Characterization of Mitochondrial Heat Shock Protein 60 variants in HEK293 Cells Transformed into Steroidogenic
Main Article Content
Abstract
Introduction: During pregnancy, P4 is essential to maintain the maternal-fetal relationship. Maternal cholesterol is the main source of P4 production, a process that takes place in the syncytiotrophoblast mitochondrion. The mechanism and proteins involved in the cholesterol transport for the steroidogenic process are still unknown in detail. The STARD3 protein could be the substitute for its STARD1 equivalent localized in all acute response tissues. However, mutation or null STARD3 mice maintain their reproductive capacity, suggesting other proteins are involved in this process. Previously, we reported that the HSP60 participates in steroidogenesis in mitochondria isolated from the placental syncytiotrophoblast, mitochondrial contact sites or JEG-3.
Also, take relevance that non-steroidogenic cells, such as the HEK293, which are human kidney embryo cells, when are transformed into steroidogenic by transfection of the steroidogenic machinery, they synthesize progesterone.
To understand better the mechanism through which HSP60 participates in placental steroidogenesis, mutation of cysteine 442, which is essential in the active site for its activity, and deletion of 146 amino acid residues of the N-terminal of HSP60 were performed. The first was implemented to determine whether the protein structure is essential to support steroidogenesis, and the second was done to elucidate whether its activity occurs outside or inside the mitochondrion.
Methods: Two mutants were obtained: a) cysteine 442 was replaced by alanine (HSP60C442A) and b) the HSP60-mature (HSP60M) without the mitochondrial-leading sequence. Human kidney cells HEK293 were transformed into steroidogenic by transfection with pECE-P450scc, pCMV-3βHSD-I. The transfected cells were transfected with the HSP60wt, HSP60C442A, or HSP60M plasmids. The transfection was validated by western blot and P4 was determined by an enzyme immunoassay kit. HSP60 without mutations was used as control (HSP60wt).
Results: The synthesis of P4 was stimulated by the wild type HSP60 (HSP60wt). However, with both mutants, steroidogenesis occurred as in the control, suggesting that mutants do not support P4 synthesis.
Discussion: The mechanism to transport cholesterol to steroidogenic mitochondria requires the full HSP60 to support P4 synthesis, which is necessary to maintain pregnancy.
Highlights
- HSP60 participates in the steroidogenesis of transformed HEK293 cells.
- Cys442 mutant of HSP60 loses its activity in steroidogenesis.
- N-terminal deletion of HSP60 is not involved in steroidogenesis.
- Native HSP60 is critical for steroidogenesis.
Keywords:
Steroidogenesis, transformed HEK293, HSP60, P4 synthesis
Article Details
How to Cite
HERNÁNDEZ-ARCIGA, Ulalume et al.
Characterization of Mitochondrial Heat Shock Protein 60 variants in HEK293 Cells Transformed into Steroidogenic.
Medical Research Archives, [S.l.], v. 11, n. 9, sep. 2023.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/4274>. Date accessed: 21 dec. 2024.
doi: https://doi.org/10.18103/mra.v11i9.4274.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
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20. Zhang M, Liu P, Dwyer NK, et al. MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria. J Biol Chem. 2002;277(36):33300-33310. doi:10.1074/jbc.M200003200
21. Esparza-Perusquia M, Olvera-Sanchez S, Flores-Herrera O, et al. Mitochondrial proteases act on STARD3 to activate P4 synthesis in human syncytiotrophoblast. Biochim Biophys Acta. 2015;1850(1):107-117. doi:10.1016/j.bbagen.2014.10.009
22. Nara A, Komiya T, STARD3/MLN64 is striving at membrane contact sites: Intracellular cholesterol trafficking for steroidogenesis in human placental cells. Ame. J. Life Sci. 2015;3(3–2);48–52. doi:10.11648/j.ajls.s.2015030302.19
23. Hayashi T, Rizzuto R, Hajnoczky G, Su TP. MAM: more than just a housekeeper. Trends Cell Biol. 2009;19(2):81-88. doi:10.1016/j.tcb.2008.12.002
24. Uribe A, Strauss JF 3rd, Martínez F. Contact sites from human placental mitochondria: characterization and role in P4 synthesis. Arch Biochem Biophys. 2003;413(2):172-181. doi:10.1016/s0003-9861(03)00097-3
25. Toulmay A, Prinz WA. Lipid transfer and signaling at organelle contact sites: the tip of the iceberg. Curr Opin Cell Biol. 2011;23(4):458-463. doi:10.1016/j.ceb.2011.04.006
26. Kishida T, Kostetskii I, Zhang Z, et al. Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism. J Biol Chem. 2004;279(18):19276-19285. doi:10.1074/jbc.M400717200
27. Olvera-Sanchez S, Espinosa-Garcia MT, Monreal J, Flores-Herrera O, Martinez F. Mitochondrial heat shock protein participates in placental steroidogenesis. Placenta. 2011;32(3):222-229. doi:10.1016/j.placenta.2010.12.018
28. Nagumo Y, Kakeya H, Shoji M, Hayashi Y, Dohmae N, Osada H. Epolactaene binds human Hsp60 Cys442 resulting in the inhibition of chaperone activity. Biochem J. 2005;387(Pt 3):835-840. doi:10.1042/BJ20041355
29. Monreal-Flores J, Espinosa-García MT, García-Regalado A, Arechavaleta-Velasco F, Martínez F. The heat shock protein 60 promotes P4 synthesis in mitochondria of JEG-3 cells. Reprod Biol. 2017;17(2):154-161. doi:10.1016/j.repbio.2017.04.001
30. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989;77(1):51-59. doi:10.1016/0378-1119(89)90358-2
31. Gómez-Manzo S, Terrón-Hernández J, De la Mora-De la Mora I, et al. The stability of G6PD is affected by mutations with different clinical phenotypes. Int J Mol Sci. 2014;15(11):21179-21201. doi:10.3390/ijms151121179
32. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-685. doi:10.1038/227680a0
33. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979;76(9):4350-4354. doi:10.1073/pnas.76.9.4350
34. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. doi:10.1006/abio.1976.9999
35. Benjamini Y, Krieger AM, Yekutieli D, Adaptive linear step-up procedures that control the false discovery rate. Biometrika. 2006;9:491–507. https://doi.org/10.1093/biomet/93.3.491
36. Rasmussen MK, Ekstrand B, Zamaratskaia G. Regulation of 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase: a review. Int J Mol Sci. 2013;14(9):17926-17942. doi:10.3390/ijms140917926
37. Payne AH, Clarke TR, Bain PA. The murine 3 beta-hydroxysteroid dehydrogenase multigene family: structure, function, and tissue-specific expression. J Steroid Biochem Mol Biol. 1995;53(1-6):111-118. doi:10.1016/0960-0760(95)00028-x
38. Gomez-Sanchez CE, Lewis M, Nanba K, Rainey WE, Kuppusamy M, Gomez-Sanchez EP. Development of monoclonal antibodies against the human 3β-hydroxysteroid dehydrogenase/isomerase isozymes. Steroids. 2017;127:56-61. doi:10.1016/j.steroids.2017.08.011
39. Pagotto MA, Roldán ML, Pagotto RM, et al. Localization and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1) in the adult rat kidney. Mol Cell Endocrinol. 2011;332(1-2):253-260. doi:10.1016/j.mce.2010.10.020
40. Rahaman MM, Sawa T, Ahtesham AK, et al. S-guanylation proteomics for redox-based mitochondrial signaling. Antioxid Redox Signal. 2014;20(2):295-307. doi:10.1089/ars.2012.4606
41. Ghosh JC, Siegelin MD, Dohi T, Altieri DC. Heat shock protein 60 regulation of the mitochondrial permeability transition pore in tumor cells. Cancer Res. 2010;70(22):8988-8993. doi:10.1158/0008-5472.CAN-10-2225
42. Spinello A, Barone G, Cappello F, Pace A, Buscemi S, Palumbo Piccionello A, The binding mechanism of epolactaene to Hsp60 unveiled by in silico modelling, Chemistry Select. 2016;1(4):759–765. https://doi.org/10.1002/slct.201600125
43. Smyth DG, Blumenfeld OO, Konigsberg W. Reactions of N-ethylmaleimide with peptides and amino acids. Biochem J. 1964;91(3):589-595. doi:10.1042/bj0910589
44. Henderson B, Fares MA, Lund PA. Chaperonin 60: a paradoxical, evolutionarily conserved protein family with multiple moonlighting functions. Biol Rev Camb Philos Soc. 2013;88(4):955-987. doi:10.1111/brv.12037
45. Rosal KG, Chen WY, Chung BC. The A'-helix of CYP11A1 remodels mitochondrial cristae. J Biomed Sci. 2022;29(1):61. doi:10.1186/s12929-022-00846-7
46. Martínez F, Kiriakidou M, Strauss JF 3rd. Structural and functional changes in mitochondria associated with trophoblast differentiation: methods to isolate enriched preparations of syncytiotrophoblast mitochondria. Endocrinology. 1997;138(5):2172-2183. doi:10.1210/endo.138.5.5133
47. De los Rios Castillo D, Zarco-Zavala M, Olvera-Sanchez S, et al. Atypical cristae morphology of human syncytiotrophoblast mitochondria: role for complex V. J Biol Chem. 2011;286(27):23911-23919. doi:10.1074/jbc.M111.252056
48. Mogk A, Ruger-Herreros C, Bukau B. Cellular Functions and Mechanisms of Action of Small Heat Shock Proteins. Annu Rev Microbiol. 2019;73:89-110. doi:10.1146/annurev-micro-020518-115515
49. Saibil H. Chaperone machines for protein folding, unfolding and disaggregation. Nat Rev Mol Cell Biol. 2013;14(10):630-642. doi:10.1038/nrm3658
50. Caruso Bavisotto C, Alberti G, Vitale AM, et al. Hsp60 post-translational modifications: functional and pathological consequences. Front Mol Biosci. 2020;7:95. doi:10.3389/fmolb.2020.00095
2. Gude NM, Roberts CT, Kalionis B, King RG. Growth and function of the normal human placenta. Thromb Res. 2004;114(5-6):397-407. doi:10.1016/j.thromres.2004.06.038
3. Burton GJ, Fowden AL. The placenta: a multifaceted, transient organ. Philos Trans R Soc Lond B Biol Sci. 2015;370(1663):20140066. doi:10.1098/rstb.2014.0066
4. Ortega MA, Fraile-Martínez O, García-Montero C, et al. The pivotal role of the placenta in normal and pathological pregnancies: A focus on preeclampsia, fetal growth restriction, and maternal chronic venous disease. Cells. 2022;11(3):568. doi:10.3390/cells11030568
5. Aspillaga MO, Whittaker PG, Grey CE, Lind T. Endocrinologic events in early pregnancy failure. Am J Obstet Gynecol. 1983;147(8):903-908. doi:10.1016/0002-9378(83)90243-0
6. Martinez F, Olvera-Sanchez S, Esparza-Perusquia M, Gomez-Chang E, Flores-Herrera O. Multiple functions of syncytiotrophoblast mitochondria. Steroids. 2015;103:11-22. doi:10.1016/j.steroids.2015.09.006
7. Dey SK, Lim H, Das SK, et al. Molecular cues to implantation. Endocr Rev. 2004;25(3):341-373. doi:10.1210/er.2003-0020
8. Halasz M, Szekeres-Bartho J. The role of P4 in implantation and trophoblast invasion. J Reprod Immunol. 2013;97(1):43-50. doi:10.1016/j.jri.2012.10.011
9. Shah NM, Lai PF, Imami N, Johnson MR. P4-related immune modulation of pregnancy and labor. Front Endocrinol (Lausanne). 2019;10:198. doi:10.3389/fendo.2019.00198
10. Sugawara T, Holt JA, Driscoll D, et al. Human steroidogenic acute regulatory protein: functional activity in COS-1 cells, tissue-specific expression, and mapping of the structural gene to 8p11.2 and a pseudogene to chromosome 13. Proc Natl Acad Sci U S A. 1995;92(11):4778-4782. doi:10.1073/pnas.92.11.4778
11. Csapo AI, Wiest WG. An examination of the quantitative relationship between P4 and the maintenance of pregnancy. Endocrinology. 1969;85(4):735-746. doi:10.1210/endo-85-4-735
12. Chatuphonprasert W, Jarukamjorn K, Ellinger I. Physiology and pathophysiology of steroid biosynthesis, transport and metabolism in the human placenta. Front Pharmacol. 2018;9:1027. doi:10.3389/fphar.2018.01027
13. Woollett LA, Shah AS. Fetal and neonatal sterol metabolism. In: Feingold KR, Anawalt B, Blackman MR, et al., eds. Endotext. South Dartmouth (MA): MDText.com, Inc.; March 25, 2023.
14. Yañez MJ, Leiva A. Human placental intracellular cholesterol transport: a focus on lysosomal and mitochondrial dysfunction and oxidative Stress. Antioxidants (Basel). 2022;11(3):500. doi:10.3390/antiox11030500
15. Moog-Lutz C, Tomasetto C, Régnier CH, et al. MLN64 exhibits homology with the steroidogenic acute regulatory protein (STAR) and is over-expressed in human breast carcinomas. Int J Cancer. 1997;71(2):183-191. doi:10.1002/(sici)1097-0215(19970410)71:2<183::aid-ijc10>3.0.co;2-j
16. Bose HS, Whittal RM, Huang MC, Baldwin MA, Miller WL. N-218 MLN64, a protein with StAR-like steroidogenic activity, is folded and cleaved similarly to StAR. Biochemistry. 2000;39(38):11722-11731. doi:10.1021/bi000911l
17. Watari H, Arakane F, Moog-Lutz C, et al. MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis. Proc Natl Acad Sci U S A. 1997;94(16):8462-8467. doi:10.1073/pnas.94.16.8462
18. Tuckey RC, Bose HS, Czerwionka I, Miller WL. Molten globule structure and steroidogenic activity of N-218 MLN64 in human placental mitochondria. Endocrinology. 2004;145(4):1700-1707. doi:10.1210/en.2003-1034
19. Charman M, Kennedy BE, Osborne N, Karten B. MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein. J Lipid Res. 2010;51(5):1023-1034. doi:10.1194/jlr.M002345
20. Zhang M, Liu P, Dwyer NK, et al. MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria. J Biol Chem. 2002;277(36):33300-33310. doi:10.1074/jbc.M200003200
21. Esparza-Perusquia M, Olvera-Sanchez S, Flores-Herrera O, et al. Mitochondrial proteases act on STARD3 to activate P4 synthesis in human syncytiotrophoblast. Biochim Biophys Acta. 2015;1850(1):107-117. doi:10.1016/j.bbagen.2014.10.009
22. Nara A, Komiya T, STARD3/MLN64 is striving at membrane contact sites: Intracellular cholesterol trafficking for steroidogenesis in human placental cells. Ame. J. Life Sci. 2015;3(3–2);48–52. doi:10.11648/j.ajls.s.2015030302.19
23. Hayashi T, Rizzuto R, Hajnoczky G, Su TP. MAM: more than just a housekeeper. Trends Cell Biol. 2009;19(2):81-88. doi:10.1016/j.tcb.2008.12.002
24. Uribe A, Strauss JF 3rd, Martínez F. Contact sites from human placental mitochondria: characterization and role in P4 synthesis. Arch Biochem Biophys. 2003;413(2):172-181. doi:10.1016/s0003-9861(03)00097-3
25. Toulmay A, Prinz WA. Lipid transfer and signaling at organelle contact sites: the tip of the iceberg. Curr Opin Cell Biol. 2011;23(4):458-463. doi:10.1016/j.ceb.2011.04.006
26. Kishida T, Kostetskii I, Zhang Z, et al. Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism. J Biol Chem. 2004;279(18):19276-19285. doi:10.1074/jbc.M400717200
27. Olvera-Sanchez S, Espinosa-Garcia MT, Monreal J, Flores-Herrera O, Martinez F. Mitochondrial heat shock protein participates in placental steroidogenesis. Placenta. 2011;32(3):222-229. doi:10.1016/j.placenta.2010.12.018
28. Nagumo Y, Kakeya H, Shoji M, Hayashi Y, Dohmae N, Osada H. Epolactaene binds human Hsp60 Cys442 resulting in the inhibition of chaperone activity. Biochem J. 2005;387(Pt 3):835-840. doi:10.1042/BJ20041355
29. Monreal-Flores J, Espinosa-García MT, García-Regalado A, Arechavaleta-Velasco F, Martínez F. The heat shock protein 60 promotes P4 synthesis in mitochondria of JEG-3 cells. Reprod Biol. 2017;17(2):154-161. doi:10.1016/j.repbio.2017.04.001
30. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989;77(1):51-59. doi:10.1016/0378-1119(89)90358-2
31. Gómez-Manzo S, Terrón-Hernández J, De la Mora-De la Mora I, et al. The stability of G6PD is affected by mutations with different clinical phenotypes. Int J Mol Sci. 2014;15(11):21179-21201. doi:10.3390/ijms151121179
32. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-685. doi:10.1038/227680a0
33. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979;76(9):4350-4354. doi:10.1073/pnas.76.9.4350
34. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. doi:10.1006/abio.1976.9999
35. Benjamini Y, Krieger AM, Yekutieli D, Adaptive linear step-up procedures that control the false discovery rate. Biometrika. 2006;9:491–507. https://doi.org/10.1093/biomet/93.3.491
36. Rasmussen MK, Ekstrand B, Zamaratskaia G. Regulation of 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase: a review. Int J Mol Sci. 2013;14(9):17926-17942. doi:10.3390/ijms140917926
37. Payne AH, Clarke TR, Bain PA. The murine 3 beta-hydroxysteroid dehydrogenase multigene family: structure, function, and tissue-specific expression. J Steroid Biochem Mol Biol. 1995;53(1-6):111-118. doi:10.1016/0960-0760(95)00028-x
38. Gomez-Sanchez CE, Lewis M, Nanba K, Rainey WE, Kuppusamy M, Gomez-Sanchez EP. Development of monoclonal antibodies against the human 3β-hydroxysteroid dehydrogenase/isomerase isozymes. Steroids. 2017;127:56-61. doi:10.1016/j.steroids.2017.08.011
39. Pagotto MA, Roldán ML, Pagotto RM, et al. Localization and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1) in the adult rat kidney. Mol Cell Endocrinol. 2011;332(1-2):253-260. doi:10.1016/j.mce.2010.10.020
40. Rahaman MM, Sawa T, Ahtesham AK, et al. S-guanylation proteomics for redox-based mitochondrial signaling. Antioxid Redox Signal. 2014;20(2):295-307. doi:10.1089/ars.2012.4606
41. Ghosh JC, Siegelin MD, Dohi T, Altieri DC. Heat shock protein 60 regulation of the mitochondrial permeability transition pore in tumor cells. Cancer Res. 2010;70(22):8988-8993. doi:10.1158/0008-5472.CAN-10-2225
42. Spinello A, Barone G, Cappello F, Pace A, Buscemi S, Palumbo Piccionello A, The binding mechanism of epolactaene to Hsp60 unveiled by in silico modelling, Chemistry Select. 2016;1(4):759–765. https://doi.org/10.1002/slct.201600125
43. Smyth DG, Blumenfeld OO, Konigsberg W. Reactions of N-ethylmaleimide with peptides and amino acids. Biochem J. 1964;91(3):589-595. doi:10.1042/bj0910589
44. Henderson B, Fares MA, Lund PA. Chaperonin 60: a paradoxical, evolutionarily conserved protein family with multiple moonlighting functions. Biol Rev Camb Philos Soc. 2013;88(4):955-987. doi:10.1111/brv.12037
45. Rosal KG, Chen WY, Chung BC. The A'-helix of CYP11A1 remodels mitochondrial cristae. J Biomed Sci. 2022;29(1):61. doi:10.1186/s12929-022-00846-7
46. Martínez F, Kiriakidou M, Strauss JF 3rd. Structural and functional changes in mitochondria associated with trophoblast differentiation: methods to isolate enriched preparations of syncytiotrophoblast mitochondria. Endocrinology. 1997;138(5):2172-2183. doi:10.1210/endo.138.5.5133
47. De los Rios Castillo D, Zarco-Zavala M, Olvera-Sanchez S, et al. Atypical cristae morphology of human syncytiotrophoblast mitochondria: role for complex V. J Biol Chem. 2011;286(27):23911-23919. doi:10.1074/jbc.M111.252056
48. Mogk A, Ruger-Herreros C, Bukau B. Cellular Functions and Mechanisms of Action of Small Heat Shock Proteins. Annu Rev Microbiol. 2019;73:89-110. doi:10.1146/annurev-micro-020518-115515
49. Saibil H. Chaperone machines for protein folding, unfolding and disaggregation. Nat Rev Mol Cell Biol. 2013;14(10):630-642. doi:10.1038/nrm3658
50. Caruso Bavisotto C, Alberti G, Vitale AM, et al. Hsp60 post-translational modifications: functional and pathological consequences. Front Mol Biosci. 2020;7:95. doi:10.3389/fmolb.2020.00095