Dynamic PRC1-CBX8 stabilizes a porous structure of chromatin condensates.

The compaction of chromatin is a prevalent paradigm in gene repression. Chromatin compaction is commonly thought to repress transcription by restricting chromatin accessibility. However, the spatial organization and dynamics of chromatin compacted by gene-repressing factors are unknown.

Single-Molecule Fingerprinting Reveals Different Growth Mechanisms in Seed Amplification Assays for Different Polymorphs of α-Synuclein Fibrils.

α-Synuclein (αSyn) aggregates, detected in the biofluids of patients with Parkinson's disease (PD), have the ability to catalyze their own aggregation, leading to an increase in the number and size of aggregates. This self-templated amplification is used by newly developed assays to diagnose Parkinson's disease and turns the presence of αSyn aggregates into a biomarker of the disease. It has become evident that αSyn can form fibrils with slightly different structures, called "strains" or polymorphs, but little is known about their differential reactivity in diagnostic assays.

Expressed Protein Ligation in Flow.

The development of a flow chemistry platform for the generation of modified protein targets via expressed protein ligation (EPL) is described. The flow EPL platform enables efficient ligation reactions with high recoveries of target protein products and superior reaction rates compared to corresponding batch processes. The utility of the flow EPL technology was first demonstrated through the semisynthesis of the tick-derived chemokine-binding protein ACA-01 containing two tyrosine sulfate modifications.

The HIV capsid mimics karyopherin engagement of FG-nucleoporins.

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides.

α-Synuclein Strains and Their Relevance to Parkinson's Disease, Multiple System Atrophy, and Dementia with Lewy Bodies.

Like many neurodegenerative diseases, Parkinson's disease (PD) is characterized by the formation of proteinaceous aggregates in brain cells. In PD, those proteinaceous aggregates are formed by the α-synuclein (αSyn) and are considered the trademark of this neurodegenerative disease. In addition to PD, αSyn pathological aggregation is also detected in atypical Parkinsonism, including Dementia with Lewy Bodies (DLB), Multiple System Atrophy (MSA), as well as neurodegeneration with brain iron accumulation, some cases of traumatic brain injuries, and variants of Alzheimer's disease.

The blood vasculature instructs lymphatic patterning in a SOX7-dependent manner.

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.

The RHIM of the Immune Adaptor Protein TRIF Forms Hybrid Amyloids with Other Necroptosis-Associated Proteins.

TIR-domain-containing adapter-inducing interferon-β (TRIF) is an innate immune protein that serves as an adaptor for multiple cellular signalling outcomes in the context of infection. TRIF is activated via ligation of Toll-like receptors 3 and 4. One outcome of TRIF-directed signalling is the activation of the programmed cell death pathway necroptosis, which is governed by interactions between proteins that contain a RIP Homotypic Interaction Motif (RHIM).

Single Molecule Fingerprinting Reveals Different Amplification Properties of α-Synuclein Oligomers and Preformed Fibrils in Seeding Assay.

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.

A dominant-negative SOX18 mutant disrupts multiple regulatory layers essential to transcription factor activity.

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.

Selectivity of Lewy body protein interactions along the aggregation pathway of α-synuclein.

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.

Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response.

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.

MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography.

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 (MAL) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88). 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).

Rapid HIV-1 Capsid Interaction Screening Using Fluorescence Fluctuation Spectroscopy.

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.

Single-Molecule Counting Coupled to Rapid Amplification Enables Detection of α-Synuclein Aggregates in Cerebrospinal Fluid of Parkinson's Disease Patients.

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.

SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species.

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.

Herpes simplex virus encoded ICP6 protein forms functional amyloid assemblies with necroptosis-associated host proteins.

The viral protein ICP6, encoded by herpes simplex virus 1 (HSV-1), harbours a RIP-homotypic interaction motif (RHIM), that plays a role in viral inhibition of host cell death pathways. Other members of the Herpesviridae family also encode RHIM-containing proteins that interfere with host-cell death pathways, including the M45 protein from murine cytomegalovirus, and ORF20 protein from varicella zoster virus. We have used amyloid assembly assays, electron microscopy and single molecule fluorescence spectroscopy to show that the ICP6 RHIM is amyloidogenic and can interact with host RHIM-containing proteins to form heteromeric amyloid complexes, in a manner similar to that of M45 and ORF20 RHIMs.

Varicella zoster virus encodes a viral decoy RHIM to inhibit cell death.

Herpesviruses are known to encode a number of inhibitors of host cell death, including RIP Homotypic Interaction Motif (RHIM)-containing proteins. Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and is responsible for causing chickenpox and shingles. We have identified a novel viral RHIM in the VZV capsid triplex protein, open reading frame (ORF) 20, that acts as a host cell death inhibitor.

Biophysical Techniques for Target Validation and Drug Discovery in Transcription-Targeted Therapy.

In the post-genome era, pathologies become associated with specific gene expression profiles and defined molecular lesions can be identified. The traditional therapeutic strategy is to block the identified aberrant biochemical activity. However, an attractive alternative could aim at antagonizing key transcriptional events underlying the pathogenesis, thereby blocking the consequences of a disorder, irrespective of the original biochemical nature.

Single-molecule detection on a portable 3D-printed microscope.

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.