ABSTRACT
The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.
Subject(s)
Immunologic Deficiency Syndromes , Nerve Tissue Proteins , Ubiquitins , Humans , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/immunology , Female , Male , NF-kappa B/metabolism , Ubiquitin-Protein Ligases/genetics , Inflammation/immunology , Inflammation/genetics , B-Lymphocytes/immunology , Loss of Function Mutation , Fibroblasts/metabolism , Fibroblasts/immunology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Animals , Mice , AllelesABSTRACT
Certain obligate parasites induce complex and substantial phenotypic changes in their hosts in ways that favor their transmission to other trophic levels. However, the mechanisms underlying these changes remain largely unknown. Here we demonstrate how SAP05 protein effectors from insect-vectored plant pathogenic phytoplasmas take control of several plant developmental processes. These effectors simultaneously prolong the host lifespan and induce witches' broom-like proliferations of leaf and sterile shoots, organs colonized by phytoplasmas and vectors. SAP05 acts by mediating the concurrent degradation of SPL and GATA developmental regulators via a process that relies on hijacking the plant ubiquitin receptor RPN10 independent of substrate ubiquitination. RPN10 is highly conserved among eukaryotes, but SAP05 does not bind insect vector RPN10. A two-amino-acid substitution within plant RPN10 generates a functional variant that is resistant to SAP05 activities. Therefore, one effector protein enables obligate parasitic phytoplasmas to induce a plethora of developmental phenotypes in their hosts.
Subject(s)
Arabidopsis/growth & development , Arabidopsis/parasitology , Host-Parasite Interactions/physiology , Parasites/physiology , Proteolysis , Ubiquitins/metabolism , Amino Acid Sequence , Animals , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Genetic Engineering , Humans , Insecta/physiology , Models, Biological , Phenotype , Photoperiod , Phylogeny , Phytoplasma/physiology , Plant Development , Plant Shoots/growth & development , Plants, Genetically Modified , Proteasome Endopeptidase Complex/metabolism , Protein Stability , Reproduction , Nicotiana , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
The anaphase-promoting complex/cyclosome (APC/C) represents a large multisubunit E3-ubiquitin ligase complex that controls the unidirectional progression through the cell cycle by the ubiquitination of specific target proteins, marking them for proteasomal destruction. Although the APC/C's role is largely conserved among eukaryotes, its subunit composition and target spectrum appear to be species specific. In this review, we focus on the plant APC/C complex, whose activity correlates with different developmental processes, including polyploidization and gametogenesis. After an introduction into proteolytic control by ubiquitination, we discuss the composition of the plant APC/C and the essential nature of its core subunits for plant development. Subsequently, we describe the APC/C activator subunits and interactors, most being plant specific. Finally, we provide a comprehensive list of confirmed and suspected plant APC/C target proteins. Identification of growth-related targets might offer opportunities to increase crop yield and resilience of plants to climate change by manipulating APC/C activity.
Subject(s)
Anaphase , Plants , Anaphase-Promoting Complex-Cyclosome/genetics , Anaphase-Promoting Complex-Cyclosome/metabolism , Cell Cycle , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Plants/genetics , Plants/metabolism , Ubiquitination , Ubiquitins/metabolismABSTRACT
Ubiquitin-like protein ISG15 (interferon-stimulated gene 15) (ISG15) is a ubiquitin-like modifier induced during infections and involved in host defense mechanisms. Not surprisingly, many viruses encode deISGylating activities to antagonize its effect. Here we show that infection by Zika, SARS-CoV-2 and influenza viruses induce ISG15-modifying enzymes. While influenza and Zika viruses induce ISGylation, SARS-CoV-2 triggers deISGylation instead to generate free ISG15. The ratio of free versus conjugated ISG15 driven by the papain-like protease (PLpro) enzyme of SARS-CoV-2 correlates with macrophage polarization toward a pro-inflammatory phenotype and attenuated antigen presentation. In vitro characterization of purified wild-type and mutant PLpro revealed its strong deISGylating over deubiquitylating activity. Quantitative proteomic analyses of PLpro substrates and secretome from SARS-CoV-2-infected macrophages revealed several glycolytic enzymes previously implicated in the expression of inflammatory genes and pro-inflammatory cytokines, respectively. Collectively, our results indicate that altered free versus conjugated ISG15 dysregulates macrophage responses and probably contributes to the cytokine storms triggered by SARS-CoV-2.
Subject(s)
COVID-19/immunology , Cytokines/metabolism , Inflammation/immunology , Macrophages/immunology , SARS-CoV-2/physiology , Ubiquitins/metabolism , Cell Differentiation , Coronavirus Papain-Like Proteases/metabolism , Cytokines/genetics , Gene Knockdown Techniques , HeLa Cells , Humans , Immune Evasion , Immunity, Innate , Influenza A virus/physiology , Influenza, Human/immunology , Pluripotent Stem Cells/cytology , Ubiquitination , Ubiquitins/genetics , Zika Virus/physiology , Zika Virus Infection/immunologyABSTRACT
Protein ubiquitination is one of the most powerful posttranslational modifications of proteins, as it regulates a plethora of cellular processes in distinct manners. Simple monoubiquitination events coexist with more complex forms of polyubiquitination, the latter featuring many different chain architectures. Ubiquitin can be subjected to further posttranslational modifications (e.g., phosphorylation and acetylation) and can also be part of mixed polymers with ubiquitin-like modifiers such as SUMO (small ubiquitin-related modifier) or NEDD8 (neural precursor cell expressed, developmentally downregulated 8). Together, cellular ubiquitination events form a sophisticated and versatile ubiquitin code. Deubiquitinases (DUBs) reverse ubiquitin signals with equally high sophistication. In this review, we conceptualize the many layers of specificity that DUBs encompass to control the ubiquitin code and discuss examples in which DUB specificity has been understood at the molecular level. We further discuss the many mechanisms of DUB regulation with a focus on those that modulate catalytic activity. Our review provides a framework to tackle lingering questions in DUB biology.
Subject(s)
Deubiquitinating Enzymes/metabolism , Eukaryotic Cells/metabolism , Protein Processing, Post-Translational , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Ubiquitins/metabolism , Acetylation , Allosteric Regulation , Deubiquitinating Enzymes/chemistry , Deubiquitinating Enzymes/genetics , Humans , Models, Molecular , NEDD8 Protein , Phosphorylation , Protein Binding , Protein Conformation , Proteolysis , Substrate Specificity , Sumoylation , Ubiquitin/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitination , Ubiquitins/geneticsABSTRACT
Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through UFMylation, a process similar to ubiquitylation. Growing lines of evidence regarding not only the structural basis of the components essential for UFMylation but also their biological properties shed light on crucial roles of the UFM1 system in the endoplasmic reticulum (ER), such as ER-phagy and ribosome-associated quality control at the ER, although there are some functions unrelated to the ER. Mouse genetics studies also revealed the indispensable roles of this system in hematopoiesis, liver development, neurogenesis, and chondrogenesis. Of critical importance, mutations of genes encoding core components of the UFM1 system in humans cause hereditary developmental epileptic encephalopathy and Schohat-type osteochondrodysplasia of the epiphysis. Here, we provide a multidisciplinary review of our current understanding of the mechanisms and cellular functions of the UFM1 system as well as its pathophysiological roles, and discuss issues that require resolution.
Subject(s)
Proteins , Ubiquitins , Humans , Animals , Mice , Proteins/metabolism , Ubiquitination , Ubiquitins/metabolism , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Cell Physiological PhenomenaABSTRACT
Mutations in Ubiquilin-2 are linked to the onset of amyotrophic lateral sclerosis, but its connection to disease processes has remained unknown. Hjerpe et. al now report that Ubiquilin-2 enables the ubiquitin proteasome system (UPS) to single-handedly clear aggregated proteins, a cellular function previously thought to rely at least partially on autophagy.
Subject(s)
Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Amyotrophic Lateral Sclerosis/genetics , Autophagy , Cell Cycle Proteins/genetics , Humans , Ubiquitins/geneticsABSTRACT
Cellular bodies such as P bodies and PML nuclear bodies (PML NBs) appear to be phase-separated liquids organized by multivalent interactions among proteins and RNA molecules. Although many components of various cellular bodies are known, general principles that define body composition are lacking. We modeled cellular bodies using several engineered multivalent proteins and RNA. In vitro and in cells, these scaffold molecules form phase-separated liquids that concentrate low valency client proteins. Clients partition differently depending on the ratio of scaffolds, with a sharp switch across the phase diagram diagonal. Composition can switch rapidly through changes in scaffold concentration or valency. Natural PML NBs and P bodies show analogous partitioning behavior, suggesting how their compositions could be controlled by levels of PML SUMOylation or cellular mRNA concentration, respectively. The data suggest a conceptual framework for considering the composition and control thereof of cellular bodies assembled through heterotypic multivalent interactions.
Subject(s)
Artificial Cells/chemistry , Cell Compartmentation , Organelles/chemistry , Proteins/chemistry , Amino Acid Motifs , Body Composition , Carrier Proteins/chemistry , Cell Line , Cell Nucleus/chemistry , Cytoplasm , Electrochemistry , HeLa Cells , Humans , In Vitro Techniques , Molecular Structure , Polypyrimidine Tract-Binding Protein/chemistry , Protein Engineering , Ubiquitins/chemistry , YeastsABSTRACT
Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation.
Subject(s)
Carrier Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Ubiquitination , Ubiquitins/metabolism , Carrier Proteins/genetics , Cullin Proteins/metabolism , Gene Knockdown Techniques , HEK293 Cells , Humans , Mutation , NEDD8 Protein , Polyubiquitin/metabolism , Proteomics , Substrate Specificity , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies.
Subject(s)
Ligases , Melanoma , Humans , HeLa Cells , Ubiquitination , UbiquitinsABSTRACT
Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.
Subject(s)
Chromatin , Transcription Factors , Humans , Ligands , Chromatin/genetics , Transcription Factors/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Ubiquitins , Ubiquitin-Protein Ligases/geneticsABSTRACT
The AAA+ ATPase Cdc48 utilizes the cofactor Ufd1/Npl4 to bind and thread polyubiquitinated substrates for their extraction from complexes or membranes and often for subsequent proteasomal degradation. Previous studies indicated that Cdc48 engages polyubiquitin chains through the Npl4-mediated unfolding of an initiator ubiquitin; yet, the underlying principles remain largely unknown. Using FRET-based assays, we revealed the mechanisms and kinetics of ubiquitin unfolding, insertion into the ATPase, and unfolding of the ubiquitin-attached substrate. We found that Cdc48 uses Ufd1's UT3 domain to bind a K48-linked ubiquitin on the initiator's proximal side of the chain, thereby directing the initiator toward rapid unfolding by Npl4 and engagement by Cdc48. Ubiquitins on the initiator's distal side increase substrate affinity and facilitate unfolding but impede substrate release from Cdc48-Ufd1/Npl4 in the absence of additional cofactors. Our findings explain how Cdc48-UN efficiently processes substrates with K48-linked chains of 4-6 ubiquitins, which represent most cellular polyubiquitinated proteins.
Subject(s)
Polyubiquitin , Saccharomyces cerevisiae Proteins , Polyubiquitin/metabolism , Saccharomyces cerevisiae Proteins/metabolism , ATPases Associated with Diverse Cellular Activities/metabolism , Valosin Containing Protein/metabolism , Vesicular Transport Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Ubiquitin/metabolism , Ubiquitins/metabolism , Cell Cycle Proteins/metabolismABSTRACT
Ubiquitination pathways have crucial roles in protein homeostasis, signalling and innate immunity1-3. In these pathways, an enzymatic cascade of E1, E2 and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues4. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism5,6, but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here we demonstrate that a bacterial operon associated with phage defence islands encodes a complete ubiquitination pathway. Two structures of a bacterial E1-E2-Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 possesses an amino-terminal inactive adenylation domain and a carboxy-terminal active adenylation domain with a mobile α-helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C terminus positioned for adenylation, and a second structure mimics an E1-to-E2 transthioesterification state with the E1 CYS domain adjacent to the bound E2. We show that a deubiquitinase in the same pathway preprocesses the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria.
Subject(s)
Bacterial Proteins , Bacteriophages , Escherichia , Ubiquitin-Activating Enzymes , Ubiquitin-Conjugating Enzymes , Ubiquitination , Ubiquitins , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacteriophages/chemistry , Bacteriophages/immunology , Bacteriophages/metabolism , Catalytic Domain , Crystallography, X-Ray , Cysteine/chemistry , Cysteine/metabolism , Deubiquitinating Enzymes/chemistry , Deubiquitinating Enzymes/metabolism , Escherichia/chemistry , Escherichia/enzymology , Escherichia/immunology , Escherichia/virology , Evolution, Molecular , Lysine/chemistry , Lysine/metabolism , Models, Molecular , Operon/genetics , Protein Domains , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitins/metabolism , Ubiquitins/chemistry , Eukaryota/enzymology , Eukaryota/metabolismABSTRACT
Several immune pathways in humans conjugate ubiquitin-like proteins to virus and host molecules as a means of antiviral defence1-5. Here we studied an antiphage defence system in bacteria, comprising a ubiquitin-like protein, ubiquitin-conjugating enzymes E1 and E2, and a deubiquitinase. We show that during phage infection, this system specifically conjugates the ubiquitin-like protein to the phage central tail fibre, a protein at the tip of the tail that is essential for tail assembly as well as for recognition of the target host receptor. Following infection, cells encoding this defence system release a mixture of partially assembled, tailless phage particles and fully assembled phages in which the central tail fibre is obstructed by the covalently attached ubiquitin-like protein. These phages show severely impaired infectivity, explaining how the defence system protects the bacterial population from the spread of phage infection. Our findings demonstrate that conjugation of ubiquitin-like proteins is an antiviral strategy conserved across the tree of life.
Subject(s)
Bacterial Proteins , Bacteriophages , Deubiquitinating Enzymes , Escherichia coli , Ubiquitin-Conjugating Enzymes , Ubiquitins , Virus Assembly , Bacteriophages/chemistry , Bacteriophages/metabolism , Bacteriophages/pathogenicity , Bacteriophages/physiology , Deubiquitinating Enzymes/metabolism , Escherichia coli/enzymology , Escherichia coli/metabolism , Escherichia coli/virology , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitins/metabolism , Viral Tail Proteins/metabolism , Viral Tail Proteins/chemistry , Bacterial Proteins/metabolism , Evolution, Molecular , Conserved SequenceABSTRACT
The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.
Subject(s)
Blastocyst/metabolism , Cell Lineage , Chromatin Assembly and Disassembly , Chromatin/metabolism , Histones/metabolism , Mouse Embryonic Stem Cells/metabolism , Animals , Blastocyst/cytology , Chromatin/genetics , Embryo Culture Techniques , Embryonic Development , Gene Expression Regulation, Developmental , HEK293 Cells , Histones/genetics , Humans , Mice , Phenotype , Small Ubiquitin-Related Modifier Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation , Ubiquitins/genetics , Ubiquitins/metabolismABSTRACT
Most E3 ligases use a RING domain to activate a thioester-linked E2â¼ubiquitin-like protein (UBL) intermediate and promote UBL transfer to a remotely bound target protein. Nonetheless, RING E3 mechanisms matching a specific UBL and acceptor lysine remain elusive, including for RBX1, which mediates NEDD8 ligation to cullins and >10% of all ubiquitination. We report the structure of a trapped RING E3-E2â¼UBL-target intermediate representing RBX1-UBC12â¼NEDD8-CUL1-DCN1, which reveals the mechanism of NEDD8 ligation and how a particular UBL and acceptor lysine are matched by a multifunctional RING E3. Numerous mechanisms specify cullin neddylation while preventing noncognate ubiquitin ligation. Notably, E2-E3-target and RING-E2â¼UBL modules are not optimized to function independently, but instead require integration by the UBL and target for maximal reactivity. The UBL and target regulate the catalytic machinery by positioning the RING-E2â¼UBL catalytic center, licensing the acceptor lysine, and influencing E2 reactivity, thereby driving their specific coupling by a multifunctional RING E3.
Subject(s)
Ubiquitins/chemistry , Ubiquitins/metabolism , Amino Acid Sequence , Carrier Proteins/metabolism , Catalytic Domain , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Crystallography, X-Ray , Cullin Proteins/chemistry , Cullin Proteins/metabolism , Humans , Models, Molecular , Molecular Sequence Data , NEDD8 Protein , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.
Subject(s)
Autophagy , Endoplasmic Reticulum Stress , Endoplasmic Reticulum , Ubiquitination , Autophagy/physiology , Endoplasmic Reticulum/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Ubiquitins/metabolism , Microtubule-Associated Proteins/metabolism , Receptors, Autocrine Motility Factor/metabolismABSTRACT
In all organisms, innate immune pathways sense infection and rapidly activate potent immune responses while avoiding inappropriate activation (autoimmunity). In humans, the innate immune receptor cyclic GMP-AMP synthase (cGAS) detects viral infection to produce the nucleotide second messenger cyclic GMP-AMP (cGAMP), which initiates stimulator of interferon genes (STING)-dependent antiviral signalling1. Bacteria encode evolutionary predecessors of cGAS called cGAS/DncV-like nucleotidyltransferases2 (CD-NTases), which detect bacteriophage infection and produce diverse nucleotide second messengers3. How bacterial CD-NTase activation is controlled remains unknown. Here we show that CD-NTase-associated protein 2 (Cap2) primes bacterial CD-NTases for activation through a ubiquitin transferase-like mechanism. A cryo-electron microscopy structure of the Cap2-CD-NTase complex reveals Cap2 as an all-in-one ubiquitin transferase-like protein, with distinct domains resembling eukaryotic E1 and E2 proteins. The structure captures a reactive-intermediate state with the CD-NTase C terminus positioned in the Cap2 E1 active site and conjugated to AMP. Cap2 conjugates the CD-NTase C terminus to a target molecule that primes the CD-NTase for increased cGAMP production. We further demonstrate that a specific endopeptidase, Cap3, balances Cap2 activity by cleaving CD-NTase-target conjugates. Our data demonstrate that bacteria control immune signalling using an ancient, minimized ubiquitin transferase-like system and provide insight into the evolution of the E1 and E2 machinery across domains of life.
Subject(s)
Bacteria , Bacterial Proteins , Immunity, Innate , Nucleotidyltransferases , Humans , Bacteria/enzymology , Bacteria/immunology , Bacteria/metabolism , Cryoelectron Microscopy , Nucleotidyltransferases/metabolism , Ubiquitins/metabolism , Bacteriophages/immunology , Second Messenger Systems , Catalytic Domain , Bacterial Proteins/metabolism , Adenosine Monophosphate/metabolismABSTRACT
Agents that induce DNA damage can cure some cancers. However, the side effects of chemotherapy are severe because of the indiscriminate action of DNA-damaging agents on both healthy and cancerous cells. DNA repair pathway inhibition provides a less toxic and targeted alternative to chemotherapy. A compelling DNA repair target is the Fanconi anemia (FA) E3 ligase core complex due to its critical-and likely singular-role in the efficient removal of specific DNA lesions. FA pathway inactivation has been demonstrated to specifically kill some types of cancer cells without the addition of exogenous DNA damage, including cells that lack BRCA1, BRCA2, ATM, or functionally related genes. In this perspective, we discuss the genetic and biochemical evidence in support of the FA core complex as a compelling drug target for cancer therapy. In particular, we discuss the genetic, biochemical, and structural data that could rapidly advance our capacity to identify and implement the use of FA core complex inhibitors in the clinic.