Cu-Induced self-assembly and amyloid formation of a cyclic D,L-α-peptide: structure and function.

In a wide spectrum of neurodegenerative diseases, self-assembly of pathogenic proteins to cytotoxic intermediates is accelerated by the presence of metal ions such as Cu. Only low concentrations of these early transient oligomeric intermediates are present in a mixture of species during fibril formation, and hence information on the extent of structuring of these oligomers is still largely unknown. Here, we investigate dimers as the first intermediates in the Cu-driven aggregation of a cyclic D,L-α-peptide architecture.

Pre-Exascale Computing of Protein-Ligand Binding Free Energies with Open Source Software for Drug Design.

Nowadays, drug design projects benefit from highly accurate protein-ligand binding free energy predictions based on molecular dynamics simulations. While such calculations have been computationally expensive in the past, we now demonstrate that workflows built on open source software packages can efficiently leverage pre-exascale computing resources to screen hundreds of compounds in a matter of days. We report our results of free energy calculations on a large set of pharmaceutically relevant targets assembled to reflect industrial drug discovery projects.

Sequence determinants of human gene regulatory elements.

DNA can determine where and when genes are expressed, but the full set of sequence determinants that control gene expression is unknown. Here, we measured the transcriptional activity of DNA sequences that represent an ~100 times larger sequence space than the human genome using massively parallel reporter assays (MPRAs). Machine learning models revealed that transcription factors (TFs) generally act in an additive manner with weak grammar and that most enhancers increase expression from a promoter by a mechanism that does not appear to involve specific TF-TF interactions.

Conformational Dynamics of Intrinsically Disordered Proteins Regulate Biomolecular Condensate Chemistry.

Motions in biomolecules are critical for biochemical reactions. In cells, many biochemical reactions are executed inside of biomolecular condensates formed by ultradynamic intrinsically disordered proteins. A deep understanding of the conformational dynamics of intrinsically disordered proteins in biomolecular condensates is therefore of utmost importance but is complicated by diverse obstacles.

Widespread occurrence of covalent lysine-cysteine redox switches in proteins.

We recently reported the discovery of a lysine-cysteine redox switch in proteins with a covalent nitrogen-oxygen-sulfur (NOS) bridge. Here, a systematic survey of the whole protein structure database discloses that NOS bridges are ubiquitous redox switches in proteins of all domains of life and are found in diverse structural motifs and chemical variants. In several instances, lysines are observed in simultaneous linkage with two cysteines, forming a sulfur-oxygen-nitrogen-oxygen-sulfur (SONOS) bridge with a trivalent nitrogen, which constitutes an unusual native branching cross-link.

Large-scale analysis of Drosophila core promoter function using synthetic promoters.

The core promoter plays a central role in setting metazoan gene expression levels, but how exactly it "computes" expression remains poorly understood. To dissect its function, we carried out a comprehensive structure-function analysis in Drosophila. First, we performed a genome-wide bioinformatic analysis, providing an improved picture of the sequence motifs architecture.

Dynamic Runx1 chromatin boundaries affect gene expression in hematopoietic development.

The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures.

The pathogenic R5L mutation disrupts formation of Tau complexes on the microtubule by altering local N-terminal structure.

The microtubule-associated protein (MAP) Tau is an intrinsically disordered protein (IDP) primarily expressed in axons, where it functions to regulate microtubule dynamics, modulate motor protein motility, and participate in signaling cascades. Tau misregulation and point mutations are linked to neurodegenerative diseases, including progressive supranuclear palsy (PSP), Pick's disease, and Alzheimer's disease. Many disease-associated mutations in Tau occur in the C-terminal microtubule-binding domain of the protein.

Modest Offset Difference Internuclear Selective Transfer via Homonuclear Dipolar Coupling.

Homonuclear dipolar recoupling is routinely used for magic-angle spinning NMR-based structure determination. In fully protonated samples, only short proton-proton distances are accessible to broadband recoupling approaches because of high proton density. Selective methods allow detection of longer distances by directing polarization to a subset of spins.

Molecular Basis of Small-Molecule Binding to α-Synuclein.

Intrinsically disordered proteins (IDPs) are implicated in many human diseases. They have generally not been amenable to conventional structure-based drug design, however, because their intrinsic conformational variability has precluded an atomic-level understanding of their binding to small molecules. Here we present long-time-scale, atomic-level molecular dynamics (MD) simulations of monomeric α-synuclein (an IDP whose aggregation is associated with Parkinson's disease) binding the small-molecule drug fasudil in which the observed protein-ligand interactions were found to be in good agreement with previously reported NMR chemical shift data.

Frequent origins of traumatic insemination involve convergent shifts in sperm and genital morphology.

Traumatic insemination is a mating behavior during which the (sperm) donor uses a traumatic intromittent organ to inject an ejaculate through the epidermis of the (sperm) recipient, thereby frequently circumventing the female genitalia. Traumatic insemination occurs widely across animals, but the frequency of its evolution, the intermediate stages via which it originates, and the morphological changes that such shifts involve remain poorly understood. Based on observations in 145 species of the free-living flatworm genus , we identify at least nine independent evolutionary origins of traumatic insemination from reciprocal copulation, but no clear indication of reversals.

Capture-C: a modular and flexible approach for high-resolution chromosome conformation capture.

Chromosome conformation capture (3C) methods measure the spatial proximity between DNA elements in the cell nucleus. Many methods have been developed to sample 3C material, including the Capture-C family of protocols. Capture-C methods use oligonucleotides to enrich for interactions of interest from sequencing-ready 3C libraries.

SNARE proteins: zip codes in vesicle targeting?

Membrane traffic in eukaryotic cells is mediated by transport vesicles that bud from a precursor compartment and are transported to their destination compartment where they dock and fuse. To reach their intracellular destination, transport vesicles contain targeting signals such as Rab GTPases and polyphosphoinositides that are recognized by tethering factors in the cytoplasm and that connect the vesicles with their respective destination compartment. The final step, membrane fusion, is mediated by SNARE proteins.

Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation.

Post-translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA-binding protein TAR DNA-binding protein (TDP-43), is hyperphosphorylated in disease on several C-terminal serine residues, a process generally believed to promote TDP-43 aggregation. Here, we however find that Casein kinase 1δ-mediated TDP-43 hyperphosphorylation or C-terminal phosphomimetic mutations reduce TDP-43 phase separation and aggregation, and instead render TDP-43 condensates more liquid-like and dynamic.

The calcium-free form of atorvastatin inhibits amyloid-β(1-42) aggregation in vitro.

Alzheimer's disease is characterized by the presence of extraneuronal amyloid plaques composed of amyloid-beta (Aβ) fibrillar aggregates in the brains of patients. In mouse models, it has previously been shown that atorvastatin (Ator), a cholesterol-lowering drug, has some reducing effect on the production of cerebral Aβ. A meta-analysis on humans showed moderate effects in the short term but no improvement in the Alzheimer's Disease Assessment Scale-Cognitive Subscale behavioral test.

VASP boosts protrusive activity of macroendocytic cups and drives phagosome rocketing after internalization.

Ena/VASP proteins are powerful actin polymerases that drive the processive elongation of actin filaments. Members of this protein family have been implicated in a variety of important cellular processes including axon guidance, cell migration and adhesion. However, the specific function of these proteins in macroendocytosis, comprising macropinocytosis and phagocytosis remain rather poorly understood.

Fluorescence Lifetime and Cross-correlation Spectroscopy for Observing Membrane Fusion of Liposome Models Containing Synaptic Proteins.

Watching events of membrane fusion in real time and distinguishing between intermediate steps of these events is useful for mechanistic insights but at the same time a challenging task. In this chapter, we describe how to use fluorescence cross-correlation spectroscopy and Förster-resonance energy transfer to resolve the tethering and fusion of membranes by SNARE proteins (syntaxin-1, SNAP-25, and synaptobrevin-2) as an example. The given protocols can easily be adapted to other membrane proteins to investigate their ability to tether or even fuse vesicular membrane.

Building and Using a Two-Photon Fluorescence Cross-Correlation Spectroscopy Setup Including Fluorescence Lifetime Analysis.

Fluorescence Cross-Correlation Spectroscopy (FCCS) is a well-established and useful tool in physics and chemistry. Furthermore, due to its hybrid character of being a bulk assay at a single molecular level, it found many applications in biophysics and molecular biochemistry. Examples may be investigating kinetics and dynamics of chemical and biochemical reactions such as protein-ligand-, protein-protein-binding, fast conformational changes, and intracellular transportation.

Isolation of Synaptic Vesicles from Mammalian Brain.

Synaptic vesicles (SVs) store neurotransmitters and undergo a fine-tuned regulatory and dynamic cycle of exo- and endocytosis, which is essential for neurotransmission at chemical synapses. The development of protocols for isolating SVs from biological extracts was a fundamental accomplishment since it allowed for characterizing the molecular properties of SVs using biochemical methods. In this chapter, we describe a modified procedure for isolating SVs from a few g of rodent brain and that can be completed within ~12 h.

Multi-omic landscaping of human midbrains identifies disease-relevant molecular targets and pathways in advanced-stage Parkinson's disease.

Background: Parkinson's disease (PD) is the second most common neurodegenerative disorder whose prevalence is rapidly increasing worldwide. The molecular mechanisms underpinning the pathophysiology of sporadic PD remain incompletely understood. Therefore, causative therapies are still elusive.

Applications of Cell-Free Synthesized Membrane Protein Precipitates.

Cell-free protein expression systems are new core platforms for membrane protein synthesis. Expression in the presence of supplied artificial hydrophobic environments such as nanomembranes or micelles allows the co-translational solubilization and folding of membrane proteins. In the absence of hydrophobic compounds, the synthesized membrane proteins quantitatively precipitate, while frequently still retaining a significant part of folded structural elements.

Proteomic profiling of cisplatin-resistant and cisplatin-sensitive germ cell tumour cell lines using quantitative mass spectrometry.

Purpose: Advanced testicular germ cell tumours (GCT) generally have a good prognosis owing to their unique sensitivity towards cisplatin-based chemotherapies. However, cisplatin-resistant GCT have a poor outcome. Further studies are mandatory to better understand resistance mechanisms and develop therapeutic strategies for refractory GCTs.

Sequence Divergence and Functional Specializations of the Ancient Spliceosomal SF3b: Implications in Flexibility and Adaptations of the Multi-Protein Complex.

Multi-protein assemblies are complex molecular systems that perform highly sophisticated biochemical functions in an orchestrated manner. They are subject to changes that are governed by the evolution of individual components. We performed a comparative analysis of the ancient and functionally conserved spliceosomal SF3b complex, to recognize molecular signatures that contribute to sequence divergence and functional specializations.

Ovarian carcinoma immunoreactive antigen-like protein 2 (OCIAD2) is a novel complex III-specific assembly factor in mitochondria.

Assembly of the dimeric complex III (CIII) in the mitochondrial inner membrane is an intricate process in which several accessory proteins are involved as assembly factors. Despite numerous studies, this process has yet to be fully understood. Here we report the identification of human OCIAD2 (ovarian carcinoma immunoreactive antigen-like protein 2) as an assembly factor for CIII.

Molecular Diffusivity of Click Reaction Components: The Diffusion Enhancement Question.

Micrometer-sized objects are widely known to exhibit chemically driven motility in systems away from equilibrium. Experimental observation of reaction-induced motility or enhancement in diffusivity at the much shorter length scale of small molecules is, however, still a matter of debate. Here, we investigate the molecular diffusivity of reactants, catalyst, and product of a model reaction, the copper-catalyzed azide-alkyne cycloaddition click reaction, and develop new NMR diffusion approaches that allow the probing of reaction-induced diffusion enhancement in nanosized molecular systems with higher accuracy than the state of the art.