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 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.
Single-molecule assays have, by definition, the ultimate sensitivity and represent the next frontier in biological analysis and diagnostics. However, many of these powerful technologies require ...dedicated laboratories and trained personnel and have therefore remained research tools for specialists. Here, we present a single-molecule confocal system built from a 3D-printed scaffold, resulting in a compact, plug and play device called the AttoBright. This device performs single photon counting and fluorescence correlation spectroscopy (FCS) in a simple format and is widely applicable to the detection of single fluorophores, proteins, liposomes or bacteria. The power of single-molecule detection is demonstrated by detecting single α-synuclein amyloid fibrils, that are currently evaluated as biomarkers for Parkinson's disease, with an improved sensitivity of >100,000-fold over bulk measurements.
The aggregation of alpha-synuclein (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. Although LB contain over 70 proteins, the potential for interactions along the aggregation pathway of α-SYN is unknown. Here we propose a map of interactions of 65 proteins against different species of α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead luminescence assay for detection of protein interactions. To access oligomeric species, we used the pathological mutants of α-SYN (A30P, G51D and A53T) which form oligomers with distinct properties. Finally, we generated amyloid fibrils from recombinant α-SYN. 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 recruited to specific steps in the aggregation of α-SYN, uncovering future targets to modulate aggregation in synucleinopathies.
Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the ...activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibition, or variation in the selectivity of binding partners. As such, dimerization and higher order oligomerization is a common feature in signaling proteins, for example, and more than 70% of enzymes have the potential to self-associate. On the other hand, protein aggregation can overcome the regulatory mechanisms of the cell and can have disastrous physiological effects. This is the case in a number of neurodegenerative diseases, where proteins, due to mutation or dysregulation later in life, start polymerizing and often fibrillate, leading to the creation of protein inclusion bodies in cells. Dimerization, well-defined oligomerization and random aggregation are often difficult to differentiate and characterize experimentally. Single molecule "counting" methods are particularly well suited to the study of self-oligomerization as they allow observation and quantification of behaviors in heterogeneous conditions. However, the extreme dilution of samples often causes weak complexes to dissociate, and rare events can be overlooked. Here, we discuss a straightforward alternative where the principles of single molecule detection are used at higher protein concentrations to quantify oligomers and aggregates in a background of monomers. We propose a practical guide for the use of confocal spectroscopy to quantify protein oligomerization status and also discuss about its use in monitoring changes in protein aggregation in drug screening assays.
α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson's disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this ...oligomerization cascade is an interesting approach to developing therapeutical strategies and β-synuclein (βS) has been described as a natural negative regulator of this process. However, the biological background and molecular mechanisms by which this inhibition occurs is unclear. Herein, we focused on assessing the effect of βS on the aggregation of five αS pathological mutants linked to early-onset PD (A30P, E46K, H50Q, G51D and A53T). By coupling single molecule fluorescence spectroscopy to a cell-free protein expression system, we validated the ability of βS to act as a chaperone of αS, effectively inhibiting its aggregation. Interestingly, we found that βS does so in a selective manner, i.e., is a more effective inhibitor for certain αS pathological mutants-A30P and G51D-as compared to E46K, H50Q and A53T. Moreover, two-color coincidence experiments proved that this discrepancy is due to a preferential incorporation of βS into smaller oligomers of αS. This was validated by showing that the chaperoning effect was lost when proteins were mixed after being expressed individually. This study highlights the potential of fluorescence spectroscopy to deconstruct αS aggregation cascade and its interplay with βS.
The use of fluorescently-tagged proteins in microscopy has become routine, and anti-GFP (Green fluorescent protein) affinity matrices are increasingly used in proteomics protocols. However, some ...protein-protein interactions assays, such as protein complementation assays (PCA), require recloning of each protein as a fusion with the different parts of the complementation system. Here we describe a generic system where the complementation is separated from the proteins and can be directly used with fluorescently-tagged proteins. By using nanobodies and performing tests in cell-free expression systems, we accelerated the development of multiple reporters, detecting heterodimers and homodimers or oligomers tagged with GFP or mCherry. We demonstrate that the system can detect interactions at a broad range of concentrations, from low nanomolar up to micromolar.
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.
Cell‐free methods of protein synthesis offer rapid access to expressed proteins. Though the amounts produced are generally only at a small scale, these are sufficient to perform protein‐protein ...interaction assays and tests of enzymatic activity. As such they are valuable tools for the biochemistry and bioengineering community. However the most complex, eukaryotic cell‐free systems are difficult to manufacture in house and can be prohibitively expensive to obtain from commercial sources. The Leishmania tarentolae system offers a relatively cheap alternative which is capable of producing difficult to express proteins, but which is simpler to produce in large scale. However, this system suffers from batch‐to‐batch variability, which has been accepted as a consequence of the complexity of the extracts. Here we show an unexpected origin for the variability observed and demonstrate that small variations in a single parameter can dramatically affect expression, such that minor pipetting errors can have major effects on yields. L. tarentolae cell‐free lysate activity is shown to be more stable to changes in Mg2+ concentration at a lower ratio of feed solution to lysate in the reaction than typically used, and a higher Mg2+ optimum. These changes essentially eliminate batch‐to‐batch variability of L. tarentolae lysate activity and permit their full potential to be realized.
Leishmania cell‐free expression systems have always shown a high degree of instability and “batch‐to‐batch” variability. Here we show an unexpected origin for this variability, as we describe an instability in protein expression depending on a single parameter, the ratio of feed solution to cell‐free lysate. In the unstable zone, pipetting errors can translate into almost complete loss of expression. The picture shows the matrix of compositions we perform to map the metastable zone for each lysate and eliminate batch‐to‐batch variability.
Abstract
During embryogenesis, vascular development relies on a handful of transcription factors that instruct cell fate in a distinct sub-population of the endothelium (1). The SOXF proteins that ...comprise SOX7, 17 and 18, are molecular switches modulating arterio-venous and lymphatic endothelial differentiation (2,3). Here, we show that, in the SOX-F family, only SOX18 has the ability to switch between a monomeric and a dimeric form. We characterized the SOX18 dimer in binding assays in vitro, and using a split-GFP reporter assay in a zebrafish model system in vivo. We show that SOX18 dimerization is driven by a novel motif located in the vicinity of the C-terminus of the DNA binding region. Insertion of this motif in a SOX7 monomer forced its assembly into a dimer. Genome-wide analysis of SOX18 binding locations on the chromatin revealed enrichment for a SOX dimer binding motif, correlating with genes with a strong endothelial signature. Using a SOX18 small molecule inhibitor that disrupts dimerization, we revealed that dimerization is important for transcription. Overall, we show that dimerization is a specific feature of SOX18 that enables the recruitment of key endothelial transcription factors, and refines the selectivity of the binding to discrete genomic locations assigned to endothelial specific genes.
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.
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