteristic extended helical lobe that is not found in other OTU-DUBs. The structural topology of an extended helical lobe is the same throughout the Lot family, and it provides an S1' ubiquitin-binding site. Moreover, the catalytic triads of Lot DUBs resemble those of the A20-type OTU-DUBs. Furthermore, we revealed a unique mechanism by which LotA OTU domains cooperate together to distinguish the length of the chain and preferentially cleave longer K48-linked polyubiquitin chains. The LotA OTU1 domain itself cleaves K6-linked ubiquitin chains, whereas it is also essential for assisting the cleavage of longer K48-linked polyubiquitin chains by the OTU2 domain. Thus, this study provides novel insights into the structure and mechanism of action of Lot DUBs.

Epstein-Barr virus regulates the life cycle and host cell biology by hijacking post-translational modification.

Protein post-translational modifications (PTMs) are reversible processes that regulate the function of target proteins without altering their sequences. High-throughput sequencing surveys have provided insights into the patterns of PTMs, such as ubiquitination, SUMOylation, and phosphorylation. After primary infection, the Epstein-Barr virus (EBV), a ubiquitous herpesvirus, establishes a life-long latent infection. EBV can establish a delicate balance to regulate its proliferation and host cell survival. Owing to the limited gene products of EBV, interfering with the host PTM machinery is an effective way to alter host immune responses and physiological status and establish infection. In this review, we focus on the current knowledge of the mechanisms by which EBV products manipulate host ubiquitination, SUMOylation, and phosphorylation to establish a latent infection or favour viral replication and pathogenesis.

The deubiquitylase-ubiquitin-specific protease 4 absence in HeLa cells leads to a reduction in Semliki Forest virus replication

The emergence of a potential epidemic of alphaviruses is a growing concern for public health officials and the global community. These highly contagious and fast-spreading viruses have the potential to cause severe illness, disability, and even death, and are a growing threat to populations worldwide. Understanding the interaction between viruses, deubiquitylases (DUBs), and other human host cell proteins during replication is crucial when developing targeted antiviral medications. Many viruses have developed strategies to manipulate the host ubiquitin system by hijacking DUBs to evade the host immune response and promote viral replication, making DUBs attractive therapeutic targets. Small interfering RNA (siRNA) knockdown of USP4 expression inhibited Semliki Forest virus (SFV) replication and reduced virus titre. These findings highlight the potential to target USP4 as an antiviral treatment during alphavirus infection.

An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins.

Sensing of pathogens by ubiquitination is a critical arm of cellular immunity. However, universal ubiquitination targets on microbes remain unidentified. Here, using in vitro, ex vivo, and in vivo studies, we identify the first protein-based ubiquitination substrates on phylogenetically diverse bacteria by unveiling a strategy that uses recognition of degron-like motifs. Such motifs form a new class of intra-cytosolic pathogen-associated molecular patterns (PAMPs). Their incorporation enabled recognition of nonubiquitin targets by host ubiquitin ligases. We find that SCFFBW7 E3 ligase, supported by the regulatory kinase, glycogen synthase kinase 3β, is crucial for effective pathogen detection and clearance. This provides a mechanistic explanation for enhanced risk of infections in patients with chronic lymphocytic leukemia bearing mutations in F-box and WD repeat domain containing 7 protein. We conclude that exploitation of this generic pathogen sensing strategy allows conservation of host resources and boosts antimicrobial immunity.

HPV upregulates MARCHF8 ubiquitin ligase and inhibits apoptosis by degrading the death receptors in head and neck cancer.

The membrane-associated RING-CH-type finger ubiquitin ligase MARCHF8 is a human homolog of the viral ubiquitin ligases Kaposi's sarcoma herpesvirus K3 and K5 that promote host immune evasion. Previous studies have shown that MARCHF8 ubiquitinates several immune receptors, such as the major histocompatibility complex II and CD86. While human papillomavirus (HPV) does not encode any ubiquitin ligase, the viral oncoproteins E6 and E7 are known to regulate host ubiquitin ligases. Here, we report that MARCHF8 expression is upregulated in HPV-positive head and neck cancer (HNC) patients but not in HPV-negative HNC patients compared to normal individuals. The MARCHF8 promoter is highly activated by HPV oncoprotein E6-induced MYC/MAX transcriptional activation. The knockdown of MARCHF8 expression in human HPV-positive HNC cells restores cell surface expression of the tumor necrosis factor receptor superfamily (TNFRSF) death receptors, FAS, TRAIL-R1, and TRAIL-R2, and enhances apoptosis. MARCHF8 protein directly interacts with and ubiquitinates the TNFRSF death receptors. Further, MARCHF8 knockout in mouse oral cancer cells expressing HPV16 E6 and E7 augments cancer cell apoptosis and suppresses tumor growth in vivo. Our findings suggest that HPV inhibits host cell apoptosis by upregulating MARCHF8 and degrading TNFRSF death receptors in HPV-positive HNC cells.

Pathogen-defending deubiquitinase possesses distinct specificity towards K6-polyubiquitination.

The bacterial pathogen Legionella pneumophila encodes numerous effectors to manipulate host ubiquitin signaling. Recently, Warren et al. revealed the structural basis of K6-polyubiquitination recognition by Legionella deubiquitinase LotA, while validating its potential as an enzymatic tool to study linkage-specific ubiquitination. During Legionella infection, LotA counteracts valosin-containing protein (VCP) recruitment to the Legionella-containing vacuole.

Legionella longbeachae Regulates the Association of Polyubiquitinated Proteins on Bacterial Phagosome with Multiple Deubiquitinases.

Legionella spp. are the causative agents of a severe pneumonia known as Legionnaires' disease. Upon being engulfed by host cells, these environmental bacteria replicate intracellularly in a plasma membrane-derived niche termed Legionella-containing vacuole (LCV) in a way that requires the defective in organelle trafficking/intracellular multiplication (Dot/Icm) protein transporter. Our understanding of interactions between Legionella and its hosts was mostly based on studies of Legionella pneumophila. In this study, we found that the LCVs created by virulent Legionella longbeachae are similarly decorated by polyubiquitinated proteins to those formed by L. pneumophila and that the ubiquitin-proteasome system (UPS) is required for optimal intracellular growth of L. longbeachae. Furthermore, we utilized bioinformatics methods and the ubiquitin-vinylmethyl ester probe to obtain potential deubiquitinases (DUBs) encoded by L. longbeachae. These efforts led to the identification of 9 L. longbeachae DUBs that displayed distinct specificity toward ubiquitin chain types. Among these, LLO_1014 and LLO_2238 are associated with the LCVs and impact the accumulation of polyubiquitinated species on the bacterial phagosome. Moreover, LLO_1014 and LLO_2238 could fully restore the phenotypes associated with Δceg23 (lotB) and Δlem27 (lotC) mutants of L. pneumophila, indicating that these DUBs have similar functions. Together, these results reveal that L. longbeachae uses multiple DUBs to construct an intracellular niche for its replication. IMPORTANCE Legionella spp. are opportunistic intracellular bacterial pathogens that cause Legionnaires' disease. Legionella utilizes the Dot/Icm type IV secretion system to deliver effector protein into host cells to modulate various cellular functions. At least 26 L. pneumophila effectors are known to hijack the host ubiquitin system via diverse mechanisms. L. longbeachae is the second leading cause of Legionnaires' disease worldwide. However, our knowledge about the interactions between L. longbeachae and its hosts is very limited. Here, we found that, similar to L. pneumophila infection, the host ubiquitin proteasome system is also important for the intracellular replication of L. longbeachae. In addition, the bacterial phagosomes harboring L. longbeachae are enriched with polyubiquitinated proteins in a Dot/Icm system-dependent manner. We further identified 9 L. longbeachae proteins that function as DUBs with distinct ubiquitin chain specificity. Of note, several of the phagosome-associated L. longbeachae DUBs regulate the recruitment of polyubiquitinated proteins to the LCV.

Ubiquitin-Mediated Regulation of Autophagy During Viral Infection

Virus infections skew the host autophagic response to meet their replication and transmission demands by tapping into the critical host regulatory mechanisms that control the autophagic flux. This review is a compendium of previous reports highlighting the mechanisms that viruses adapt to hijack the host ubiquitination machinery to repurpose autophagy for their sustenance. Emerging evidence suggests a critical role of host ubiquitin machinery in the manifestation of the antiviral or proviral functions of autophagy. Lately, more emphasis has been laid to identify specific host E3 ubiquitin ligases, their targets (viral or host), and characterizing corresponding ubiquitin linkages by biochemical or genome-wide genetic screening approaches. Here, we highlight how viruses ingeniously engage and subvert the host ubiquitin-autophagy system to promote virus replication and antagonize intracellular innate immune responses.

Structural basis for HIV-1 antagonism of host APOBEC3G via Cullin E3 ligase.

Human APOBEC3G (A3G) is a virus restriction factor that inhibits HIV-1 replication and triggers lethal hypermutation on viral reverse transcripts. HIV-1 viral infectivity factor (Vif) breaches this host A3G immunity by hijacking a cellular E3 ubiquitin ligase complex to target A3G for ubiquitination and degradation. The molecular mechanism of A3G targeting by Vif-E3 ligase is unknown, limiting the antiviral efforts targeting this host-pathogen interaction crucial for HIV-1 infection. Here, we report the cryo-electron microscopy structures of A3G bound to HIV-1 Vif in complex with T cell transcription cofactor CBF-β and multiple components of the Cullin-5 RING E3 ubiquitin ligase. The structures reveal unexpected RNA-mediated interactions of Vif with A3G primarily through A3G's noncatalytic domain, while A3G's catalytic domain is poised for ubiquitin transfer. These structures elucidate the molecular mechanism by which HIV-1 Vif hijacks the host ubiquitin ligase to specifically target A3G to establish infection and offer structural information for the rational development of antiretroviral therapeutics.

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.

Distinct Molecular Features of NleG Type 3 Secreted Effectors Allow for Different Roles during Citrobacter rodentium Infection in Mice.

The NleGs are the largest family of type 3 secreted effectors in attaching and effacing (A/E) pathogens, such as enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and Citrobacter rodentium. NleG effectors contain a conserved C-terminal U-box domain acting as a ubiquitin protein ligase and target host proteins via a variable N-terminal portion. The specific roles of these effectors during infection remain uncertain. Here, we demonstrate that the three NleG effectors-NleG1Cr, NleG7Cr, and NleG8Cr-encoded by C. rodentium DBS100 play distinct roles during infection in mice. Using individual nleGCr knockout strains, we show that NleG7Cr contributes to bacterial survival during enteric infection while NleG1Cr promotes the expression of diarrheal symptoms and NleG8Cr contributes to accelerated lethality in susceptible mice. Furthermore, the NleG8Cr effector contains a C-terminal PDZ domain binding motif that enables interaction with the host protein GOPC. Both the PDZ domain binding motif and the ability to engage with host ubiquitination machinery via the intact U-box domain proved to be necessary for NleG8Cr function, contributing to the observed phenotype during infection. We also establish that the PTZ binding motif in the EHEC NleG8 (NleG8Ec) effector, which shares 60% identity with NleG8Cr, is engaged in interactions with human GOPC. The crystal structure of the NleG8Ec C-terminal peptide in complex with the GOPC PDZ domain, determined to 1.85 Å, revealed a conserved interaction mode similar to that observed between GOPC and eukaryotic PDZ domain binding motifs. Despite these common features, nleG8Ec does not complement the ΔnleG8Cr phenotype during infection, revealing functional diversification between these NleG effectors.

SARS-COV-2 Coronavirus Papain-like Protease PLpro as an Antiviral Target for Inhibitors of Active Site and Protein-Protein Interactions.

The papain-like protease PLpro of the SARS-CoV-2 coronavirus is a multifunctional enzyme that catalyzes the proteolytic processing of two viral polyproteins, pp1a and pp1ab. PLpro also cleaves peptide bonds between host cell proteins and ubiquitin (or ubiquitin-like proteins), which is associated with a violation of immune processes. Nine structures of the most effective inhibitors of the PLpro active center were prioritized according to the parameters of biochemical (IC 50) and cellular tests to assess the suppression of viral replication (EC 50) and cytotoxicity (CC 50). A literature search has shown that PLpro can interact with at least 60 potential protein partners in cells, 23 of which are targets for other viral proteins (human papillomavirus and Epstein-Barr virus). The analysis of protein-protein interactions showed that the proteins USP3, UBE2J1, RCHY1, and FAF2 involved in deubiquitinylation and ubiquitinylation processes contain the largest number of bonds with other proteins; the interaction of viral proteins with them can affect the architecture of the entire network of protein-protein interactions. Using the example of a spatial model of the PLpro/ubiquitin complex and a set of 154 naturally occurring compounds with known antiviral activity, 13 compounds (molecular masses in the range of 454-954 Da) were predicted as potential PLpro inhibitors. These compounds bind to the "hot" amino acid residues of the protease at the positions Gly163, Asp164, Arg166, Glu167, and Tyr264 involved in the interaction with ubiquitin. Thus, pharmacological effects on peripheral PLpro sites, which play important roles in binding protein substrates, may be an additional target-oriented antiviral strategy.

GarD-ing the pathogen-containing vacuole from destruction

The human pathogen Chlamydia trachomatis evades killing by IFNγ-activated mechanisms, yet how this occurs remains unclear. In this issue of Cell Host & Microbe, Walsh etal. identify an IFNγ-dependent antimicrobial mechanism mediated by the host ubiquitin ligase RNF213 that is evaded by the Chlamydia effector GarD.

VpdC is a ubiquitin-activated phospholipase effector that regulates Legionella vacuole expansion during infection

Intravacuolar pathogens need to gradually expand their surrounding vacuole to accommodate the growing number of bacterial offspring during intracellular replication. Here we found that Legionella pneumophila controls vacuole expansion by fine-tuning the generation of lysophospholipids within the vacuolar membrane. Upon allosteric activation by binding to host ubiquitin, the type IVB (Dot/Icm) effector VpdC converts phospholipids into lysophospholipids which, at moderate concentrations, are known to promote membrane fusion but block it at elevated levels by generating excessive positive membrane curvature. Consequently, L. pneumophila overproducing VpdC were prevented from adequately expanding their surrounding membrane, trapping the replicating bacteria within spatially confined vacuoles and reducing their capability to proliferate intracellularly. Quantitative lipidomics confirmed a VpdC-dependent increase in several types of lysophospholipids during infection, and VpdC production in transiently transfected cells caused tubulation of organelle membranes as well as mitochondria fragmentation, processes that can be phenocopied by supplying cells with exogenous lysophospholipids. Together, these results demonstrate an important role for bacterial phospholipases in vacuolar expansion.

Pathogenesis of Zika Virus Infection.

Zika virus (ZIKV) is an emerging virus from the Flaviviridae family that is transmitted to humans by mosquito vectors and represents an important health problem. Infections in pregnant women are of major concern because of potential devastating consequences during pregnancy and have been associated with microcephaly in newborns. ZIKV has a unique ability to use the host machinery to promote viral replication in a tissue-specific manner, resulting in characteristic pathological disorders. Recent studies have proposed that the host ubiquitin system acts as a major determinant of ZIKV tropism by providing the virus with an enhanced ability to enter new cells. In addition, ZIKV has developed mechanisms to evade the host immune response, thereby allowing the establishment of viral persistence and enhancing viral pathogenesis. We discuss recent reports on the mechanisms used by ZIKV to replicate efficiently, and we highlight potential new areas of research for the development of therapeutic approaches.

Helicobacter pylori pathogen inhibits cellular responses to oncogenic stress and apoptosis.

Helicobacter pylori (H. pylori) is a common gastric pathogen that infects approximately half of the world’s population. Infection with H. pylori can lead to diverse pathological conditions, including chronic gastritis, peptic ulcer disease, and cancer. The latter is the most severe consequence of H. pylori infection. According to epidemiological studies, gastric infection with H. pylori is the strongest known risk factor for non-cardia gastric cancer (GC), which remains one of the leading causes of cancer-related deaths worldwide. However, it still remains to be poorly understood how host-microbe interactions result in cancer development in the human stomach. Here we focus on the H. pylori bacterial factors that affect the host ubiquitin proteasome system. We investigated E3 ubiquitin ligases SIVA1 and ULF that regulate p14ARF (p19ARF in mice) tumor suppressor. ARF plays a key role in regulation of the oncogenic stress response and is frequently inhibited during GC progression. Expression of ARF, SIVA1 and ULF proteins were investigated in gastroids, H. pylori-infected mice and human gastric tissues. The role of the H. pylori type IV secretion system was assessed using various H. pylori isogenic mutants. Our studies demonstrated that H. pylori infection results in induction of ULF, decrease in SIVA1 protein levels, and subsequent ubiquitination and degradation of p14ARF tumor suppressor. Bacterial CagA protein was found to sequentially bind to SIVA1 and ULF proteins. This process is regulated by CagA protein phosphorylation at the EPIYA motifs. Downregulation of ARF protein leads to inhibition of cellular apoptosis and oncogenic stress response that may promote gastric carcinogenesis.

Genomes of six viruses that infect Asgard archaea from deep-sea sediments.

Asgard archaea are globally distributed prokaryotic microorganisms related to eukaryotes; however, viruses that infect these organisms have not been described. Here, using metagenome sequences recovered from deep-sea hydrothermal sediments, we characterize six relatively large (up to 117 kb) double-stranded DNA (dsDNA) viral genomes that infected two Asgard archaeal phyla, Lokiarchaeota and Helarchaeota. These viruses encode Caudovirales-like structural proteins, as well as proteins distinct from those described in known archaeal viruses. Their genomes contain around 1-5% of genes associated with eukaryotic nucleocytoplasmic large DNA viruses (NCLDVs) and appear to be capable of semi-autonomous genome replication, repair, epigenetic modifications and transcriptional regulation. Moreover, Helarchaeota viruses may hijack host ubiquitin systems similar to eukaryotic viruses. Genomic analysis of these Asgard viruses reveals that they contain features of both prokaryotic and eukaryotic viruses, and provides insights into their potential infection and host interaction mechanisms.

Theoretical modeling and design of some pyrazolopyrimidine derivatives as Wolbachia inhibitors, targeting lymphatic filariasis and onchocerciasis.

Lymphatic filariasis and onchocerciasis are two common filarial diseases caused by a group of parasitic nematodes called filarial worms, which play host to the bacteria organism Wolbachia. One good treatment approach seeks Wolbachia as drug target. Here, a QSAR study was conducted to investigate the anti-wolbachia activities (pEC50) of 52 pyrazolopyrimidine analogues, while using the built model to predict the pEC50 values of the newly designed analogues. Density Functional Theory was used for the structural optimization, while the model building was based on Genetic Function Algorithm approach. The built QSAR model was validated thus: R2 = 0.8104, R2 adj = 0.7629, Q2 cv = 0.6981, R2 test = 0.7501 and cRp2 = 0.7476. The predicted pEC50 of all newly designed compounds were higher than that of the template (43). The new compounds were; observed to pass the drug-likeness criteria, uniformly distributed to the brain, and found to be non-mutagenic. Also, the new compounds and the reference drug (doxycycline), were docked onto Ovarian Tumor (OTU) deubiquitinase receptor (PDB ID: 6W9O) using iGEMDOCK tool. This protein is known to help Wolbachia subvert host ubiquitin signaling. The resulting binding scores of the newly designed compounds except A5 were higher than that of doxycycline, while the protein-ligand interactions were majorly characterized by Hydrogen-bonding and hydrophobic interaction types. Therefore, the newly designed molecules could be developed as potential drug candidates for the treatment of lymphatic filariasis and onchocerciasis.

The Roles of Ubiquitination in Pathogenesis of Influenza Virus Infection.

The ubiquitin system denotes a potent post-translational modification machinery that is capable of activation or deactivation of target proteins through reversible linkage of a single ubiquitin or ubiquitin chains. Ubiquitination regulates major cellular functions such as protein degradation, trafficking and signaling pathways, innate immune response, antiviral defense, and virus replication. The RNA sensor RIG-I ubiquitination is specifically induced by influenza A virus (IAV) to activate type I IFN production. Influenza virus modulates the activity of major antiviral proteins in the host cell to complete its full life cycle. Its structural and non-structural proteins, matrix proteins and the polymerase complex can regulate host immunity and antiviral response. The polymerase PB1-F2 of mutated 1918 IAV, adapts a novel IFN antagonist function by sending the DDX3 into proteasomal degradation. Ultimately the fate of virus is determined by the outcome of interplay between viral components and host antiviral proteins and ubiquitination has a central role in the encounter of virus and its host cell.