翻訳後修飾とタンパク質分解 Posttranslational Modulation and Proteolysis
P1-2-30 ユビキチンリガーゼDorfinがアミロイド前駆体タンパク質輸送を介したアミロイドβ 産生に関与する可能性 Possible involvement of the ubiquitin ligase Dorfin in Aβ generation mediated by protein trafficking ○金子雅幸1,2, 山森正嗣2, 保住功1, 野村靖幸3, 大熊康修2 ○Masayuki Kaneko1,2, Masashi Yamamori2, Isao Hozumi1, Yasuyuki Nomura3, Yasunobu Okuma2 岐阜薬大・薬・薬物治療1, 千葉科学大・薬・薬理2, 横浜薬大・薬・薬物治療3 Lab Med Ther & Mol Ther, Gifu Pharmceut Univ, Gifu1, Dept Pharmacol, Fac Pharmaceut Sci, Chiba Inst Sci, Choshi2, Lab Pharmacother, Yokohama Col Pharm, Yokohama3 Amyloid β (Aβ) is believed to be the cause of Alzheimer's disease (AD). The amyloid precursor protein (APP) is cleaved by β-secretase to generate APP–C-terminal fragment β (APP–CTFβ), which in turn is cleaved by γ-secretase to generate Aβ. Therefore, the γ-secretase inhibitor is expected to be a therapeutic drug for AD. Here we report the possible role of the ubiquitin ligase Dorfin in Aβ production. Inhibition of Dorfin expression elevated APP levels but decreased Aβ levels. Thus, we speculate that Dorfin is involved in Aβ production through the APP-processing pathway. To investigate the involvement of Dorfin in APP processing, we determined APP–CTFβ levels. Inhibition of Dorfin expression increased APP–CTFβ levels. These results suggest that the decrease in Aβ levels caused by suppression of Dorfin expression is the result of inhibition of the APP–CTFβ cleavage by γ-secretase. Furthermore, to determine the involvement of Dorfin in intracellular trafficking of APP, we examined the localization of APP–CTFβ in SH-SY5Y cells stably expressing APP–CTFβ. Immunofluorescent staining revealed that APP–CTFβ was partially concentrated to late endosomes and lysosomes under suppression of Dorfin expression. We suggest that Dorfin is specifically involved in γ-secretase-mediated APP–CTF cleavage associated with APP trafficking.	P1-2-31 低分子量Gタンパク質Rab5の活性化因子、ALS2とRabGEF1は、EEA1-LC3陽性小胞の成熟と分解効率を調節する Rab5 activator ALS2 and RabGEF1, regulate amphisome formation and degradation ○大友麻子1,2, 潘雷1,2, 小川温子3, 平塚結衣5, 秦野伸二1,2,3 ○Asako Otomo1,2, Lei Pan1,2, Haruko Ogawa3, Yui Hiratsuka5, Shinji Hadano1,2,3 東海大学医学部基礎医学系分子生命科学1, 東海大学総合医学研究所2, 東海大学医学研究科脳神経疾患研究センター3, 東海大学医学部教育研究支援センター4, 東海大学工学研究科5 Department of Molecular Life Sciences, Tokai University School of Medicine1, The Institute of Medical Sciences, Tokai University2, Research Center for Brain and Nervous Diseases, Tokai University Graduate School of Medicine3, Education and Research Support Center, Tokai University School of Medicine4, Graduate School of Engineering5 ALS2 mutations account for a number of recessive motor neuron diseases including forms of amyotrophic lateral sclerosis, primary lateral sclerosis and hereditary spastic paraplegia. ALS2 acts as an activator for Rab5, and regulates endosome fusion and maturation. Rab5 is a multi-functional protein which controls early steps in endocytosis and following events (e.g., internalization, endosome fusion, maturation, signal transduction, and endolysosome formation). Since nine Rab5 activators (Rab5GEFs) are conserved in human, they might share the regulatory function of Rab5 on the particular membranous compartment in the cells, and cooperatively regulates Rab5 function. Indeed, ALS2 is localized in membrane ruffles, macropinosomes and endosomes, and plays a role in endosome fusion and maturation. Recently we reported that expression of ALS2 enhanced amphisome (EEA1-LC3 double positive vesicles) formation, suggesting that ALS2 may contribute to the regulation of the autophagy-endolysosomal degradative pathways. However, it remains unclear how Rab5-dependent system is involved in this process. In order to understand the molecular basis of Rab5-dependent system associated with autophagy-endolysosomal degradation, we investigated Rab5GEFs that regulate autophagy-endolysosomal degradation. The results of our Immunocytochemical analysis demonstrate that among nine Rab5GEFs, ALS2 and RabGEF1 are localized in EGFP-LC3 positive vesicles in HeLa cells. Further, ectopic expression of either ALS2 or RabGEF1 enhances amphisome formation. In contrast, siRNA-mediated knockdown of ALS2 or RabGEF1 resulted in the increase of endogenous LC-3 positive vesicles in the cells, although there is no difference in the number of LC3-positive vesicles under the treatment of Chloroquin. Collectively, our results indicate that Rab5 activation by particular Rab5GEFs on early endosomes is essential to regulate amphisome maturation and its degradation.
P1-2-32 初代培養神経細胞におけるリソソームとオートファジー形成の関連性について Relationship of lysosomes and autophagy formation in primary cultured cortical neurons ○七尾友久1, 内山安男1 ○Tomohisa Nanao1, Yasuo Uchiyama1 順天堂大学大学院　医学研究科　神経生物学・形態学講座1 Dept Cell Biol and Neurosci, Juntendo Univ, Tokyo1 Neurons are known to have the distinct polarity concerning the shape, structure and function of neurons. It is very interesting to know the difference in the degradation system between somatodendritic and axonal compartments. For this, we have analyzed cathepsin D (CD)-deficient mice, an excellent animal model of neuronal ceroid lipofuscinosis (NCL), and found that autophagosomes without lysosomal enzymes and with double membranes accumulate in axons forming spheroids. However, granular osmiophilic deposits, one of structural features of neurons with NCL that contain lysosomal enzymes and are provided with lamp-1/2 cannot be detected in these axons. From these results it is worthy to suppose that autophagosome formation is carried out in axons/axon terminals, and nascent autophagosomes are transported retrogradely to cell soma where sequestrated materials are degraded after receiving lysosomal enzymes. To further understand characteristic features of the axon and its terminal, the relationship between lysosomes and autophagosome formation in the axon was examined by using primary culture of cortical neurons obtained at E17. During culturing for two weeks, lysosomes visualized by lamp 1 and/or Lysotracker decreased in number in Tau-1-positive axons and largely disappeared from the axon and its terminal at 14 days of culturing. This indicates that degradation in lysosomes is performed in cell soma after maturation of neurons. Then, the presence of Atg proteins and formation of autophagosomes were also analyzed by using real time imaging. LC3-positive ring formation by real time imaging and the presence of Atg9, together with synaptophysin, were demonstrated in Tau-1-positive axons. As has been shown by in vivo analysis of CD-deficient neurons, our data suggest that autophagy occurs in the axon and its terminal, while degradation of sequestrated materials occurs in cell soma after receiving lysosomal enzymes.

上部に戻る

前に戻る