TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS.

Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS.

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2.

The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.

NLRP1 variant M1184V decreases inflammasome activation in the context of DPP9 inhibition and asthma severity.

BACKGROUND: NLRP1 is an innate immune sensor that can form cytoplasmic inflammasome complexes. Polymorphisms in NLRP1 are linked to asthma; however, there is currently no functional or mechanistic explanation for this. OBJECTIVE: We sought to clarify the role of NLRP1 in asthma pathogenesis. METHODS: Results from the GALA II cohort study were used to identify a link between NLRP1 and asthma in Mexican Americans. In vitro and in vivo models for NLRP1 activation were applied to investigate the role of this inflammasome in asthma at the molecular level. RESULTS: We document the association of an NLRP1 haplotype with asthma for which the single nucleotide polymorphism rs11651270 (M1184V) individually is the most significant. Surprisingly, M1184V increases NLRP1 activation in the context of N-terminal destabilization, but decreases NLRP1 activation on dipeptidyl peptidase 9 inhibition. In vitro studies demonstrate that M1184V increases binding to dipeptidyl peptidase 9, which can account for its inhibitory role in this context. In addition, in vivo data from a mouse model of airway inflammation reveal a protective role for NLRP1 inflammasome activation reducing eosinophilia in this setting. CONCLUSIONS: Linking our in vitro and in vivo results, we found that the NLRP1 variant M1184V reduces inflammasome activation in the context of dipeptidyl peptidase 9 inhibition and could thereby increase asthma severity. Our studies may have implications for the treatment of asthma in patients carrying this variant of NLRP1.

Understanding SOCS protein specificity.

The development and activity of our immune system are largely controlled by the action of pleiotropic cytokines and growth factors, small secreted proteins, which bind to receptors on the surface of immune cells to initiate an appropriate physiological response. Cytokine signalling is predominantly executed by intracellular proteins known as the Janus kinases (JAKs) and the signal transducers and activators of transcriptions (STATs). Although the 'nuts and bolts' of cytokine-activated pathways have been well established, the nuanced way in which distinct cellular outcomes are achieved and the precise molecular details of the proteins that regulate these pathways are still being elucidated. This is highlighted by the intricate role of the suppressor of cytokine signalling (SOCS) proteins. The SOCS proteins act as negative feedback inhibitors, dampening specific cytokine signals to prevent excessive cellular responses and returning the cell to a homeostatic state. A great deal of study has demonstrated their ability to inhibit these pathways at the receptor complex, either through direct inhibition of JAK activity or by targeting the receptor complex for proteasomal degradation. Detailed analysis of individual SOCS proteins is slowly revealing the complex and highly controlled manner by which they can achieve specificity for distinct substrates. However, for many of the SOCS, a level of detail is still lacking, including confident identification of the full suite of tyrosine phosphorylated targets of their SH2 domain. This review will highlight the general mechanisms which govern SOCS specificity of action and discuss the similarities and differences between selected SOCS proteins, focusing on CIS, SOCS1 and SOCS3. Because of the functional and sequence similarities within the SOCS family, we will also discuss the evidence for functional redundancy.

Mutational profiling of SARS-CoV-2 papain-like protease reveals requirements for function, structure, and drug escape.

Papain-like protease (PLpro) is an attractive drug target for SARS-CoV-2 because it is essential for viral replication, cleaving viral poly-proteins pp1a and pp1ab, and has de-ubiquitylation and de-ISGylation activities, affecting innate immune responses. We employ Deep Mutational Scanning to evaluate the mutational effects on PLpro enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify mutations in neighboring residues that alter activity. We characterize residues responsible for substrate binding and demonstrate that although residues in the blocking loop are remarkably tolerant to mutation, blocking loop flexibility is important for function. We additionally find a connected network of mutations affecting activity that extends far from the active site. We leverage our library to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process.

Characterisation of the OTU domain deubiquitinase complement of Toxoplasma gondii.

The phylum Apicomplexa contains several parasitic species of medical and agricultural importance. The ubiquitination machinery remains, for the most part, uncharacterised in apicomplexan parasites, despite the important roles that it plays in eukaryotic biology. Bioinformatic analysis of the ubiquitination machinery in apicomplexan parasites revealed an expanded ovarian tumour domain-containing (OTU) deubiquitinase (DUB) family in Toxoplasma, potentially reflecting functional importance in apicomplexan parasites. This study presents comprehensive characterisation of Toxoplasma OTU DUBs. AlphaFold-guided structural analysis not only confirmed functional orthologues found across eukaryotes, but also identified apicomplexan-specific enzymes, subsequently enabling discovery of a cryptic OTU DUB in Plasmodium species. Comprehensive biochemical characterisation of 11 Toxoplasma OTU DUBs revealed activity against ubiquitin- and NEDD8-based substrates and revealed ubiquitin linkage preferences for Lys6-, Lys11-, Lys48-, and Lys63-linked chain types. We show that accessory domains in Toxoplasma OTU DUBs impose linkage preferences, and in case of apicomplexan-specific TgOTU9, we discover a cryptic ubiquitin-binding domain that is essential for TgOTU9 activity. Using the auxin-inducible degron (AID) to generate knockdown parasite lines, TgOTUD6B was found to be important for Toxoplasma growth.

Inhibitors of SARS-CoV-2 PLpro.

The emergence of SARS-CoV-2 causing the COVID-19 pandemic, has highlighted how a combination of urgency, collaboration and building on existing research can enable rapid vaccine development to fight disease outbreaks. However, even countries with high vaccination rates still see surges in case numbers and high numbers of hospitalized patients. The development of antiviral treatments hence remains a top priority in preventing hospitalization and death of COVID-19 patients, and eventually bringing an end to the SARS-CoV-2 pandemic. The SARS-CoV-2 proteome contains several essential enzymatic activities embedded within its non-structural proteins (nsps). We here focus on nsp3, that harbours an essential papain-like protease (PLpro) domain responsible for cleaving the viral polyprotein as part of viral processing. Moreover, nsp3/PLpro also cleaves ubiquitin and ISG15 modifications within the host cell, derailing innate immune responses. Small molecule inhibition of the PLpro protease domain significantly reduces viral loads in SARS-CoV-2 infection models, suggesting that PLpro is an excellent drug target for next generation antivirals. In this review we discuss the conserved structure and function of PLpro and the ongoing efforts to design small molecule PLpro inhibitors that exploit this knowledge. We first discuss the many drug repurposing attempts, concluding that it is unlikely that PLpro-targeting drugs already exist. We next discuss the wealth of structural information on SARS-CoV-2 PLpro inhibition, for which there are now ∼30 distinct crystal structures with small molecule inhibitors bound in a surprising number of distinct crystallographic settings. We focus on optimisation of an existing compound class, based on SARS-CoV PLpro inhibitor GRL-0617, and recapitulate how new GRL-0617 derivatives exploit different features of PLpro, to overcome some compound liabilities.

Insights Into Drug Repurposing, as Well as Specificity and Compound Properties of Piperidine-Based SARS-CoV-2 PLpro Inhibitors.

The COVID-19 pandemic continues unabated, emphasizing the need for additional antiviral treatment options to prevent hospitalization and death of patients infected with SARS-CoV-2. The papain-like protease (PLpro) domain is part of the SARS-CoV-2 non-structural protein (nsp)-3, and represents an essential protease and validated drug target for preventing viral replication. PLpro moonlights as a deubiquitinating (DUB) and deISGylating enzyme, enabling adaptation of a DUB high throughput (HTS) screen to identify PLpro inhibitors. Drug repurposing has been a major focus through the COVID-19 pandemic as it may provide a fast and efficient route for identifying clinic-ready, safe-in-human antivirals. We here report our effort to identify PLpro inhibitors by screening the ReFRAME library of 11,804 compounds, showing that none inhibit PLpro with any reasonable activity or specificity to justify further progression towards the clinic. We also report our latest efforts to improve piperidine-scaffold inhibitors, 5c and 3k, originally developed for SARS-CoV PLpro. We report molecular details of binding and selectivity, as well as in vitro absorption, distribution, metabolism and excretion (ADME) studies of this scaffold. A co-crystal structure of SARS-CoV-2 PLpro bound to inhibitor 3k guides medicinal chemistry efforts to improve binding and ADME characteristics. We arrive at compounds with improved and favorable solubility and stability characteristics that are tested for inhibiting viral replication. Whilst still requiring significant improvement, our optimized small molecule inhibitors of PLpro display decent antiviral activity in an in vitro SARS-CoV-2 infection model, justifying further optimization.

Protein kinase R is an innate immune sensor of proteotoxic stress via accumulation of cytoplasmic IL-24.

Proteasome dysfunction can lead to autoinflammatory disease associated with elevated type I interferon (IFN-αß) and NF-κB signaling; however, the innate immune pathway driving this is currently unknown. Here, we identified protein kinase R (PKR) as an innate immune sensor for proteotoxic stress. PKR activation was observed in cellular models of decreased proteasome function and in multiple cell types from patients with proteasome-associated autoinflammatory disease (PRAAS). Furthermore, genetic deletion or small-molecule inhibition of PKR in vitro ameliorated inflammation driven by proteasome deficiency. In vivo, proteasome inhibitor-induced inflammatory gene transcription was blunted in PKR-deficient mice compared with littermate controls. PKR also acted as a rheostat for proteotoxic stress by triggering phosphorylation of eIF2α, which can prevent the translation of new proteins to restore homeostasis. Although traditionally known as a sensor of RNA, under conditions of proteasome dysfunction, PKR sensed the cytoplasmic accumulation of a known interactor, interleukin-24 (IL-24). When misfolded IL-24 egress into the cytosol was blocked by inhibition of the endoplasmic reticulum-associated degradation pathway, PKR activation and subsequent inflammatory signaling were blunted. Cytokines such as IL-24 are normally secreted from cells; therefore, cytoplasmic accumulation of IL-24 represents an internal danger-associated molecular pattern. Thus, we have identified a mechanism by which proteotoxic stress is detected, causing inflammation observed in the disease PRAAS.

Discovery of an exosite on the SOCS2-SH2 domain that enhances SH2 binding to phosphorylated ligands.

Suppressor of cytokine signaling (SOCS)2 protein is a key negative regulator of the growth hormone (GH) and Janus kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) signaling cascade. The central SOCS2-Src homology 2 (SH2) domain is characteristic of the SOCS family proteins and is an important module that facilitates recognition of targets bearing phosphorylated tyrosine (pTyr) residues. Here we identify an exosite on the SOCS2-SH2 domain which, when bound to a non-phosphorylated peptide (F3), enhances SH2 affinity for canonical phosphorylated ligands. Solution of the SOCS2/F3 crystal structure reveals F3 as an α-helix which binds on the opposite side of the SH2 domain to the phosphopeptide binding site. F3:exosite binding appears to stabilise the SOCS2-SH2 domain, resulting in slower dissociation of phosphorylated ligands and consequently, enhances binding affinity. This biophysical enhancement of SH2:pTyr binding affinity translates to increase SOCS2 inhibition of GH signaling.

TBK1 and IKKε Act Redundantly to Mediate STING-Induced NF-κB Responses in Myeloid Cells.

Stimulator of Interferon Genes (STING) is a critical component of host innate immune defense but can contribute to chronic autoimmune or autoinflammatory disease. Once activated, the cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS)-STING pathway induces both type I interferon (IFN) expression and nuclear factor-κB (NF-κB)-mediated cytokine production. Currently, these two signaling arms are thought to be mediated by a single upstream kinase, TANK-binding kinase 1 (TBK1). Here, using genetic and pharmacological approaches, we show that TBK1 alone is dispensable for STING-induced NF-κB responses in human and mouse immune cells, as well as in vivo. We further demonstrate that TBK1 acts redundantly with IκB kinase ε (IKKε) to drive NF-κB upon STING activation. Interestingly, we show that activation of IFN regulatory factor 3 (IRF3) is highly dependent on TBK1 kinase activity, whereas NF-κB is significantly less sensitive to TBK1/IKKε kinase inhibition. Our work redefines signaling events downstream of cGAS-STING. Our findings further suggest that cGAS-STING will need to be targeted directly to effectively ameliorate the inflammation underpinning disorders associated with STING hyperactivity.

Stressing out the mitochondria: Mechanistic insights into NLRP3 inflammasome activation.

Inflammasomes are multimeric protein complexes that induce the cleavage and release of bioactive IL-1ß and cause a lytic form of cell death, termed pyroptosis. Due to its diverse triggers, ranging from infectious pathogens and host danger molecules to environmental irritants, the NOD-like receptor protein 3 (NLRP3) inflammasome remains the most widely studied inflammasome to date. Despite intense scrutiny, a universal mechanism for its activation remains elusive, although, recent research has focused on mitochondrial dysfunction or potassium (K+ ) efflux as key events. In this review, we give a general overview of NLRP3 inflammasome activation and explore the recently emerging noncanonical and alternative pathways to NLRP3 activation. We highlight the role of the NLRP3 inflammasome in the pathogenesis of metabolic disease that is associated with mitochondrial and oxidative stress. Finally, we interrogate the mechanisms proposed to trigger NLRP3 inflammasome assembly and activation. A greater understanding of how NLRP3 inflammasome activation is triggered may reveal new therapeutic targets for the treatment of inflammatory disease.