• Bulletin of the Korean Chemical Society
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• v.27 no.7
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• pp.1001-1004
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• 2006

The m-calpain activity hydrolyzing a fluorogenic substrate of N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcourmarin (LLVY-AMC) was significantly stimulated by more than two-fold in the presence of 5$\mu$M $\alpha$synuclein at $15{^{\circ}C}$. The stimulation was also confirmed with azocasein. The stimulation of the peptide hydrolyzing activity required structural intactness of $\alpha$-synuclein since the C-terminally or N-terminally modified proteins such as $\beta$-synuclein, $\alpha$-syn1-97, and $\alpha$-syn61-140 did not increase the proteolytic activity. Instead, however, the N-terminally truncated $\alpha$-syn61-140 was shown to drastically suppress the calpain activity. Since the N-terminal truncation was known to be the primary cleaving event of calpain-mediated proteolysis of $\alpha$-synuclein and the $\alpha$-syn61-140 has been demonstrated to be resistant against the calpain digestion, it has been proposed that the intracellular calpain activity could be regulated in a reciprocal manner by $\alpha$-synuclein and its proteolyzed C-terminal fragment. Based on the results, a possible physiological function of $\alpha$-synuclein has been suggested as a calpain regulator which contains both stimulatory and inhibitory activities.

• BMB Reports
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• v.39 no.3
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• pp.253-262
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• 2006

Parkinson's disease (PD) is a common neurodegenerative disorder and is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Although many studies showed that the aggregation of $\alpha$-synuclein might be involved in the pathogenesis of PD, its protective properties against oxidative stress remain to be elucidated. In this study, human wild type and mutant $\alpha$-synuclein genes were fused with a gene fragment encoding the nine amino acid trans activator of transcription (Tat) protein transduction domain of HIV-l in a bacterial expression vector to produce a genetic in-frame WT Tat-$\alpha$-synuclein (wild type) and mutant Tat-a-synucleins (mutants; A30P and A53T), respectively, and we investigated the protective effects of wild type and mutant Tat-$\alpha$-synucleins in vitro and in vivo. WT Tat-$\alpha$-synuclein rapidly transduced into an astrocyte cells and protected the cells against paraquat induced cell death. However, mutant Tat-$\alpha$-synucleins did not protect at all. In the mice models exposed to the herbicide paraquat, the WT Tat-$\alpha$-synuclein completely protected against dopaminergic neuronal cell death, whereas mutants failed in protecting against oxidative stress. We found that these protective effects were characterized by increasing the expression level of heat shock protein 70 (HSP70) in the neuronal cells and this expression level was dependent on the concentration of transduced WT Tat-$\alpha$-synuclein. These results suggest that transduced Tat-$\alpha$-synuclein might protect cell death from oxidative stress by increasing the expression level of HSP70 in vitro and in vivo and this may be of potential therapeutic benefit in the pathogenesis of PD.

• Bulletin of the Korean Chemical Society
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• v.35 no.12
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• pp.3542-3546
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• 2014

Relationship between molecular freedom of amyloidogenic protein and its self-assembly into amyloid fibrils has been evaluated with ${\alpha}$-synuclein, an intrinsically unfolded protein related to Parkinson's disease, by restricting its structural plasticity through an end-to-end disulfide bond formation between two newly introduced cysteine residues on the N- and C-termini. Although the resulting circular form of ${\alpha}$-synuclein exhibited an impaired fibrillation propensity, the restriction did not completely block the protein's interactive core since co-incubation with wild-type ${\alpha}$-synuclein dramatically facilitated the fibrillation by producing distinctive forms of amyloid fibrils. The suppressed fibrillation propensity was instantly restored as the structural restriction was unleashed with ${\beta}$-mercaptoethanol. Conformational flexibility of the accreting amyloidogenic protein to pre-existing seeds has been demonstrated to be critical for fibrillar extension process by exerting structural adjustment to a complementary structure for the assembly.

• Bulletin of the Korean Chemical Society
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• v.35 no.2
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• pp.425-430
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• 2014

Amyloidogenic proteins often exhibit fibrillar polymorphism through alternative assembly processes, which has been considered to have possible pathological implications. Here, firefly luciferase (LUC) is shown to induce amyloid polymorphism of ${\alpha}$-synuclein, the major constituent of Lewy bodies found in Parkinson's disease, by acting as a novel template. The drastically accelerated fibrillation kinetics of ${\alpha}$-synuclein with LUC required the nucleation center produced by the active enzyme of LUC. Fluorescent dye binding, transmission electron microscopy, and Fourier transformed infrared spectroscopy revealed the morphologically distinctive amyloid fibrils of ${\alpha}$-synuclein prepared in the absence or presence of LUC. As the altered morphological characteristics became inherent to the mature fibrils, those properties were inherited to next-generations via nucleation-dependent fibrillation process. The seed control, therefore, would be an effective means to modify amyloid fibrils with different biochemical characteristics. In addition, the LUC-directed amyloid fibrillar polymorphism also suggests that other cellular biomolecules including enzymes in general are able to diversify amyloid fibrils, which could be self-propagated with diversified biological activities, if any, inside cells.

• Journal of Microbiology and Biotechnology
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• v.17 no.12
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• pp.2027-2032
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• 2007

[ ${\alpha}$ ]-Synuclein is the major component of Lewy bodies and responsible for the amyloid deposits observed in Parkinson's disease. Ordered filamentous aggregate formation of the natively unfolded ${\alpha}$-synuclein was investigated in vitro with the periodic ultrasonication. The ultrasonication induced the fibrillation of ${\alpha}$-synuclein, as the random structure gradually converted into a ${\beta}$-sheet structure. The resulting fibrils obtained at the stationary phase appeared heterogeneous in their size distribution, with the average length and height of $0.28\;{\mu}m{\pm}0.21\;{\mu}m$ and $5.6\;nm{\pm}1.9\;nm$, respectively. After additional extensive ultrasonication in the absence of monomeric ${\alpha}$-synuclein, the equilibrium between the fibril formation and its breakdown shifted to the disintegration of the preexisting fibrils. The resulting fragments served as nucleation centers for the subsequent seed-dependent accelerated fibrillation under a quiescent incubation condition. This self-seeding amplification process depended on the seed formation and subsequent alterations in their properties by the ultrasonication to a state that accretes the monomeric soluble protein more effectively than their reassociation of the seeds back to the original fibrils. Since many neurodegenerative disorders have been considered to be propagated via the seed-dependent amyloidosis, this study would provide a novel aspect of the significance of the seed structure and its properties leading to the acce]erated amyloid formation.

• BMB Reports
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• v.56 no.12
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• pp.657-662
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• 2023

Neurodegenerative diseases are characterized by distinct protein aggregates, such as those of α-synuclein and tau. Lysosomal defect is a key contributor to the accumulation and propagation of aberrant protein aggregates in these diseases. The discoveries of common proteinopathies in multiple forms of lysosomal storage diseases (LSDs) and the identification of some LSD genes as susceptible genes for those proteinopathies suggest causative links between LSDs and the proteinopathies. The present study hypothesized that defects in lysosomal genes will differentially affect the propagation of α-synuclein and tau proteins, thereby determining the progression of a specific proteinopathy. We established an imaging-based high-contents screening (HCS) system in Caenorhabditis elegans (C. elegans) model, by which the propagation of α-synuclein or tau is measured by fluorescence intensity. Using this system, we performed RNA interference (RNAi) screening to induce a wide range of lysosomal malfunction through knock down of 79 LSD genes, and to obtain the candidate genes with significant change in protein propagation. While some LSD genes commonly affected both α-synuclein and tau propagation, our study identified the distinct sets of LSD genes that differentially regulate the propagation of either α-synuclein or tau. The specificity and efficacy of these LSD genes were retained in the disease-related phenotypes, such as pharyngeal pumping behavior and life span. This study suggests that distinct lysosomal genes differentially regulate the propagation of α-synuclein and tau, and offer a steppingstone to understanding disease specificity.

• Bulletin of the Korean Chemical Society
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• v.30 no.3
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• pp.623-629
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• 2009

We have performed replica-exchange molecular dynamics simulations on 41 residue peptide mainly composed of NAC (non A$\beta$ component) sequence in $\alpha$-Synuclein. To investigate conformational characteristics of intrinsically unstructured peptides, we carried out structural analysis on the ‘representative structures’ for ensemble of structures occurring at different temperatures. The secondary structure profile obtained from our simulations suggests that the NAC region of $\alpha$-synuclein can be divided into roughly three helical-like segments. It is found that the overall helix-turn-helix like topology is conserved even though the conformational fluctuations grow as the temperature increases. The coordinate-based and the distance-based representative structures exhibit noticeable differences at higher temperatures while they are similar at lower temperatures. It is found that structural variations for the coordinate-based representative structures are much larger, suggesting that distance-based representative structures provide more reliable information concerning characteristic features of intrinsically unstructured proteins. The present analysis also indicates that the conformational features of representative structures at high temperatures might be related to those in membrane or low pH environment.

• Bulletin of the Korean Chemical Society
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• v.30 no.8
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• pp.1845-1850
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• 2009

We have performed replica-exchange molecular dynamics (REMD) simulations on the dimer formation of fibrilforming segments of $\alpha$-Synuclein (residues 71 - 82) using implicit solvation models with two kinds of force fields- AMBER parm99SB and parm96. We observed spontaneous formation of dimers from the extensive simulations, demonstrating the self-aggregating and fibril forming properties of the peptides. Secondary structure profile and clustering analysis showed that dimers with antiparallel $\beta$-sheet conformations, stabilized by well-defined hydrogen boding, are major species corresponding to global free energy minimum. Parallel dimers with partial $\beta$-sheets are found to be off-pathway intermediates. The relative instability of the parallel arrangements is due to the repulsive interactions between bulky and polar side chains as well as weaker backbone hydrogen bonds.

• BMB Reports
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• v.52 no.6
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• pp.349-359
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• 2019

After the first research declaring the generation of human induced pluripotent stem cells (hiPSCs) in 2007, several attempts have been made to model neurodegenerative disease in vitro during the past decade. Parkinson's disease (PD) is the second most common neurodegenerative disorder, which is mainly characterized by motor dysfunction. The formation of unique and filamentous inclusion bodies called Lewy bodies (LBs) is the hallmark of both PD and dementia with LBs. The key pathology in PD is generally considered to be the alpha-synuclein (${\alpha}$-syn) accumulation, although it is still controversial whether this protein aggregation is a cause or consequence of neurodegeneration. In the present work, the recently published researches which recapitulated the ${\alpha}$-syn aggregation phenomena in sporadic and familial PD hiPSC models were reviewed. Furthermore, the advantages and potentials of using patient-derived PD hiPSC with focus on ${\alpha}$-syn aggregation have been discussed.

• Journal of the Korean Magnetic Resonance Society
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• v.26 no.2
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• pp.21-23
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• 2022

High-pressure (HP) NMR is a powerful method to elucidate various structural features of amyloidogenic proteins. Following the previous mini-review recapitulating the HP-NMR application for amyloid-β peptides of the last issue [J. H. Kim, J. Kor. Mag. Reson. Soc. 26, 17 (2022)], the recent advancements in the HP NMR application for α-synuclein (α-Syn) are briefly summarized and discussed here. Although α-Syn is a well-known intrinsically disordered protein (IDP), several studies have shown that it can also exhibit heterogeneous yet partially folded conformations, which may correlate with its amyloid-forming propensity. HP NMR has been a valuable tool for investigating the dynamic and transient structural features of α-Syn and has provided unique insights to appreciate its aggregation-prone characters.