Kunihiro Hongo

Amyloid Fibril Formation of α-Synuclein Is Accelerated by Preformed Amyloid Seeds of Other Proteins IMPLICATIONS FOR THE MECHANISM OF TRANSMISSIBLE CONFORMATIONAL DISEASES

α-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 α-synuclein but also aggregates of other proteins. This fact raises a question as to whether the formation of α-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 α-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 α-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 α-synuclein may allow binding to various protein particles, which in turn triggers the formation (extension) of α-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.

Hydrophilic Residues 526KNDAAD531 in the Flexible C-terminal Region of the Chaperonin GroEL Are Critical for Substrate Protein Folding within the Central Cavity

The final 23 residues in the C-terminal region of Escherichia coli GroEL are invisible in crystallographic analyses due to high flexibility. To probe the functional role of these residues in the chaperonin mechanism, we generated and characterized C-terminal truncated, double ring, and single ring mutants of GroEL. The ability to assist the refolding of substrate proteins rhodanese and malate dehydrogenase decreased suddenly when 23 amino acids were truncated, indicating that a sudden change in the environment within the central cavity had occurred. From further experiments and analyses of the hydropathy of the C-terminal region, we focused on the hydrophilicity of the sequence region 526KNDAAD531 and generated two GroEL mutants where these residues were changed to a neutral hydropathy sequence (526GGGAAG531) and a hydrophobic sequence (526IGIAAI531), respectively. Very interestingly, the two mutants were found to be defective in function both in vitro and in vivo. Deterioration of function was not observed in mutants where this region was replaced by a scrambled (526NKADDA531) or homologous (526RQEGGE531) sequence, indicating that the hydrophilicity of this sequence was important. These results highlight the importance of the hydrophilic nature of 526KNDAAD531 residues in the flexible C-terminal region for proper protein folding within the central cavity of GroEL.

Chaperonin GroE and ADP facilitate the folding of various proteins and protect against heat inactivation

In the presence of ADP, the molecular chaperones GroEL and GroES from Escherichia coli not only facilitated the refolding of various proteins, but also prevented their irreversible heat inactivation in vitro. Without nucleotides the refolding reactions were arrested by GroEL. Addition of GroES and ADP to the reaction mixture initiated at the refolding reactions and the enzyme activities were regained efficiently. The presence of GroE(GroEL and GroES) and ADP also protected against heat inactivation of native enzymes at various temperatures. These findings suggest that in the presence of GroES, nucleotide binding is an important event in the mechanism of GroEL‐facilitated protein folding.

A novel ATP/ADP hydrolysis activity of hyperthermostable group II chaperonin in the presence of cobalt or manganese ion

A novel ATPase activity that was strongly activated in the presence of either cobalt or manganese ion was discovered in the chaperonin from hyperthermophilic Pyrococcus furiosus (Pfu‐cpn). Surprisingly, a significant ADPase activity was also detected under the same conditions. A more extensive search revealed similar nucleotide hydrolysis activities in other thermostable chaperonins. Chaperonin activity, i.e., thermal stabilization and refolding of malate dehydrogenase from the guanidine‐hydrochloride unfolded state were also detected for Pfu‐cpn under the same conditions. We propose that the novel cobalt/manganese‐dependent ATP/ADPase activity may be a common trait of various thermostable chaperonins.

Role of C-terminal negative charges and tyrosine residues in fibril formation of α-synuclein

α‐Synuclein (140 amino acids), one of the causative proteins of Parkinsons disease, forms amyloid fibrils in brain neuronal cells. In order to further explore the contributions of the C‐terminal region of α‐synuclein in fibril formation and also to understand the overall mechanism of fibril formation, we reduced the number of negatively charged residues in the C‐terminal region using mutagenesis. Mutants with negative charges deleted displayed accelerated fibril formation compared with wild‐type α‐synuclein, demonstrating that negative charges located in the C‐terminal region of α‐synuclein modulate fibril formation. Additionally, when tyrosine residues located at position 125, 133, and 136 in the C‐terminal region were changed to alanine residue(s), we found that all mutants containing the Tyr136Ala mutation showed delays in fibril formation compared with wild type. Mutation of Tyr136 to various amino acids revealed that aromatic residues located at this position act favorably toward fibril formation. In mutants where charge neutralization and tyrosine substitution were combined, we found that these two factors influence fibril formation in complex fashion. These findings highlight the importance of negative charges and aromatic side chains in the C‐terminal region of α‐synuclein in fibril formation.

Fibril Formation of hsp10 Homologue Proteins and Determination of Fibril Core Regions: Differences in Fibril Core Regions Dependent on Subtle Differences in Amino Acid Sequence

Heat shock protein 10 (hsp10) is a member of the molecular chaperones and works with hsp60 in mediating various protein folding reactions. GroES is a representative protein of hsp10 from Escherichia coli. Recently, we found that GroES formed a typical amyloid fibril from a guanidine hydrochloride (Gdn-HCl) unfolded state at neutral pH. Here, we report that other hsp10 homologues, such as human hsp10 (Hhsp10), rat mitochondrial hsp10 (Rhsp10), Gp31 from T4 phage, and hsp10 from the hyperthermophilic bacteria Thermotoga maritima, also form amyloid fibrils from an unfolded state. Interestingly, whereas GroES formed fibrils from either the Gdn-HCl unfolded state (at neutral pH) or the acidic unfolded state (at pH 2.0-3.0), Hhsp10, Rhsp10, and Gp31 formed fibrils from only the acidic unfolded state. Core peptide regions of these protein fibrils were determined by proteolysis treatment followed by a combination of Edman degradation and mass spectroscopy analyses of the protease-resistant peptides. The core peptides of GroES fibrils were identical for fibrils formed from the Gdn-HCl unfolded state and those formed from the acidic unfolded state. However, a peptide with a different sequence was isolated from fibrils of Hhsp10 and Rhsp10. With the use of synthesized peptides of the determined core regions, it was also confirmed that the identified regions were capable of fibril formation. These findings suggested that GroES homologues formed typical amyloid fibrils under acidic unfolding conditions but that the fibril core structures were different, perhaps owing to differences in local amino acid sequences.

Anthocyanin suppresses the toxicity of Aβ deposits through diversion of molecular forms in in vitro and in vivo models of Alzheimer's disease

Objectives: The pathogenesis of Alzheimers disease (AD) is strongly correlated with the aggregation and deposition of the amyloid beta (Aβ1–42) peptide in fibrillar form, and many studies have shown that plant-derived polyphenols are capable of attenuating AD progression in various disease models. In this study, we set out to correlate the effects of anthocyanoside extracts (Vaccinium myrtillus anthocyanoside (VMA)) obtained from bilberry on the in vitro progression of Aβ fibril formation with the in vivo effects of this compound on AD pathogenesis. Methods: Thioflavin T fluorescence assays and atomic force microscopy were used to monitor Aβ amyloid formation in in vitro assays. Effects of Aβ amyloids on cellular viability were assayed using cultured Neuro2a cells. Cognitive effects were probed using mice that simultaneously expressed mutant human Aβ precursor and mutant presenilin-2. Results: Addition of VMA inhibited the in vitro formation of Aβ peptide fibrils and also reduced the toxicity of these aggregates toward Neuro2a cells. A diet containing 1% VMA prevented the cognitive degeneration in AD mice. Curiously, this diet-derived retention of cognitive ability was not accompanied by a reduction in aggregate deposition in brains; rather, an increase in insoluble deposits was observed compared with mice raised on a control diet. Discussion: The paradoxical increase in insoluble deposits caused by VMA suggests that these polyphenols divert Aβ aggregation to an alternate, non-toxic form. This finding underscores the complex effects that polyphenol compounds may exert on amyloid deposition in vivo.

Isolation of short peptide fragments from α-synuclein fibril core identifies a residue important for fibril nucleation: A possible implication for diagnostic applications

alpha-Synuclein is one of the causative proteins of the neurodegenerative disorder, Parkinsons disease. Deposits of alpha-synuclein called Lewy bodies are a hallmark of this disorder, which is implicated in its progression. In order to understand the mechanism of amyloid fibril formation of alpha-synuclein in more detail, in this study we have isolated a specific, ~20 residue peptide region of the alpha-synuclein fibril core, using a combination of Edman degradation and mass-spectroscopy analyses of protease-resistant samples. Starting from this core peptide sequence, we then synthesized a series of peptides that undergo aggregation and fibril formation under similar conditions. Interestingly, in a derivative peptide where a crucial phenylalanine residue was changed to a glycine, the ability to initiate spontaneous fibril formation was abolished, while the ability to extend from preexisting fibril seeds was conserved. This fibril extension occurred irrespective of the source of the initial fibril seed, as demonstrated in experiments using fibril seeds of insulin, lysozyme, and GroES. This interesting ability suggests that this peptide might form the basis for a possible diagnostic tool useful in detecting the presence of various fibrillogenic factors.

Covalent Structural Changes in Unfolded GroES That Lead to Amyloid Fibril Formation Detected by NMR INSIGHT INTO INTRINSICALLY DISORDERED PROTEINS

Co-chaperonin GroES from Escherichia coli works with chaperonin GroEL to mediate the folding reactions of various proteins. However, under specific conditions, i.e. the completely disordered state in guanidine hydrochloride, this molecular chaperone forms amyloid fibrils similar to those observed in various neurodegenerative diseases. Thus, this is a good model system to understand the amyloid fibril formation mechanism of intrinsically disordered proteins. Here, we identified a critical intermediate of GroES in the early stages of this fibril formation using NMR and mass spectroscopy measurements. A covalent rearrangement of the polypeptide bond at Asn45-Gly46 and/or Asn51-Gly52 that eventually yield β-aspartic acids via deamidation of asparagine was observed to precede fibril formation. Mutation of these asparagines to alanines resulted in delayed nucleus formation. Our results indicate that peptide bond rearrangement at Asn-Gly enhances the formation of GroES amyloid fibrils. The finding provides a novel insight into the structural process of amyloid fibril formation from a disordered state, which may be applicable to intrinsically disordered proteins in general.

Functional Characterization of the Recombinant Group II Chaperonin α from Thermoplasma acidophilum

The functional characteristics of group II chaperonins, especially those from archaea, have not been elucidated extensively. Here, we performed a detailed functional characterization of recombinant chaperonin alpha subunits (16-mer) (Ta-cpn alpha) from the thermophilic archaea Thermoplasma acidophilum as a model protein of archaeal group II chaperonins. Recombinant Ta-cpn alpha formed an oligomeric ring structure similar to that of native protein, and displayed an ATP hydrolysis activity (optimal temperature: 60 degrees C) in the presence of either magnesium, manganese or cobalt ions. Ta-cpn alpha was able to bind refolding intermediates of Thermus MDH and GFP in the absence of ATP, and to promote the refolding of Thermus MDH at 50 degrees C in the presence of Mg2+-, Mn2+-, or Co2+-ATP. Ta-cpn alpha also prevented thermal aggregation of rhodanese and luciferase at 50 degrees C. Interestingly, Ta-cpn alpha in the presence of Mn2+ ion showed an increased hydrophobicity, which correlated with an increased efficiency in substrate protein binding. Our finding that Ta-cpn alpha chaperonin system displays folding assistance ability with ATP-dependent substrate release may provide a detailed look at the potential functional capabilities of archaeal chaperonins.