Hyo Jin Park

Autocatalytic Processing of HtrA2/Omi Is Essential for Induction of Caspase-dependent Cell Death through Antagonizing XIAP

A mature form of nuclear-encoded mitochondrial serine protease HtrA2/Omi is pivotal in regulating apoptotic cell death; however, the underlying mechanism of the processing event of HtrA2/Omi and its relevant biological function remain to be clarified. Here, we describe that HtrA2/Omi is autocatalytically processed to the 36-kDa protein fragment, which is required for the cytochrome c-dependent caspase activation along with neutralizing XIAP-mediated inhibition of caspases through interaction with XIAP, eventually promoting apoptotic cell death. We have shown that the autocatalytic processing of HtrA2/Omi occurs via an intermolecular event, demonstrated by incubating an in vitro translated HtrA2/Omi (S306A) mutant with the enzymatically active glutathione S-transferase-HtrA2/Omi protein. Using N-terminal amino acid sequencing and mutational analysis, we identified that the autocatalytic cleavage site is the carboxyl side of alanine 133 of HtrA2/Omi, resulting in exposure of an inhibitor of apoptosis protein binding motif in its N terminus. Our study provides evidence that the autocatalytic processing of HtrA2/Omi is crucial for regulating HtrA2/Omi-mediated apoptotic cell death.

A single center's 30 years' experience of esophageal adenocarcinoma.

Background Adenocarcinoma of the esophagus has been reported to be increasing in incidence in a number of regions throughout the world, while the incidence of squamous cell carcinoma (SCCA) of the esophagus is mostly stable or decreasing. To evaluate the increasing tendency of adenocarcinoma of the esophagus. Methods we studied retrospectively the records of patients with histologically proven esophageal cancer between 1970 and 1999 at the Yonsei Medical Center. Results Total cases of esophageal cancer were 969 patients of which the cases of adenocarcinoma and SCCA were 27 patients and 918 patients, respectively. The ratio of esophageal adenocarcinoma to SCCA was 0.0375 in the 1970s, 0.0241 in the 1980s and 0.0292 in the 1990s. There was no statistical difference (p = 0.811) in the ratios of adenocarcinoma of the esophagus between the three consecutive 10-year groups. Conclusion In conclusion, unlike the US and other western countries, it seems that the ratio of esophageal adenocarcinoma compared to SCCA has not increased among patients with esophageal carcinoma at the Yonsei Medical Center.

Rapid purification and analysis of α‐synuclein proteins: C‐terminal truncation promotes the conversion of α‐synuclein into a protease‐sensitive form in Escherichia coli

Parkinsons disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons and the formation of eosinophilic intracytoplasmic inclusion bodies known as Lewy bodies. Although α‐synuclein is known to be a pivotal factor implicated in the pathogenesis of PD, its function remains to be elucidated. We used the pGEX expression system to develop a simple and rapid method for purifying α‐synuclein proteins in suitable forms for biochemical studies of their functions. The wild‐type α‐synuclein protein was overexpressed in Escherichia coli and purified to approx. 80% purity with relatively high yields. We also used this expression system to investigate the expression pattern of the various domains of α‐synuclein. With the exception of the α‐synuclein protein that was truncated at amino acid residue 95, all domain constructs of α‐synuclein were purified at similar levels with relatively high yields. Unexpectedly, removal of amino acid residues 96–140 in the C‐terminal acidic region of α‐synuclein promotes its conversion to a protease‐sensitive form during expression and purification in E. coli. Our study suggests a method for generating useful reagents to investigate the molecular mechanism by which α‐synuclein regulates the pathogenesis of PD.

Intracellular Aβ and C99 aggregates induce mitochondria-dependent cell death in human neuroglioma H4 cells through recruitment of the 20S proteasome subunits

Recent studies have reported that neuronal apoptosis is induced not only by extracellular Abeta but also by intracellular Abeta; however, the mechanism by which intracellular Abeta contributes to the regulation of cell death associated with the pathogenesis of AD remains to be elucidated. Using immunological assays and a short-lived enhanced green fluorescent protein (d2EGFP) system, we showed that intracellular Abeta and C99 form perinuclear aggregates in the cytosol, and the resulting aggregates attenuate the activity of the 26S proteasome. In addition, the immunofluorescence assays (IFA) revealed that the 20S proteasome alpha-subunits are recruited into perinuclear aggregates in both human embryonic kidney (HEK293) and human neuroglioma H4 (H4) cells. Interestingly, we observed an increase in the levels of Bax, cleavage of PARP-1, and mitochondrial release of proapoptotic proteins, such as cytochrome c and HtrA2, in H4 cells with intracellular Abeta or C99 aggregates, but not in HEK293 cells with those aggregates. The results of the present study indicate that intracellular Abeta and C99 aggregates induce mitochondria-dependent apoptotic cell death via elevation of Bax levels as a result of proteasome inhibition in a cell type-specific manner.

The homotrimeric structure of HtrA2 is indispensable for executing its serine protease activity.

Serine protease activity of high temperature requrement 2 (HtrA2) is essential for promoting cell death, as well as for protecting against cellular stresses. An X-ray crystallographic study described the formation of a pyramid shaped homotrimer that is a proteolytically competent form of HtrA2; however, little is known about effects of the trimeric structure of HtrA2 on the natural substrates. In this study, we generated the HtrA2 protein that has a single point mutation at the homotrimerization motif to assess relationship between structure and the proteolytic activity of HtrA2 on its substrates. Using gel filtration, a native gel electrophoresis system, and a co-precipitation assay, we confirm that phenylalanine 149 in HtrA2 is a crucial determinant for the formation of the HtrA2 homotrimeric structure. Moreover, we described that the HtrA2 monomeric form abolished not only autoproteolytic activity, but also the proteolytic activity against XIAP (X-linked inhibitor of apoptosis protein) known as the HtrA2 substrate. Taken together, the results indicate that the homotrimeric structure of HtrA2 is required for executing its serine protease activity.

A 5-year retrospective clinical study of the Dentium implants

PURPOSE The aim of this retrospective study was to evaluate cumulative survival rate (CSR) of Implantium implants followed for 5 years and association between risk factors and the CSR. MATERIALS AND METHODS A total of two hundred forty-nine Implantium Implants System (Dentium, Seoul, Korea) placed in ninety-five patients from 2004 to 2009 were investigated with several identified risk factors (sex, systemic disease, smoking, alchohol, reason of tooth loss, length, arch (maxilla or mandible), replace tooth type (incisor, canine, premolar or molar) Kennedy classification, prosthodontic type, prosthodontic design, opposite dentition, abutment type, occlusal material, occlusal unit, splint to tooth, cantilever, other surgery). Clinical examination (mobility, percussion, screw loosening, discomfort, etc.) and radiographic examination data were collected from patient records including all problems during follow-up period according to protocols described earlier. Life table analysis was undertaken to examine the CSR. Cox regression method was conducted to assess the association between potential risk factors and overall CSR. RESULTS Five of 249 implants were failed. Four of these were lost before loading. The 5-year implant cumulative survival rate was 97.37%. Cox regression analysis demonstrated a significant predictive association between overall CSR and systemic disease, smoking, reason of tooth loss, arch, Kennedy classification and prosthodontic design (P<.05). The screw related complication was rare. Two abutment screw fractures were found. Another complications of prosthetic components were porcelain fracture, resin facing fracture and denture fracture (n=19). CONCLUSION The 5-year CSR of Implantium implants was 97.37%. Implant survival may be dependent upon systemic disease, smoking reason of tooth loss, arch, Kennedy classification and prosthodontic design (P<.05). The presence of systemic diseases and combination of other surgical procedures may be associated with increased implant failure.

CD43 cross-linking increases the Fas-induced apoptosis through induction of Fas aggregation in Jurkat T-cells

CD43 (sialophorin, leukosialin) is a heavily sialylated surface protein expressed on most leukocytes and platelets including T cells. Although CD43 antigen is known to have multiple and complex structure, exact function of CD43 in each cell type is not completely understood. Here we evaluated the role of CD43 in Fas (CD95)-induced cell death in human T lymphoblastoid cell line, Jurkat. Crosslinking CD43 antigen by K06 mAb increased the Fas-mediated Jurkat cell apoptosis and the augmentation was inhibited by treatment with caspase inhibitors. Further, CD43 signaling of Jurkat cells induced Fas oligomerization on the cell surfaces implying that CD43 ligation have effects on early stage of Fas-induced T cell death. These also suggest that CD43 might play an important role in contraction of the immune response by promotion of Fas-induced apoptosis in human T cells.

PLZF+ Innate T Cells Support the TGF-β-Dependent Generation of Activated/Memory-Like Regulatory T Cells

PLZF-expressing invariant natural killer T cells and CD4 T cells are unique subsets of innate T cells. Both are selected via thymocyte-thymocyte interaction, and they contribute to the generation of activated/memory-like CD4 and CD8 T cells in the thymus via the production of IL-4. Here, we investigated whether PLZF+ innate T cells also affect the development and function of Foxp3+ regulatory CD4 T cells. Flow cytometry analysis of the thymus and spleen from both CIITA transgenic C57BL/6 and wild-type BALB/c mice, which have abundant PLZF+ CD4 T cells and invariant natural killer T cells, respectively, revealed that Foxp3+ T cells in these mice exhibited a CD103+ activated/memory-like phenotype. The frequency of CD103+ regulatory T cells was considerably decreased in PLZF+ cell-deficient CIITATgPlzflu/lu and BALB/c.CD1d−/− mice as well as in an IL-4-deficient background, such as in CIITATgIL-4−/− and BALB/c.lL-4−/− mice, indicating that the acquisition of an activated/memory-like phenotype was dependent on PLZF+ innate T cells and IL-4. Using fetal thymic organ culture, we further demonstrated that IL-4 in concert with TGF-β enhanced the acquisition of the activated/memory-like phenotype of regulatory T cells. In functional aspects, the activated/memory-like phenotype of Treg cells was directly related to their suppressive function; regulatory T cells of CIITATgPIV−/− mice more efficiently suppressed ovalbumin-induced allergic airway inflammation compared with their counterparts from wild-type mice. All of these findings suggest that PLZF+ innate T cells also augmented the generation of activated/memory-like regulation via IL-4 production.

Correction: In situ induction of dendritic cell–based T cell tolerance in humanized mice and nonhuman primates

Vol. 208, No. 12, November 21, 2011. Pages [2477–2488][1]. The authors regret that a measurement was incorrect and that the source of the 5C8 clone was left out of the original publication. The corrected sentence in the Results section MD-3 efficiently induces antigen-specific T cell tolerance in