Primary Nucleation of Polymorphic α-Synuclein Dimers Depends on Copper Concentrations and Definite Copper-Binding Site.

The primary nucleation process of α-synuclein (AS) that forms toxic oligomeric species is the early stage of the pathological cause of Parkinson's disease. It is well-known that copper influences this primary nucleation process. While significant efforts have been made to solve the structures of polymorphic AS fibrils, the structures of AS oligomers and the copper-bound AS oligomers at the molecular level and the effect of copper concentrations on the primary nucleation are elusive. Here, we propose and demonstrate new molecular mechanism pathways of primary nucleation of AS that are tuned by distinct copper concentrations and by a specific copper-binding site. We present the polymorphic AS dimers bound to different copper-binding sites at the atomic resolution in high- and low-copper concentrations, using extensive molecular dynamics simulations. Our results show the complexity of the primary nucleation pathways that rely on the copper concentrations and the copper binding site. From a broader perspective, our study proposes a new strategy to control the primary nucleation of other toxic amyloid oligomers in other neurodegenerative diseases.

Structural packing of the non-amyloid component core domain in α-synuclein plays a role in the stability of the fibrils.

Parkinson's disease (PD) is one of many neurodegenerative diseases. The protein associated with PD is α-synuclein (AS). Aggregation of AS protein into oligomers, protofilaments, and finally to fibrils yields to the development of PD. The aggregation process of AS leads to the formation of polymorphic AS fibrils. Herein, we compared four polymorphic full-length AS1-140 fibrils, using extensive computational tools. The main conclusion of this study emphasizes the role of the structurally packed non-amyloid component (NAC) core domain in AS fibrils. Polymorphic AS fibrils that presented a packed NAC core domain, exhibited more ß-sheets and fewer fluctuations in the NAC domain. Hence, these AS fibrils are more stable and populated than the AS fibrils, by which the NAC domains are more exposed, more fluctuate and less packed in the fibrillary structure. Therefore, this study emphasizes the importance of the NAC domain packing in the morphology of AS fibrils. The results obtained in this study will initiate future studies to develop compounds to prevent and inhibit AS aggregation.

Insights into Non-Proteolytic Inhibitory Mechanisms of Polymorphic Early-Stage Amyloid ß Oligomers by Insulin Degrading Enzyme.

Insulin degrading enzyme (IDE) has been detected in the cerebrospinal fluid media and plays a role in encapsulating and degrading the amyloid ß (Aß) monomer, thus regulating the levels of Aß monomers. The current work illustrates a first study by which IDE encapsulates polymorphic early-stage Aß oligomers. The main goal of this study was to investigate the molecular mechanisms of IDE activity on the encapsulated early-stage Aß dimers: fibril-like and random coil/α-helix dimers. Our work led to several findings. First, when the fibril-like Aß dimer interacts with IDE-C domain, IDE does not impede the contact between the monomers, but plays a role as a 'dead-end' chaperone protein. Second, when the fibril-like Aß dimer interacts with the IDE-N domain, IDE successfully impedes the contacts between monomers. Third, the inhibitory activity of IDE on random coil/α-helix dimers depends on the stability of the dimer. IDE could impede the contacts between monomers in relatively unstable random coil/α-helix dimers, but gets hard to impede in stable dimers. However, IDE encapsulates stable dimers and could serve as a 'dead-end' chaperone. Our results examine the molecular interactions between IDE and the dimers, and between the monomers within the dimers. Hence, this study provides insights into the inhibition mechanisms of the primary nucleation of Aß aggregation and the basic knowledge for rational design to inhibit Aß aggregation.

Distinct Antifouling Mechanisms on Different Chain Densities of Zwitterionic Polymers.

Antifouling polymer coating surfaces are used in widespread industries applications. Zwitterionic polymers have been identified as promising materials in developing polymer coating surfaces. Importantly, the density of the polymer chains is crucial for acquiring superior antifouling performance. This study introduces two different zwitterionic polymer density surfaces by applying molecular modeling tools. To assess the antifouling performance, we mimic static adsorption test, by placing the foulant model bovine serum albumin (BSA) on the surfaces. Our findings show that not only the density of the polymer chain affect antifouling performance, but also the initial orientation of the BSA on the surface. Moreover, at a high-density surface, the foulant either detaches from the surface or anchor on the surface. At low-density surface, the foulant does not detach from the surface, but either penetrates or anchors on the surface. The anchoring and the penetrating mechanisms are elucidated by the electrostatic interactions between the foulant and the surface. While the positively charged ammonium groups of the polymer play major role in the interactions with the negatively charged amino acids of the BSA, in the penetrating mechanism the ammonium groups play minor role in the interactions with the contact with the foulant. The sulfonate groups of the polymer pull the foulant in the penetrating mechanism. Our work supports the design of a high-density polymer chain surface coating to prevent fouling phenomenon. Our study provides for the first-time insights into the molecular mechanism by probing the interactions between BSA and the zwitterion surface, while testing high- and low-densities polymer chains.

Cross-seeding between Aß and SEVI indicates a pathogenic link and gender difference between alzheimer diseases and AIDS.

Amyloid-ß (Aß) and semen-derived enhancer of viral infection (SEVI) are considered as the two causative proteins for central pathogenic cause of Alzheimer's disease (AD) and HIV/AIDS, respectively. Separately, Aß-AD and SEVI-HIV/AIDS systems have been studied extensively both in fundamental research and in clinical trials. Despite significant differences between Aß-AD and SEVI-HIV/AIDS systems, they share some commonalities on amyloid and antimicrobial characteristics between Aß and SEVI, there are apparent overlaps in dysfunctional neurological symptoms between AD and HIV/AIDS. Few studies have reported a potential pathological link between Aß-AD and SEVI-HIV/AIDS at a protein level. Here, we demonstrate the cross-seeding interactions between Aß and SEVI proteins using in vitro and in vivo approaches. Cross-seeding of SEVI with Aß enabled to completely prevent Aß aggregation at sub-stoichiometric concentrations, disaggregate preformed Aß fibrils, reduce Aß-induced cell toxicity, and attenuate Aß-accumulated paralysis in transgenic AD C. elegans. This work describes a potential crosstalk between AD and HIV/AIDS via the cross-seeding between Aß and SEVI, identifies SEVI as Aß inhibitor for possible treatment or prevention of AD, and explains the role of SEVI in the gender difference in AD.

Prodrug-Based Targeting Approach for Inflammatory Bowel Diseases Therapy: Mechanistic Study of Phospholipid-Linker-Cyclosporine PLA2-Mediated Activation.

Therapeutics with activity specifically at the inflamed sites throughout the gastrointestinal tract (GIT) would be a major advance in our therapeutic approach to inflammatory bowel disease (IBD). We aimed to develop the prodrug approach that can allow such site-specific drug delivery. Currently, using cyclosporine as a drug of choice in IBD is limited to the most severe cases due to substantial systemic toxicities and narrow therapeutic index of this drug. Previously, we synthesized a series of a phospholipid-linker-cyclosporine (PLC) prodrugs designed to exploit the overexpression of phospholipase A2 (PLA2) in the inflamed intestinal tissues, as the prodrug-activating enzyme. Nevertheless, the extent and rate of prodrug activation differed significantly. In this study we applied in-vitro and modern in-silico tools based on molecular dynamics (MD) simulation, to gain insight into the dynamics and mechanisms of the PLC prodrug activation. We aimed to elucidate the reason for the significant activation change between different linker lengths in our prodrug design. Our work reveals that the PLC conjugate with the 12-carbon linker length yields the optimal prodrug activation by PLA2 in comparison to shorter linker length (6-carbons). This optimized length efficiently allows cyclosporine to be released from the prodrug to the active pocket of PLA2. This newly developed mechanistic approach, presented in this study, can be applied for future prodrug optimization to accomplish optimal prodrug activation and drug targeting in various conditions that include overexpression of PLA2.

PLA2-Triggered Activation of Cyclosporine-Phospholipid Prodrug as a Drug Targeting Approach in Inflammatory Bowel Disease Therapy.

Oral medication with activity specifically at the inflamed sites throughout the gastrointestinal tract and limited systemic exposure would be a major advance in our therapeutic approach to inflammatory bowel disease (IBD). For this purpose, we have designed a prodrug by linking active drug moiety to phospholipid (PL), the substrate of phospholipase A2 (PLA2). PLA2 expression and activity is significantly elevated in the inflamed intestinal tissues of IBD patients. Since PLA2 enzyme specifically hydrolyses the sn-2 bond within PLs, in our PL-based prodrug approach, the sn-2 positioned FA is replaced with cyclosporine, so that PLA2 may be exploited as the prodrug-activating enzyme, releasing the free drug from the PL-complex. Owing to the enzyme overexpression, this may effectively target free cyclosporine to the sites of inflammation. Four PL-cyclosporine prodrugs were synthesized, differing by their linker length between the PL and the drug moiety. To study the prodrug activation, a novel enzymatically enriched model was developed, the colonic brush border membrane vesicles (cBBMVs); in this model, tissue vesicles were produced from colitis-induced (vs. healthy) rat colons. PLA2 overexpression (3.4-fold) was demonstrated in diseased vs. healthy cBBMVs. Indeed, while healthy cBBMVs induced only marginal activation, substantial prodrug activation was evident by colitis-derived cBBMVs. Together with the PLA2 overexpression, these data validate our drug targeting strategy. In the diseased cBBMVs, quick and complete activation of the entire dose was obtained for the 12-carbon linker prodrug, while slow and marginal activation was obtained for the 6/8-carbon linkers. The potential to target the actual sites of inflammation and treat any localizations throughout the GIT, together with the extended therapeutic index, makes this orally delivered prodrug approach an exciting new therapeutic strategy for IBD treatment.

Molecular insights into the primary nucleation of polymorphic amyloid ß dimers in DOPC lipid bilayer membrane.

Alzheimer's disease (AD) pathology is characterized by loss of memory cognitive and behavioral deterioration. One of the hallmarks of AD is amyloid ß (Aß) plaques in the brain that consists of Aß oligomers and fibrils. It is accepted that oligomers, particularly dimers, are toxic species that are produced extracellularly and intracellularly in membranes. It is believed that the disruption of membranes by polymorphic Aß oligomers is the key for the pathology of AD. This is a first study that investigate the effect of polymorphic "α-helix/random coil" and "fibril-like" Aß dimers on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane. It has been found that the DOPC membrane promotes Aß1-42 "fibril-like" dimers and impedes Aß1-42 "α-helix/random coil" dimers. The N-termini domains within Aß1-42 dimers play a role in Aß aggregation in membrane milieus. In addition, the aromatic π-π interactions (involving residues F19 and F20 in Aß1-42 ) are the driving forces for the hydrophobic interactions that initiate the primary nucleation of polymorphic Aß1-42 dimers within DOPC membrane. Finally, the DOPC bilayer membrane thickness is locally decreased, and it is disrupted by an embedded distinct Aß1-42 dimer, due to relatively large contacts between Aß1-42 monomers and the DOPC membrane. This study reveals insights into the molecular mechanisms by which polymorphic early-stage Aß1-42 dimers have distinct impacts on DOPC membrane.

Molecular Insights into the Effect of Metals on Amyloid Aggregation.

Amyloid diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and type 2 diabetes (T2D) are characterized by accumulation of misfolded proteins' species, e.g., oligomers and fibrils. The formation of these species occurs via self-assemble of the misfolded proteins in a process which is named "aggregation." It is known that essential divalent metal ions initiate the aggregation of these misfolded proteins, and that specific concentrations of these metal ions may be implicated in the pathology of amyloid diseases. This chapter focuses on the effects of two of the most common divalent metal ions in the brain-Zn2+ and Cu2+, and while Zn2+ ion is known as a metal that is release from the pancreas. Specifically, the spotlight of this chapter illustrates recent computational molecular modelling studies that investigate the effect of the concentrations of metal ions on aggregation of the misfolded proteins amylin, amyloid ß, and α-synuclein. The challenges for computational molecular modeling and future perspectives are discussed.

Insights into the Interactions that Trigger the Primary Nucleation of Polymorphic α-Synuclein Dimers.

Parkinson's disease is associated with the accumulation of α-synuclein (AS) aggregates that include polymorphic AS oligomers and polymorphic fibrils. There have been advances in solving the polymorphic state of AS fibrils, both by experimental techniques and molecular modeling tools. Yet, the polymorphic AS oligomers are now considered as the neurotoxic species, thus current and future studies making efforts to solve their structures at the molecular level. Importantly, it is crucial to explore the specific interactions between AS monomers within the dimer that stabilize the dimer and yield nucleation. Herein, we present a first work that probes at the molecular level the specific interactions between monomers in polymorphic AS dimers are derived from AS fibrils by applying molecular modeling tools. Our work reveals that both N-terminal and the non-amyloidogenic component domains play a role in the dimerization of all polymorphic AS dimers. In addition, helices along the N-terminal of AS monomers impede the contacts between AS monomers, thus preventing the nucleation or the dimerization of AS. This work provides insights into several mechanisms of the production of polymorphic AS dimers. Thus, the findings obtained in this work may assist in developing new therapeutic strategies for inhibiting the formation of the early-stage neurotoxic AS dimers.

Advancements and future directions in research of the roles of insulin in amyloid diseases.

Amyloid diseases, such as type 2 diabetes, Alzheimer's disease and Parkinson's disease are characterized by amyloid aggregates. Insulin is released from the pancreas, and it is known that insulin downstream signaling molecules are located majorly in the regions of cortex and hippocampus. Therefore, insulin plays crucial roles not only in the pancreas, but also in the brain. Recent studies have focused on the role of insulin in amyloid diseases. This review demonstrates the recent studies in which insulin affects amyloid aggregation. Specifically, molecular modeling studies provide insights into the molecular mechanisms of the effects of insulin in amyloid aggregates. Still, experimental studies are required to provide insights into the kinetics effects. This review opens new avenues for future studies on insulin molecules and amyloid aggregation.

Mechanism of Side Chain-Controlled Proton Conductivity in Bioinspired Peptidic Nanostructures.

Bioinspired peptide assemblies are promising candidates for use as proton-conducting materials in electrochemical devices and other advanced technologies. Progress toward applications requires establishing foundational structure-function relationships for transport in these materials. This experimental-theoretical study sheds light on how the molecular structure and proton conduction are linked in three synthetic cyclic peptide nanotube assemblies that comprise the three canonical basic amino acids (lysine, arginine, and histidine). Experiments find an order of magnitude higher proton conductivity for lysine-containing peptide assemblies compared to histidine and arginine containing assemblies. The simulations indicate that, upon peptide assembly, the basic amino acid side chains are close enough to enable direct proton transfer. The proton transfer kinetics is determined in the simulations to be governed by the structure and flexibility of the side chains. Together, experiments and theory indicate that the proton mobility is the main determinant of proton conductivity, critical for the performance of peptide-based devices.

Insights into the Mechanistic Perspective Effect of Insulin on the Nonamyloidogenic Component (NAC) and α-Synuclein Aggregation.

Insulin plays important functions in the brain, such as neuroprotective effects on neurons, and it is also involved in cognitive functions (e.g., attention, learning and memory). It is proposed that a lack of insulin in the brain may initiate development of neurodegenerative diseases. Herein, we examined the effect of insulin on aggregates of α-synuclein (AS), a protein that is related to Parkinson's disease (PD), and its segment nonamyloidogenic component (NAC), which is known to play a crucial role in AS aggregation. The molecular modeling tools assist us to provide insights into the molecular mechanism of the effect of insulin on fibrillation of NAC and AS. Our research leads to three conclusions. First, the preferred interactions between insulin chain B and the "zipper domain" sequence within both NAC and AS appear at the central domain across the fibril axis or at the edge of the fibril. Second, these interactions do not disrupt the cross-ß structure of NAC fibril-like oligomers but disrupt the cross-ß structure of AS fibril-like oligomers. Thus, insulin does not inhibit the fibrillation of NAC but may inhibit AS fibrillation. Third, some of the polymorphic NAC and AS fibril-like oligomers bind to chain A in insulin. This is the first study that demonstrates that insulin chain A can also participate in the interactions with amyloid fibril-like oligomers. Our study proposes that insulin plays a crucial role in impeding AS aggregation in the brain and consequently could inhibit the development of PD.

Amyloid Oligomers: A Joint Experimental/Computational Perspective on Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, and Amyotrophic Lateral Sclerosis.

Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, in vitro, in vivo, and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aß, tau), α-synuclein, IAPP, and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D), and amyotrophic lateral sclerosis (ALS) research, respectively, for many years.

Neuropeptides: Roles and Activities as Metal Chelators in Neurodegenerative Diseases.

Neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by deposits of amyloid proteins. The homeostasis of metal ions is crucial for the normal biological functions in the brain. However, in AD and PD, the imbalance of metal ions leads to formation of amyloid deposits. In the past four decades, there has been extensive effort to design compound agents than can chelate metal ions with the aim of preventing the formation of the amyloid deposits. Unfortunately, the compounds to date that were designed were not successful candidates to be used in clinical trials. Neuropeptides are small molecules that are produced and released by neurons. It has been shown that neuropeptides have neuroprotective effects in the brain and reduce the formation of amyloid deposits. This Review Article is focused on the function of neuropeptides as metal chelators. Experimental and computational studies demonstrated that neuropeptides could bind metal ions, such as Cu2+ and Zn2+. This Review Article provides perspectives and initiates future studies to investigate the role of neuropeptides as metal chelators in neurodegenerative diseases.

Molecular Mechanisms and Aspects on the Role of Neuropeptide Y as a Zn2+ and Cu2+ Chelator.

The concept of metal chelation is based on simple coordination chemistry. The development of an ideal metal chelator that completely and selectively removes toxic metals from a specific metal binding site in proteins is required to prevent and or inhibit a variety of diseases, among them neurodegenerative diseases. This work examines neuropeptide Y (NPY) as a Zn2+ and Cu2+ chelator agent. NPY is a natural peptide that is produced in the human body; therefore, it is not a toxic agent and the complex that it forms is not toxic as well. Our simulations reveal that NPY has an efficient Zn2+ chelation activity but is less effective in chelating Cu2+. Moreover, while NPY demonstrates several conformations, the metal chelation occurs more efficiently in its native structure. Beyond the exploration of the activity of NPY as a Zn2+ and Cu2+ chelator agent, this work provides an insight into the molecular mechanisms of the chelation of these metals at the molecular level. The outcomes from this work may guide future experimental studies to examine NPY in metal chelation therapy for neurodegenerative diseases.

Inhibition of Osteoclast Differentiation by Carotenoid Derivatives through Inhibition of the NF-ƙB Pathway.

The bone protective effects of carotenoids have been demonstrated in several studies, and the inhibition of RANKL-induced osteoclast differentiation by lycopene has also been demonstrated. We previously reported that carotenoid oxidation products are the active mediators in the activation of the transcription factor Nrf2 and the inhibition of the NF-ƙB transcription system by carotenoids. Here, we demonstrate that lycopene oxidation products are more potent than intact lycopene in inhibiting osteoclast differentiation. We analyzed the structure-activity relationship of a series of dialdehyde carotenoid derivatives (diapocarotene-dials) in inhibiting osteoclastogenesis. We found that the degree of inhibition depends on the electron density of the carbon atom that determines the reactivity of the conjugated double bond in reactions such as Michael addition to thiol groups in proteins. Moreover, the carotenoid derivatives attenuated the NF-ƙB signal through inhibition of IƙB phosphorylation and NF-ƙB translocation to the nucleus. In addition, we show a synergistic inhibition of osteoclast differentiation by combinations of an active carotenoid derivative with the polyphenols curcumin and carnosic acid with combination index (CI) values < 1. Our findings suggest that carotenoid derivatives inhibit osteoclast differentiation, partially by inhibiting the NF-ƙB pathway. In addition, carotenoid derivatives can synergistically inhibit osteoclast differentiation with curcumin and carnosic acid.

Assessments of the Effect of Neurokinin B on Toxic Aß Aggregates in Alzheimer's Disease with the Molecular Mechanisms' Action.

Clinical trials of past and current treatments for Alzheimer's disease (AD) patients on the market suffer from the dual drawbacks of a lack of efficacy and side effects. Neuropeptides have been highlighted by their potential to protect cells against AD and can reverse the toxic effect induced by Aß in cultured neurons. One of the neuropeptides that has insufficient attention in the literature as a potential treatment for prevention of the progression of AD is neurokinin B (NKB). There are critical and unresolved questions concerning the activation, and the molecular mechanisms underlying NKB effect on prevention of Aß aggregation remain unknown. The current work identifies for the first time the specific interactions that contribute to the inhibition and prevention of initial seeding of polymorphic early-stage dimers. Three main conclusions are observed in this work. First, NKB inhibits formation of polymorphic early-stage fibrillar Aß dimers. The efficiency of the inhibition depends on the concentration of NKB (i.e., NKB:Aß ratio). Second, NKB has an excellent effect of preventing the formation of initial seeding of early-stage nonfibrillar Aß dimers. Third, NKB peptides may self-assemble to form cross-α fibril-like structure during the inhibition activity of the polymorphic early-stage fibrillar Aß dimers but not during the prevention activity of early-stage nonfibrillar Aß dimers. The work provides crucial information for future experimental studies to approve the functional effect of NKB on inhibition and prevention of Aß polymorphic early-stage oligomers.

Controlling the properties and self-assembly of helical nanofibrils by engineering zinc-binding ß-hairpin peptides.

This work illustrates a series of novel peptides that have the capability to bind Zn2+ ions and to produce fibrillar structures. The location and the type of the residues along the peptide sequence can determine the nature of the fibril. This work presents a proof-of-concept milestone for designing peptides with different properties to produce diverse materials.