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Quantification of Protein Expression by Proximity Ligation Assay in the Nonhuman Primate in Response to Estrogen

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Estrogen Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2418))

Abstract

In the field of protein biology, immunology-based techniques are continuously evolving for the detection and quantification of individual protein levels, protein–protein interaction, and protein modifications in cells and tissues. The proximity ligation assay (PLA), a method of detection that combines immunologic and PCR-based approaches, was developed to overcome some of the drawbacks that are inherent with other detection methods. The PLA allows for very sensitive and discretely quantifiable measures of unmodified, native protein levels and protein–protein interaction/modification complexes in situ in both fixed tissues and cultured cells. We describe herein the PLA method and its applicability to quantify the effects of estrogen on expression of angioregulatory factors, e.g., endothelial nitric oxide synthase (eNOS) in the vasculature, vascular endothelial growth factor (VEGF) in the placenta, and melanocortin 2 receptor (MC2R)/accessory protein (MRAP) in the fetal adrenal of the nonhuman primate.

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References

  1. Weibrecht I, Leuchowius KJ, Clausson CM et al (2010) Proximity ligation assays: a recent addition to the proteomics toolbox. Expert Rev Proteomics 7:401–409

    Article  CAS  Google Scholar 

  2. Eisenstein M (2005) Westward expansion. Nat Methods 2(10):796

    Article  CAS  Google Scholar 

  3. Lequin R (2005) Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem 51(12):2415–2418

    Article  CAS  Google Scholar 

  4. Akercan F, Cirpan T, Terek MC et al (2008) The immunohistochemical evaluation of VEGF in placental biopsies of pregnancies complicated by preeclampsia. Arch Gynecol Obstet 277:109–114

    Article  CAS  Google Scholar 

  5. Lievens S, Tavernier J (2006) Single protein complex visualization: seeing is believing. Nat Methods 3:971–972

    Article  CAS  Google Scholar 

  6. Remy I, Michnick SW (2006) A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nat Methods 3:977–979

    Article  CAS  Google Scholar 

  7. Rochette S, Diss G, Filteau M et al (2015) Genome-wide protein-protein interaction screening by protein-fragment complementation assay (PCA) in living cells. Biology 97:e52255

    Google Scholar 

  8. Kurihara M, Ohmuro-Matsuyama Y, Ayabe K, Yamashita T et al (2016) Ultra sensitive firefly luciferase-based protein-protein interaction assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions. Biotechnol J 11:91–99

    Article  CAS  Google Scholar 

  9. Yurlova L, Derks M, Buchfellner A et al (2014) The fluorescent two-hybrid assay to screen for protein–protein interaction inhibitors in live cells: targeting the interaction of p53 with Mdm2 and Mdm4. J Biomol Screen 19(4):516–525

    Article  CAS  Google Scholar 

  10. Chrétien A-E, Gagnon-Arsenault I, Dubé A et al (2018) Extended linkers improve the detection of protein-protein interactions (PPIs) by dihydrofolate reductase protein-fragment complementation assay (DHFR PCA) in living cells. Mol Cell Proteomics 17(2):373–383

    Article  Google Scholar 

  11. Barcena M, Koster A (2009) Electron tomography in life science. Semin Cell Dev Biol 20(8):920–923

    Article  CAS  Google Scholar 

  12. Dudkina N, Kouril R, Bultema J et al (2010) Imaging of organelles by electron microscopy reveals protein–protein interactions in mitochondria and chloroplasts. FEBS Lett 524(12):2510–2515

    Article  Google Scholar 

  13. Keskin O, Tuncbag N, Gursoy A (2016) Predicting protein–protein interactions from the molecular to the proteome level. Chem Rev 116(8):4884–4909

    Article  CAS  Google Scholar 

  14. Kovacs I, Luck K, Spirohn K et al (2019) Network-based prediction of protein interactions. Nat Commun 10:1240

    Article  Google Scholar 

  15. Ding Z, Kihara D (2019) Computational identification of protein-protein interactions in model plant proteomes. Sci Rep 9:8740

    Article  Google Scholar 

  16. Gullberg M, Gustafsdottir S, Schallmeiner E et al (2004) Cytokine detection by antibody-based proximity ligation. Proc Natl Acad Sci U S A 101:8420–8424

    Article  CAS  Google Scholar 

  17. Gustafsdottir S, Schallmeiner E, Fredriksson S et al (2005) Proximity ligation assays for sensitive and specific protein analyses. Anal Biochem 345:2–9

    Article  CAS  Google Scholar 

  18. Soderberg O, Gullberg M, Jarvius M et al (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3:995–1000

    Article  Google Scholar 

  19. Kerppola TK (2006) Complementary methods for studies of protein interactions in living cells. Nat Methods 3:969–971

    Article  CAS  Google Scholar 

  20. Bonagura TW, Aberdeen GW, Babischkin JS et al (2010) Divergent regulation of angiopoietin-1 and -2, Tie-2, and thrombospondin-1 expression by estrogen in the baboon endometrium. Mol Reprod Dev 77:430–438

    Article  CAS  Google Scholar 

  21. Bonagura TW, Babischkin JS, Aberdeen GW et al (2012) Prematurely elevating estradiol in early baboon pregnancy suppresses uterine artery remodeling and expression of extravillous placental vascular endothelial growth factor and α1β1 and α5β1 integrins. Endocrinology 153:2897–2906

    Article  CAS  Google Scholar 

  22. Hansen MC, Nederby L, Henriksen MO et al (2014) Sensitive ligand-based protein quantification using immuno-PCR: a critical review of single-probe and proximity ligation assays. BioTechniques 56:217–228

    Article  CAS  Google Scholar 

  23. Trifilieff P, Rives ML, Urizar E et al (2011) Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum. BioTechniques 51:111–118

    Article  CAS  Google Scholar 

  24. Babischkin JS, Aberdeen GW, Lindner JR et al (2019) Vascular endothelial growth factor delivery to placental basal plate promotes uterine artery remodeling in the primate. Endocrinology 160:1492–1505. Reprinted with permission from Oxford University Press

    Article  CAS  Google Scholar 

  25. Babischkin JS, Aberdeen GW, Pepe GJ et al (2016) Estrogen suppresses interaction of melanocortin 2 receptor and its accessory protein in the primate fetal adrenal cortex. Endocrinology 157(12):4588–4601. Reprinted with permission from Oxford University Press

    Article  CAS  Google Scholar 

  26. Darmanis S, Kahler A, Spangberg L et al (2007) Self-assembly of proximity probes for flexible and modular proximity ligation assays. BioTechniques 43:443–450

    Article  CAS  Google Scholar 

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Acknowledgments

The authors appreciate the contributions of Dr. Graham Aberdeen in the collection of tissues used in this chapter. This work was supported by NIH Research Grants R01 HD 93070 and R01 DK 120513.

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Authors and Affiliations

  1. Department of Biology, Buena Vista University, Storm Lake, IA, USA

    Thomas W. Bonagura

  2. Department of Obstetrics and Gynecology, Reproductive Sciences, University of Maryland School of Medicine, Baltimore, MD, USA

    Jeffery S. Babischkin & Eugene D. Albrecht

  3. Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA, USA

    Gerald J. Pepe

Authors
  1. Thomas W. Bonagura
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  2. Jeffery S. Babischkin
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  3. Gerald J. Pepe
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  4. Eugene D. Albrecht
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Corresponding author

Correspondence to Eugene D. Albrecht .

Editor information

Editors and Affiliations

  1. Sanford School of Medicine, University of South Dakota, Vermillion, SD, USA

    Kathleen M. Eyster

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Bonagura, T.W., Babischkin, J.S., Pepe, G.J., Albrecht, E.D. (2022). Quantification of Protein Expression by Proximity Ligation Assay in the Nonhuman Primate in Response to Estrogen. In: Eyster, K.M. (eds) Estrogen Receptors. Methods in Molecular Biology, vol 2418. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1920-9_6

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  • DOI: https://doi.org/10.1007/978-1-0716-1920-9_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1919-3

  • Online ISBN: 978-1-0716-1920-9

  • eBook Packages: Springer Protocols

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