최용준 허재학 2020-02 29698 https://aurora.ajou.ac.kr/handle/2018.oak/19529 학위논문(석사)--아주대학교 일반대학원 :의생명과학과,2020. 2 세포고사유래 엑소좀(ApoExos)은 건강한 세포에서 분비되는 엑소좀과 마찬가지로 CD63를 발현하며, 특이적인 표지자인, Spingoshine-1-phosphate Receptor 1과 3를 갖는다. 세포고사유래 엑소좀은 세포간 상호작용과 면역반응에 관여한다고 보고되고 있지만, 이들이 어떻게 생합성 되는지에 대해서는 자세히 보고되지 않았다. 이에 본 연구는 세포막에 구멍을 뚫는다고 보고된 게스더민 단백질들이 세포막을 재구성하여 세포고사유래 엑소좀의 생합성에 관여하는지 알아보는 연구를 수행하였다. 본 연구의 결과에 의하면, GSDMB와 DFNB59을 제외한 게스더민 단백질들은 케스페이즈에 의해 절단되며 세포고사유래 엑소좀의 생합성을 증가시켰는데, 절단 후 만들어진 cleaved fragments와 온전한 형태의 단백질 모두 엑소좀의 막으로 이동하는 것이 관찰되었다. 특히, DFNA5는 케스페이즈에 의한 절단 여부와 상관없이 세포고사유래 엑소좀의 생합성을 증가시켰는데, 이는 DFNA5의 pore-forming ability가 다른 게스더민 단백질들과는 다르게 케이페이즈에 의한 절단 여부와 상관없이 기능할 수 있음을 제시한다. 1. Introduction 1 2. Materials and Methods 7 2.1. Cells and reagents 7 2.2. Lentiviral infection and generation of stable cell lines 8 2.3. CRISPR/Cas9 Knock-out 8 2.4 ApoExos preparation 9 2.5 Preparation of cell lysates and Western Blots 9 2.6 LDH assay 9 3. Results 10 3.1 Expression of GSDMD and DFNA5 is in accordance with the biogenesis of ApoExos 10 3.2 Knock-out of DFNA5 or GSDMD inhibits the biogenesis of ApoExos 13 3.3 Overexpression of DFNA5 increase the biogenesis of ApoExos 16 3.4 Gasdermins as full-length or cleaved forms increase the biogenesis of ApoExos 18 3.5 Cleaved gasdermins are required for the biogenesis of ApoExos 26 3.6 GSDMA, GSDMC, and GSDMD require caspase-3-mediated cleavage for the biogenesis of ApoExos 29 3.7 Ability for ApoExos release of DFNA5 remains functional without cleavage 32 3.8 Pore-forming ability of DFNA5 needs to be regulated to increase the release of ApoExos 35 4. Discussion 39 5. Conclusion 42 6. References 43 7. 국문요약 52 eng The Graduate School, Ajou University 아주대학교 논문은 저작권에 의해 보호받습니다. Roles of Gasdermin family in the Biogenesis of Apoptotic cell-derived Exosomes Jaehark Hur Thesis 아주대학교 일반대학원 Jaehark Hur 일반대학원 의생명과학과 2020. 2 Master I804:41038-000000029698 http://dcoll.ajou.ac.kr:9080/dcollection/common/orgView/000000029698 Pyroptosis Apoptotic-cell derived exosomes (ApoExos), characterized by exosomal marker proteins, CD63 as well as their unique markers, S1PR1 and S1PR3 have been proposed to be associated with intercellular communication, and mediate immunological responses. Detailed mechanisms in the release of ApoExos, however, remain unclear. This study therefore, investigates the roles of gasdermin family proteins in regards to their pore-forming ability to mediate plasma membrane remodelling that may lead to the release of ApoExos. As results, gasdermin proteins, except GSDMB and DFNB59, increased the release of ApoExos in a caspase 3 dependent manner, in which cleavage of gasdermin and the subsequent migration of full-length as well as cleaved fragments to the membranous portion of the vesicles were observed. DFNA5, in particular, was localized at the vesicles in both caspase-dependent and -independent manner, and increased the release of ApoExos, possibly due to its pore-forming ability remain functional to mediate remodelling of the plasma membrane contributing to the biogenesis of ApoExos.