muramidase and Parkinson-Disease

The abrupt aggregation of misfolded proteins is linked to the onset and spread of amyloidogenic diseases, including diabetes type 2, systemic amyloidosis, and Alzheimer's (AD) and Parkinson's diseases (PD). Although the exact cause of these pathological processes is unknown, a growing body of evidence suggests that amyloid diseases are triggered by misfolded or unfolded proteins, forming highly toxic oligomers. These transient species exhibit high structural and morphological heterogeneity. Protein oligomers can also propagate into β-sheet-rich filaments that braid and coil with other filaments to form amyloid fibrils and supramolecular structures with both flat and twisted morphologies. Microscopic examination of protein deposits formed in the brains of both AD and PD patients revealed the presence of fragments of lipid membranes. Furthermore, nanoscale infrared analysis of

Topics: Amyloid; Amyloidogenic Proteins; Animals; Humans; Insulins; Muramidase; Parkinson Disease; Phospholipids; Rats

Alpha-synuclein (α-syn) aggregation and mitochondrial dysfunction are considered as two of the main factors associated with Parkinson's disease (PD). In the present investigation, the effectiveness of the amyloid fibrils obtained from α-syn with those of hen egg white lysozyme (HEWL), as disease-related and-unrelated proteins, to damage rat brain and rat liver mitochondria have been investigated. This was extended by looking at SH-SY5Y human neuroblastoma cells and erythrocytes, thereby investigating the significance of structural characteristics of amyloid fibrils related to their interactions with biomembranes obtained from various sources. Results presented clearly demonstrate substantial differences in the response of tested biomembranes to toxicity induced by α-syn/HEWL amyloid fibrils, highlighting a structure-function relationship. We found that fibrillar aggregates of α-syn, but not HEWL, caused a significant increase in mitochondrial ROS, loss of membrane potential, and mitochondrial swelling, in a dose-dependent manner. Toxicity was found to be more pronounced in brain mitochondria, as compared to liver mitochondria. For SH-SY5Y cells and erythrocytes, however, both α-syn and HEWL amyloid fibrils showed the capacity to induce toxicity. Taken together, these results may suggest selective toxicity of α-syn amyloid fibrils to mitochondria mediated likely by their direct interaction with the outer mitochondrial membrane, indicating a correlation between specific structural characteristics of α-syn fibrils and an organelle strongly implicated in PD pathology.

Topics: alpha-Synuclein; Amyloid; Animals; Brain; Cell Line, Tumor; Cell Membrane; Chickens; Egg White; Erythrocytes; Humans; Membrane Potential, Mitochondrial; Mitochondria, Liver; Muramidase; Parkinson Disease; Rats; Structure-Activity Relationship

Extensive research has shown that assembling of α-synuclein amyloid aggregates on mitochondria is an important mechanistic feature of Parkinson's disease (PD) and other Lewy body disorders. However, the molecular mechanism(s) of its neuronal toxicity remain unclear. Type 1 Hexokinase (HKI), a key enzyme in the control of brain glucose metabolism, plays an important role in protecting against mitochondrially-regulated apoptosis through reducing generation of reactive oxygen species (ROS). The release of mitochondrially-bound HKI causes a significant decrease in enzyme activity and triggers oxidative stress. Here, we have investigated the potency of amyloid fibrillation products arising from α-synuclein and hen egg white lysozyme (HEWL) for the release of HKI and ROS content enhancement in mitochondria isolated from rat brain. Results clearly indicate the capacity of the fibrillation products of α-synuclein, and not HEWL, to trigger release of HKI from the Type A binding site of mitochondria for the enzyme and to induce mitochondrial ROS enhancement in a dose-dependent manner. Moreover, we found that curcumin was very effective in preventing mitochondrial HKI release and ROS enhancement induced by α-synuclein fibrillation products. The pathophysiological significance of mitochondrial HKI activity and localization in pathogenesis of neurodegenerative disorders including PD are discussed. Taken together, these results may offer insight into a possible mechanism by which disease-related peptides and proteins may exert their neuronal toxicity.

Topics: alpha-Synuclein; Amyloid; Animals; Apoptosis; Brain; Chickens; Curcumin; Hexokinase; Humans; Mitochondria; Muramidase; Oxidative Stress; Parkinson Disease; Protective Agents; Rats; Reactive Oxygen Species

More than 20 unique diseases such as diabetes, Alzheimer's disease, Parkinson's disease are caused by the abnormal aggregations of pathogenic proteins such as amylin, β-amyloid (Aβ), and α-synuclein. All pathogenic proteins differ from each other in biological function, primary sequences, and morphologies; however, the proteins are toxic when aggregated. Here, we investigated the cellular toxicity of pathogenic or non-pathogenic protein aggregates. In this study, six proteins were selected and they were incubated at acid pH and high temperature. The aggregation kinetic and cellular toxicity of protein species with time were characterized. Three non-pathogenic proteins, bovine serum albumin (BSA), catalase, and pepsin at pH 2 and 65 °C were stable in protein structure and non-toxic at a lower concentration of 1 mg/mL. They formed aggregates at a higher concentration of 20 mg/mL with time and they induced the toxicity in short incubation time points, 10 min and 20 min only and they became non-toxic after 30 min. Other three pathogenic proteins, lysozyme, superoxide dismutase (SOD), and insulin, also produced the aggregates with time and they caused cytotoxicity at both 1 mg/mL and 20 mg/mL after 10 min. TEM images and DSC analysis demonstrated that fibrils or aggregates at 1 mg/mL induced cellular toxicity due to low thermal stability. In DSC data, fibrils or aggregates of pathogenic proteins had low thermal transition compared to fresh samples. The results provide useful information to understand the aggregation and cellular toxicity of pathogenic and non-pathogenic proteins.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Catalase; Cell Line; Diabetes Mellitus; Humans; Insulin; Islet Amyloid Polypeptide; Models, Molecular; Muramidase; Parkinson Disease; Pepsin A; Protein Aggregates; Protein Aggregation, Pathological; Protein Structure, Secondary; Serum Albumin, Bovine; Superoxide Dismutase

Increasing evidence proposed that amyloid deposition by proteins play a crucial role in an array of neurotoxic and degenerative disorders like Parkinson's disease, systemic amyloidosis etc, that could be controlled by anti-aggregation methodologies which either inhibit or disaggregate such toxic aggregates. The present work targets the amyloid inhibiting and disaggregating potential of promethazine (PRM) against human insulin (HI) and human lysozyme (HL) fibrillogenesis. Biophysical techniques like Rayleigh scattering measurements (RLS), Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence measurement, circular dichroism (CD) and dynamic light scattering (DLS) measurements illustrated the inhibitory action of PRM. The half maximal inhibitory concentration (IC

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Anilino Naphthalenesulfonates; Benzothiazoles; Circular Dichroism; Dynamic Light Scattering; Fluorescence; Humans; Insulin; Muramidase; Parkinson Disease; Promethazine; Protein Aggregates; Protein Aggregation, Pathological; Thiazoles

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; beta 2-Microglobulin; Humans; Immunoglobulins; Models, Molecular; Muramidase; Parkinson Disease; Prealbumin; Protein Folding; Protein Interaction Domains and Motifs; Protein Multimerization; Superoxide Dismutase

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most prevalent neurodegenerative diseases of the central nervous system. These two diseases share a common feature in that a normally soluble peptide (amyloid-beta) or protein (alpha-synuclein) aggregates into an ordered fibrillar structure. As well as structural similarities observed between fibrillar aggregates related to these diseases, common pathological processes of increased oxidative injury, excitotoxicity and altered cell cycle are also evident. It was the aim of this study to identify novel interacting proteins to the amyloid-like motif and therefore identify common potential pathways between neurodegenerative diseases that share biophysical properties common to classical amyloid fibrils. Optimal ageing of recombinant proteins to form amyloid-like fibrils was determined by electron microscopy, Congo red birefringement and photo-induced cross-linking. Using pull-down assays the strongest detected interacting protein to the amyloid-like motifs of amyloid-beta, alpha-synuclein and lysozyme was identified as histone H1. The interaction with the amyloid-like motif was confirmed by techniques including surface plasmon resonance and immunohistochemistry. Histone H1 is known to be an integral part of chromatin within the nucleus, with a primary role of binding DNA that enters and exits from the nucleosome, and facilitating the shift in equilibrium of chromatin towards a more condensed form. However, phosphorylated histone H1 is predominantly present in the cytoplasm and as yet the functional significance of this translocation is unknown. This study also found that histone H1 is localised within the cytoplasm of neurons and astrocytes from areas affected by disease as well as amyloid plaques, supporting the hypothesis that histone H1 favoured binding to an ordered fibrillar motif. We conclude that the binding of histone H1 to a general amyloid-like motif indicates that histone H1 may play an important common role in diseases associated with amyloid-like fibrils.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Animals; Astrocytes; Brain; Cells, Cultured; Histones; Humans; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Muramidase; Neurons; Parkinson Disease; Plaque, Amyloid; Protein Binding; Recombinant Proteins; Surface Plasmon Resonance

Alpha-synuclein is one of the causative proteins of familial Parkinson disease, which is characterized by neuronal inclusions named Lewy bodies. Lewy bodies include not only alpha-synuclein but also aggregates of other proteins. This fact raises a question as to whether the formation of alpha-synuclein amyloid fibrils in Lewy bodies may occur via interaction with fibrils derived from different proteins. To probe this hypothesis, we investigated in vitro fibril formation of human alpha-synuclein in the presence of preformed fibril seeds of various different proteins. We used three proteins, Escherichia coli chaperonin GroES, hen lysozyme, and bovine insulin, all of which have been shown to form amyloid fibrils. Very surprisingly, the formation of alpha-synuclein amyloid fibril was accelerated markedly in the presence of preformed seeds of GroES, lysozyme, and insulin fibrils. The structural characteristics of the natively unfolded state of alpha-synuclein may allow binding to various protein particles, which in turn triggers the formation (extension) of alpha-synuclein amyloid fibrils. This finding is very important for understanding the molecular mechanism of Parkinson disease and also provides interesting implications into the mechanism of transmissible conformational diseases.

Topics: alpha-Synuclein; Amyloid; Animals; Benzothiazoles; Biochemistry; Cattle; Chaperonin 10; Chickens; Circular Dichroism; Escherichia coli; Humans; Insulin; Lewy Bodies; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Models, Biological; Molecular Chaperones; Muramidase; Parkinson Disease; Peptides; Protein Binding; Protein Conformation; Scattering, Radiation; Thiazoles; X-Rays

Recently, impairment of the ubiquitin-proteasome system is suggested to be responsible for the neuronal death in ageing and Parkinson's disease. The specific degeneration of dopamine neurons containing neuromelanin (NM) suggests that NM itself may be involved in the cellular dysfunction and death, even though the direct link has never been reported. We examined the effects of NM isolated from the human substantia nigra on the proteasome activity in human dopaminergic SH-SY5Y cells. NM reduced the activities of 26S proteasome, as shown in situ using a green fluorescent protein homologue targeted to 26S proteasome and also in vitro using ubiquitinated lysozyme as a substrate. However, NM did not affect 20S proteasome activity in vitro. NM reduced the amount of PA700 regulatory subunit of 26S proteasome, but did not affect that of alpha- and beta-subunits of 20S proteasome. These results suggest that NM may inhibit the ubiquitin-26S proteasome system, and determine the selective vulnerability of dopamine neurons in ageing and related disorders.

Topics: Adult; Aging; Cell Death; Chymotrypsin; Dopamine; Genetic Vectors; Green Fluorescent Proteins; Humans; In Vitro Techniques; Melanins; Microscopy, Phase-Contrast; Muramidase; Ornithine Decarboxylase; Parkinson Disease; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Spectrometry, Fluorescence; Ubiquitin

Parkinson's disease is the second most common age-related neurodegenerative disease, resulting from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of alpha-synuclein has been implicated as a causative factor in the disease, and the process of fibril formation has been intensively studied in vitro with dilute protein solutions. However, the intracellular environment of proteins is crowded with other macromolecules, whose concentration can reach 400 g/l. To address this discrepancy, the effect of molecular crowding on alpha-synuclein fibrillation has being studied. The addition of high concentrations of different polymers (proteins, polysaccharides and polyethylene glycols) dramatically accelerated alpha-synuclein fibrillation in vitro. The magnitude of the accelerating effect depended on the nature of the polymer, its length and concentration. Our results suggest that the major factor responsible for the accelerated fibrillation under crowded conditions is the excluded volume.

Topics: alpha-Synuclein; Chemical Phenomena; Chemistry, Physical; Dose-Response Relationship, Drug; Humans; Macromolecular Substances; Muramidase; Nerve Tissue Proteins; Parkinson Disease; Polyethylene Glycols; Polysaccharides; Protein Binding; Serum Albumin, Bovine; Synucleins