High-Content Imaging Platform to Discover Chemical Modulators of Plasma Membrane Rafts.

Plasma membrane organization profoundly impacts cellular functionality. A well-known mechanism underlying this organization is through nanoscopic clustering of distinct lipids and proteins in membrane rafts. Despite their physiological importance, rafts remain a difficult-to-study aspect of membrane organization, in part because of the paucity of chemical tools to experimentally modulate their properties.

Recombinant SARS-CoV-2 envelope protein traffics to the trans-Golgi network following amphipol-mediated delivery into human cells.

The severe acute respiratory syndrome coronavirus 2 envelope protein (S2-E) is a conserved membrane protein that is important for coronavirus (CoV) assembly and budding. Here, we describe the recombinant expression and purification of S2-E in amphipol-class amphipathic polymer solutions, which solubilize and stabilize membrane proteins, but do not disrupt membranes. We found that amphipol delivery of S2-E to preformed planar bilayers results in spontaneous membrane integration and formation of viroporin cation channels.

The C99 domain of the amyloid precursor protein resides in the disordered membrane phase.

Processing of the amyloid precursor protein (APP) via the amyloidogenic pathway is associated with the etiology of Alzheimer's disease. The cleavage of APP by β-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by γ-secretase to yield amyloid-β (Aβ) peptides are essential steps in this pathway. Biochemical evidence suggests that amyloidogenic processing of C99 occurs in cholesterol- and sphingolipid-enriched liquid-ordered phase membrane rafts.

Delivery of recombinant SARS-CoV-2 envelope protein into human cells.

SARS-CoV-2 envelope protein (S2-E) is a conserved membrane protein that is essential to coronavirus assembly and budding. Here, we describe the recombinant expression and purification of S2-E into amphipol-class amphipathic polymer solutions. The physical properties of amphipols underpin their ability to solubilize and stabilize membrane proteins without disrupting membranes.

The small heat shock proteins, HSPB1 and HSPB5, interact differently with lipid membranes.

Increasing evidence shows that heat shock proteins (hsp) escape the cytosol gaining access to the extracellular environment, acting as signaling agents. Since the majority of these proteins lack the information necessary for their export via the classical secretory pathway, attention has been focused on alternative releasing mechanisms. Crossing the plasma membrane is a major obstacle to the secretion of a cytosolic protein into the extracellular milieu.

Modulation of Alzheimer's amyloid β peptide oligomerization and toxicity by extracellular Hsp70.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to dementia caused by advanced neuronal dysfunction and death. The most significant symptoms of AD are observed at late stages of the disease when interventions are most likely too late to ameliorate the condition. Currently, the predominant theory for AD is the "amyloid hypothesis," which states that abnormally increased levels of amyloid β (Aβ) peptides result in the production of a variety of aggregates that are neurotoxic.

Small molecule NPT-440-1 inhibits ionic flux through Aβ pores: Implications for Alzheimer's disease therapeutics.

Increased levels of soluble amyloid-beta (Aβ) oligomers are suspected to underlie Alzheimer's disease (AD) pathophysiology. These oligomers have been shown to form multi-subunit Aβ pores in bilayers and induce uncontrolled, neurotoxic, ion flux, particularly calcium ions, across cellular membranes that might underlie cognitive impairment in AD. Small molecule interventions that modulate pore activity could effectively prevent or ameliorate their toxic activity.

All-d-Enantiomer of β-Amyloid Peptide Forms Ion Channels in Lipid Bilayers.

Alzheimer's disease (AD) is the most common type of senile dementia in aging populations. Amyloid β (Aβ)-mediated dysregulation of ionic homeostasis is the prevailing underlying mechanism leading to synaptic degeneration and neuronal death. Aβ-dependent ionic dysregulation most likely occurs either directly via unregulated ionic transport through the membrane or indirectly via Aβ binding to cell membrane receptors and subsequent opening of existing ion channels or transporters.

Probing structural features of Alzheimer's amyloid-β pores in bilayers using site-specific amino acid substitutions.

A current hypothesis for the pathology of Alzheimer's disease (AD) proposes that amyloid-β (Aβ) peptides induce uncontrolled, neurotoxic ion flux across cellular membranes. The mechanism of ion flux is not fully understood because no experiment-based Aβ channel structures at atomic resolution are currently available (only a few polymorphic states have been predicted by computational models). Structural models and experimental evidence lend support to the view that the Aβ channel is an assembly of loosely associated mobile β-sheet subunits.

Atomic force microscopy and MD simulations reveal pore-like structures of all-D-enantiomer of Alzheimer's β-amyloid peptide: relevance to the ion channel mechanism of AD pathology.

Alzheimer's disease (AD) is a protein misfolding disease characterized by a buildup of β-amyloid (Aβ) peptide as senile plaques, uncontrolled neurodegeneration, and memory loss. AD pathology is linked to the destabilization of cellular ionic homeostasis and involves Aβ peptide-plasma membrane interactions. In principle, there are two possible ways through which disturbance of the ionic homeostasis can take place: directly, where the Aβ peptide either inserts into the membrane and creates ion-conductive pores or destabilizes the membrane organization, or, indirectly, where the Aβ peptide interacts with existing cell membrane receptors.

Antimicrobial properties of amyloid peptides.

More than two dozen clinical syndromes known as amyloid diseases are characterized by the buildup of extended insoluble fibrillar deposits in tissues. These amorphous Congo red staining deposits known as amyloids exhibit a characteristic green birefringence and cross-β structure. Substantial evidence implicates oligomeric intermediates of amyloids as toxic species in the pathogenesis of these chronic disease states.

Mutant SOD1 forms ion channel: implications for ALS pathophysiology.

Point mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) impart a gain-of-function to this protein that underlies 20-25% of all familial amyotrophic lateral sclerosis (FALS) cases. However, the specific mechanism of mutant SOD1 toxicity has remained elusive. Using the complementary techniques of atomic force microscopy (AFM), electrophysiology, and cell and molecular biology, here we examine the structure and activity of A4VSOD1, a mutant SOD1.

Antimicrobial protegrin-1 forms amyloid-like fibrils with rapid kinetics suggesting a functional link.

Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich β-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability.

β-Barrel topology of Alzheimer's β-amyloid ion channels.

Emerging evidence supports the ion channel mechanism for Alzheimer's disease pathophysiology wherein small β-amyloid (Aβ) oligomers insert into the cell membrane, forming toxic ion channels and destabilizing the cellular ionic homeostasis. Solid-state NMR-based data of amyloid oligomers in solution indicate that they consist of a double-layered β-sheets where each monomer folds into β-strand-turn-β-strand and the monomers are stacked atop each other. In the membrane, Aβ peptides are proposed to be β-type structures.

Structural convergence among diverse, toxic beta-sheet ion channels.

Recent studies show that an array of beta-sheet peptides, including N-terminally truncated Abeta peptides (Abeta(11-42/17-42)), K3 (a beta(2)-microglobulin fragment), and protegrin-1 (PG-1) peptides form ion channel-like structures and elicit single channel ion conductance when reconstituted in lipid bilayers and induce cell damage through cell calcium overload. Striking similarities are observed in the dimensions of these toxic channels irrespective of their amino acid sequences. However, the intriguing question of preferred channel sizes is still unresolved.

Antimicrobial protegrin-1 forms ion channels: molecular dynamic simulation, atomic force microscopy, and electrical conductance studies.

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among beta-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood.

Treponema denticola PrcB is required for expression and activity of the PrcA-PrtP (dentilisin) complex.

The Treponema denticola surface protease complex, consisting of PrtP protease (dentilisin) and two auxiliary polypeptides (PrcA1 and PrcA2), is believed to contribute to periodontal disease by degrading extracellular matrix components and disrupting host intercellular signaling. Previously, we showed that transcription of the protease operon initiates upstream of TDE0760 (herein designated prcB), the open reading frame immediately 5' of prcA-prtP. The prcB gene is conserved in T.

Truncated beta-amyloid peptide channels provide an alternative mechanism for Alzheimer's Disease and Down syndrome.

Full-length amyloid beta peptides (Abeta(1-40/42)) form neuritic amyloid plaques in Alzheimer's disease (AD) patients and are implicated in AD pathology. However, recent transgenic animal models cast doubt on their direct role in AD pathology. Nonamyloidogenic truncated amyloid-beta fragments (Abeta(11-42) and Abeta(17-42)) are also found in amyloid plaques of AD and in the preamyloid lesions of Down syndrome, a model system for early-onset AD study.

Misfolded amyloid ion channels present mobile beta-sheet subunits in contrast to conventional ion channels.

In Alzheimer's disease, calcium permeability through cellular membranes appears to underlie neuronal cell death. It is increasingly accepted that calcium permeability involves toxic ion channels. We modeled Alzheimer's disease ion channels of different sizes (12-mer to 36-mer) in the lipid bilayer using molecular dynamics simulations.

A semi-synthetic ion channel platform for detection of phosphatase and protease activity.

Sensitive methods to probe the activity of enzymes are important for clinical assays and for elucidating the role of these proteins in complex biochemical networks. This paper describes a semi-synthetic ion channel platform for detecting the activity of two different classes of enzymes with high sensitivity. In the first case, this method uses single ion channel conductance measurements to follow the enzyme-catalyzed hydrolysis of a phosphate group attached to the C-terminus of gramicidin A (gA, an ion channel-forming peptide) in the presence of alkaline phosphatase (AP).

K3 fragment of amyloidogenic beta(2)-microglobulin forms ion channels: implication for dialysis related amyloidosis.

Beta(2)-microglobulin (beta(2)m) amyloid deposits are linked to dialysis-related amyloidosis (DRA) in hemodialysis patients. The mechanism by which beta(2)m causes DRA is not understood. It is also unclear whether only the full-length beta(2)m induces pathophysiology or if proteolytic fragments are sufficient for inducing this effect.

Amyloid-beta-induced ion flux in artificial lipid bilayers and neuronal cells: resolving a controversy.

Understanding the pathogenicity of amyloid-beta (Abeta) peptides constitutes a major goal in research on Alzheimer's disease (AD). One hypothesis entails that Abeta peptides induce uncontrolled, neurotoxic ion flux through cellular membranes. The exact biophysical mechanism of this ion flux is, however, a subject of an ongoing controversy which has attenuated progress toward understanding the importance of Abeta-induced ion flux in AD.

Films of agarose enable rapid formation of giant liposomes in solutions of physiologic ionic strength.

This paper describes a method to form giant liposomes in solutions of physiologic ionic strength, such as phosphate buffered saline (PBS) or 150 mM KCl. Formation of these cell-sized liposomes proceeded from hybrid films of partially dried agarose and lipids. Hydrating the films of agarose and lipids in aqueous salt solutions resulted in swelling and partial dissolution of the hybrid films and in concomitant rapid formation of giant liposomes in high yield.

Chemically reactive derivatives of gramicidin A for developing ion channel-based nanoprobes.

Ion channel-forming peptides and proteins offer tremendous opportunities for fundamental and applied studies of function on individual molecules. An ongoing challenge in ion channel research is the lack of simple and accessible synthetic methods to engineer pores with tailored chemical and physical properties. This paper describes a practical synthetic route to rapidly generate C-terminally modified derivatives of gramicidin A (gA), an ion channel-forming peptide, through the use of two chemically reactive gA-based building blocks.

Designing nanosensors based on charged derivatives of gramicidin A.

Detection of chemical processes on a single molecule scale is the ultimate goal of sensitive analytical assays. We recently reported the possibility to detect chemical modifications on individual molecules by monitoring a change in the single ion channel conductance of derivatives of gramicidin A (gA) upon reaction with analytes in solution. These peptide-based nanosensors detect reaction-induced changes in the charge of gA derivatives that were engineered to carry specific functional groups near their C-terminus.