Reactivation of anti-human alpha-enolase antibody-positive Behçet's disease by carbon dioxide laser treatment.

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Evaluation of the role of hexokinase type II in cellular proliferation and apoptosis using human hepatocellular carcinoma cell lines.

The (18)F-FDG uptake pattern on PET could be an indicator of the prognosis and aggressiveness of various tumors, including hepatocellular carcinoma (HCC). Hexokinase, especially hexokinase type II (HKII), plays a critical role in (18)F-FDG uptake in rapidly growing tumors. We established a stable cell line overexpressing HKII by the transfection of full DNA of HKII to HCC cells (SNU449) that express low levels of HKII and investigated how (18)F-FDG uptake mechanisms, especially overexpression of HKII, are linked to tumor proliferation mechanisms. METHODS: The HKII gene was stably transfected to SNU449 cells with an expression vector. HKII expression in the cells was verified by reverse-transcriptase polymerase chain reaction, Western blot analysis, adenosine triphosphate (ATP) and lactate production, (18)F-FDG uptake measurement, and confocal microscopy. Cellular proliferation activity and response to the anticancer drug cisplatin were evaluated by cell counting using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. For the evaluation of molecular pathways involved in tumor proliferation, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was investigated. RESULTS: The stable cell line produced HKII effectively, but expression of other enzymes or transporters for glycolysis, such as glucose-6-phosphatase (G6Pase), HKI and III, and glucose transporter type 1 and 2 (Glut-1 and Glut-2), did not show any changes. (18)F-FDG uptake was significantly increased after transfection. ATP and lactate production was also increased after transfection. Overexpressed HKII was associated with mitochondria on confocal microscopy. Cells with overexpression of HKII, compared with the nontransfected cell line, showed 1.5- to 2-fold higher cell survival and resistance to the anticancer agent cisplatin (2- to 8-fold). In the molecular study, the activated form of Akt was increased after transfection, and PI3K inhibitor dissociated the mitochondrial HKII to the cytoplasm. In addition, the adenosine monophosphate-activated protein kinase (AMPK) pathway is also involved in Akt signaling. CONCLUSION: HKII plays an important role in (18)F-FDG uptake and tumor proliferation by both the PI3K-dependent and the PI3K-independent Akt signal pathways; therefore, the (18)F-FDG uptake pattern on a PET scan can be a surrogate marker of prognosis in HCC.ope

Enzymatic properties of the N- and C-terminal halves of human hexokinase II.

Although previous studies on hexokinase (HK) II indicate both the N- and C-terminal halves are catalytically active, we show in this study the N-terminal half is significantly more catalytic than the C-terminal half in addition to having a significantly higher Km for ATP and Glu. Furthermore, truncated forms of intact HK II lacking its first N-terminal 18 amino acids (delta18) and a truncated N-terminal half lacking its first 18 amino acids (delta18N) have higher catalytic activity than other mutants tested. Similar results were obtained by PET-scan analysis using (18)FFDG. Our results collectively suggest that each domain of HK II possesses enzyme activity, unlike HK I, with the N-terminal half showing higher enzyme activity than the C-terminal halfope

α-synuclein의 세포막 전위에서의 아미노산 서열 모티프와 기전적 특징

Dept. of Medical Science/박사[한글] α-Synuclein에 세포막 전위(주; 세포 외부로의 분비나 세포 내부로의 투과)와 관련해서 많은 증거들이 제시되고 있는 반면, 그러한 일련에 과정에 대한 세분화된 기전에 대해서는 아직까지 알려져 있지 않다. 이에 본 연구자는 α-synuclein의 세포막 전위에 중요하게 작용하는 아미노산 모티프를 찾고 이를 바탕으로 세포막 전위에 세분화된 기전을 찾고자 하였다. 우리는 α-synuclein 뿐만 아니라 β-와 γ-synuclein 역시 세포 내로 투과되는 현상을 관찰할 수 있었다. 이러한 현상은 synuclein 그룹간 높은 유사성을 갖는 N-말단 부위에 의한 것으로 생각된다. α-Synuclein 구조를 기초로 제작한 결실 변이형을 이용한 실험 결과, α-synuclein의 세포막 전위는 N-말단 쪽에 존재하는 불완전한 11개의 아미노산 반복 모티프에 의한 것임을 확인 할 수 있었다. 또한 이러한 11개의 불완전 아미노산 모티프 만으로 구성된 펩타이드들 역시 세포막 전위와 관련해서 중요한 역할을 하는 것을 알 수 있었다. 더욱이 세포막 전위에 있어 가장 효율적인 펩타이드는 두 개의 불완전 모티프를 가지는 경우로 나타났다. 또한 α-synuclein의 세포막 전위는 단백질 처리 5분 후 세포질에서 관찰 할 수 있을 정도로 비교적 빠른 시간 내에 세포 내로 투과됨을 확인 할 수 있었다. 흥미로운 것은 37°C와 비교해서 4°C에서도 별다른 영향 없이 세포 내로 투과됨을 확인하였다. 게다가 endocytosis inhibitor를 처리한 후 살펴본 α-synuclein의 세포막 전위 역시 아무런 영향을 받지 않는 것을 확인 할 수 있었다.이상에 결과들을 종합해 보면 α-synuclein의 세포막 전위는 정상적인 endocytosis 외에 다른 경로를 통해 energy-independent하며 매우 빠르게 일어나는 것으로 생각된다.이러한 결과들을 볼 때 α-synuclein은 향후 PTD (Protein Transduction Domain)와 유사한 방식으로 여러 가지 물질들을 세포 내로 유입시키는 하나의 도구가 될 것으로 사료된다. [영문]Many lines of evidence suggest that α-synuclein can be secreted from cells and can penetrate into them, although the detailed mechanism is not known. In this study, we investigated the amino acid sequence motifs required for the membrane translocation of α-synuclein, and the mechanistic features of the phenomenon. We first showed that not only α-synuclein but also β- and γ-synucleins penetrated into live cells, indicating that the conserved N-terminal region might be responsible for the membrane translocation. Using a series of deletion mutants, we demonstrated that the 11-amino acid imperfect repeats found in synuclein family members play a critical role in the membrane translocation of these proteins. We further demonstrated that fusion peptides containing the 11-amino acid imperfect repeats of α-synuclein can transverse the plasma membrane, and that the membrane translocation efficiency is optimal when the peptide contains two repeat motifs. α-Synuclein appeared to be imported rapidly and efficiently into cells, with detectable protein in the cytoplasm within 5 min after exogenous treatment. Interestingly, the import of α-synuclein at 4°C was comparable to the import observed at 37°C. Furthermore, membrane translocation of α-synuclein was not significantly affected by treatment with inhibitors of endocytosis.These results suggest that the internalization of α-synuclein is temperature-insensitive and occurs very rapidly via a mechanism distinct from normal endocytosis.ope

The chaperone activity of α-synuclein: Utilizing deletion mutants to map its interaction with target proteins

α-Synuclein is the principal component of the Lewy body deposits that are characteristic of Parkinson's disease. In vivo, and under physiological conditions in vitro, α-synuclein aggregates to form amyloid fibrils, a process that is likely to be associated with the development of Parkinson's disease. α-Synuclein also possesses chaperone activity to prevent the precipitation of amorphously aggregating target proteins, as demonstrated in vitro. α-Synuclein is an intrinsically disordered (i.e., unstructured) protein of 140 amino acids in length, and therefore studies on its fragments can be correlated directly to the functional role of these regions in the intact protein. In this study, the fragment containing residues 61-140 [α-syn(61-140)] was observed to be highly amyloidogenic and was as effective a chaperone in vitro as the full-length protein, while the N- and C-terminal fragments α-syn(1-60) and α-syn(96-140) had no intrinsic chaperone activity. Interestingly, full-length fibrillar α-synuclein had greater chaperone activity than nonfibrillar α-synuclein. It is concluded that the amyloidogenic NAC region (residues 61-95) contains the chaperone-binding site which is optimized for target protein binding as a result of its β-sheet formation and/or ordered aggregation by α-synuclein. On the other hand, the first 60 residues of α-synuclein modulate the protein's chaperone-active site, while at the same time protecting α-synuclein from fibrillation. On its own, however, this fragment [α-syn(1-60)] had a tendency to aggregate amorphously. As a result of this study, the functional roles of the various regions of α-synuclein in its chaperone activity have been delineated.ope

Identification of the amino acid sequence motif of alpha-synuclein responsible for macrophage activation

Alpha-synuclein (Syn) is implicated in the pathogenesis of PD and related neurodegenerative disorders. Recent studies have also shown that alpha-synuclein can activate microglia and enhance dopaminergic neurodegeneration. The mechanisms of microglia activation by alpha-synuclein, however, are not well understood. In this study, we found that not only alpha-synuclein but also beta- and gamma-synucleins activated macrophages (RAW 264.7) in vitro. Macrophages treated with synuclein proteins secreted TNF-alpha in a dose-dependent manner. Synuclein family proteins also increased mRNA transcription of COX-2 and iNOS. Two alpha-synuclein deletion mutants, SynDeltaNAC and Syn61-140, activated macrophages, while deletion mutants Syn1-60 and Syn96-140 did not significantly activate them. Finally, we demonstrated that macrophage activation by alpha-synuclein was accompanied by phosphorylation of ERK. These results suggest that synuclein family proteins can activate macrophages, and that macrophage activation needs both the N-terminal and C-terminal domains of alpha-synuclein, but not the central NAC region.ope

Detection of melanocyte autoantigens reacting with autoantibodies in vitiligo patients by proteomics

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Amino acid sequence motifs and mechanistic features of the membrane translocation of alpha-synuclein

Many lines of evidence suggest that α-synuclein can be secreted from cells and can penetrate into them, although the detailed mechanism is not known. In this study, we investigated the amino acid sequence motifs required for the membrane translocation of α-synuclein, and the mechanistic features of the phenomenon. We first showed that not only α-synuclein but also β- and γ-synucleins penetrated into live cells, indicating that the conserved N-terminal region might be responsible for the membrane translocation. Using a series of deletion mutants, we demonstrated that the 11-amino acid imperfect repeats found in synuclein family members play a critical role in the membrane translocation of these proteins. We further demonstrated that fusion peptides containing the 11-amino acid imperfect repeats of α-synuclein can transverse the plasma membrane, and that the membrane translocation efficiency is optimal when the peptide contains two repeat motifs. α-Synuclein appeared to be imported rapidly and efficiently into cells, with detectable protein in the cytoplasm within 5 min after exogenous treatment. Interestingly, the import of α-synuclein at 4°C was comparable with the import observed at 37°C. Furthermore, membrane translocation of α-synuclein was not significantly affected by treatment with inhibitors of endocytosis. These results suggest that the internalization of α-synuclein is temperature-insensitive and occurs very rapidly via a mechanism distinct from normal endocytosis.ope

Role of reactive oxygen species-mediated mitochondrial dysregulation in 3-bromopyruvate induced cell death in hepatoma cells : ROS-mediated cell death by 3-BrPA

Hexokinase type II (HK II) is the key enzyme for maintaining increased glycolysis in cancer cells where it is overexpressed. 3-bromopyruvate (3-BrPA), an inhibitor of HK II, induces cell death in cancer cells. To elucidate the molecular mechanism of 3-BrPA-induced cell death, we used the hepatoma cell lines SNU449 (low expression of HKII) and Hep3B (high expression of HKII). 3-BrPA induced ATP depletion-dependent necrosis and apoptosis in both cell lines. 3-BrPA increased intracellular reactive oxygen species (ROS) leading to mitochondrial dysregulation. NAC (N-acetyl-L: -cysteine), an antioxidant, blocked 3-BrPA-induced ROS production, loss of mitochondrial membrane potential and cell death. 3-BrPA-mediated oxidative stress not only activated poly-ADP-ribose (PAR) but also translocated AIF from the mitochondria to the nucleus. Taken together, 3-BrPA induced ATP depletion-dependent necrosis and apoptosis and mitochondrial dysregulation due to ROS production are involved in 3-BrPA-induced cell death in hepatoma cells.ope