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The phospholipid and free fatty acid (FFA) composition of neuronal membranes plays a crucial role in learning and memory, but the mechanisms through which neuronal activity affects the brain’s lipid landscape remain largely unexplored. Saturated FFAs, particularly myristic acid (C14:0), strongly increase during neuronal stimulation and memory acquisition, suggesting the involvement of phospholipase A1 (PLA1) activity in synaptic plasticity. Here, we show that genetic ablation of the DDHD2 isoform of PLA1 in mice reduced memory performance in reward-based learning and spatial memory models prior to the development of neuromuscular deficits, and markedly reduced saturated FFAs across the brain. DDHD2 was shown to bind to the key synaptic protein STXBP1. Using STXBP1/2 knockout neurosecretory cells and a haploinsufficientSTXBP1+/-mouse model of STXBP1 encephalopathy that is also associated with intellectual disability and motor dysfunction, we show that STXBP1 controls the targeting of DDHD2 to the plasma membrane and the generation of saturated FFAs in the brain. Our findings suggest key roles for DDHD2 and STXBP1 in the lipid metabolism underlying synaptic plasticity, learning and memory.

The quantification of α-synuclein aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinson's disease. However, multiple species, such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein; these species are likely to coexist in biological samples, and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify individual contributions to the amplification process, recombinant oligomers and preformed fibrils were produced and purified to characterize their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent, but our results reveal that their seeding behavior is very different compared to that of preformed fibrils, in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples is likely to dominate the response in seeding assays.

The SOX transcription factor family is pivotal in controlling aspects of development. To identify genotype–phenotype relationships of SOX proteins, we performed a non-biased study of SOX using 1890 open-reading frame and 6667 amino acid sequences in combination with structural dynamics to interpret 3999 gnomAD, 485 ClinVar, 1174 Geno2MP, and 4313 COSMIC human variants. We identified, within the HMG (High Mobility Group)- box, twenty-seven amino acids with changes in multiple SOX proteins annotated to clinical pathologies. These sites were screened through Geno2MP medical phenotypes, revealing novel SOX15 R104G associated with musculature abnormality and SOX8 R159G with intellectual disability. Within gnomAD, SOX18 E137K (rs201931544), found within the HMG box of ~0.8% of Latinx individuals, is associated with seizures and neurological complications, potentially through blood–brain barrier alterations. A total of 56 highly conserved variants were found at sites outside the HMG-box, including several within the SOX2 HMG-box-flanking region with neurological associations, several in the SOX9 dimerization region associated with Campomelic Dysplasia, SOX14 K88R (rs199932938) flanking the HMG box associated with cardiovascular complications within European populations, and SOX7 A379V (rs143587868) within an SOXF conserved far C-terminal domain heterozygous in 0.716% of African individuals with associated eye phenotypes. This SOX data compilation builds a robust genotype-to-phenotype association for a gene family through more robust ortholog data integration.

Refereed/Peer-reviewed Despite a growing catalog of secreted factors critical for lymphatic network assembly, little is known about the mechanisms that modulate the expression level of these molecular cues in blood vascular endothelial cells (BECs). Here, we show that a BEC-specific transcription factor, SOX7, plays a crucial role in a non-cell-autonomous manner by modulating the transcription of angiocrine signals to pattern lymphatic vessels. While SOX7 is not expressed in lymphatic endothelial cells (LECs), the conditional loss of SOX7 function in mouse embryos causes a dysmorphic dermal lymphatic phenotype. We identify novel distant regulatory regions in mice and humans that contribute to directly repressing the transcription of a major lymphangiogenic growth factor (Vegfc) in a SOX7-dependent manner. Further, we show that SOX7 directly binds HEY1, a canonical repressor of the Notch pathway, suggesting that transcriptional repression may also be modulated by the recruitment of this protein partner at Vegfc genomic regulatory regions. Our work unveils a role for SOX7 in modulating downstream signaling events crucial for lymphatic patterning, at least in part via the transcriptional repression of VEGFC levels in the blood vascular endothelium.

Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged Opossum and the human genetic disorder Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome. Combining three single-molecule imaging assays in living cells together with genomics and proteomics analysis, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its target gene selection process. The dominant-negative effect is further amplified by poisoning the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular aetiology of dominant-negative transcription factor function.

α‐Synuclein aggregation is a hallmark of Parkinson's disease and a promising biomarker for early detection and assessment of disease progression. The prospect of a molecular test for Parkinson's disease is materializing with the recent developments of detection methods based on amplification of synuclein seeds (e.g. RT‐QuIC or PMCA). Here we adapted single‐molecule counting methods for the detection of α‐synuclein aggregates in cerebrospinal fluid (CSF), using a simple 3D printed microscope. Single‐molecule methods enable to probe the early events in the amplification process used in RT‐QuIC and a precise counting of ThT‐positive aggregates. Importantly, the use of single‐molecule counting also allows a refined characterization of the samples and fingerprinting of the protein aggregates present in CSF of patients. The fingerprinting of size and reactivity of individual aggregate shows a unique signature for each PD patients compared to controls and may provide new insights on synucleinopathies in the future., A new single‐molecule method was established for the detection of α‐synuclein fibrils, a promising biomarker of Parkinson's disease (PD). Individual ThT(+) aggregates were fingerprinted and amplified to improve detection limits by 3 orders of magnitude, compared to direct detection. A difference of behaviour was detected between PD and control samples.

The HIV capsid is a multifunctional protein capsule that mediates the delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here, we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labeled capsid-binding analytes ("prey" molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using the fluorescent capsid as the bait further allows the quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for the discovery and characterization of molecules used by the HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays, utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms.

Prion-like behaviour is an abrupt process, an "all-or-nothing" transition between a monomeric species and an "infinite" fibrillated form. Once a nucleation point is formed, the process is unstoppable as fibrils self-propagate by recruiting and converting all monomers into the amyloid fold. After the "mad cow" episode, prion diseases have made the headlines, but more and more prion-like behaviours have emerged in neurodegenerative diseases, where formation of fibrils and large conglomerates of proteins deeply disrupt the cell homeostasis. More interestingly, in the last decade, examples emerged to suggest that prion-like conversion can be used as a positive gain of function, for memory storage or structural scaffolding. More recent experiments show that we are only seeing the tip of the iceberg and that, for example, prion-like amplification is found in many pathways of the immune response. In innate immunity, receptors on the cellular surface or within the cells 'sense' danger and propagate this information as signal, through protein-protein interactions (PPIs) between 'receptor', 'adaptor' and 'effector' proteins. In innate immunity, the smallest signal of a foreign element or pathogen needs to trigger a macroscopic signal output, and it was found that adaptor polymerize to create an extreme signal amplification. Interestingly, our body uses multiple structural motifs to create large signalling platform; a few innate proteins use amyloid scaffolds but most of the polymers discovered are composed by self-assembly in helical filaments. Some of these helical assemblies even have intercellular "contamination" in a "true" prion action, as demonstrated for ASC specks and MyD88 filaments. Here, we will describe the current knowledge in neurodegenerative diseases and innate immunity and show how these two very different fields can cross-seed discoveries.

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19., During SARS-CoV-2 replication, viral and cellular proteases play crucial roles and have been shown to be promising anti-viral targets. Here, Meyer et al. apply mass spectrometry to characterize the proteolytic cleavage profile of viral and cellular proteins in vitro.

The quantification of α-synuclein (α-syn) aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinson’s disease. However, multiple species such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein and these species are likely to co-exist in biological samples and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify which species can be detected in seeding assays, recombinant oligomers and preformed fibrils were produced and purified to characterise their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent but our results reveal that their seeding behaviour is very different compared to preformed fibrils in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples are likely to dominate the response in seeding assays.

Despite a growing catalogue of secreted factors critical for lymphatic network assembly, little is known about the mechanisms that modulate the expression level of these molecular cues in blood vascular endothelial cells (BECs). Here, we show that a BEC-specific transcription factor, SOX7, plays a crucial role in lymphatic vessel patterning by modulating the transcription of lymphangiocrine signals. While SOX7 is not expressed in lymphatic endothelial cells (LECs), loss of SOX7 function in mouse embryos causes a dysmorphic dermal lymphatic phenotype. We identify novel distant regulatory regions in mice and humans that contribute to directly repressing the transcription of a major lymphangiogenic growth factor (Vegfc) in a SOX7-dependent manner. Further, we show that SOX7 directly binds HEY1, a canonical repressor of the Notch pathway, suggesting that transcriptional repression may also be modulated by the recruitment of this protein partner at Vegfc genomic regulatory regions. Our work unveils a role for SOX7 in modulating downstream signalling events crucial for lymphatic patterning, at least in part via the transcriptional repression of VEGFC levels in the blood vascular endothelium.

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques., MAL and MyD88 are downstream adaptors of Toll-like receptors (TLR) and the MAL TIR domain forms filaments in vitro, which in turn nucleate the assembly of crystalline arrays of the MyD88 TIR domain. Here, the authors present the structure of these MyD88 TIR crystalline arrays solved by both microcrystal electron diffraction and serial femtosecond crystallography, and they show with mutagenesis experiments that MyD88 interface residues are important for TLR4 signaling in vivo.

Caveolae-associated protein 3 (cavin3) is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair, with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays revealed a direct interaction between BRCA1 and cavin3 that occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. Supporting a role in DNA repair, cavin3-deficient cells were sensitive to PARP inhibition, where concomitant depletion of 53BP1 restored BRCA1-dependent sensitivity to PARP inhibition. We conclude that cavin3 functions together with BRCA1 in multiple cancer-related pathways. The loss of cavin3 function may provide tumor cell survival by attenuating apoptotic sensitivity and hindering DNA repair under chronic stress conditions., eLife digest When cells become cancerous they often stop making certain proteins. This includes a protein known as cavin3 which resides in bulb-shaped pits of the membrane that surrounds the cell called caveolae. These structures work like stress detectors, picking up changes in the membrane and releasing proteins, such as cavin3, into the cell’s interior. Past studies suggest that cavin3 might interact with a protein called BRCA1 that suppresses the formation of tumors. Cells with mutations in the gene for BRCA1 struggle to fix damage in their DNA, and have to rely on other repair proteins, such as PARPs (short for poly (ADP-ribose) polymerases). Blocking PARP proteins with drugs can kill cancer cells with problems in their BRCA1 proteins. However, it was unclear what role cavin3 plays in this mechanism. To investigate this, McMahon et al. exposed cells grown in the laboratory to DNA-damaging UV light to stimulate the release of cavin3 from caveolae. This revealed that cavin3 interacts with BRCA1 when cells are under stress, and helps stabilize the protein so it can perform DNA repairs. Cells without cavin3 showed decreased levels of the BRCA1 protein, but compensated for the loss of BRCA1 by increasing the levels of their PARP proteins. These cells also had increased DNA damage following treatment with drugs that block PARPs, similar to cancer cells carrying mutations in the gene for BRCA1. These findings suggest that cavin3 helps BRCA1 to suppress the formation of tumors, and therefore should be considered when developing new anti-cancer treatments.

Macrophage-expressed gene 1 (MPEG1/Perforin-2) is a perforin-like protein that functions within the phagolysosome to damage engulfed microbes. MPEG1 is thought to form pores in target membranes, however, its mode of action remains unknown. We use cryo-Electron Microscopy (cryo-EM) to determine the 2.4 Å structure of a hexadecameric assembly of MPEG1 that displays the expected features of a soluble prepore complex. We further discover that MPEG1 prepore-like assemblies can be induced to perforate membranes through acidification, such as would occur within maturing phagolysosomes. We next solve the 3.6 Å cryo-EM structure of MPEG1 in complex with liposomes. These data reveal that a multi-vesicular body of 12 kDa (MVB12)-associated β-prism (MABP) domain binds membranes such that the pore-forming machinery of MPEG1 is oriented away from the bound membrane. This unexpected mechanism of membrane interaction suggests that MPEG1 remains bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets., Macrophage-expressed gene 1 (MPEG1) functions within the phagolysosome to damage engulfed microbes, presumably via forming pores in target membranes. In order to provide insights into the mechanism of MPEG1 function and membrane binding, the authors present structures of hexadecameric MPEG1 prepores both in solution and in complex with liposomes.

Caveolae are specialized domains of the plasma membrane. Formation of these invaginations is dependent on the expression of Caveolin-1 or -3 and proteins of the cavin family. In response to stress, caveolae disassemble and cavins are released from caveolae, allowing cavins to potentially interact with intracellular targets. Here, we describe the intracellular (non-plasma membrane) cavin interactome using biotin affinity proteomics and mass spectrometry. We validate 47 potential cavin-interactor proteins using a cell-free expression system and protein-protein binding assays. These data, together with pathway analyses, reveal unknown roles for cavin proteins in metabolism and stress signaling. We validated the interaction between one candidate interactor protein, protein phosphatase 1 alpha (PP1α), and Cavin-1 and -3 and show that UV treatment causes release of Cavin3 from caveolae allowing interaction with, and inhibition of, PP1α. This interaction increases H2AX phosphorylation to stimulate apoptosis, identifying a pro-apoptotic signaling pathway from surface caveolae to the nucleus., Caveolae are plasma membrane invaginations containing cavin proteins that are disrupted upon stress stimuli, causing cavin release inside the cell. Here, McMahon et al. identify cavin interacting proteins using proteomic analyses and reveal functions in stress signaling that can promote apoptosis.

Background Higher-order self-assembly of proteins, or “prion-like” polymerisation, is now emerging as a simple and robust mechanism for signal amplification, in particular within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns needs to trigger a strong, binary response within cells. MyD88, an important adaptor protein downstream of TLRs, is one of the most recent candidates for involvement in signalling by higher order self-assembly. In this new light, we set out to re-interpret the role of polymerisation in MyD88-related diseases and study the impact of disease-associated point mutations L93P, R196C, and L252P/L265P at the molecular level. Results We first developed new in vitro strategies to characterise the behaviour of polymerising, full-length MyD88 at physiological levels. To this end, we used single-molecule fluorescence fluctuation spectroscopy coupled to a eukaryotic cell-free protein expression system. We were then able to explore the polymerisation propensity of full-length MyD88, at low protein concentration and without purification, and compare it to the behaviours of the isolated TIR domain and death domain that have been shown to have self-assembly properties on their own. These experiments demonstrate that the presence of both domains is required to cooperatively lead to efficient polymerisation of the protein. We then characterised three pathological mutants of MyD88. Conclusion We discovered that all mutations block the ability of MyD88 to polymerise fully. Interestingly, we show that, in contrast to L93P and R196C, L252P is a gain-of-function mutation, which allows the MyD88 mutant to form extremely stable oligomers, even at low nanomolar concentrations. Thus, our results shed new light on the digital “all-or-none” responses by the myddosomes and the behaviour of the oncogenic mutations of MyD88.

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.

The aggregation of α-SYN follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinson’s Disease in neurons. Whilst α-synuclein is a major contributor to LB, these dense accumulations of protein aggregates and tangles of fibrils contain over 70 different proteins. However, the potential for interactions between these proteins and the different aggregated species of α-SYN is largely unknown. We hypothesized that the proteins present in the Lewy Bodies are trapped or pulled into the aggregates in a hierarchical manner, by binding at specific stages of the aggregation of α-SYN.In this study we uncover a map of interactions of a total of 65 proteins, against different species formed by α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead assay for detection of protein-protein interactions. To access different oligomeric species, we made use of the pathological mutants of α-SYN (A30P, G51D and A53T), which form oligomeric species with distinct properties. Finally, we used bacterially expressed recombinant α-SYN to generate amyloid fibrils and measure interactions with a pool of GFP-tagged potential partners. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are selectively recruited to specific steps in the formation of the LB, explaining their presence in the inclusions. Only a few proteins were found to interact with α-SYN monomers at detectable levels, and only a subset recognizes the oligomeric α-SYN including autophagosomal proteins. We therefore propose a new model for the formation of Lewy Bodies, where selectivity of protein partners at different steps drives the arrangement of these structures, uncovering new ways to modulate aggregation.Significance StatementThe molecular complexity of the Lewy Bodies has been a major hindrance to a bottom-up reconstruction of these inclusions, protein by protein. This work presents an extensive dataset of protein-protein interactions, showing that despite its small size and absence of structure, α-SYN binds to specific partners in the LB, and that there is a clear selectivity of interactions between the different α-SYN species along the self-assembly pathway. We use single-molecule methods to deconvolute number and size of the co-aggregates, to gain detailed information about the mechanisms of interaction. These observations constitute the basis for the elaboration of a global interactome of α-SYN.

The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory responThe genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.

Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged opossum and the human genetic disorder Hypotrichosis-Lymphedema-Telangiectasia-Renal Syndrome. Combing three single-molecule imaging assays in living cells, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its chromatin-binding dynamics. The dominant-negative effect is further amplified by recruiting the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular etiology of dominant-negative transcription factor function.

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy. Here we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal to noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins and cytoskeletal proteins. The method can be combined with different electron microscopic techniques including fast freezing and freeze substitution, focussed ion beam scanning electron microscopy, and electron tomography. The method allows detection of endogenously expressed proteins in genome-edited cells. We make use of a cell-free expression system to generate membrane particles with a defined quantum of an APEX-fusion protein. These particles can be added to cells to provide an internal standard for estimating absolute density of expressed APEX-fusion proteins.