Michal Richman

In vitro and mechanistic studies of an antiamyloidogenic self-assembled cyclic D,L-α-peptide architecture.

Misfolding of the Aβ protein and its subsequent aggregation into toxic oligomers are related to Alzheimers disease. Although peptides of various sequences can self-assemble into amyloid structures, these structures share common three-dimensional features that may promote their cross-reaction. Given the significant similarities between amyloids and the architecture of self-assembled cyclic D,L-α-peptide, we hypothesized that the latter may bind and stabilize a nontoxic form of Aβ, thereby preventing its aggregation into toxic forms. By screening a focused library of six-residue cyclic D,L-α-peptides and optimizing the activity of a lead peptide, we found one cyclic D,L-α-peptide (CP-2) that interacts strongly with Aβ and inhibits its aggregation. In transmission electron microscopy, optimized thioflavin T and cell survival assays, CP-2 inhibits the formation of Aβ aggregates, entirely disassembles preformed aggregated and fibrillar Aβ, and protects rat pheochromocytoma PC12 cells from Aβ toxicity, without inducing any toxicity by itself. Using various immunoassays, circular dichroism spectroscopy, photoinduced cross-linking of unmodified proteins (PICUP) combined with SDS/PAGE, and NMR, we probed the mechanisms underlying CP-2s antiamyloidogenic activity. NMR spectroscopy indicates that CP-2 interacts with Aβ through its self-assembled conformation and induces weak secondary structure in Aβ. Upon coincubation, CP-2 changes the aggregation pathway of Aβ and alters its oligomer distribution by stabilizing small oligomers (1-3 mers). Our results support studies suggesting that toxic early oligomeric states of Aβ may be composed of antiparallel β-peptide structures and that the interaction of Aβ with CP-2 promotes formation of more benign parallel β-structures. Further studies will show whether these kinds of abiotic cyclic D,L-α-peptides are also beneficial as an intervention in related in vivo models.

Surface‐Modified Protein Microspheres Capture Amyloid‐β and Inhibit its Aggregation and Toxicity

The biocompatible and biodegradable properties of protein microspheres and the recent advances in their preparation have generated considerable interest of utilizing these core-shell structures for drug delivery and diagnostic applications. However, effective targeting of protein microspheres to desirable cells or loci still remains a challenge. Here, we describe for the first time a facile one-pot sonochemical approach for covalent modification of protein microspheres made from serum albumin; the surface of which is covalently decorated with a short recognition peptide to target amyloid-β (Aβ) as the main pathogenic protein in Alzheimers disease (AD). The microspheres were characterized for their morphology, size, and entrapment efficacy by electron microscopy, dynamic light scattering and confocal microscopy. Fluorescence-activated cell-sorting analysis and Thioflavin-T binding assay demonstrated that the conjugated microspheres bind with high affinity and selectivity to Aβ, sequester it from the medium and reduce its aggregation. Upon incubation with Aβ, the microspheres induced formation of amorphous aggregates on their surface with no apparent fibrillar structure. Moreover, the microspheres directly reduced the Aβ-induced toxicity toward neuron like PC12 cells. The conjugated microspheres are smaller than unmodified microspheres and remained stable throughout the incubation under physiological conditions.

Effective targeting of Aβ to macrophages by sonochemically prepared surface-modified protein microspheres.

Imbalanced homeostasis and oligomerization of the amyloid-β (Aβ) peptide in the brain are hallmarks of Alzheimers disease (AD). Microglia and macrophages play a critical role in the etiology of AD either by clearing Aβ from the brain or inducing inflammation. Recent evidence suggests that clearance of Aβ by microglia/macrophages via the phagocytic pathway is defective in AD, which can contribute to the accumulation of Aβ in the brain. We have recently demonstrated that protein microspheres modified at their surface with multiple copies of an Aβ-recognition motif can strongly bind Aβ, inhibit its aggregation, and directly reduce its toxicity by sequestering it from the medium. Here, we describe how microsphere-bound Aβ can stimulate microglial cells and be phagocytosed through a mechanism that is distinct from that of Aβ removal and, thus, contribute to the clearance of Aβ, even by defective microglial cells. The phagocytosis was most effective, with microspheres having a diameter of <1 μm. The introduction of polyethylene glycol to the surface of the microspheres changed the kinetics of the phagocytosis. Moreover, while aggregated Aβ induced a significant inflammatory response that was manifested by the release of TNF-α, the microsphere-bound Aβ dramatically reduced the amount of cytokine released from microglial cells.

Multifunctional Cyclic d,l-α-Peptide Architectures Stimulate Non-Insulin Dependent Glucose Uptake in Skeletal Muscle Cells and Protect Them Against Oxidative Stress

Oxidative stress directly correlates with the early onset of vascular complications and the progression of peripheral insulin resistance in diabetes. Accordingly, exogenous antioxidants augment insulin sensitivity in type 2 diabetic patients and ameliorate its clinical signs. Herein, we explored the unique structural and functional properties of the abiotic cyclic D,L-α-peptide architecture as a new scaffold for developing multifunctional agents to catalytically decompose ROS and stimulate glucose uptake. We showed that His-rich cyclic D,L-α-peptide 1 is very stable under high H2O2 concentrations, effectively self-assembles to peptide nanotubes, and increases the uptake of glucose by increasing the translocation of GLUT1 and GLUT4. It also penetrates cells and protects them against oxidative stress induced under hyperglycemic conditions at a much lower concentration than α-lipoic acid (ALA). In vivo studies are now required to probe the mode of action and efficacy of these abiotic cyclic D,L-α-peptides as a novel class of antihyperglycemic compounds.

Selective Inhibition of Aggregation and Toxicity of a Tau-Derived Peptide using Its Glycosylated Analogues.

Protein glycosylation is a ubiquitous post-translational modification that regulates the folding and function of many proteins. Misfolding of protein monomers and their toxic aggregation are the hallmark of many prevalent diseases. Thus, understanding the role of glycans in protein aggregation is highly important and could contribute both to unraveling the pathology of protein misfolding diseases as well as providing a means for modifying their course for therapeutic purposes. Using β-O-linked glycosylated variants of the highly studied Tau-derived hexapeptide motif VQIVYK, which served as a simplified amyloid model, we demonstrate that amyloid formation and toxicity can be strongly attenuated by a glycan unit, depending on the nature of the glycan itself. Importantly, we show for the first time that not only do glycans hinder self-aggregation, but the glycosylated peptides are capable of inhibiting aggregation of the non-modified corresponding amyloid scaffold.

Distinct Effects of O-GlcNAcylation and Phosphorylation of a Tau-Derived Amyloid Peptide on Aggregation of the Native Peptide

Protein phosphorylation and O-GlcNAcylation are very common nucleoplasmic post-translational modifications. Mono-addition of either the phosphate or the O-GlcNAc group were shown to inhibit the self-aggregation of amyloidogenic proteins and peptides, which is the hallmark of various protein misfolding diseases. However, their comparable effect upon co-incubation with a native non-modified amyloid scaffold has not been reported. O-linked glycans and phosphate variants of the tau protein-derived VQIVYK hexapeptide motif were generated as a simplified amyloid scaffold model and demonstrate that, while self-aggregation can be attenuated by either a single glycan or a phosphate unit, only co-incubation with the O-GlcNAc variant inhibits aggregation of the native peptide. These results shed light on the role of post-translational modifications in protein aggregation and suggest a novel therapeutic approach to protein misfolding diseases.

Anti-amyloidogenic Heterocyclic Peptides

The amyloid fibril is a highly ordered proteinous aggregate, originally discovered in the context of the self-assembly of soluble proteins into insoluble extracellular plaques. The molecular structure of amyloidosis, an energetically stable conformation with a thermodynamic local minimum, is of particular interest because of its possible pathogenicity. Amyloidogenic diseases (amyloidoses) are often fatal, widely heterogenic, and caused by sporadic, genetic, or infectious pathogens. Consequently, major effort has been directed in the past few decades to identify and develop agents that decrease the concentration of the pathogenic aggregates either by interfering with the self-assembly of the proteins or by modulating their physiological concentration. Heterocyclic peptides of natural and synthetic origin have gained special attention because of their demonstrated ability to interact with various amyloids. In addition to interfering with the amyloid aggregation process, many of the discovered peptides also inhibit the formation of fibrils, disassemble preformed fibrils, and prevent the pathogenic seeding effect. Others are instrumental candidates for passive vaccination against amyloid deposits. The promising preliminary results described here, together with the broad chemical diversity of heterocyclic peptides and their amenability to large-scale production, make these compounds promising for anti-amyloidogenic research and for pharmacological therapy of amyloidoses.