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.

Targeted inhibition of branched-chain amino acid metabolism drives apoptosis of glioblastoma by facilitating ubiquitin degradation of Mfn2 and oxidative stress.

Glioblastoma is one of the most challenging malignancies with high aggressiveness and invasiveness and its development and progression of glioblastoma highly depends on branched-chain amino acid (BCAA) metabolism. The study aimed to investigate effects of inhibition of BCAA metabolism with cytosolic branched-chain amino acid transaminase (BCATc) Inhibitor 2 on glioblastoma, elucidate its underlying mechanisms, and explore therapeutic potential of targeting BCAA metabolism. The expression of BCATc was upregulated in glioblastoma and BCATc Inhibitor 2 precipitated apoptosis both in vivo and in vitro with the activation of Bax/Bcl2/Caspase-3/Caspase-9 axis. In addition, BCATc Inhibitor 2 promoted K63-linkage ubiquitination of mitofusin 2 (Mfn2), which subsequently caused lysosomal degradation of Mfn2, and then oxidative stress, mitochondrial fission and loss of mitochondrial membrane potential. Furthermore, BCATc Inhibitor 2 treatment resulted in metabolic reprogramming, and significant inhibition of expression of ATP5A, UQCRC2, SDHB and COX II, indicative of suppressed oxidative phosphorylation. Moreover, Mfn2 overexpression or scavenging mitochondria-originated reactive oxygen species (ROS) with mito-TEMPO ameliorated BCATc Inhibitor 2-induced oxidative stress, mitochondrial membrane potential disruption and mitochondrial fission, and abrogated the inhibitory effect of BCATc Inhibitor 2 on glioblastoma cells through PI3K/AKT/mTOR signaling. All of these findings indicate suppression of BCAA metabolism promotes glioblastoma cell apoptosis via disruption of Mfn2-mediated mitochondrial dynamics and inhibition of PI3K/AKT/mTOR pathway, and suggest that BCAA metabolism can be targeted for developing therapeutic agents to treat glioblastoma.

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.

Dysfunction of Avo3, an essential component of target of rapamycin complex 2, induces ubiquitin-proteasome-dependent downregulation of Avo2 in Saccharomyces cerevisiae.

The ubiquitin-proteasome system (UPS) plays a key role in maintaining cellular protein homeostasis and participates in modulating various cellular functions. Target of rapamycin (TOR), a highly conserved Ser/Thr kinase found across species from yeasts to humans, forms two multi-protein complexes, TORC1 and TORC2, to orchestrate cellular processes crucial for optimal growth, survival, and stress responses. While UPS-mediated regulation of mammalian TOR complexes has been documented, the ubiquitination of yeast TOR complexes remains largely unexplored. Here we report a functional interplay between the UPS and TORC2 in Saccharomyces cerevisiae. Using avo3-2ts, a temperature-sensitive mutant of the essential TORC2 component Avo3 exhibiting TORC2 defects at restrictive temperatures, we obtained evidence for UPS-dependent protein degradation and downregulation of the TORC2 component Avo2. Our results established the involvement of the E3 ubiquitin ligase Ubr1 and its catalytic activity in mediating Avo2 degradation in cells with defective Avo3. Coimmunoprecipitation revealed the interaction between Avo2 and Ubr1, indicating Avo2 as a potential substrate of Ubr1. Furthermore, depleting Ubr1 rescued the growth of avo3-2ts cells at restrictive temperatures, suggesting an essential role of Avo2 in sustaining cell viability under heat stress and/or TORC2 dysfunction. This study uncovers a role of UPS in yeast TORC2 regulation, highlighting the impact of protein degradation control on cellular signaling.

Targeting the Plasmodium falciparum UCHL3 ubiquitin hydrolase using chemically constrained peptides.

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.

N4BP1 coordinates ubiquitin-dependent crosstalk within the IκB kinase family to limit Toll-like receptor signaling and inflammation.

The ubiquitin-binding endoribonuclease N4BP1 potently suppresses cytokine production by Toll-like receptors (TLRs) that signal through the adaptor MyD88 but is inactivated via caspase-8-mediated cleavage downstream of death receptors, TLR3, or TLR4. Here, we examined the mechanism whereby N4BP1 limits inflammatory responses. In macrophages, deletion of N4BP1 prolonged activation of inflammatory gene transcription at late time points after TRIF-independent TLR activation. Optimal suppression of inflammatory cytokines by N4BP1 depended on its ability to bind polyubiquitin chains, as macrophages and mice-bearing inactivating mutations in a ubiquitin-binding motif in N4BP1 displayed increased TLR-induced cytokine production. Deletion of the noncanonical IκB kinases (ncIKKs), Tbk1 and Ikke, or their adaptor Tank phenocopied N4bp1 deficiency and enhanced macrophage responses to TLR1/2, TLR7, or TLR9 stimulation. Mechanistically, N4BP1 acted in concert with the ncIKKs to limit the duration of canonical IκB kinase (IKKα/ß) signaling. Thus, N4BP1 and the ncIKKs serve as an important checkpoint against over-exuberant innate immune responses.

Deep mutational scanning reveals a correlation between degradation and toxicity of thousands of aspartoacylase variants.

Unstable proteins are prone to form non-native interactions with other proteins and thereby may become toxic. To mitigate this, destabilized proteins are targeted by the protein quality control network. Here we present systematic studies of the cytosolic aspartoacylase, ASPA, where variants are linked to Canavan disease, a lethal neurological disorder. We determine the abundance of 6152 of the 6260 ( ~ 98%) possible single amino acid substitutions and nonsense ASPA variants in human cells. Most low abundance variants are degraded through the ubiquitin-proteasome pathway and become toxic upon prolonged expression. The data correlates with predicted changes in thermodynamic stability, evolutionary conservation, and separate disease-linked variants from benign variants. Mapping of degradation signals (degrons) shows that these are often buried and the C-terminal region functions as a degron. The data can be used to interpret Canavan disease variants and provide insight into the relationship between protein stability, degradation and cell fitness.

VCP/p97 mediates nuclear targeting of non-ER-imported prion protein to maintain proteostasis.

Mistargeting of secretory proteins in the cytosol can trigger their aggregation and subsequent proteostasis decline. We have identified a VCP/p97-dependent pathway that directs non-ER-imported prion protein (PrP) into the nucleus to prevent the formation of toxic aggregates in the cytosol. Upon impaired translocation into the ER, PrP interacts with VCP/p97, which facilitates nuclear import mediated by importin-ß. Notably, the cytosolic interaction of PrP with VCP/p97 and its nuclear import are independent of ubiquitination. In vitro experiments revealed that VCP/p97 binds non-ubiquitinated PrP and prevents its aggregation. Inhibiting binding of PrP to VCP/p97, or transient proteotoxic stress, promotes the formation of self-perpetuating and partially proteinase resistant PrP aggregates in the cytosol, which compromised cellular proteostasis and disrupted further nuclear targeting of PrP. In the nucleus, RNAs keep PrP in a soluble and non-toxic conformation. Our study revealed a novel ubiquitin-independent role of VCP/p97 in the nuclear targeting of non-imported secretory proteins and highlights the impact of the chemical milieu in triggering protein misfolding.

Specific isoforms of the ubiquitin ligase gene WWP2 are targets of osteoarthritis genetic risk via a differentially methylated DNA sequence.

BACKGROUND: Transitioning from a genetic association signal to an effector gene and a targetable molecular mechanism requires the application of functional fine-mapping tools such as reporter assays and genome editing. In this report, we undertook such studies on the osteoarthritis (OA) risk that is marked by single nucleotide polymorphism (SNP) rs34195470 (A > G). The OA risk-conferring G allele of this SNP associates with increased DNA methylation (DNAm) at two CpG dinucleotides within WWP2. This gene encodes a ubiquitin ligase and is the host gene of microRNA-140 (miR-140). WWP2 and miR-140 are both regulators of TGFß signaling. METHODS: Nucleic acids were extracted from adult OA (arthroplasty) and foetal cartilage. Samples were genotyped and DNAm quantified by pyrosequencing at the two CpGs plus 14 flanking CpGs. CpGs were tested for transcriptional regulatory effects using a chondrocyte cell line and reporter gene assay. DNAm was altered using epigenetic editing, with the impact on gene expression determined using RT-qPCR. In silico analysis complemented laboratory experiments. RESULTS: rs34195470 genotype associates with differential methylation at 14 of the 16 CpGs in OA cartilage, forming a methylation quantitative trait locus (mQTL). The mQTL is less pronounced in foetal cartilage (5/16 CpGs). The reporter assay revealed that the CpGs reside within a transcriptional regulator. Epigenetic editing to increase their DNAm resulted in altered expression of the full-length and N-terminal transcript isoforms of WWP2. No changes in expression were observed for the C-terminal isoform of WWP2 or for miR-140. CONCLUSIONS: As far as we are aware, this is the first experimental demonstration of an OA association signal targeting specific transcript isoforms of a gene. The WWP2 isoforms encode proteins with varying substrate specificities for the components of the TGFß signaling pathway. Future analysis should focus on the substrates regulated by the two WWP2 isoforms that are the targets of this genetic risk.

Systematic HOIP interactome profiling reveals critical roles of linear ubiquitination in tissue homeostasis.

Linear ubiquitination catalyzed by HOIL-1-interacting protein (HOIP), the key component of the linear ubiquitination assembly complex, plays fundamental roles in tissue homeostasis by executing domain-specific regulatory functions. However, a proteome-wide analysis of the domain-specific interactome of HOIP across tissues is lacking. Here, we present a comprehensive mass spectrometry-based interactome profiling of four HOIP domains in nine mouse tissues. The interaction dataset provides a high-quality HOIP interactome resource with an average of approximately 90 interactors for each bait per tissue. HOIP tissue interactome presents a systematic understanding of linear ubiquitination functions in each tissue and also shows associations of tissue functions to genetic diseases. HOIP domain interactome characterizes a set of previously undefined linear ubiquitinated substrates and elucidates the cross-talk among HOIP domains in physiological and pathological processes. Moreover, we show that linear ubiquitination of Integrin-linked protein kinase (ILK) decreases focal adhesion formation and promotes the detachment of Shigella flexneri-infected cells. Meanwhile, Hoip deficiency decreases the linear ubiquitination of Smad ubiquitination regulatory factor 1 (SMURF1) and enhances its E3 activity, finally causing a reduced bone mass phenotype in mice. Overall, our work expands the knowledge of HOIP-interacting proteins and provides a platform for further discovery of linear ubiquitination functions in tissue homeostasis.

SUMO and the DNA damage response.

The preservation of genome integrity requires specialised DNA damage repair (DDR) signalling pathways to respond to each type of DNA damage. A key feature of DDR is the integration of numerous post-translational modification signals with DNA repair factors. These modifications influence DDR factor recruitment to damaged DNA, activity, protein-protein interactions, and ultimately eviction to enable access for subsequent repair factors or termination of DDR signalling. SUMO1-3 (small ubiquitin-like modifier 1-3) conjugation has gained much recent attention. The SUMO-modified proteome is enriched with DNA repair factors. Here we provide a snapshot of our current understanding of how SUMO signalling impacts the major DNA repair pathways in mammalian cells. We highlight repeating themes of SUMO signalling used throughout DNA repair pathways including the assembly of protein complexes, competition with ubiquitin to promote DDR factor stability and ubiquitin-dependent degradation or extraction of SUMOylated DDR factors. As SUMO 'addiction' in cancer cells is protective to genomic integrity, targeting components of the SUMO machinery to potentiate DNA damaging therapy or exacerbate existing DNA repair defects is a promising area of study.

TRIM47-CDO1 axis dictates hepatocellular carcinoma progression by modulating ferroptotic cell death through the ubiquitin‒proteasome system.

Hepatocellular carcinoma (HCC) is the predominant form of liver cancer, characterized by high morbidity and mortality rates, as well as unfavorable treatment outcomes. Tripartite motif-containing protein 47 (TRIM47) has been implicated in various diseases including tumor progression with the activity of E3 ubiquitin ligase. However, the precise regulatory mechanisms underlying the involvement of TRIM47 in HCC remain largely unexplored. Here, we provide evidence that TRIM47 exhibits heightened expression in tumor tissues, and its expression is in intimate association with clinical staging and patient prognosis. TRIM47 promotes HCC proliferation, migration, and invasion as an oncogene by in vitro gain- and loss-of-function experiments. TRIM47 knockdown results in HCC ferroptosis induction, primarily through CDO1 involvement to regulate GSH synthesis. Subsequent experiments confirm the interaction between TRIM47 and CDO1 dependent on B30.2 domain, wherein TRIM47 facilitates K48-linked ubiquitination, leading to a decrease in CDO1 protein abundance in HCC. Furthermore, CDO1 is able to counteract the promotional effect of TRIM47 on HCC biological functions. Overall, our research provides novel insight into the mechanism of TRIM47 in CDO1-mediated ferroptosis in HCC cells, highlighting its value as a potential target candidate for HCC therapeutic approaches.

Viral deubiquitinating proteases and the promising strategies of their inhibition.

Several viruses are now known to code for deubiquitinating proteases in their genomes. Ubiquitination is an essential post-translational modification of cellular substrates involved in many processes in the cell, including in innate immune signalling. This post-translational modification is regulated by the ubiquitin conjugation machinery, as well as various host deubiquitinating enzymes. The conjugation of ubiquitin chains to several innate immune related factors is often needed to induce downstream signalling, shaping the antiviral response. Viral deubiquitinating proteins, besides often having a primary function in the viral replication cycle by cleaving the viral polyprotein, are also able to cleave ubiquitin chains from such host substrates, in that way exerting a function in innate immune evasion. The presence of viral deubiquitinating enzymes has been firmly established for numerous animal-infecting viruses, such as some well-researched and clinically important nidoviruses, and their presence has now been confirmed in several plant viruses as well. Viral proteases in general have long been highlighted as promising drug targets, with a current focus on small molecule inhibitors. In this review, we will discuss the range of viral deubiquitinating proteases known to date, summarise the various avenues explored to inhibit such proteases and discuss novel strategies and models intended to inhibit and study these specific viral enzymes.

An evolutionarily conserved ubiquitin ligase drives infection and transmission of flaviviruses.

Mosquito-borne flaviviruses such as dengue (DENV) and Zika (ZIKV) cause hundreds of millions of infections annually. The single-stranded RNA genome of flaviviruses is translated into a polyprotein, which is cleaved equally into individual functional proteins. While structural proteins are packaged into progeny virions and released, most of the nonstructural proteins remain intracellular and could become cytotoxic if accumulated over time. However, the mechanism by which nonstructural proteins are maintained at the levels optimal for cellular fitness and viral replication remains unknown. Here, we identified that the ubiquitin E3 ligase HRD1 is essential for flaviviruses infections in both mammalian hosts and mosquitoes. HRD1 directly interacts with flavivirus NS4A and ubiquitylates a conserved lysine residue for ER-associated degradation. This mechanism avoids excessive accumulation of NS4A, which otherwise interrupts the expression of processed flavivirus proteins in the ER. Furthermore, a small-molecule inhibitor of HRD1 named LS-102 effectively interrupts DENV2 infection in both mice and Aedes aegypti mosquitoes, and significantly disturbs DENV transmission from the infected hosts to mosquitoes owing to reduced viremia. Taken together, this study demonstrates that flaviviruses have evolved a sophisticated mechanism to exploit the ubiquitination system to balance the homeostasis of viral proteins for their own advantage and provides a potential therapeutic target to interrupt flavivirus infection and transmission.

Decoding the ubiquitin landscape by cutting-edge ubiquitinomic approaches.

Functional consequences of protein ubiquitination have gone far beyond the degradation regulation as was initially imagined during its discovery 40 years back. The state-of-the-art has revealed the plethora of signaling pathways that are largely regulated by ubiquitination process in eukaryotes. To no surprise, ubiquitination is often dysregulated in many human diseases, including cancer, neurodegeneration and infection. Hence it has become a major focus with high-gain research value for many investigators to unravel new proteoforms, that are the targets of this ubiquitination modification. Despite many biochemical or proteomic approaches available for ubiquitination detection, mass-spectrometry stood out to be the most efficient and transformative technology to read this complex modification script. Here in this review, we have discussed how different ubiquitin codes can be decoded qualitatively and quantitatively following various sequential proteomic approaches to date reported and indicated the current limitations with scope for improvements.

Proteasome-Associated Syndromes: Updates on Genetics, Clinical Manifestations, Pathogenesis, and Treatment.

The ubiquitin-proteasome system (UPS) has a critical role in post-translational protein modification that is essential for the maintenance of all cellular functions, including immune responses. The proteasome complex is ubiquitously expressed and is responsible for degradation of short-lived structurally abnormal, misfolded and not-needed proteins that are targeted for degradation via ubiquitin conjugation. Over the last 14 years, an increasing number of human diseases have been linked to pathogenic variants in proteasome subunits and UPS regulators. Defects of the proteasome complex or its chaperons - which have a regulatory role in the assembly of the proteasome - disrupt protein clearance and cellular homeostasis, leading to immune dysregulation, severe inflammation, and neurodevelopmental disorders in humans. Proteasome-associated diseases have complex inheritance, including monogenic, digenic and oligogenic disorders and can be dominantly or recessively inherited. In this review, we summarize the current known genetic causes of proteasomal disease, and discuss the molecular pathogenesis of these conditions based on the function and cellular expression of mutated proteins in the proteasome complex.

Rhes, a striatal enriched protein, regulates post-translational small-ubiquitin-like-modifier (SUMO) modification of nuclear proteins and alters gene expression.

Rhes (Ras homolog enriched in the striatum), a multifunctional protein that regulates striatal functions associated with motor behaviors and neurological diseases, can shuttle from cell to cell via the formation of tunneling-like nanotubes (TNTs). However, the mechanisms by which Rhes mediates diverse functions remain unclear. Rhes is a small GTPase family member which contains a unique C-terminal Small Ubiquitin-like Modifier (SUMO) E3-like domain that promotes SUMO post-translational modification of proteins (SUMOylation) by promoting "cross-SUMOylation" of the SUMO enzyme SUMO E1 (Aos1/Uba2) and SUMO E2 ligase (Ubc-9). Nevertheless, the identity of the SUMO substrates of Rhes remains largely unknown. Here, by combining high throughput interactome and SUMO proteomics, we report that Rhes regulates the SUMOylation of nuclear proteins that are involved in the regulation of gene expression. Rhes increased the SUMOylation of histone deacetylase 1 (HDAC1) and histone 2B, while decreasing SUMOylation of heterogeneous nuclear ribonucleoprotein M (HNRNPM), protein polybromo-1 (PBRM1) and E3 SUMO-protein ligase (PIASy). We also found that Rhes itself is SUMOylated at 6 different lysine residues (K32, K110, K114, K120, K124, and K245). Furthermore, Rhes regulated the expression of genes involved in cellular morphogenesis and differentiation in the striatum, in a SUMO-dependent manner. Our findings thus provide evidence for a previously undescribed role for Rhes in regulating the SUMOylation of nuclear targets and in orchestrating striatal gene expression via SUMOylation.

Malvidin attenuates trauma-induced heterotopic ossification of tendon in rats by targeting Rheb for degradation via the ubiquitin-proteasome pathway.

The pathogenesis of trauma-induced heterotopic ossification (HO) in the tendon remains unclear, posing a challenging hurdle in treatment. Recognizing inflammation as the root cause of HO, anti-inflammatory agents hold promise for its management. Malvidin (MA), possessing anti-inflammatory properties, emerges as a potential agent to impede HO progression. This study aimed to investigate the effect of MA in treating trauma-induced HO and unravel its underlying mechanisms. Herein, the effectiveness of MA in preventing HO formation was assessed through local injection in a rat model. The potential mechanism underlying MA's treatment was investigated in the tendon-resident progenitor cells of tendon-derived stem cells (TDSCs), exploring its pathway in HO formation. The findings demonstrated that MA effectively hindered the osteogenic differentiation of TDSCs by inhibiting the mTORC1 signalling pathway, consequently impeding the progression of trauma-induced HO of Achilles tendon in rats. Specifically, MA facilitated the degradation of Rheb through the K48-linked ubiquitination-proteasome pathway by modulating USP4 and intercepted the interaction between Rheb and the mTORC1 complex, thus inhibiting the mTORC1 signalling pathway. Hence, MA presents itself as a promising candidate for treating trauma-induced HO in the Achilles tendon, acting by targeting Rheb for degradation through the ubiquitin-proteasome pathway.

Structural insights into the functional mechanism of the ubiquitin ligase E6AP.

E6AP dysfunction is associated with Angelman syndrome and Autism spectrum disorder. Additionally, the host E6AP is hijacked by the high-risk HPV E6 to aberrantly ubiquitinate the tumor suppressor p53, which is linked with development of multiple types of cancer, including most cervical cancers. Here we show that E6AP and the E6AP/E6 complex exist, respectively, as a monomer and a dimer of the E6AP/E6 protomer. The short α1-helix of E6AP transforms into a longer helical structure when in complex with E6. The extended α1-helices of the dimer intersect symmetrically and contribute to the dimerization. The two protomers sway around the crossed region of the two α1-helices to promote the attachment and detachment of substrates to the catalytic C-lobe of E6AP, thus facilitating ubiquitin transfer. These findings, complemented by mutagenesis analysis, suggest that the α1-helix, through conformational transformations, controls the transition between the inactive monomer and the active dimer of E6AP.