Co-opting templated aggregation to degrade pathogenic tau assemblies and improve motor function.

Protein aggregation causes a wide range of neurodegenerative diseases. Targeting and removing aggregates, but not the functional protein, is a considerable therapeutic challenge. Here, we describe a therapeutic strategy called "RING-Bait," which employs an aggregating protein sequence combined with an E3 ubiquitin ligase.

Aggregate-selective removal of pathological tau by clustering-activated degraders.

Selective degradation of pathological protein aggregates while sparing monomeric forms is of major therapeutic interest. The E3 ligase tripartite motif-containing protein 21 (TRIM21) degrades antibody-bound proteins in an assembly state-specific manner due to the requirement of TRIM21 RING domain clustering for activation, yet effective targeting of intracellular assemblies remains challenging. Here, we fused the RING domain of TRIM21 to a target-specific nanobody to create intracellularly expressed constructs capable of selectively degrading assembled proteins.

The type-I interferon response potentiates seeded tau aggregation and exacerbates tau pathology.

Introduction: Signatures of a type-I interferon (IFN-I) response are observed in the post mortem brain in Alzheimer's disease (AD) and other tauopathies. However, the effect of the IFN-I response on pathological tau accumulation remains unclear.

Methods: We examined the effects of IFN-I signaling in primary neural culture models of seeded tau aggregation and P301S-tau transgenic mouse models in the context of genetic deletion of the IFN-I receptor (IFNAR).

Cytosolic antibody receptor TRIM21 is required for effective tau immunotherapy in mouse models.

Aggregates of the protein tau are proposed to drive pathogenesis in neurodegenerative diseases. Tau can be targeted by using passively transferred antibodies (Abs), but the mechanisms of Ab protection are incompletely understood. In this work, we used a variety of cell and animal model systems and showed that the cytosolic Ab receptor and E3 ligase TRIM21 (T21) could play a role in Ab protection against tau pathology.

IP6-stabilised HIV capsids evade cGAS/STING-mediated host immune sensing.

HIV-1 uses inositol hexakisphosphate (IP6) to build a metastable capsid capable of delivering its genome into the host nucleus. Here, we show that viruses that are unable to package IP6 lack capsid protection and are detected by innate immunity, resulting in the activation of an antiviral state that inhibits infection. Disrupting IP6 enrichment results in defective capsids that trigger cytokine and chemokine responses during infection of both primary macrophages and T-cell lines.

Cholesterol determines the cytosolic entry and seeded aggregation of tau.

Assemblies of tau can transit between neurons, seeding aggregation in a prion-like manner. To accomplish this, tau must cross cell-limiting membranes, a process that is poorly understood. Here, we establish assays for the study of tau entry into the cytosol as a phenomenon distinct from uptake, in real time, and at physiological concentrations.

Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice.

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. Here, we show that ferritin-like Dps from hyperthermophilic Sulfolobus islandicus, covalently coupled with SARS-CoV-2 antigens via the SpyCatcher system, forms stable multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) elicited a higher antibody titre and an enhanced neutralising antibody response compared to monomeric RBD.

A functional assay for serum detection of antibodies against SARS-CoV-2 nucleoprotein.

The humoral immune response to SARS-CoV-2 results in antibodies against spike (S) and nucleoprotein (N). However, whilst there are widely available neutralization assays for S antibodies, there is no assay for N-antibody activity. Here, we present a simple in vitro method called EDNA (electroporated-antibody-dependent neutralization assay) that provides a quantitative measure of N-antibody activity in unpurified serum from SARS-CoV-2 convalescents.

Tau assemblies do not behave like independently acting prion-like particles in mouse neural tissue.

A fundamental property of infectious agents is their particulate nature: infectivity arises from independently-acting particles rather than as a result of collective action. Assemblies of the protein tau can exhibit seeding behaviour, potentially underlying the apparent spread of tau aggregation in many neurodegenerative diseases. Here we ask whether tau assemblies share with classical pathogens the characteristic of particulate behaviour.

Target-induced clustering activates Trim-Away of pathogens and proteins.

Trim-Away is a recently developed technology that exploits off-the-shelf antibodies and the RING E3 ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically activated upon target engagement, either during its normal immune function or when repurposed for targeted protein degradation, is unknown. Here we show that a mechanism of target-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce virus neutralization or drive Trim-Away.

Viral nucleoprotein antibodies activate TRIM21 and induce T cell immunity.

Nucleoprotein (N) is an immunodominant antigen in many enveloped virus infections. While the diagnostic value of anti-N antibodies is clear, their role in immunity is not. This is because while they are non-neutralising, they somehow clear infection by coronavirus, influenza and LCMV in vivo.

Intracellular neutralisation of rotavirus by VP6-specific IgG.

Rotavirus is a major cause of gastroenteritis in children, with infection typically inducing high levels of protective antibodies. Antibodies targeting the middle capsid protein VP6 are particularly abundant, and as VP6 is only exposed inside cells, neutralisation must be post-entry. However, while a system of poly immune globulin receptor (pIgR) transcytosis has been proposed for anti-VP6 IgAs, the mechanism by which VP6-specific IgG mediates protection remains less clear.

Cellular IP Levels Limit HIV Production while Viruses that Cannot Efficiently Package IP Are Attenuated for Infection and Replication.

HIV-1 hijacks host proteins to promote infection. Here we show that HIV is also dependent upon the host metabolite inositol hexakisphosphate (IP) for viral production and primary cell replication. HIV-1 recruits IP into virions using two lysine rings in its immature hexamers.

Antibody and DNA sensing pathways converge to activate the inflammasome during primary human macrophage infection.

Inflammasomes are potent innate immune signalling complexes that couple cytokine release with pro-inflammatory cell death. However, pathogens have evolved strategies to evade this cell autonomous system. Here, we show how antibodies combine with innate sensors in primary human macrophages to detect viral infection and activate the inflammasome.

Complement C4 Prevents Viral Infection through Capsid Inactivation.

The complement system is vital for anti-microbial defense. In the classical pathway, pathogen-bound antibody recruits the C1 complex (C1qC1rC1s) that initiates a cleavage cascade involving C2, C3, C4, and C5 and triggering microbial clearance. We demonstrate a C4-dependent antiviral mechanism that is independent of downstream complement components.

Trivalent RING Assembly on Retroviral Capsids Activates TRIM5 Ubiquitination and Innate Immune Signaling.

TRIM5 is a RING domain E3 ubiquitin ligase with potent antiretroviral function. TRIM5 assembles into a hexagonal lattice on retroviral capsids, causing envelopment of the infectious core. Concomitantly, TRIM5 initiates innate immune signaling and orchestrates disassembly of the viral particle, yet how these antiviral responses are regulated by capsid recognition is unclear.

TRIM21 mediates antibody inhibition of adenovirus-based gene delivery and vaccination.

Adenovirus has enormous potential as a gene-therapy vector, but preexisting immunity limits its widespread application. What is responsible for this immune block is unclear because antibodies potently inhibit transgene expression without impeding gene transfer into target cells. Here we show that antibody prevention of adenoviral gene delivery in vivo is mediated by the cytosolic antibody receptor TRIM21.

Intracellular antibody signalling is regulated by phosphorylation of the Fc receptor TRIM21.

Cell surface Fc receptors activate inflammation and are tightly controlled to prevent autoimmunity. Antibodies also simulate potent immune signalling from inside the cell via the cytosolic antibody receptor TRIM21, but how this is regulated is unknown. Here we show that TRIM21 signalling is constitutively repressed by its B-Box domain and activated by phosphorylation.

Cytosolic Fc receptor TRIM21 inhibits seeded tau aggregation.

Alzheimer's disease (AD) and other neurodegenerative disorders are associated with the cytoplasmic aggregation of microtubule-associated protein tau. Recent evidence supports transcellular transfer of tau misfolding (seeding) as the mechanism of spread within an affected brain, a process reminiscent of viral infection. However, whereas microbial pathogens can be recognized as nonself by immune receptors, misfolded protein assemblies evade detection, as they are host-derived.

TRIM21 Promotes cGAS and RIG-I Sensing of Viral Genomes during Infection by Antibody-Opsonized Virus.

Encapsidation is a strategy almost universally employed by viruses to protect their genomes from degradation and from innate immune sensors. We show that TRIM21, which targets antibody-opsonized virions for proteasomal destruction, circumvents this protection, enabling the rapid detection and degradation of viral genomes before their replication. TRIM21 triggers an initial wave of cytokine transcription that is antibody, rather than pathogen, driven.

Subunit disassembly and inhibition of TNFα by a semi-synthetic bicyclic peptide.

Macrocyclic peptides are potentially a source of powerful drugs, but their de novo discovery remains challenging. Here we describe the discovery of a high-affinity (Kd = 10 nM) peptide macrocycle (M21) against human tumor necrosis factor-alpha (hTNFα), a key drug target in the treatment of inflammatory disorders, directly from diverse semi-synthetic phage peptide repertoires. The bicyclic peptide M21 (ACPPCLWQVLC) comprises two loops covalently anchored to a 2,4,6-trimethyl-mesitylene core and upon binding induces disassembly of the trimeric TNFα cytokine into dimers and monomers.

Intracellular antibody receptor TRIM21 prevents fatal viral infection.

Host species have evolved mechanisms that can inhibit pathogen replication even after a cell has been successfully invaded. Here we show that tripartite-motif protein 21 (TRIM21), a ubiquitously expressed E3 ubiquitin ligase that targets viruses inside the cytosol, protects mice against fatal viral infection. Upon infection with mouse adenovirus-1, naive mice lacking TRIM21 succumb to encephalomyelitis within 7 d.

Structure of human MDM4 N-terminal domain bound to a single-domain antibody.

The N-terminal domain of MDM4 binds to the N-terminal transactivation domain of the tumor suppressor p53 and is an important negative regulator of its transactivation activity. As such, inhibition of the binding of MDM4 to p53 is a target for anticancer therapy. The protein has not been crystallized satisfactorily for structural studies without the addition of an N-terminal p53 peptide.