Kyunghee Lee

Copper-Catalyzed Azide–Alkyne Cycloaddition Reaction in Water Using Cyclodextrin as a Phase Transfer Catalyst

1,4-Disubstituted-1,2,3-triazoles were obtained in excellent yields from azides and terminal alkynes in H(2)O in the presence of catalytic amount of β-cyclodextrin as a phase transfer catalyst. Also, a one-pot CuAAC reaction was carried out successfully, affording 1,4-disubstituted-1,2,3-triazoles in good to high yields starting from an alkyl bromide, sodium azide, and terminal alkyne.

Simulation Studies on the Stabilities of Aggregates Formed by Fibril-Forming Segments of α-Synuclein

Abstract We performed molecular dynamics simulations for various oligomers with different β-sheet conformations consisting of α-Synuclein 71–82 residues using an all atom force field and explicit water model. Tetramers of antiparallel β-sheet are shown to be stable, whereas parallel sheets are highly unstable due to the repulsive interactions between bulky and polar side chains as well as the weaker backbone hydrogen bonds. We also investigated the stabilities of double antiparallel β-sheets stacked with asymmetric and symmetric geometries. Our results show that this 12 amino acid residue peptide can form stable β-sheet conformers at 320K and higher temperatures. The backbone hydrogen bonds in β-sheet and the steric packing between hydrophobic side chains between β-sheets are shown to give conformational stabilities.

Human α-synuclein modulates vesicle trafficking through its interaction with prenylated Rab acceptor protein 1

α-Synuclein has been implicated in the pathogenesis of Parkinsons disease. Although it is highly conserved, its physiological function has not yet been elucidated in detail. In an effort to define the function of α-synuclein, interacting proteins were screened in phage display assays. Prenylated Rab acceptor protein 1 (PRA1) was identified as an interacting partner. A selective interaction between α-synuclein and PRA1 was confirmed by coimmunoprecipitation and GST pull-down assays. PRA1 and α-synuclein were colocalized in N2a neuronal cells. Cotransfection of α-synuclein and PRA1 caused vesicles to accumulate in the periphery of the cytosol in neuronal cells, suggesting that overexpression of α-synuclein hinders proper vesicle trafficking and recycling as a result of the interaction between α-synuclein and PRA1.

Conformational Characteristics of Unstructured Peptides: α-Synuclein

Abstract We have performed replica-exchange molecular dynamics simulations on 41 residue peptides containing NAC region of α-synuclein in various force fields and solvent conditions. Alpha-synuclein is known to be the major cause of Parkinsons disease by amyloid-like aggregation, and one of the natively unfolded proteins. To investigate conformational characteristics of intrinsically unstructured peptides, we carried out structural analysis by introducing ‘representative structure’ for ensemble of structures occurring during the overall trajectory. Representative structures may be defined by using either coordinate averaging or distance averaging. When applied to the natively folded proteins such as villin headpiece, structural analysis based on representative structure was found to yield consistent results with those obtained from conventional analysis. Individual conformations obtained from the simulations of NAC peptide for various conditions show flexible structures close to random coil. Secondary structure contents and free energy surfaces showed dependency on solvent conditions, which may be interpreted as another manifestation of structural diversity. It is found that representative structures can provide useful information about structural characteristics of intrinsically unstructured proteins.

Regulation Of In Vitro Fibril Formation Of Synuclein Mutant Proteins By Hsp104p

Hsp104p, as an anti-oxidative protector of ROS generation, was examined to inquire if it prevents aggregation of synuclein mutants, A30P or A53T upon aging, in vitro. The role of Hsp104p was also addressed in dissociation of pre-formed aggregates of synuclein mutants. Significant protection in fibril formation was observed by wild-type Hsp104p regardless of ATP presence, not by mutant Hsp104p. To a lesser extent, the dissociation effect of wild-type Hsp104p was observed only in the presence of ATP. These results will be discussed in relation to the development of an antioxidant approach to prevent amyloid fibril formation in several neurodegenerative diseases.

Theoretical Studies of the Response of a Protein Structure to Cavity-Creating Mutations

We have investigated the response of a protein structure to cavity-creating mutations by molecular dynamics (MD) simulations for the wild-type and the five mutants of phage T4 lysozyme. Essential dynamics (ED) analysis and the methods for calculating different components of local interaction energies are used to examine the structural and energetic characteristics associated with the mutations. In agreement with the x-ray results, it is found that the structural changes due to the replacements of a bulky side chain such as Leu or Phe with Ala within the hydrophobic core can be characterized as slight adjustments rather than substantial reorganization of the protein. The relative stability of different mutant structures can be related with the extent of structural readjustments in response to the mutation. The destabilization of the mutant Leu-->Ala proteins relative to the wild-type is closely related with the loss of van der Waals contacts due to the cavity-creating mutations.

α-Synuclein modulates neurite outgrowth by interacting with SPTBN1.

α-Synuclein is the major component of Lewy bodies and Lewy neurites, the pathological hallmarks of surviving neuronal cells in Parkinsons disease patients. However, the physiological role played by α-synuclein remains unclear. In this study, spectrin beta non-erythrocyte 1 (SPTBN1) interacted with α-synuclein in phage display assays using a normalized human brain cDNA library. A direct interaction between α-synuclein and SPTBN1 was confirmed by GST pull-down and co-immunoprecipitation assays. SPTBN1 and α-synuclein proteins colocalized in N2a neuronal cells. Transfection of SPTBN1 caused human SH-SY5Y dopaminergic neuron cells to inappropriately induce neurites, which extended from cell bodies. Cotransfection with α-synuclein reversed SPTBN1-induced excessive neurite branching in SH-SY5Y cells, and only a single neurite extended from each neuron. These results suggest that α-synuclein modulates neurite outgrowth by interacting with cytoskeletal proteins such as SPTBN1.

Phase II evaluation of CKD-602, a camptothecin analog, administered on a 5-day schedule to patients with platinum-sensitive or -resistant ovarian cancer

BACKGROUND To evaluate the toxicity and efficacy of a newly developed topoisomerase I inhibitor, CKD-602 in second-line therapy of ovarian cancer. METHODS We enrolled 24 patients with recurrent ovarian cancer, of median age 54 years (range, 39-64). Eleven patients had measurable lesions on CT scan, and the other 13 had increased serum CA-125 levels. Eighteen patients had platinum-sensitive disease (minimum treatment free interval > or =6 months) and 6 had platinum-resistant disease (minimum treatment free interval <6 months). CKD-602 (0.5 mg/m(2)/day) was administered intravenously for 5 days every 3 weeks. The median number of courses per patient was 6 (range, 1 to 12). Response was evaluated by the evaluation of the size of the mass by CT scan and CA-125 response. RESULTS The overall response rate was 45.0% (9/20), with 4 patients exhibiting partial responses and 5 patients exhibiting 75% CA-125 responses in 20 evaluable patients. Of the 9 responsive patients, 8 were platinum-sensitive (8/15, 53.3%) and 1 was platinum-resistant (1/5, 20.0%). An additional 5 patients showed stable disease, whereas 6 patients exhibited progressive lesions. Of 24 patients, the most common toxicity was hematological, with grades 3 or 4 neutropenia developing in all 24 patients (100%) and in 94 cycles (71.7%). Grade 3 thrombocytopenia developed in 4 patients (16.7%) and 6 cycles (4.6%). None of the patients experienced grades 3 and 4 gastrointestinal toxicities, including nausea, vomiting, and anorexia. CONCLUSIONS The newly developed topoisomerase I inhibitor, CKD-602, showed activity against both platinum-sensitive and -resistant ovarian cancer, with acceptable toxicity.

Detection of Recurrence by 18F-FDG PET in Patients with Endometrial Cancer Showing No Evidence of Disease

This study assessed the feasibility of F-18-fluorodeoxyglucose positron-emission tomography (18F-FDG PET) in the post-therapy surveillance for patients with endometrial cancer showing no evidence of disease (NED). From April 1997 to June 2007, 127 patients with endometrial cancer showing NED were performed 18F-FDG PET scan. The feasibility of 18F-FDG PET for the early detection of recurrence in patients with endometrial cancer was evaluated retrospectively. Of the 127 patients, 32 patients showed positive lesions on 18F-FDG PET scan. Nineteen (19/127 cases, 15%) of them were confirmed to have a recurrence clinically or histologically. The sensitivity, specificity and positive and negative predictive value of 18F-FDG PET for detecting recurrences in patients with endometrial cancer were 100%, 88%, 59% and 100%, respectively. In conclusion, 18F-FDG PET may be a useful method for the post-therapy surveillance in patients with endometrial cancer.

Protein Production By Stationary Phase Induction (Spi)

An alternative method for expressing the recombinant proteins in Escherichia coli is proposed. Unlike the ordinary induction protocol where the cells in the early- to mid-log phase are induced for the protein production, this alternative protocol utilizes the cells in the stationary phase. By using a glutathione S-transferase fusion protein as an example, the protocol proposed in this report yielded a higher amount of the desired protein than that from the ordinary protocol. This protocol also suppressed the proteolytic cleavage of the desired protein in the Escherichia coli cytoplasm, thus it should be particularly useful to produce proteins that undergo unwanted cleavages.