mNeonGreen aggregates when overexpressed in neurons.

The optical transparency of the nematode makes it possible to monitor the behaviour of fluorescently labelled proteins in a living multicellular organism. This study investigates the suitability of mNeonGreen as a fluorescent tag for studying proteins of interest in the nervous system of adult . Despite its reported brightness, stability, and monomeric nature, our findings reveal that mNeonGreen forms solid aggregates in neurons, particularly upon plasmid overexpression.

Islet amyloid polypeptide tagged with green fluorescent protein localises to mitochondria and forms filamentous aggregates in Caenorhabditis elegans.

Type 2 diabetes (T2D) is the most common form of diabetes and represents a growing health concern. A characteristic feature of T2D is the aggregation of islet amyloid polypeptide (IAPP), which is thought to be associated with the death of pancreatic β-cells. Inhibiting IAPP aggregation is a promising therapeutic avenue to treat T2D, but the mechanisms of aggregation and toxicity are not yet fully understood.

Fibril elongation by human islet amyloid polypeptide is the main event linking aggregation to membrane damage.

The aggregation of human islet amyloid polypeptide (hIAPP) is linked to the death of pancreatic β-cells in type II diabetes. The process of fibril formation by hIAPP is thought to cause membrane damage, but the precise mechanisms are still unclear. Previously, we showed that the aggregation of hIAPP in the presence of membranes containing anionic lipids is dominated by secondary nucleation events, which occur at the interface between existing fibrils and the membrane surface.

Molecular mechanisms of amyloid formation in living systems.

Fibrillar protein aggregation is a hallmark of a variety of human diseases. Examples include the deposition of amyloid-β and tau in Alzheimer's disease, and that of α-synuclein in Parkinson's disease. The molecular mechanisms by which soluble proteins form amyloid fibrils have been extensively studied in the test tube.

Membrane-Catalyzed Aggregation of Islet Amyloid Polypeptide Is Dominated by Secondary Nucleation.

Type II diabetes is characterized by the loss of pancreatic β-cells. This loss is thought to be a consequence of membrane disruption, caused by the aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils. However, the molecular mechanisms of IAPP aggregation in the presence of membranes have remained unclear.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans.

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy.

Kinetic analysis reveals that independent nucleation events determine the progression of polyglutamine aggregation in .

Protein aggregation is associated with a wide range of degenerative human diseases with devastating consequences, as exemplified by Alzheimer's, Parkinson's, and Huntington's diseases. In vitro kinetic studies have provided a mechanistic understanding of the aggregation process at the molecular level. However, it has so far remained largely unclear to what extent the biophysical principles of amyloid formation learned in vitro translate to the complex environment of living organisms.

Comparative Studies in the A30P and A53T α-Synuclein Strains to Investigate the Molecular Origins of Parkinson's Disease.

The aggregation of α-synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression.

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies.

Misfolded α-synuclein is a major component of Lewy bodies, which are a hallmark of Parkinson's disease (PD). A large body of evidence shows that α-synuclein can aggregate into amyloid fibrils, but the relationship between α-synuclein self-assembly and Lewy body formation remains unclear. Here, we show, both in vitro and in a Caenorhabditis elegans model of PD, that α-synuclein undergoes liquid‒liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel with Lewy-body-like properties.

Biophysical studies of protein misfolding and aggregation in models of Alzheimer's and Parkinson's diseases.

Neurodegenerative disorders, including Alzheimer's (AD) and Parkinson's diseases (PD), are characterised by the formation of aberrant assemblies of misfolded proteins. The discovery of disease-modifying drugs for these disorders is challenging, in part because we still have a limited understanding of their molecular origins. In this review, we discuss how biophysical approaches can help explain the formation of the aberrant conformational states of proteins whose neurotoxic effects underlie these diseases.

Challenging Proteostasis: Role of the Chaperone Network to Control Aggregation-Prone Proteins in Human Disease.

Protein homeostasis (Proteostasis) is essential for correct and efficient protein function within the living cell. Among the critical components of the Proteostasis Network (PN) are molecular chaperones that serve widely in protein biogenesis under physiological conditions, and prevent protein misfolding and aggregation enhanced by conditions of cellular stress. For Alzheimer's, Parkinson's, Huntington's diseases and ALS, multiple classes of molecular chaperones interact with the highly aggregation-prone proteins amyloid-β, tau, α-synuclein, huntingtin and SOD1 to influence the course of proteotoxicity associated with these neurodegenerative diseases.

Fast Fluorescence Lifetime Imaging Reveals the Aggregation Processes of α-Synuclein and Polyglutamine in Aging .

The nematode worm has emerged as an important model organism in the study of the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation, and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterization of different types of aggregates.

Expression of the amyloid-β peptide in a single pair of C. elegans sensory neurons modulates the associated behavioural response.

Although the aggregation of the amyloid-β peptide (Aβ) into amyloid fibrils is a well-established hallmark of Alzheimer's disease, the complex mechanisms linking this process to neurodegeneration are still incompletely understood. The nematode worm C. elegans is a valuable model organism through which to study these mechanisms because of its simple nervous system and its relatively short lifespan.

Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in .

Reduced protein homeostasis leading to increased protein instability is a common molecular feature of aging, but it remains unclear whether this is a cause or consequence of the aging process. In neurodegenerative diseases and other amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological aggregates in different tissues. More recently, widespread protein aggregation has been described during normal aging.

Formation of the β-barrel assembly machinery complex in lipid bilayers as seen by solid-state NMR.

The β-barrel assembly machinery (BAM) is a pentameric complex (BamA-E), which catalyzes the essential process of β-barrel protein insertion into the outer membrane of E. coli. Thus far, a detailed understanding of the insertion mechanism has been elusive but recent results suggest that local protein motion, in addition to the surrounding membrane environment, may be of critical relevance.

Combined H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments.

Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane proteins from their native environment, but this removal can significantly alter the structure and function of these proteins. Here, we overcome these challenges with a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample.

An Efficient Labelling Approach to Harness Backbone and Side-Chain Protons in (1) H-Detected Solid-State NMR Spectroscopy.

(1) H-detection can greatly improve spectral sensitivity in biological solid-state NMR (ssNMR), thus allowing the study of larger and more complex proteins. However, the general requirement to perdeuterate proteins critically curtails the potential of (1) H-detection by the loss of aliphatic side-chain protons, which are important probes for protein structure and function. Introduced herein is a labelling scheme for (1) H-detected ssNMR, and it gives high quality spectra for both side-chain and backbone protons, and allows quantitative assignments and aids in probing interresidual contacts.

Conformational Plasticity of the POTRA 5 Domain in the Outer Membrane Protein Assembly Factor BamA.

BamA is the main component of the β-barrel assembly machinery (BAM) that folds and inserts outer membrane proteins in Gram-negative bacteria. Crystal structures have suggested that this process involves conformational changes in the transmembrane β-barrel of BamA that allow for lateral opening, as well as large overall rearrangements of its periplasmic POTRA domains. Here, we identify local dynamics of the BamA POTRA 5 domain by solution and solid-state nuclear magnetic resonance.

Magic-angle-spinning solid-state NMR of membrane proteins.

Solid-state NMR spectroscopy (ssNMR) provides increasing possibilities to examine membrane proteins in different molecular settings, ranging from synthetic bilayers to whole cells. This flexibility often enables ssNMR experiments to be directly correlated with membrane protein function. In this contribution, we discuss experimental aspects of such studies starting with protein expression and labeling, leading to membrane protein isolation or to membrane proteins in a cellular environment.

Insight into the conformational stability of membrane-embedded BamA using a combined solution and solid-state NMR approach.

The β-barrel assembly machinery (BAM) is involved in folding and insertion of outer membrane proteins in Gram-negative bacteria, a process that is still poorly understood. With its 790 residues, BamA presents a challenge to current NMR methods. We utilized a "divide and conquer" approach in which we first obtained resonance assignments for BamA's periplasmic POTRA domains 4 and 5 by solution NMR.

Solid-state NMR studies of full-length BamA in lipid bilayers suggest limited overall POTRA mobility.

The outer membrane protein BamA is the key player in β-barrel assembly in Gram-negative bacteria. Despite the availability of high-resolution crystal structures, the dynamic behavior of the transmembrane domain and the large periplasmic extension consisting of five POTRA (POlypeptide-TRansport-Associated) domains remains unclear. We demonstrate reconstitution of full-length BamA in proteoliposomes at low lipid-to-protein ratio, leading to high sensitivity and resolution in solid-state NMR (ssNMR) experiments.

Proton clouds to measure long-range contacts between nonexchangeable side chain protons in solid-state NMR.

We show that selective labeling of proteins with protonated amino acids embedded in a perdeuterated matrix, dubbed 'proton clouds', provides general access to long-range contacts between nonexchangeable side chain protons in proton-detected solid-state NMR, which is important to study protein tertiary structure. Proton-cloud labeling significantly improves spectral resolution by simultaneously reducing proton line width and spectral crowding despite a high local proton density in clouds. The approach is amenable to almost all canonical amino acids.

Expressed protein ligation for a large dimeric protein.

Expressed protein ligation (EPL) is a protein engineering tool for post-translational ligation of protein or peptide fragments. This technique allows modification of specific parts of proteins, opening possibilities for incorporating probes for biophysical applications such as nuclear magnetic resonance (NMR) or fluorescence spectroscopy. The application for oligomeric proteins, however, is restricted by the need to obtain a large excess of active dimer over reactants and intermediates.

N-terminal domain of human Hsp90 triggers binding to the cochaperone p23.

The molecular chaperone Hsp90 is a protein folding machine that is conserved from bacteria to man. Human, cytosolic Hsp90 is dedicated to folding of chiefly signal transduction components. The chaperoning mechanism of Hsp90 is controlled by ATP and various cochaperones, but is poorly understood and controversial.