In Situ Proximity Ligation Assay for Detection of Proteins, Their Interactions and Modifications

Česká verzia

Authors: V. Brychtová; B. Vojtěšek
Authors place of work: Regionální centrum aplikované molekulární onkologie, Masarykův onkologický ústav, Brno
Published in the journal: Klin Onkol 2014; 27(Supplementum): 87-91

Summary

To understand cellular processes and events responsible for their perturbations, proteomic analyses are needed in biomedical research and clinical diagnostics. Several techniques based on specifically binding reagents (antibodies) or recombinant proteins (GFP fusion protein, methods of fluorescence/ bioluminescence resonance energy transfer) are generally used to study protein location and activity resulting from secondary modifications and interactions. The in situ proximity ligation assay represents a novel technique of in situ protein imaging using DNA as a reporter molecule and DNA amplification processes. This method enables direct visualization of single molecules, their levels, modifications and pattern of interactions in individual fixed cells and tissues. Proximity probes consist of specific antibody with attached oligonucleotides that are used as reporter molecules for identification of such events. Proximity probes guide the formation of a circular DNA strand when bound in close proximity. The DNA circle after that serves as a template for rolling‑ circle amplification allowing the interaction to be visualized. Compared to available proteomic techniques benefiting from genetic engineering, in situ PLA enables study of endogenous proteins in their natural environment and thus can be used for clinical specimens. The areas of applicability where proximity ligation procedure can be used include any research field where protein interaction measurements are important, such as signaling pathway studies, monitoring of pharmacological treatment targets and oncological diagnostics.

Key words:
in situ PLA – protein interaction – protein detection methods – proximity ligation

This work was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101) and by MH CZ – DRO (MMCI, 00209805) and BBMRI_CZ (LM2010004).

The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.

The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers.

Submitted:
31. 1. 2014

Accepted:
25. 3. 2014

Zdroje

1. Hung MC, Link W. Protein localization in disease and therapy. J Cell Sci 2011; 124(20): 3381– 3392. doi: 10.1242/ jcs.089110.

2. Fabbro M, Henderson BR. Regulation of tumor suppressors by nuclear‑ cytoplasmic shuttling. Exp Cell Res 2003(2); 282: 59– 69.

3. Hu MC, Lee DF, Xia W et al. IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a. Cell 2011; 117(2): 225– 237.

4. Chen MF, Fang FM, Lu C et al. Significance of nuclear accumulation of Foxo3a in esophageal squamous cell carcinoma. Int J Radiat Oncol Biol Phys 2008; 71: 1220– 1229. doi: 10.1016/ j.ijrobp.2008.02.077.

5. Xia W, Chen J, Zhou X et al. Phosphorylation/ cytoplasmic localization of p21Cip1/ WAF1 is associated with HER2/ neu overexpression and provides a novel combination predictor for poor prognosis in breast cancer patients. Clin Cancer Res 2004; 10(11): 3815– 3824.

6. Xia X, Ma Q, Li X et al. Cytoplasmic p21 is a potential predictor for cisplatin sensitivity in ovarian cancer. BMC Cancer 2011; 11: 399. doi: 10.1186/ 1471- 2407- 11- 399.

7. Liang J, Zubovitz J, Petrocelli T et al. PKB/ Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27- mediated G1 arrest. Nat Med 2002; 8(10): 1153– 1160.

8. Nan KJ, Jing Z, Gong L. Expression and altered subcellular localization of the cyclin‑dependent kinase inhibitor p27Kip1 in hepatocellular carcinoma. World J Gastroenterol 2004; 10(10): 1425– 1430.

9. Rosen DG, Yang G, Cai KQ et al. Subcellular localization of p27kip1 expression predicts poor prognosis in human ovarian cancer. Clin Cancer Res 2005; 11(1): 632– 637.

10. Ogino S, Shima K, Nosho K et al. A cohort study of p27 localization in colon cancer, body mass index, and patient survival. Cancer Epidemiol Biomarkers Prev 2009; 18(6): 1849– 1858. doi: 10.1158/ 1055-9965.EPI‑ 09- 0181.

11. Li C, Iida M, Dunn E et al. Nuclear EGFR contributes to acquired resistance to cetuximab. Oncogene 2009; 28: 3801– 3813. doi: 10.1038/ onc.2009.234.

12. Hsu S, Miller S, Wang Y et al. Nuclear EGFR is required for cisplatin resistance and DNA repair. Am J Transl Res 2009; 1(3): 249– 258.

13. Weibrecht I, Leuchowius KJ, Clausson CM et al. Proximity ligation assays: a recent addition to the proteomics toolbox. Expert Rev Proteomics 2010; 7(3): 401– 409. doi: 10.1586/ epr.10.10.

14. Pfleger KD, Eidne KA. Illuminating insights into protein‑protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 2006; 3(3): 165– 174.

15. Fredriksson S, Gullberg M, Jarvius J et al. Protein detection using proximity‑ dependent DNA ligation assays. Nat Biotechnol 2002; 20(5): 473– 477.

16. Gullberg M, Gústafsdóttir SM, Schallmeiner E et al. Cytokine detection by antibody‑based proximity ligation. Proc Natl Acad Sci USA 2004; 101(22): 8420– 8424.

17. Nong RY, Wu D, Yan J et al. Solid‑ phase proximity ligation assays for individual or parallel protein analyses with readout via real‑ time PCR or sequencing. Nat Protoc 2013; 8(6): 1234– 1248. doi: 10.1038/ nprot.2013.070.

18. Soderberg O, Gullberg M, Jarvius M et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 2006; 3(12): 995– 1000.

19. Jarvius M, Paulsson J, Weibrecht I et al. In situ detection of phosphorylated platelet‑ derived growth factor receptor beta using a generalized proximity ligation method. Mol Cell Proteomics 2007; 6(9): 1500– 1509.

20. Gu GJ, Friedman M, Jost C et al. Protein tag‑ mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation. N Biotechnol 2013; 144– 152. doi: 10.1016/j.nbt.2012.05.005.

21. Gustafsdottir SM, Schlingemann J, Rada‑ Iglesias A et al. In vitro analysis of DNA‑ protein interactions by proximity ligation. Proc Natl Acad Sci USA 2007; 104(9): 3067– 3072.

22. Gomez D, Shankman LS, Nguyen AT et al. Detection of histone modifications at specific gene loci in single cells in histological sections. Nat Methods 2013; 10(2): 171– 177. doi: 10.1038/ nmeth.2332.

23. Koos B, Andersson L, Clausson CM et al. Analysis of protein interactions in situ by proximity ligation assays. Curr Top Microbiol Immunol 2014; 377: 111– 126. doi: 10.1007/ 82_2013_334.

24. Weibrecht I, Gavrilovic M, Lindbom L et al. Visualising individual sequence‑ specific protein‑DNA interactions in situ. N Biotechnol 2012; 29(5): 589– 598. doi: 10.1016/ j.nbt.2011.08.002.

25. Koos B, Paulsson J, Jarvius M et al. Platelet‑ derived growth factor receptor expression and activation in choroid plexus tumors. Am J Pathol 2009; 175(4): 1631– 1637. doi: 10.2353/ ajpath.2009.081022.

26. Pinto R, Carvalho AS, Conze T et al. Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation. J Cell Mol Med 2012; 16(7): 1474– 1484. doi: 10.1111/ j.1582- 4934.2011.01436.x.

27. Renfrow JJ, Scheck AC, Dhawan NS et al. Gene‑ protein correlation in single cells. Neuro Oncol 2011; 13(8): 880– 885. doi: 10.1093/ neuonc/ nor071.

28. Nilsson M, Malmgren H, Samiotaki M et al. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 1994; 265(5181): 2085– 2088.

29. Leuchowius KJ, Jarvius M, Wickström M et al. High content screening for inhibitors of protein interactions and post‑translational modifications in primary cells by proximity ligation. Mol Cell Proteomics 2010; 9(1): 178– 183. doi: 10.1074/ mcp.M900331-MCP200.