CARD14 signalosome formation is associated with its endosomal relocation and mTORC1-induced keratinocyte proliferation.

Rare mutations in CARD14 promote psoriasis by inducing CARD14-BCL10-MALT1 complexes that activate NF-κB and MAP kinases. Here, the downstream signalling mechanism of the highly penetrant CARD14E138A alteration is described. In addition to BCL10 and MALT1, CARD14E138A associated with several proteins important in innate immune signalling. Interactions with M1-specific ubiquitin E3 ligase HOIP, and K63-specific ubiquitin E3 ligase TRAF6 promoted BCL10 ubiquitination and were essential for NF-κB and MAP kinase activation. In contrast, the ubiquitin binding proteins A20 and ABIN1, both genetically associated with psoriasis development, negatively regulated signalling by inducing CARD14E138A turnover. CARD14E138A localized to early endosomes and was associated with the AP2 adaptor complex. AP2 function was required for CARD14E138A activation of mTOR complex 1 (mTORC1), which stimulated keratinocyte metabolism, but not for NF-κB nor MAP kinase activation. Furthermore, rapamycin ameliorated CARD14E138A-induced keratinocyte proliferation and epidermal acanthosis in mice, suggesting that blocking mTORC1 may be therapeutically beneficial in CARD14-dependent psoriasis.

Cyclophilin J limits linear ubiquitin signaling and controls colorectal cancer progression.

Exorbitant sustained inflammation is closely linked to inflammation-associated disorders, including cancer. The initiation of gastrointestinal cancers such as colorectal cancer is frequently accelerated by uncontrollable chronic inflammation which is triggered by excessive activation of nuclear factor kappa-B (NF-κB) signaling. Linear ubiquitin chains play an important role in activating canonical NF-κB pathway. The only known E3 complex, linear ubiquitin chain assembly complex is responsible for the synthesis of linear ubiquitin chains, thus leading to the activation of NF-κB axis and promoting the development of inflammation and inflammation-associated cancers. We report here cyclophilin J (CYPJ) which is a negative regulator of the linear ubiquitin chain assembly complex. The N terminus of CYPJ binds to the second Npl4 zinc finger (NZF) domain of HOIL-1-interacting protein and the ubiquitin-like domain of Shank-associated RH domain-interacting protein to disrupt the interaction between HOIL-1-interacting protein and Shank-associated RH domain-interacting protein and thus restrains linear ubiquitin chain synthesis and NF-κB activation. Cypj-deficient mice are highly susceptible to dextran sulfate sodium-induced colitis and dextran sulfate sodium plus azoxymethane-induced colon cancer. Moreover, CYPJ expression is induced by hypoxia. Patients with high expression of both CYPJ and hypoxia-inducible factor-1α have longer overall survival and progression-free survival. These results implicate CYPJ as an unexpected robust attenuator of inflammation-driven tumorigenesis that exerts its effects by controlling linear ubiquitin chain synthesis in NF-κB signal pathway.

A novel HOIP frameshift variant alleviates NF-kappaB signalling and sensitizes cells to TNF-induced death.

BACKGROUND: HOIP is the catalytic subunit of the E3 ligase complex (linear ubiquitin chain assembly complex), which is able to generate linear ubiquitin chains. However, the role of rare HOIP functionally deficient variants remains unclear. The pathogenic mechanism and the relationship with immune deficiency phenotypes remain to be clarified. METHODS: Based on a next-generation sequencing panel of 270 genes, we identified a HOIP deletion variant that causes common variable immunodeficiency disease. Bioinformatics analysis and cell-based experiments were performed to study the molecular mechanism by which the variant causes immunodeficiency diseases. FINDINGS: A homozygous loss-of-function variant in HOIP was identified. The variant causes a frameshift and generates a premature termination codon in messenger RNA, resulting in a C-terminal truncated HOIP mutant, that is, the loss of the linear ubiquitin chain-specific catalytic domain. The truncated HOIP mutant has impaired E3 ligase function in linear ubiquitination, leading to the suppression of canonical NF-κB signalling and increased TNF-induced multiple forms of cell death. INTERPRETATION: The loss-of-function HOIP variant accounts for the immune deficiencies. The canonical NF-κB pathway and cell death are involved in the pathogenesis of the disease. FUNDING: This study was funded by the National Natural Science Foundation of China (No. 82270444 and 81501851). RESEARCH IN CONTEXT: Evidence before this study LUBAC is the only known linear ubiquitin chain assembly complex for which HOIP is an essential catalytic subunit. Three HOIP variants have now been identified in two immunodeficient patients and functionally characterised. However, there have been no reports on the pathogenicity of only catalytic domain deletion variants in humans, or the pathogenic mechanisms of catalytic domain deletion variants. Added value of this study We report the first case of an autosomal recessive homozygous deletion variant that results in deletion of the HOIP catalytic structural domain. We demonstrate that this variant is a loss-of-function variant using a heterologous expression system. The variant has impaired E3 ligase function. It can still bind to other subunits of LUBAC, but it fails to generate linear ubiquitin chains. We also explored the underlying mechanisms by which this variant leads to immunodeficiency. The variant attenuates the canonical NF-κB and MAPK signalling cascades and increases the sensitivity of TNFα-induced diverse cell death and activation of mitochondrial apoptosis pathways. These findings provide support for the treatment and drug development of patients with inborn errors of immunity in HOIP and related signalling pathways. Implications of all the available evidence First, this study expands the HOIP pathogenic variant database and phenotypic spectrum. Furthermore, studies on the biological functions of pathogenic variants in relation to the NF-κB signalling pathway and cell death provided new understanding into the genetic basis and pathogenesis of HOIP-deficient immune disease, indicating the necessity of HOIP and related signalling pathway variants as diagnostic targets in patients with similar genetic deficiency phenotypes..

AlphaFold2 assists in providing novel mechanistic insights into the interactions among the LUBAC subunits.

The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ligase complex in which the ubiquitin-like (UBL) domains of SHARPIN and HOIL-1L interact with HOIP to determine the structural stability of LUBAC. The interactions between subunits within LUBAC have been a topic of extensive research. However, the impact of the LTM motif on the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP remains unclear. Here, we discover that the absence of the LTM motif in the AlphaFold2-predicted LUBAC structure alters the HOIP-UBA structure. We employ GeoPPI to calculate the changes in binding free energy (ΔG) caused by single-point mutations between subunits, simulating their protein-protein interactions. The results reveal that the presence of the LTM motif decreases the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP, leading to a decrease in the structural stability of LUBAC. Furthermore, using the AlphaFold2-predicted results, we find that HOIP (629‒695) and HOIP-UBA bind to both sides of HOIL-1L-UBL, respectively. The experiments of Gromacs molecular dynamics simulations, SPR and ITC demonstrate that the elongated domain formed by HOIP (629‒695) and HOIP-UBA, hereafter referred to as the HOIP (466‒695) structure, interacts with HOIL-1L-UBL to form a structurally stable complex. These findings illustrate the collaborative interaction between HOIP-UBA and HOIP (629‒695) with HOIL-1L-UBL, which influences the structural stability of LUBAC.

Mechanisms underlying linear ubiquitination and implications in tumorigenesis and drug discovery.

Linear ubiquitination is a distinct type of ubiquitination that involves attaching a head-to-tail polyubiquitin chain to a substrate protein. Early studies found that linear ubiquitin chains are essential for the TNFα- and IL-1-mediated NF-κB signaling pathways. However, recent studies have discovered at least sixteen linear ubiquitination substrates, which exhibit a broader activity than expected and mediate many other signaling pathways beyond NF-κB signaling. Dysregulation of linear ubiquitination in these pathways has been linked to many types of cancers, such as lymphoma, liver cancer, and breast cancer. Since the discovery of linear ubiquitin, extensive effort has been made to delineate the molecular mechanisms of how dysregulation of linear ubiquitination causes tumorigenesis and cancer development. In this review, we highlight newly discovered linear ubiquitination-mediated signaling pathways, recent advances in the role of linear ubiquitin in different types of cancers, and the development of linear ubiquitin inhibitors. Video Abstract.

Mechanistic insights into the homo-dimerization of HOIL-1L and SHARPIN.

HOIL-1L and SHARPIN are two essential regulatory subunits of the linear ubiquitin chain assembly complex (LUBAC), which is the only known E3 ligase complex generating linear ubiquitin chains. In addition to their LUBAC-dependent functions, HOIL-1L and SHARPIN alone play crucial roles in many LUBAC-independent cellular processes. Importantly, deficiency of HOIL-1L or SHARPIN leads to severe disorders in humans or mice. However, the mechanistic bases underlying the multi-functions of HOIL-1L and SHARPIN are still largely unknown. Here, we uncover that HOIL-1L and SHARPIN alone can form homo-dimers through their LTM motifs. We solve two crystal structures of the dimeric LTM motifs of HOIL-1L and SHARPIN, which not only elucidate the detailed molecular mechanism underpinning the dimer formations of HOIL-1L and SHARPIN, but also reveal a general mode shared by the LTM motifs of HOIL-1L and SHARPIN for forming homo-dimer or hetero-dimer. Furthermore, we elucidate that the polyglucosan body myopathy-associated HOIL-1L A18P mutation disturbs the structural folding of HOIL-1L LTM, and disrupts the dimer formation of HOIL-1L. In summary, our study provides mechanistic insights into the homo-dimerization of HOIL-1L and SHARPIN mediated by their LTM motifs, and expands our understandings of the multi-functions of HOIL-1L and SHARPIN as well as the etiology of relevant human disease caused by defective HOIL-1L.

Solution structure of the HOIL-1L NZF domain reveals a conformational switch regulating linear ubiquitin affinity.

Attachment of polyubiquitin (poly-Ub) chains to proteins is a major posttranslational modification in eukaryotes. Linear ubiquitin chain assembly complex, consisting of HOIP (HOIL-1-interacting protein), HOIL-1L (heme-oxidized IRP2 Ub ligase 1), and SHARPIN (Shank-associated RH domain-interacting protein), specifically synthesizes "head-to-tail" poly-Ub chains, which are linked via the N-terminal methionine α-amino and C-terminal carboxylate of adjacent Ub units and are thus commonly called "linear" poly-Ub chains. Linear ubiquitin chain assembly complex-assembled linear poly-Ub chains play key roles in immune signaling and suppression of cell death and have been associated with immune diseases and cancer; HOIL-1L is one of the proteins known to selectively bind linear poly-Ub via its Npl4 zinc finger (NZF) domain. Although the structure of the bound form of the HOIL-1L NZF domain with linear di-Ub is known, several aspects of the recognition specificity remain unexplained. Here, we show using NMR and orthogonal biophysical methods, how the NZF domain evolves from a free to the specific linear di-Ub-bound state while rejecting other potential Ub species after weak initial binding. The solution structure of the free NZF domain revealed changes in conformational stability upon linear Ub binding, and interactions between the NZF core and tail revealed conserved electrostatic contacts, which were sensitive to charge modulation at a reported phosphorylation site: threonine-207. Phosphomimetic mutations reduced linear Ub affinity by weakening the integrity of the linear di-Ub-bound conformation. The described molecular determinants of linear di-Ub binding provide insight into the dynamic aspects of the Ub code and the NZF domain's role in full-length HOIL-1L.

LUBAC-mediated linear ubiquitination in tissue homeostasis and disease.

In addition to its role in the ubiquitin-proteasome system of protein degradation, polyubiquitination is involved in the regulation of intracellular events. Depending on the type of ubiquitin-ubiquitin linkage used, polyubiquitin can assume several types of structures. The spatiotemporal dynamics of polyubiquitin involve multiple adaptor proteins and induce different downstream outputs. Linear ubiquitination, in which the N-terminal methionine on the acceptor ubiquitin serves as the site for ubiquitin-ubiquitin conjugation, is a rare and atypical type of polyubiquitin modification. The production of linear ubiquitin chains is dependent on various external inflammatory stimuli and leads to the transient activation of the downstream NF-κB signalling pathway. This in turn suppresses extrinsic programmed cell death signals and protects cells from activation-induced cell death under inflammatory conditions. Recent evidence has revealed the role of linear ubiquitination in various biological processes under both physiological and pathological conditions. This led us to propose that linear ubiquitination may be pivotal in the 'inflammatory adaptation' of cells, and consequently in tissue homeostasis and inflammatory disease. In this review, we focused on the physiological and pathophysiological roles of linear ubiquitination in vivo in response to a changing inflammatory microenvironment.

Linear ubiquitination-induced necrotic tumor remodeling elicits immune evasion.

Tumor-elicited inflammation confers tumorigenic properties, including cell death resistance, proliferation, or immune evasion. To focus on inflammatory signaling in tumors, we investigated linear ubiquitination, which enhances the nuclear factor-κB signaling pathway and prevents extrinsic programmed cell death under inflammatory environments. Here, we showed that linear ubiquitination was augmented especially in tumor cells around a necrotic core. Linear ubiquitination allowed melanomas to tolerate the hostile tumor microenvironment and to extend a necrosis-containing morphology. Loss of linear ubiquitination resulted in few necrotic lesions and growth regression, further leading to repression of innate anti-PD-1 therapy resistance signatures in melanoma as well as activation of interferon responses and antigen presentation that promote immune-mediated tumor eradication. Collectively, linear ubiquitination promotes tumor-specific tissue remodeling and the ensuing immune evasion.

Novel role for linear ubiquitination in regulating NFAT1 stability.

Linear ubiquitination is an important post-translational modification regulating the activation of numerous proinflammatory signalling mediators. Deregulated linear ubiquitination has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. In this issue, Miao et al. have identified a novel role for linear ubiquitination in the stabilisation of the NFAT1 transcription factor, leading to enhanced NFAT1-mediated gene expression, which might have functional implications in patients with Kawasaki disease.

Involvement of heterologous ubiquitination including linear ubiquitination in Alzheimer's disease and amyotrophic lateral sclerosis.

In neurodegenerative diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), the progressive accumulation of ubiquitin-positive cytoplasmic inclusions leads to proteinopathy and neurodegeneration. Along with the seven types of Lys-linked ubiquitin chains, the linear ubiquitin chain assembly complex (LUBAC)-mediated Met1-linked linear ubiquitin chain, which activates the canonical NF-κB pathway, is also involved in cytoplasmic inclusions of tau in AD and TAR DNA-binding protein 43 in ALS. Post-translational modifications, including heterologous ubiquitination, affect proteasomal and autophagic degradation, inflammatory responses, and neurodegeneration. Single nucleotide polymorphisms (SNPs) in SHARPIN and RBCK1 (which encodes HOIL-1L), components of LUBAC, were recently identified as genetic risk factors of AD. A structural biological simulation suggested that most of the SHARPIN SNPs that cause an amino acid replacement affect the structure and function of SHARPIN. Thus, the aberrant LUBAC activity is related to AD. Protein ubiquitination and ubiquitin-binding proteins, such as ubiquilin 2 and NEMO, facilitate liquid-liquid phase separation (LLPS), and linear ubiquitination seems to promote efficient LLPS. Therefore, the development of therapeutic approaches that target ubiquitination, such as proteolysis-targeting chimeras (PROTACs) and inhibitors of ubiquitin ligases, including LUBAC, is expected to be an additional effective strategy to treat neurodegenerative diseases.

Linear ubiquitination improves NFAT1 protein stability and facilitates NFAT1 signalling in Kawasaki disease.

NFAT1 is known for its roles in T cell development and activation. So far, the phosphorylation of NFAT1 has been extensively studied, but the other post-translational modifications of NFAT1 remain largely unknown. In this study, we reported that NFAT1 is a linearly ubiquitinated substrate of linear ubiquitin chain assembly complex (LUBAC). LUBAC promoted NFAT1 linear ubiquitination, which in turn inhibited K48-linked polyubiquitination of NFAT1 and therefore increased NFAT1 protein stability. Interestingly, the linear ubiquitination levels of NFAT1 in patients with the Kawasaki disease were upregulated. Further studies demonstrated that the patients with the Kawasaki disease had increased mRNA levels of HOIL-1L. These findings revealed a linearly ubiquitinated substrate of LUBAC and an important biological function of NFAT1 linear ubiquitination in the Kawasaki disease and therefore may provide a novel strategy for the treatment of the Kawasaki disease.

SPATA2 restricts OTULIN-dependent LUBAC activity independently of CYLD.

SPATA2 mediates the recruitment of CYLD to immune receptor complexes by bridging the interaction of CYLD with the linear ubiquitylation assembly complex (LUBAC) component HOIP. Whether SPATA2 exhibits functions independently of CYLD is unclear. Here, we show that, while Cyld-/- and Spata2-/- mice are viable, double mutants exhibit highly penetrant perinatal lethality, indicating independent functions of SPATA2 and CYLD. Cyld-/-Spata2-/- fibroblasts show increased M1-linked TNFR1-SC ubiquitylation and, similar to Cyld-/-Spata2-/- macrophages and intestinal epithelial cells, elevated pro-inflammatory gene expression compared with Cyld-/- or Spata2-/- cells. We show that SPATA2 competes with OTULIN for binding to HOIP via its PUB-interacting motif (PIM) and its zinc finger domain, thereby promoting autoubiquitylation of LUBAC. Consistently, increased pro-inflammatory signaling in Cyld-/-Spata2-/- cells depends on the presence of OTULIN. Our data therefore indicate that SPATA2 counteracts, independently of CYLD, the deubiquitylation of LUBAC by OTULIN and thereby attenuates LUBAC activity and pro-inflammatory signaling.

Role of linear ubiquitination in inflammatory responses and tissue homeostasis.

Polyubiquitination is a post-translational modification involved in a wide range of immunological events, including inflammatory responses, immune cell differentiation, and development of inflammatory diseases. The versatile functions of polyubiquitination are based on different types of ubiquitin linkage, which enable various UBD (ubiquitin binding domain)-containing adaptor proteins to associate and induce distinct biological outputs. A unique and atypical type of polyubiquitin chain comprising a conjugation between the N-terminal methionine of the proximal ubiquitin moiety and the C-terminal glycine of the distal ubiquitin moiety, referred to as a linear or M1-linked ubiquitin chain, has been studied exclusively within the field of immunology because it is distinct from other polyubiquitin forms: linear ubiquitin chains are generated predominantly by various inflammatory stimulants, including tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and act as a critical modulator of transient and optimal signal transduction. Moreover, accumulating evidence suggests that linear ubiquitin chains are of physiological significance. Dysregulation of linear ubiquitination triggers chronic inflammation and immunodeficiency via downregulation of linear ubiquitin-dependent nuclear factor-kappa B (NF-κB) signaling and by triggering TNF-α-induced cell death, suggesting that linear ubiquitination is a homeostatic regulator of tissue-specific functions. In this review, we focus on our current understating of the molecular and cellular mechanisms by which linear ubiquitin chains control inflammatory environments. Furthermore, we review the role of linear ubiquitination on T cell development, differentiation, and function, thereby providing insight into its direct association with maintaining the immune system.

Differential involvement of LUBAC-mediated linear ubiquitination in intestinal epithelial cells and macrophages during intestinal inflammation.

Disruption of the intestinal epithelial barrier and dysregulation of macrophages are major factors contributing to the pathogenesis of inflammatory bowel diseases (IBDs). Activation of NF-κB and cell death are involved in maintaining intestinal homeostasis in a cell type-dependent manner. Although both are regulated by linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination, the physiological relevance of linear ubiquitination to intestinal inflammation remains unexplored. Here, we used two experimental mouse models of IBD (intraperitoneal LPS and oral dextran sodium sulfate [DSS] administration) to examine the role of linear ubiquitination in intestinal epithelial cells (IECs) and macrophages during intestinal inflammation. We did this by deleting the linear ubiquitination activity of LUBAC specifically from IECs or macrophages. Upon LPS administration, loss of ligase activity in IECs induced mucosal inflammation and augmented IEC death. LPS-mediated death of LUBAC-defective IECs was triggered by TNF. IEC death was rescued by an anti-TNF antibody, and TNF (but not LPS) induced apoptosis of organoids derived from LUBAC-defective IECs. However, augmented TNF-mediated IEC death did not overtly affect the severity of colitis after DSS administration. By contrast, defective LUBAC ligase activity in macrophages ameliorated DSS-induced colitis by attenuating both infiltration of macrophages and expression of inflammatory cytokines. Decreased production of macrophage chemoattractant MCP-1/CCL2, as well as pro-inflammatory IL-6 and TNF, occurred through impaired activation of NF-κB and ERK via loss of ligase activity in macrophages. Taken together, these results indicate that both intraperitoneal LPS and oral DSS administrations are beneficial for evaluating epithelial integrity under inflammatory conditions, as well as macrophage functions in the event of an epithelial barrier breach. The data clarify the cell-specific roles of linear ubiquitination as a critical regulator of TNF-mediated epithelial integrity and macrophage pro-inflammatory responses during intestinal inflammation. © 2022 The Pathological Society of Great Britain and Ireland.

Normal lymphocyte homeostasis and function in MALT1 protease-resistant HOIL-1 knock-in mice.

The uniqueness of MALT1 protease activity in controlling several aspects of immunity in humans has made it a very attractive therapeutic target for multiple autoimmune diseases and lymphoid malignancies. Despite several encouraging preclinical studies with MALT1 inhibitors, severe reduction in regulatory T cells and immune-mediated pathology seen in MALT1 protease-dead (MALT1-PD) mice and some, but not all, studies analysing the effect of prolonged pharmacological MALT1 protease inhibition, indicates the need to further unravel the mechanism of MALT1 protease function. Notably, the contribution of individual MALT1 substrates to the immune defects seen in MALT1-PD mice is still unclear. Previous in vitro studies indicated a role for MALT1-mediated cleavage of the E3 ubiquitin ligase HOIL-1 in the modulation of nuclear factor-κB (NF-κB) signalling and inflammatory gene expression in lymphocytes. Here, we addressed the immunological consequences of inhibition of HOIL-1 cleavage by generating and immunophenotyping MALT1 cleavage-resistant HOIL-1 knock-in (KI) mice. HOIL-1 KI mice appear healthy and have no overt phenotype. NF-κB activation in T or B cells, as well as IL-2 production and in vitro T-cell proliferation, is comparable between control and HOIL-1 KI cells. Inhibition of HOIL-1 cleavage in mice has no effect on thymic T-cell development and conventional T-cell homeostasis. Likewise, B-cell development and humoral immune responses are not affected. Together, these data exclude an important role of MALT1-mediated HOIL-1 cleavage in T- and B-cell development and function in mice.

HOIP modulates the stability of GPx4 by linear ubiquitination.

LUBAC-mediated linear ubiquitination plays a pivotal role in regulation of cell death and inflammatory pathways. Genetic deficiency in LUBAC components leads to severe immune dysfunction or embryonic lethality. LUBAC has been extensively studied for its role in mediating TNF signaling. However, Tnfr1 knockout is not able to fully rescue the embryonic lethality of LUBAC deficiency, suggesting that LUBAC may modify additional key cellular substrates in promoting cell survival. GPx4 is an important selenoprotein involved in regulating cellular redox homeostasis in defense against lipid peroxidation-mediated cell death known as ferroptosis. Here we demonstrate that LUBAC deficiency sensitizes to ferroptosis by promoting GPx4 degradation and downstream lipid peroxidation. LUBAC binds and stabilizes GPx4 by modulating its linear ubiquitination both in normal condition and under oxidative stress. Our findings identify GPx4 as a key substrate of LUBAC and a previously unrecognized role of LUBAC-mediated linear ubiquitination in regulating cellular redox status and cell death.

Non-lysine ubiquitylation: Doing things differently.

The post-translational modification of proteins with ubiquitin plays a central role in nearly all aspects of eukaryotic biology. Historically, studies have focused on the conjugation of ubiquitin to lysine residues in substrates, but it is now clear that ubiquitylation can also occur on cysteine, serine, and threonine residues, as well as on the N-terminal amino group of proteins. Paradigm-shifting reports of non-proteinaceous substrates have further extended the reach of ubiquitylation beyond the proteome to include intracellular lipids and sugars. Additionally, results from bacteria have revealed novel ways to ubiquitylate (and deubiquitylate) substrates without the need for any of the enzymatic components of the canonical ubiquitylation cascade. Focusing mainly upon recent findings, this review aims to outline the current understanding of non-lysine ubiquitylation and speculate upon the molecular mechanisms and physiological importance of this non-canonical modification.

SHARPIN S146 phosphorylation mediates ARP2/3 interaction, cancer cell invasion and metastasis.

SHARPIN is involved in several cellular processes and promotes cancer progression. However, how the choice between different functions of SHARPIN is post-translationally regulated is unclear. Here, we characterized SHARPIN phosphorylation by mass spectrometry and in vitro kinase assay. Focusing on S131 and S146, we demonstrate that they have a role in SHARPIN-ARP2/3 complex interaction, but play no role in integrin inhibition or LUBAC activation. Consistent with its novel role in ARP2/3 regulation, S146 phosphorylation of SHARPIN promoted lamellipodia formation. We also demonstrate that SHARPIN S146 phosphorylation-mediated ARP2/3 interaction is sensitive to inhibition of ERK1/2 or reactivation of protein phosphatase 2A (PP2A). Notably, CRISPR/Cas9-mediated knockout of SHARPIN abrogated three-dimensional (3D) invasion of several cancer cell lines. The 3D invasion of cancer cells was rescued by overexpression of the wild-type SHARPIN, but not by SHARPIN S146A mutant. Finally, we demonstrate that inhibition of phosphorylation at S146 significantly reduces in vivo metastasis in a zebrafish model. Collectively, these results map SHARPIN phosphorylation sites and identify S146 as a novel phosphorylation switch defining ARP2/3 interaction and cancer cell invasion. This article has an associated First Person interview with the first author of the paper.

Interferon-Inducible E3 Ligase RNF213 Facilitates Host-Protective Linear and K63-Linked Ubiquitylation of Toxoplasma gondii Parasitophorous Vacuoles.

The obligate intracellular protozoan pathogen Toxoplasma gondii infects a wide range of vertebrate hosts and frequently causes zoonotic infections in humans. Whereas infected immunocompetent individuals typically remain asymptomatic, toxoplasmosis in immunocompromised individuals can manifest as a severe, potentially lethal disease, and congenital Toxoplasma infections are associated with adverse pregnancy outcomes. The protective immune response of healthy individuals involves the production of lymphocyte-derived cytokines such as interferon gamma (IFN-γ), which elicits cell-autonomous immunity in host cells. IFN-γ-inducible antiparasitic defense programs comprise nutritional immunity, the production of noxious gases, and the ubiquitylation of the Toxoplasma-containing parasitophorous vacuole (PV). PV ubiquitylation prompts the recruitment of host defense proteins to the PV and the consequential execution of antimicrobial effector programs, which reduce parasitic burden. However, the ubiquitin E3 ligase orchestrating these events has remained unknown. Here, we demonstrate that the IFN-γ-inducible E3 ligase RNF213 translocates to Toxoplasma PVs and facilitates PV ubiquitylation in human cells. Toxoplasma PVs become decorated with linear and K63-linked ubiquitin and recruit ubiquitin adaptor proteins in a process that is RNF213 dependent but independent of the linear ubiquitin chain assembly complex (LUBAC). IFN-γ priming fails to restrict Toxoplasma growth in cells lacking RNF213 expression, thus identifying RNF213 as a potent executioner of ubiquitylation-driven antiparasitic host defense. IMPORTANCE Globally, approximately one out of three people become infected with the obligate intracellular parasite Toxoplasma. These infections are typically asymptomatic but can cause severe disease and mortality in immunocompromised individuals. Infections can also be passed on from mother to fetus during pregnancy, potentially causing miscarriage or stillbirth. Therefore, toxoplasmosis constitutes a substantial public health burden. A better understanding of mechanisms by which healthy individuals control Toxoplasma infections could provide roadmaps toward novel therapies for vulnerable groups. Our work reveals a fundamental mechanism controlling intracellular Toxoplasma infections. Cytokines produced during Toxoplasma infections instruct human cells to produce the enzyme RNF213. We find that RNF213 labels intracellular vacuoles containing Toxoplasma with the small protein ubiquitin, which functions as an "eat-me" signal, attracting antimicrobial defense programs to fight off infection. Our work therefore identified a novel antiparasitic protein orchestrating a central aspect of the human immune response to Toxoplasma.