AIFM3 Has Pro-apoptotic, Whereas LZTR1 Has Bilateral Functions In The Intrinsic Apoptosis Pathway

Gökhan Yıldız; Soner Karabulut; Tuba Dinçer; Bayram Toraman; Ersan Kalay

doi:10.37989/gumussagbil.1665825

Research Article

AIFM3 Has Pro-apoptotic, Whereas LZTR1 Has Bilateral Functions In The Intrinsic Apoptosis Pathway

Year 2025, Volume: 14 Issue: 3, 932 - 937, 25.09.2025

Gökhan Yıldız , Soner Karabulut , Tuba Dinçer , Bayram Toraman , Ersan Kalay

https://doi.org/10.37989/gumussagbil.1665825

Abstract

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.

Keywords

AIFM3 , Apoptosis , LZTR1

Project Number

This work was supported by the Office of Scientific Research Projects of Karadeniz Technical University, project Numbers TSA-2023-10539 and TSA-2024-11015 and the Scientific and Technological Research Council of Turkey (TÜBİTAK), project Number 119S356.

References

1. Johnston JJ, van der Smagt JJ, Rosenfeld JA, Pagnamenta AT, Alswaid A, Baker EH, et al. “Autosomal recessive Noonan syndrome associated with biallelic LZTR1 variants”. Genet Med. 2018; 20(10), 1175-85. https://doi.org/10.1038/gim.2017.249
2. Kehrer-Sawatzki H, Farschtschi S, Mautner VF, Cooper DN. “The molecular pathogenesis of schwannomatosis, a paradigm for the co-involvement of multiple tumour suppressor genes in tumorigenesis”. Hum Genet. 2017; 136(2), 129-48. https://doi.org/10.1007/s00439-016-1753-8
3. Kurahashi H, Akagi K, Inazawa J, Ohta T, Niikawa N, Kayatani F, et al. “Isolation and characterization of a novel gene deleted in DiGeorge syndrome”. Hum Mol Genet. 1995; 4(4), 541-9. https://doi.org/10.1093/hmg/4.4.541
4. Frattini V, Trifonov V, Chan JM, Castano A, Lia M, Abate F, et al. “The integrated landscape of driver genomic alterations in glioblastoma”. Nat Genet. 2013; 45(10), 1141-9. https://doi.org/10.1038/ng.2734
5. Cancer Genome Atlas Research Network. “Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma”. Cell. 2017; 169(7), 1327-41 e23. https://doi.org/10.1016/j.cell.2017.05.046
6. Bigenzahn JW, Collu GM, Kartnig F, Pieraks M, Vladimer GI, Heinz LX, et al. “LZTR1 is a regulator of RAS ubiquitination and signaling”. Science. 2018; 362(6419), 1171-7. https://doi.org/10.1126/science.aap8210
7. Steklov M, Pandolfi S, Baietti MF, Batiuk A, Carai P, Najm P, et al. “Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination”. Science. 2018; 362 (6419), 1177-82. https://doi.org/10.1126/science.aap7607
8. Castel P, Cheng A, Cuevas-Navarro A, Everman DB, Papageorge AG, Simanshu DK, et al. “RIT1 oncoproteins escape LZTR1-mediated proteolysis”. Science. 2019; 363 (6432), 1226-30. https://doi.org/10.1126/science.aav1444
9. Motta M, Fidan M, Bellacchio E, Pantaleoni F, Schneider-Heieck K, Coppola S, et al. “Dominant Noonan syndrome-causing LZTR1 mutations specifically affect the Kelch domain substrate-recognition surface and enhance RAS-MAPK signaling”. Hum Mol Genet. 2019; 28(6), 1007-22. https://doi.org/10.1093/hmg/ddy412
10. Abe T, Umeki I, Kanno SI, Inoue SI, Niihori T, Aoki Y. “LZTR1 facilitates polyubiquitination and degradation of RAS-GTPases”. Cell Death Differ. 2020; 27(3), 1023-35. https://doi.org/10.1038/s41418-019-0395-5
11. Yi T, Luo H, Qin F, Jiang Q, He S, Wang T, et al. “LncRNA LL22NC03-N14H11.1 promoted hepatocellular carcinoma progression through activating MAPK pathway to induce mitochondrial fission”. Cell Death Dis. 2020; 11(10), 832. https://doi.org/10.1038/s41419-020-2584-z
12. Damnernsawad A, Bottomly D, Kurtz SE, Eide CA, McWeeney SK, Tyner JW, et al. “Genome-wide CRISPR screen identifies regulators of MAPK and MTOR pathways mediating sorafenib resistance in acute myeloid leukemia”. Haematologica. 2022; 107(1), 77-85. https://doi.org/10.3324/haematol.2020.257964
13. Chen S, Vedula RS, Cuevas-Navarro A, Lu B, Hogg SJ, Wang E, et al. “Impaired Proteolysis of Noncanonical RAS Proteins Drives Clonal Hematopoietic Transformation”. Cancer Discov. 2022; 12 (10), 2434-53. https://doi.org/10.1158/2159-8290.CD-21-1631
14. Nacak TG, Leptien K, Fellner D, Augustin HG, Kroll J. “The BTB-kelch protein LZTR-1 is a novel Golgi protein that is degraded upon induction of apoptosis”. J Biol Chem. 2006; 281 (8), 5065-71. https://doi.org/10.1074/jbc.M509073200
15. Huttlin EL, Bruckner RJ, Paulo JA, Cannon JR, Ting L, Baltier K, et al. “Architecture of the human interactome defines protein communities and disease networks”. Nature. 2017; 545 (7655), 505-9. https://doi.org/10.1038/nature22366
16. Huttlin EL, Bruckner RJ, Navarrete-Perea J, Cannon JR, Baltier K, Gebreab F, et al. “Dual proteome-scale networks reveal cell-specific remodeling of the human interactome”. Cell. 2021; 184(11), 3022-40 e28. https://doi.org/10.1016/j.cell.2021.04.011
17. Xie Q, Lin T, Zhang Y, Zheng J, Bonanno JA. “Molecular cloning and characterization of a human AIF-like gene with ability to induce apoptosis”. J Biol Chem. 2005; 280(20), 19673-81. https://doi.org/10.1074/jbc.M409517200
18. Yuan J, Ofengeim D. “A guide to cell death pathways”. Nat Rev Mol Cell Biol. 2024; 25(5), 379-95. https://doi.org/10.1038/s41580-023-00689-6
19. Vitale I, Pietrocola F, Guilbaud E, Aaronson SA, Abrams JM, Adam D, et al. “Apoptotic cell death in disease-Current understanding of the NCCD 2023”. Cell Death Differ. 2023; 30(5), 1097-154. https://doi.org/10.1038/s41418-023-01153-w
20. Spitzer J, Landthaler M, Tuschl T. “Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway® recombination cloning technology and Flp-In T-REx(R) lines”. Methods Enzymol. 2013; 529, 99-124. https://doi.org/10.1016/B978-0-12-418687-3.00008-2
21. Kingston RE, Chen CA, Okayama H. “Calcium phosphate transfection”. Curr Protoc Cell Biol. 2003;
Chapter 20: Unit 20 3. https://doi.org/10.1002/0471143030.cb2003s19 22. Han S, Cui Y, Helbing DL. “Inactivation of Horseradish Peroxidase by Acid for Sequential Chemiluminescent Western Blot”. Biotechnol J. 2020; 15(3), e1900397. https://doi.org/10.1002/biot.201900397
23. Chaitanya GV, Steven AJ, Babu PP. “PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration”. Cell Commun Signal. 2010; 8, 31. https://doi.org/10.1186/1478-811X-8-31
24. Liebl MC, Hofmann TG. “Cell Fate Regulation upon DNA Damage: p53 Serine 46 Kinases Pave the Cell Death Road”. Bioessays. 2019; 41(12), e1900127. https://doi.org/10.1002/bies.201900127
25. Ruvolo PP, Deng X, May WS. “Phosphorylation of Bcl2 and regulation of apoptosis”. Leukemia. 2001; 15(4), 515-22. https://doi.org/10.1038/sj.leu.2402090

İçsel Apoptoz Yolağında AIFM3 Pro-apoptotik, LZTR1 Çift Yönlü Fonksiyona Sahiptir

Year 2025, Volume: 14 Issue: 3, 932 - 937, 25.09.2025

Gökhan Yıldız , Soner Karabulut , Tuba Dinçer , Bayram Toraman , Ersan Kalay

https://doi.org/10.37989/gumussagbil.1665825

Abstract

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. Sonuç olarak, bu çalışmada elde edilen veriler AIFM3'ün içsel apoptoz yolağında pro-apoptotik, LZTR1’in ise hem anti- hem de pro-apoptotik rollere sahip olduğunu ortaya koymaktadır.

Keywords

AIFM3 , Apoptoz , LZTR1

Project Number

References

1. Johnston JJ, van der Smagt JJ, Rosenfeld JA, Pagnamenta AT, Alswaid A, Baker EH, et al. “Autosomal recessive Noonan syndrome associated with biallelic LZTR1 variants”. Genet Med. 2018; 20(10), 1175-85. https://doi.org/10.1038/gim.2017.249
2. Kehrer-Sawatzki H, Farschtschi S, Mautner VF, Cooper DN. “The molecular pathogenesis of schwannomatosis, a paradigm for the co-involvement of multiple tumour suppressor genes in tumorigenesis”. Hum Genet. 2017; 136(2), 129-48. https://doi.org/10.1007/s00439-016-1753-8
3. Kurahashi H, Akagi K, Inazawa J, Ohta T, Niikawa N, Kayatani F, et al. “Isolation and characterization of a novel gene deleted in DiGeorge syndrome”. Hum Mol Genet. 1995; 4(4), 541-9. https://doi.org/10.1093/hmg/4.4.541
4. Frattini V, Trifonov V, Chan JM, Castano A, Lia M, Abate F, et al. “The integrated landscape of driver genomic alterations in glioblastoma”. Nat Genet. 2013; 45(10), 1141-9. https://doi.org/10.1038/ng.2734
5. Cancer Genome Atlas Research Network. “Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma”. Cell. 2017; 169(7), 1327-41 e23. https://doi.org/10.1016/j.cell.2017.05.046
6. Bigenzahn JW, Collu GM, Kartnig F, Pieraks M, Vladimer GI, Heinz LX, et al. “LZTR1 is a regulator of RAS ubiquitination and signaling”. Science. 2018; 362(6419), 1171-7. https://doi.org/10.1126/science.aap8210
7. Steklov M, Pandolfi S, Baietti MF, Batiuk A, Carai P, Najm P, et al. “Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination”. Science. 2018; 362 (6419), 1177-82. https://doi.org/10.1126/science.aap7607
8. Castel P, Cheng A, Cuevas-Navarro A, Everman DB, Papageorge AG, Simanshu DK, et al. “RIT1 oncoproteins escape LZTR1-mediated proteolysis”. Science. 2019; 363 (6432), 1226-30. https://doi.org/10.1126/science.aav1444
9. Motta M, Fidan M, Bellacchio E, Pantaleoni F, Schneider-Heieck K, Coppola S, et al. “Dominant Noonan syndrome-causing LZTR1 mutations specifically affect the Kelch domain substrate-recognition surface and enhance RAS-MAPK signaling”. Hum Mol Genet. 2019; 28(6), 1007-22. https://doi.org/10.1093/hmg/ddy412
10. Abe T, Umeki I, Kanno SI, Inoue SI, Niihori T, Aoki Y. “LZTR1 facilitates polyubiquitination and degradation of RAS-GTPases”. Cell Death Differ. 2020; 27(3), 1023-35. https://doi.org/10.1038/s41418-019-0395-5
11. Yi T, Luo H, Qin F, Jiang Q, He S, Wang T, et al. “LncRNA LL22NC03-N14H11.1 promoted hepatocellular carcinoma progression through activating MAPK pathway to induce mitochondrial fission”. Cell Death Dis. 2020; 11(10), 832. https://doi.org/10.1038/s41419-020-2584-z
12. Damnernsawad A, Bottomly D, Kurtz SE, Eide CA, McWeeney SK, Tyner JW, et al. “Genome-wide CRISPR screen identifies regulators of MAPK and MTOR pathways mediating sorafenib resistance in acute myeloid leukemia”. Haematologica. 2022; 107(1), 77-85. https://doi.org/10.3324/haematol.2020.257964
13. Chen S, Vedula RS, Cuevas-Navarro A, Lu B, Hogg SJ, Wang E, et al. “Impaired Proteolysis of Noncanonical RAS Proteins Drives Clonal Hematopoietic Transformation”. Cancer Discov. 2022; 12 (10), 2434-53. https://doi.org/10.1158/2159-8290.CD-21-1631
14. Nacak TG, Leptien K, Fellner D, Augustin HG, Kroll J. “The BTB-kelch protein LZTR-1 is a novel Golgi protein that is degraded upon induction of apoptosis”. J Biol Chem. 2006; 281 (8), 5065-71. https://doi.org/10.1074/jbc.M509073200
15. Huttlin EL, Bruckner RJ, Paulo JA, Cannon JR, Ting L, Baltier K, et al. “Architecture of the human interactome defines protein communities and disease networks”. Nature. 2017; 545 (7655), 505-9. https://doi.org/10.1038/nature22366
16. Huttlin EL, Bruckner RJ, Navarrete-Perea J, Cannon JR, Baltier K, Gebreab F, et al. “Dual proteome-scale networks reveal cell-specific remodeling of the human interactome”. Cell. 2021; 184(11), 3022-40 e28. https://doi.org/10.1016/j.cell.2021.04.011
17. Xie Q, Lin T, Zhang Y, Zheng J, Bonanno JA. “Molecular cloning and characterization of a human AIF-like gene with ability to induce apoptosis”. J Biol Chem. 2005; 280(20), 19673-81. https://doi.org/10.1074/jbc.M409517200
18. Yuan J, Ofengeim D. “A guide to cell death pathways”. Nat Rev Mol Cell Biol. 2024; 25(5), 379-95. https://doi.org/10.1038/s41580-023-00689-6
19. Vitale I, Pietrocola F, Guilbaud E, Aaronson SA, Abrams JM, Adam D, et al. “Apoptotic cell death in disease-Current understanding of the NCCD 2023”. Cell Death Differ. 2023; 30(5), 1097-154. https://doi.org/10.1038/s41418-023-01153-w
20. Spitzer J, Landthaler M, Tuschl T. “Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway® recombination cloning technology and Flp-In T-REx(R) lines”. Methods Enzymol. 2013; 529, 99-124. https://doi.org/10.1016/B978-0-12-418687-3.00008-2
21. Kingston RE, Chen CA, Okayama H. “Calcium phosphate transfection”. Curr Protoc Cell Biol. 2003;
Chapter 20: Unit 20 3. https://doi.org/10.1002/0471143030.cb2003s19 22. Han S, Cui Y, Helbing DL. “Inactivation of Horseradish Peroxidase by Acid for Sequential Chemiluminescent Western Blot”. Biotechnol J. 2020; 15(3), e1900397. https://doi.org/10.1002/biot.201900397
23. Chaitanya GV, Steven AJ, Babu PP. “PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration”. Cell Commun Signal. 2010; 8, 31. https://doi.org/10.1186/1478-811X-8-31
24. Liebl MC, Hofmann TG. “Cell Fate Regulation upon DNA Damage: p53 Serine 46 Kinases Pave the Cell Death Road”. Bioessays. 2019; 41(12), e1900127. https://doi.org/10.1002/bies.201900127
25. Ruvolo PP, Deng X, May WS. “Phosphorylation of Bcl2 and regulation of apoptosis”. Leukemia. 2001; 15(4), 515-22. https://doi.org/10.1038/sj.leu.2402090

There are 25 citations in total.

Details

Primary Language	English
Subjects	Signal Transduction
Journal Section	Original Article
Authors	Gökhan Yıldız 0000-0002-6714-3343 Soner Karabulut 0000-0001-9969-4409 Tuba Dinçer 0000-0001-5054-168X Bayram Toraman 0000-0003-4500-5668 Ersan Kalay 0000-0002-1467-2268
Project Number	This work was supported by the Office of Scientific Research Projects of Karadeniz Technical University, project Numbers TSA-2023-10539 and TSA-2024-11015 and the Scientific and Technological Research Council of Turkey (TÜBİTAK), project Number 119S356.
Early Pub Date	September 25, 2025
Publication Date	September 25, 2025
Submission Date	March 26, 2025
Acceptance Date	July 22, 2025
Published in Issue	Year 2025 Volume: 14 Issue: 3

Cite

APA	Yıldız, G., Karabulut, S., Dinçer, T., … Toraman, B. (2025). AIFM3 Has Pro-apoptotic, Whereas LZTR1 Has Bilateral Functions In The Intrinsic Apoptosis Pathway. Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi, 14(3), 932-937. https://doi.org/10.37989/gumussagbil.1665825
AMA	Yıldız G, Karabulut S, Dinçer T, Toraman B, Kalay E. AIFM3 Has Pro-apoptotic, Whereas LZTR1 Has Bilateral Functions In The Intrinsic Apoptosis Pathway. Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi. September 2025;14(3):932-937. doi:10.37989/gumussagbil.1665825
Chicago	Yıldız, Gökhan, Soner Karabulut, Tuba Dinçer, Bayram Toraman, and Ersan Kalay. “AIFM3 Has Pro-Apoptotic, Whereas LZTR1 Has Bilateral Functions In The Intrinsic Apoptosis Pathway”. Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi 14, no. 3 (September 2025): 932-37. https://doi.org/10.37989/gumussagbil.1665825.
EndNote	Yıldız G, Karabulut S, Dinçer T, Toraman B, Kalay E (September 1, 2025) AIFM3 Has Pro-apoptotic, Whereas LZTR1 Has Bilateral Functions In The Intrinsic Apoptosis Pathway. Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi 14 3 932–937.
IEEE	G. Yıldız, S. Karabulut, T. Dinçer, B. Toraman, and E. Kalay, “AIFM3 Has Pro-apoptotic, Whereas LZTR1 Has Bilateral Functions In The Intrinsic Apoptosis Pathway”, Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi, vol. 14, no. 3, pp. 932–937, 2025, doi: 10.37989/gumussagbil.1665825.
ISNAD	Yıldız, Gökhan et al. “AIFM3 Has Pro-Apoptotic, Whereas LZTR1 Has Bilateral Functions In The Intrinsic Apoptosis Pathway”. Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi 14/3 (September2025), 932-937. https://doi.org/10.37989/gumussagbil.1665825.
JAMA	Yıldız G, Karabulut S, Dinçer T, Toraman B, Kalay E. AIFM3 Has Pro-apoptotic, Whereas LZTR1 Has Bilateral Functions In The Intrinsic Apoptosis Pathway. Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi. 2025;14:932–937.
MLA	Yıldız, Gökhan et al. “AIFM3 Has Pro-Apoptotic, Whereas LZTR1 Has Bilateral Functions In The Intrinsic Apoptosis Pathway”. Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi, vol. 14, no. 3, 2025, pp. 932-7, doi:10.37989/gumussagbil.1665825.
Vancouver	Yıldız G, Karabulut S, Dinçer T, Toraman B, Kalay E. AIFM3 Has Pro-apoptotic, Whereas LZTR1 Has Bilateral Functions In The Intrinsic Apoptosis Pathway. Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi. 2025;14(3):932-7.

Download Cover Image

Article Files

Full Text