TY - JOUR T1 - AIFM3 Has Pro-apoptotic, Whereas LZTR1 Has Bilateral Functions In The Intrinsic Apoptosis Pathway TT - İçsel Apoptoz Yolağında AIFM3 Pro-apoptotik, LZTR1 Çift Yönlü Fonksiyona Sahiptir AU - Yıldız, Gökhan AU - Karabulut, Soner AU - Dinçer, Tuba AU - Toraman, Bayram AU - Kalay, Ersan PY - 2025 DA - September Y2 - 2025 DO - 10.37989/gumussagbil.1665825 JF - Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi JO - Gümüşhane Sağlık Bilimleri Dergisi PB - Gümüşhane Üniversitesi WT - DergiPark SN - 2146-9954 SP - 932 EP - 937 VL - 14 IS - 3 LA - en AB - Leucine Zipper-Like Post-Translational Regulator 1 (LZTR1) is associated with several congenital diseases and cancers. The degradation of LZTR1 upon apoptosis induction in HeLa cells was reported. In addition, mass spectrometry analyses indicate that LZTR1 interacts with Apoptosis Inducing Factor Mitochondria Associated 3 (AIFM3) protein in HEK293 cells. However, the functions of LZTR1 and AIFM3 in apoptosis are not known. In this study, the characterization of the roles of LZTR1 and AIFM3 in the intrinsic apoptosis mechanism was aimed. For this purpose, firstly, FLAG-LZTR1 and Myc-AIFM3 expression plasmids had been prepared and stably FLAG-LZTR1-expressing HEK293 Flp-In cells were established. Myc-AIFM3 expression plasmids were transiently transfected into parental and stably FLAG-LZTR1-expressing HEK293 cells. Apoptosis was triggered by treating cells with hydrogen peroxide (H2O2), and the levels of apoptosis-associated target proteins were analyzed using western blotting. Our analyses firstly revealed that H2O2 treatment triggered intrinsic apoptosis through p53/p21/BCL2/Caspase 8/Caspase 9/Caspase 3 axis of the intrinsic apoptosis pathway in HEK293 cells. In addition, increased cleaved Caspase 9, Caspase 3, and PARP1 protein levels were observed in AIFM3-overexpressing cells. LZTR1 was found to have pro-apoptotic roles by increasing AIFM3 and cleaved Caspase 3 proteins and anti-apoptotic roles by decreasing cleaved Caspase 8, Caspase 9, and PARP1 proteins. In conclusion, the data obtained in this study indicate that AIFM3 has pro-apoptotic, whereas LZTR1 has both anti- and pro-apoptotic roles in the intrinsic apoptosis pathway. KW - AIFM3 KW - Apoptosis KW - LZTR1 N2 - Leucine Zipper-Like Post-Translational Regulator 1 (LZTR1), çeşitli konjenital hastalıklar ve kanserler ile ilişkilendirilmiştir. HeLa hücrelerinde apoptoz indüklendiğinde LZTR1’in parçalandığı bildirilmiştir. Ayrıca kütle spektrometrisi analizleri, LZTR1'in HEK293 hücrelerinde Apoptosis Inducing Factor Mitochondria Associated 3 (AIFM3) proteiniyle etkileştiğini göstermektedir. Bununla birlikte, LZTR1’in ve AIFM3'ün apoptozdaki işlevleri bilinmemektedir. Bu çalışmada, LZTR1’in ve AIFM3’ün içsel apoptoz mekanizmasındaki rollerinin belirlenmesi amaçlanmıştır. Bu amaçla, ilk olarak, FLAG-LZTR1 ve Myc-AIFM3 ekspresyon plazmidleri hazırlandı ve kalıcı olarak FLAG-LZTR1 ifade eden HEK293 Flp-In hücreleri oluşturuldu. Myc-AIFM3 ekspresyon plazmidleri geçici olarak parental hücrelere ve FLAG-LZTR1’i kalıcı olarak ifade eden HEK293 hücrelerine transfekte edildi. Hidrojen peroksit (H2O2) ile apoptozun tetiklendiği bu hücrelerde apoptoz ile ilişkisi bilinen hedef proteinlerin düzeyleri western blotlama yöntemi ile analiz edildi. Analizlerimiz, ilk olarak, H2O2 maruziyetinin HEK293 hücrelerinde p53/p21/BCL2/Kaspaz 8/Kaspaz 9/Kaspaz 3 aksı üzerinden içsel apoptozu tetiklediğini ortaya koydu. Ek olarak, AIFM3’i aşırı ifade eden hücrelerde kesilmiş Kaspaz 9, Kaspaz 3 ve PARP1 protein seviyelerini arttığı belirlendi. İlaveten, LZTR1'in AIFM3 ve kesilmiş Kaspaz 3 proteinlerini artırarak pro-apoptotik, kesilmiş Kaspaz 8, Kaspaz 9 ve PARP1 proteinlerini azaltarak anti-apoptotik rollerinin olduğu tespit edildi. 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