Probing differences among Aß oligomers with two triangular trimers derived from Aß.

The assembly of the ß-amyloid peptide (Aß) to form oligomers and fibrils is closely associated with the pathogenesis and progression of Alzheimer's disease. Aß is a shape-shifting peptide capable of adopting many conformations and folds within the multitude of oligomers and fibrils the peptide forms. These properties have precluded detailed structural elucidation and biological characterization of homogeneous, well-defined Aß oligomers. In this paper, we compare the structural, biophysical, and biological characteristics of two different covalently stabilized isomorphic trimers derived from the central and C-terminal regions Aß. X-ray crystallography reveals the structures of the trimers and shows that each trimer forms a ball-shaped dodecamer. Solution-phase and cell-based studies demonstrate that the two trimers exhibit markedly different assembly and biological properties. One trimer forms small soluble oligomers that enter cells through endocytosis and activate capase-3/7-mediated apoptosis, while the other trimer forms large insoluble aggregates that accumulate on the outer plasma membrane and elicit cellular toxicity through an apoptosis-independent mechanism. The two trimers also exhibit different effects on the aggregation, toxicity, and cellular interaction of full-length Aß, with one trimer showing a greater propensity to interact with Aß than the other. The studies described in this paper indicate that the two trimers share structural, biophysical, and biological characteristics with oligomers of full-length Aß. The varying structural, assembly, and biological characteristics of the two trimers provide a working model for how different Aß trimers can assemble and lead to different biological effects, which may help shed light on the differences among Aß oligomers.

Effects of Familial Alzheimer's Disease Mutations on the Assembly of a ß-Hairpin Peptide Derived from Aß16-36.

Familial Alzheimer's disease (FAD) is associated with mutations in the ß-amyloid peptide (Aß) or the amyloid precursor protein (APP). FAD mutations of Aß were incorporated into a macrocyclic peptide that mimics a ß-hairpin to study FAD point mutations K16N, A21G, E22Δ, E22G, E22Q, E22K, and L34V and their effect on assembly, membrane destabilization, and cytotoxicity. The X-ray crystallographic structures of the four E22 mutant peptides reveal that the peptides assemble to form the same compact hexamer. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments reveal that the mutant FAD peptides assemble as trimers or hexamers, with peptides that have greater positive charge assembling as more stable hexamers. Mutations that increase the positive charge also increase the cytotoxicity of the peptides and their propensity to destabilize lipid membranes.

Expression of N-Terminal Cysteine Aß42 and Conjugation to Generate Fluorescent and Biotinylated Aß42.

Fluorescent derivatives of the ß-amyloid peptides (Aß) are valuable tools for studying the interactions of Aß with cells. Facile access to labeled expressed Aß offers the promise of Aß with greater sequence and stereochemical integrity, without impurities from amino acid deletion and epimerization. Here, we report methods for the expression of Aß42 with an N-terminal cysteine residue, Aß(C1-42), and its conjugation to generate Aß42 bearing fluorophores or biotin. The methods rely on the hitherto unrecognized observation that expression of the Aß(MC1-42) gene yields the Aß(C1-42) peptide, because the N-terminal methionine is endogenously excised by Escherichia coli. Conjugation of Aß(C1-42) with maleimide-functionalized fluorophores or biotin affords the N-terminally labeled Aß42. The expression affords ∼14 mg of N-terminal cysteine Aß from 1 L of bacterial culture. Subsequent conjugation affords ∼3 mg of labeled Aß from 1 L of bacterial culture with minimal cost for labeling reagents. High-performance liquid chromatography analysis indicates the N-terminal cysteine Aß to be >97% pure and labeled Aß peptides to be 94-97% pure. Biophysical studies show that the labeled Aß peptides behave like unlabeled Aß and suggest that labeling of the N-terminus does not substantially alter the properties of the Aß. We further demonstrate applications of the fluorophore-labeled Aß peptides by using fluorescence microscopy to visualize their interactions with mammalian cells and bacteria. We anticipate that these methods will provide researchers convenient access to useful N-terminally labeled Aß, as well as Aß with an N-terminal cysteine that enables further functionalization.

Interpenetrating Cubes in the X-ray Crystallographic Structure of a Peptide Derived from Medin19-36.

Amyloidogenic peptides and proteins are rich sources of supramolecular assemblies. Sequences derived from well-known amyloids, including Aß, human islet amyloid polypeptide, and tau have been found to assemble as fibrils, nanosheets, ribbons, and nanotubes. The supramolecular assembly of medin, a 50-amino acid peptide that forms fibrillary deposits in aging human vasculature, has not been heavily investigated. In this work, we present an X-ray crystallographic structure of a cyclic ß-sheet peptide derived from the 19-36 region of medin that assembles to form interpenetrating cubes. The edge of each cube is composed of a single peptide, and each vertex is occupied by a divalent metal ion. This structure may be considered a metal-organic framework (MOF) containing a large peptide ligand. This work demonstrates that peptides containing Glu or Asp that are preorganized to adopt ß-hairpin structures can serve as ligands and assemble with metal ions to form MOFs.

Effects of N-Terminal Residues on the Assembly of Constrained ß-Hairpin Peptides Derived from Aß.

This paper describes the synthesis, solution-phase biophysical studies, and X-ray crystallographic structures of hexamers formed by macrocyclic ß-hairpin peptides derived from the central and C-terminal regions of Aß, which bear "tails" derived from the N-terminus of Aß. Soluble oligomers of the ß-amyloid peptide, Aß, are thought to be the synaptotoxic species responsible for neurodegeneration in Alzheimer's disease. Over the last 20 years, evidence has accumulated that implicates the N-terminus of Aß as a region that may initiate the formation of damaging oligomeric species. We previously studied, in our laboratory, macrocyclic ß-hairpin peptides derived from Aß16-22 and Aß30-36, capable of forming hexamers that can be observed by X-ray crystallography and SDS-PAGE. To better mimic oligomers of full length Aß, we use an orthogonal protecting group strategy during the synthesis to append residues from Aß1-14 to the parent macrocyclic ß-hairpin peptide 1, which comprises Aß16-22 and Aß30-36. The N-terminally extended peptides N+1, N+2, N+4, N+6, N+8, N+10, N+12, and N+14 assemble to form dimers, trimers, and hexamers in solution-phase studies. X-ray crystallography reveals that peptide N+1 assembles to form a hexamer that is composed of dimers and trimers. These observations are consistent with a model in which the assembly of Aß oligomers is driven by hydrogen bonding and hydrophobic packing of the residues from the central and C-terminal regions, with the N-terminus of Aß accommodated by the oligomers as an unstructured tail.

Macrocyclic Peptides Derived from Familial Alzheimer's Disease Mutants Show Charge-Dependent Oligomeric Assembly and Toxicity.

This work probes the role of charge in the oligomeric assembly, toxicity, and membrane destabilization of a series of peptides derived from Aß and the E22Q and E22K familial mutants. In the mutant Aß peptides, an acidic residue (E) is replaced with either a neutral or basic residue (Q or K), thus altering the net charge of the peptide. Acetylation at peripheral positions permits modulation of charge of the peptides and allows investigation of the role of charge in their oligomeric assembly, cytotoxicity, and membrane disruption. Peptides with the same net charge generally behave similarly even if the amino acid residue at position 22 differs. As the net charge of the peptide decreases, so does the extent of assembly, cytotoxicity, and membrane destabilization, which were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lactate dehydrogenase (LDH)-release assays with SH-SY5Y cells, and dye leakage assays using liposomes. These findings suggest that the charge of the amino acid side chain, rather than its size or hydrophobicity, accounts for the differences in the oligomeric assembly and toxicity of the E22 familial mutants of Aß.

Receptor-Ligand Kinetics Influence the Mechanism of Action of Covalently Linked TLR Ligands.

We report a mechanistic study comparing the immune activation of conjugated Toll-like receptor (TLR) agonists and their unlinked mixtures. Herein, we synthesized a set of six linked dual agonists with different ligands, molecular structures, receptor locations, and biophysical characteristics. With these dimers, we ran a series of in vitro cell-based assays, comparing initial and overall NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation, cytokine expression profiles, as well as time-resolved TNF-α (Tumor Necrosis Factor alpha) expression. We show that initial activation kinetics, ligand specificity, and the dose of the agonist influence the activity of these linked TLR systems. These results can help improve vaccine design by showing how linked TLR agonists can improve their potency with the appropriate selection of key criteria.

Correction to "Immunomodulation of the NLRP3 Inflammasome through Structure-Based Activator Design and Functional Regulation via Lysosomal Rupture".

[This corrects the article DOI: 10.1021/acscentsci.8b00218.].

Immunomodulation of the NLRP3 Inflammasome through Structure-Based Activator Design and Functional Regulation via Lysosomal Rupture.

The NLRP3 inflammasome plays a role in the inflammatory response to vaccines, in antimicrobial host defense, and in autoimmune diseases. However, its mechanism of action remains incompletely understood. NLRP3 has been shown to be activated by diverse stimuli including microbial toxins, ATP, particulate matter, etc. that activate multiple cellular processes. There have been two major challenges in translating inflammasome activators into controlled adjuvants. Both stem from their chemical and structural diversity. First, it is difficult to identify a minimum requirement for inflammasome activation. Second, no current activator can be tuned to generate a desired degree of activation. Thus, in order to design such immunomodulatory biomaterials, we developed a new tunable lysosomal rupture probe that leads to significant differences in inflammasome activation owing to structural changes as small as a single amino acid. Using these probes, we conduct experiments that suggest that rupturing lysosomes is a critical, initial step necessary to activate an inflammasome and that it precedes other pathways of activation. We demonstrate that each molecule differentially activates the inflammasome based solely on their degree of lysosomal rupture. We have employed this understanding of chemical control in structure-based design of immunomodulatory NLRP3 agonists on a semipredictive basis. This information may guide therapeutic interventions to prevent or mitigate lysosomal rupture and will also provide a predictive framework for dosable activation of the NLRP3 inflammasome for potential applications in vaccines and immunotherapies.