Treatment Of Amyloid Diseases Research Articles

Autophagy Modulation as a Treatment of Amyloid Diseases.

Amyloids are fibrous proteins aggregated into toxic forms that are implicated in several chronic disorders. More than 30 diseases show deposition of fibrous amyloid proteins associated with cell loss and degeneration in the affected tissues. Evidence demonstrates that amyloid diseases result from protein aggregation or impaired amyloid clearance, but the connection between amyloid accumulation and tissue degeneration is not clear. Common examples of amyloid diseases are Alzheimer’s disease (AD), Parkinson’s disease (PD) and tauopathies, which are the most common forms of neurodegenerative diseases, as well as polyglutamine disorders and certain peripheral metabolic diseases. In these diseases, increased accumulation of toxic amyloid proteins is suspected to be one of the main causative factors in the disease pathogenesis. It is therefore important to more clearly understand how these toxic amyloid proteins accumulate as this will aide in the development of more effective preventive and therapeutic strategies. Protein homeostasis, or proteostasis, is maintained by multiple cellular pathways—including protein synthesis, quality control, and clearance—which are collectively responsible for preventing protein misfolding or aggregation. Modulating protein degradation is a very complex but attractive treatment strategy used to remove amyloid and improve cell survival. This review will focus on autophagy, an important clearance pathway of amyloid proteins, and strategies for using it as a potential therapeutic target for amyloid diseases. The physiological role of autophagy in cells, pathways for its modulation, its connection with apoptosis, cell models and caveats in developing autophagy as a treatment and as a biomarker is discussed.

Disassembly of Alzheimer’s amyloid fibrils by functional upconversion nanoparticles under near-infrared light irradiation

Alzheimer's disease (AD) is a common neurodegenerative disease characterized by cognitive and memory function impairment. Studies have shown that the overproduction and further misfolding of amyloid polypeptide (Aβ) are the main causes of AD. Therefore, how to reduce Aβ species, especially to disassemble the preformed amyloid aggregates, has become the focus of related research. Photodynamic therapy (PDT) using normal photosensitizers eg. porphyrins, is a traditional way of inhibiting amyloid aggregation or degrading the amyloid aggregates, but UV light irradiation presents a side effect for the damage of normal tissue, which limits its medical application. Upconversion nanoparticles (UCNPs) is a luminous material which can be modified by photodynamic agent to form the complex generating reactive oxygen species (ROS) with noninvasive light irradiation. It presents a good advantage in the disassembly of amyloid aggregates via ROS in noninvasive light irradiation. Herein, we prepared β-NaYF4:Yb/Er@SiO2@RB by combing UCNPs with photosensitizer to disassemble preformed Aβ aggregates under near-infrared light. The results displayed upconversion nanomaterials could degrade Aβ1–42 fibrils effectively by photo-generating ROS, which demonstrated that the functional UCNPs may have potential applications in the treatment of amyloid diseases in future.

Inhibition of lysozyme fibrillogenesis by hydroxytyrosol and dopamine: An Atomic Force Microscopy study

Protein aggregation underlies many human diseases characterized by the deposition of normally soluble proteins in both fibrillar and amorphous aggregates. Here, Atomic Force Microscopy (AFM) has been applied to investigate the ability to inhibit hen egg white lysozyme (HEWL) fibrillogenesis by hydroxytyrosol (HT), one of the main phenolic components of olive oil. In this framework, HEWL is a useful and well-studied model protein whose amyloid-like fibril formation can be induced under experimental conditions where HT is more stable. HEWL fibrils, obtained at pH 1.6 and at 65 °C, exhibited a height of about 3 nm and a fibril length on average of about 3 μm. The presence of HT reduced the HEWL fibril number and length with respect to the control sample. Interestingly, also dopamine, a compound with a chemical structure similar to HT, decreased both the fibril number and the fibril length. AFM experimental data were supported by Thioflavin T assay and Fourier transform infrared spectroscopy. Our results show that HT is an effective inhibitor of HEWL aggregation, thus suggesting possible future applications of this natural compound for potential prevention or treatment of amyloid diseases, or as a lead molecular structure for the design of improved modulators.

Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation

Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on the surface of Escherichia coli, as well as many other enteric bacteria, and are involved in cell colonization and biofilm formation. CsgE is a periplasmic accessory protein that plays a crucial role in curli biogenesis. CsgE binds to both CsgA and the nonameric pore protein CsgG. The CsgG-CsgE complex is the curli secretion channel and is essential for the formation of the curli fibril in vivo. To better understand the role of CsgE in curli formation, we have determined the solution NMR structure of a double mutant of CsgE (W48A/F79A) that appears to be similar to the wild-type (WT) protein in overall structure and function but does not form mixed oligomers at NMR concentrations similar to the WT. The well-converged structure of this mutant has a core scaffold composed of a layer of two α-helices and a layer of three-stranded antiparallel β-sheet with flexible N and C termini. The structure of CsgE fits well into the cryoelectron microscopy density map of the CsgG-CsgE complex. We highlight a striking feature of the electrostatic potential surface in CsgE structure and present an assembly model of the CsgG-CsgE complex. We suggest a structural mechanism of the interaction between CsgE and CsgA. Understanding curli formation can provide the information necessary to develop treatments and therapeutic agents for biofilm-related infections and may benefit the prevention and treatment of amyloid diseases. CsgE could establish a paradigm for the regulation of amyloidogenesis because of its unique role in curli formation.

Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases.

Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer’s, Parkinson’s and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases.

Rigid Organization of Fluorescence-Active Ligands by Artificial Macrocyclic Receptor to Achieve the Thioflavin T-Amyloid Fibril Level Association.

The push-pull molecules with an intramolecular charge transfer from donor to acceptor sides upon excitation exhibit a wide variety of biological and electronic activities, as exemplified by the in vivo fluorescence imaging probes for amyloid fibrils in the diagnosis and treatment of amyloid diseases. Interestingly, the structurally much simpler bis(4,8-disulfonato-1,5-naphtho)-32-crown-8 (DNC), in keen contrast to the conventional macrocyclic receptors, was found to dramatically enhance the fluorescence of twisted intramolecular charge-transfer molecules possessing various benzothiazolium and stilbazolium fluorophores upon complexation. Spectroscopic and microcalorimetric titrations jointly demonstrated the complex structures and the interactions that promote the extremely strong complexation, revealing that the binding affinity in these artificial host-guest pairs could reach up to a nearly 10(7) M(-1) order of magnitude in water, and the sandwich-type complexation is driven by electrostatic, hydrophobic, π-stacking, and hydrogen-bonding interactions. Quantum chemical calculations on free molecules and their DNC-bound species in both the ground and excited states elucidated that the encapsulation by DNC could greatly deter the central single and double chemical bonds from free intramolecular rotation in the singlet excited state, thus leading to the unique and unprecedented fluorescence enhancement upon sandwich-type complexation. This complexation-induced structural reorganization mechanism may also apply to the binding of other small-molecule ligands by functional receptors and contribute to the molecular-level understanding of the receptor-ligand interactions in many biology-related systems.

A fluorimetric study on the interaction between a Trp-containing beta-strand peptide and amphiphilic polymer-coated gold nanoparticles.

Owing to the inevitability of nanoparticles encountering proteins/peptides in current bio-nano-medicine development, it is important to know how they interact with each other in vitro before developing in vivo applications. To this end, a model de novo β-sheet-forming peptide and typical biocompatible nanoparticles were selected to study thermodynamic aspects of their interactions via a fluorescence quenching method. The results showed that Pep11 and AuNPs spontaneously formed conjugates, mainly driven by a coulombic interaction with a binding affinity of ~ 0.1 µM(-1); the physical adsorption process was cooperative. These results deepen our quantitative understanding of nanoparticle-peptide interactions. The results may also be helpful in further nanoparticle-peptide hybrid nanofabrication and also useful for the application of nanoparticles in the treatment of amyloid diseases.

Interaction of human laminin receptor with Sup35, the [PSI⁺] prion-forming protein from S. cerevisiae: a yeast model for studies of LamR interactions with amyloidogenic proteins.

The laminin receptor (LamR) is a cell surface receptor for extracellular matrix laminin, whereas the same protein within the cell interacts with ribosomes, nuclear proteins and cytoskeletal fibers. LamR has been shown to be a receptor for several bacteria and viruses. Furthermore, LamR interacts with both cellular and infectious forms of the prion protein, PrPC and PrPSc. Indeed, LamR is a receptor for PrPC. Whether LamR interacts with PrPSc exclusively in a capacity of the PrP receptor, or LamR specifically recognizes prion determinants of PrPSc, is unclear. In order to explore whether LamR has a propensity to interact with prions and amyloids, we examined LamR interaction with the yeast prion-forming protein, Sup35. Sup35 is a translation termination factor with no homology or functional relationship to PrP. Plasmids expressing LamR or LamR fused with the green fluorescent protein (GFP) were transformed into yeast strain variants differing by the presence or absence of the prion conformation of Sup35, respectively [PSI +] and [psi −]. Analyses by immunoprecipitation, centrifugal fractionation and fluorescent microscopy reveal interaction between LamR and Sup35 in [PSI +] strains. The presence of [PSI +] promotes LamR co-precipitation with Sup35 as well as LamR aggregation. In [PSI +] cells, LamR tagged with GFP or mCherry forms bright fluorescent aggregates that co-localize with visible [PSI +] foci. The yeast prion model will facilitate studying the interaction of LamR with amyloidogenic prions in a safe and easily manipulated system that may lead to a better understanding and treatment of amyloid diseases.

Design of an N-Methylated Peptide Inhibitor of α-Synuclein Aggregation Guided by Solid-State NMR

Many neurodegenerative diseases are associated with the aggregation of misfolded proteins into amyloid oligomers or fibrils that are deposited as pathological lesions within areas of the brain. An attractive therapeutic strategy for preventing or ameliorating amyloid formation is to identify agents that inhibit the onset or propagation of protein aggregation. Here we demonstrate how solid-state nuclear magnetic resonance (ssNMR) may be used to identify key residues within amyloidogenic protein sequences that may be targeted to inhibit the aggregation of the host protein. For alpha-synuclein, the major protein component of Lewy bodies associated with Parkinson's disease, we have used a combination of ssNMR and biochemical data to identify the key region for self-aggregation of the protein as residues 77-82 (VAQKTV). We used our new structural information to design a peptide derived from residues 77 to 82 of alpha-synuclein with an N-methyl group at the C-terminal residue, which was able to disrupt the aggregation of alpha-synuclein. Thus, we have shown how structural data obtained from ssNMR can guide the design of modified peptides for use as amyloid inhibitors, as a primary step toward developing therapeutic compounds for prevention and/or treatment of amyloid diseases.

Probing the origins, diagnosis and treatment of amyloid diseases using antibodies

The deposition of proteins in the form of amyloid fibrils is the characteristic feature of more than 20 medical conditions affecting the central nervous system or a variety of peripheral tissues. These disorders, which include Alzheimer’s disease, the prion diseases and type II diabetes, are of enormous importance in the context of present-day human health and welfare. Extensive research is therefore being carried out to define the molecular details of the mechanism of the pathological conversion of amyloidogenic proteins from their soluble forms into fibrillar structures. This review focuses on recent studies that demonstrate the power of using antibodies or antibody fragments to probe the process of fibril formation, and discusses the emerging potential of these species as diagnostic and therapeutic agents.

Detecting hidden sequence propensity for amyloid fibril formation.

The preponderance of evidence implicates protein misfolding in many unrelated human diseases. In all cases, normal correctly folded proteins transform from their proper native structure into an abnormal beta-rich structure known as amyloid fibril. Here we introduce a computational algorithm to detect nonnative (hidden) sequence propensity for amyloid fibril formation. Analyzing sequence-structure relationships in terms of tertiary contact (TC), we find that the hidden beta-strand propensity of a query local sequence can be quantitatively estimated from the secondary structure preferences of template sequences of known secondary structure found in regions of high TC. The present method correctly pinpoints the minimal peptide fragment shown experimentally as the likely local mediator of amyloid fibril formation in beta-amyloid peptide, islet amyloid polypeptide (hIAPP), alpha-synuclein, and human acetylcholinesterase (AChE). It also found previously unrecognized beta-strand propensities in the prototypical helical protein myoglobin that has been reported as amyloidogenic. Analysis of 2358 nonhomologous protein domains provides compelling evidence that most proteins contain sequences with significant hidden beta-strand propensity. The present method may find utility in many medically relevant applications, such as the engineering of protein sequences and the discovery of therapeutic agents that specifically target these sequences for the prevention and treatment of amyloid diseases.

A New Technology for Treatment of Amyloid Diseases

A New Technology for Treatment of Amyloid Diseases

Amyloid fibril formation in microwell plates for screening of inhibitors

Fibril formation is the basis of amyloid production in a number of disease states, such as Alzheimer's disease, diabetes and immunocytic dyscrasias. Compounds that inhibit fibril formation could be directly relevant to the treatment of amyloid diseases, and may also provide a foundation for the development of interventions in other molecular condensation diseases ranging from sickle cell anemia to atherosclerosis.We developed an economical and convenient high-throughput method for screening compounds against fibril formation in microwell plates. Chalcones, Jlavonoids and bijluvonoids were screened against fibril formation by a recombinant antibody variable domain (VL). Chalcones 6 and 14 were found to demonstrate inhibition at 0.1 M in 79 M of protein solution in both test tube and microwell plate assays. The concentration of protein in the microwell plate assay could be as low as 5 M using ThTas a monitoring agent. Molecular modeling studies indicated that both compounds could be individually docked into a binding site at the monomer-monomer interface of the VLprotein dimer. These studies suggested that these compounds could potentialty stabilize the VL dimer and therefore reduce its fendency to form fibrils. These findings may provide the basis for a new therapeutic approach to prevent or treat amyloid diseases.

Seeding “one-dimensional crystallization” of amyloid: A pathogenic mechanism in Alzheimer's disease and scrapie?

Joseph T. Jerrett and Peter T. Lansbury, Jr. Department of Chemistry Massachusetts Institute of Technology Cambridge, Massachusetts 02139 Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the presence of cerebral amyloid plaque (reviewed by Selkoe, 1991), a highly ordered protein ag- gregate defined by its insolubility and fibrillar structure (Lansbury, 1992). The AD amyloid protein (p protein) is a secreted protein of unknown function that is overproduced in some but not all AD cases (Seubert et al., 1992; Shoji et al., 1992). Acausal relationship between amyloid forma- tion and AD has not been proven, but the slow onset of symptoms appears to parallel the gradual deposition of amyloid. Therefore, it is important to understand the mo- lecular mechanism of amyloid formation and to explain why the p protein aggregates in diseased individuals (Cit- ron et al., 1992; Cai et al., 1993). In this review, a simple chemical explanation is proposed, based on the observa- tion that in vitro amyloid formation bears a mechanistic resemblance to processes involving ordered protein ag- gregation (such as protein crystallization and microtubule formation), which will be referred to as nucleation- dependent polymerizations. Like AD, the human prion diseases, Creutzfeldt-Jakob disease and Gertsmann-StrBussler-Scheinker disease, are characterized by the slow onset of neurodegeneration. Brain pathology in these diseases resembles that of AD (Prusiner, 1964; Baker and Ridley, 1992) and is also char- acterized by aggregation of a normal cellular protein, prion protein (PrP) (rather than the p protein), often in amyloid plaques (reviewed by Prusiner, 1991). In contrast with AD, the pathogenic nature of PrP aggregates has been estab- lished, thanks to extensive work on the transmissible prion disease, scrapie. The infective agent of scrapie may oper- ate by accelerating the step in amyloid formation that is normally rate determining (Griffith, 1967; Prusiner, 1991). We propose that this step is mechanistically relevant to amyloid formation in human prion disease and in AD; it is the formation of an ordered nucleus, which is the defining characteristic of a nucleation-dependent polymerization. According to this hypothesis, the transmission of scrapie and the initiation of AD may both involve the seeding of amyloid formation. Protein Solubility Is Normally Operationally Defined The measurement of protein solubility often reflects a ki- netic effect rather than true thermodynamic solubility. For instance, when a protein solution appears to be clear throughout the course of an experiment, the protein is defined as soluble, although precipitation may eventually occur. The rate at which a protein polymerizes and precipi- tates is not necessarily related to its thermodynamic solu- bility. However, both properties may be relevant to the pathogenesis and treatment of amyloid diseases. Proteins can form different types of insoluble aggre- gates. Amorphous aggregates have multiple protein con- formations and ill-defined intermolecular interactions. In contrast, protein crystals are often characterized by a sin- gle protein conformation and a single well-defined intermo- lecular packing arrangement. Ordered noncrystalline polymers such as amyloid share these properties. In fact, amyloid can be thought of as a one-dimensional crystal in which packing in the plane perpendicular to the direction of fibril growth is nonuniform (Lansbury, 1992). Amor- phous aggregates can form rapidly when the protein con- centration exceeds the solubility. However, crystal formation requires time, owing to the kinetic barrier im- posed by nucleus formation, the rate-determining step. Ordered noncrystalline protein polymers such as amyloid share this requirement for nucleation. Nucleation-Dependent Polymerization Is Common Nucleation-dependent protein polymerization describes many well-characterized processes, including protein crystallization, microtubule assembly, flagellum assem- bly, sickle-cell hemoglobin fibril formation, bacteriophage procapsid assembly, and actin polymerization. A simple general mechanism is illustrated for the formation of a helical protein polymer in Figure 1. Nucleus formation re- quires a series of association steps that are thermodynam- ically unfavorable (K, > 1) because monomers contact the growing polymer at multiple sites, resulting in rapid polymerization/ growth. A distinctive feature of a nucleation-dependent polymer-