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Detection of APOBEC3 Proteins and Catalytic Activity in Urothelial Carcinoma

  • Ananda Ayyappan Jaguva Vasudevan
  • Wolfgang Goering
  • Dieter Häussinger
  • Carsten MünkEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1655)

Abstract

Members of the APOBEC3 (A3) family of enzymes were shown to act in an oncogenic manner in several cancer types. Immunodetection of APOBEC3A (A3A), APOBEC3B (A3B), and APOBEC3G (A3G) proteins is particularly challenging due to the large sequence homology of these proteins and limited availability of antibodies. Here we combine independent immunoblotting with an in vitro activity assay technique, to detect and categorize specific A3s expressed in urothelial bladder cancer and other cancer cells.

Key words

APOBEC3 Cytidine deaminase Urothelial bladder cancer Mutation Deamination assay Cancer cell 

Notes

Acknowledgments

We would like to thank Klaus Strebel and the NIH AIDS Research and Reference Reagent Program for anti-ApoC17 (A3G) antibody. We are grateful to W. A. Schulz for his constant support. CM is supported by the Heinz Ansmann foundation.

References

  1. 1.
    Sheehy AM, Gaddis NC, Choi JD, Malim MH (2002) Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418(6898):646–650. doi: 10.1038/nature00939 CrossRefPubMedGoogle Scholar
  2. 2.
    Bishop KN, Holmes RK, Sheehy AM, Davidson NO, Cho SJ, Malim MH (2004) Cytidine deamination of retroviral DNA by diverse APOBEC proteins. Curr Biol 14(15):1392–1396. doi: 10.1016/j.cub.2004.06.057 CrossRefPubMedGoogle Scholar
  3. 3.
    Zhang H, Yang B, Pomerantz RJ, Zhang C, Arunachalam SC, Gao L (2003) The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. Nature 424(6944):94–98. doi: 10.1038/nature01707 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Harris RS, Dudley JP (2015) APOBECs and virus restriction. Virology 479-480:131–145. doi: 10.1016/j.virol.2015.03.012 CrossRefPubMedGoogle Scholar
  5. 5.
    Chiu YL, Greene WC (2008) The APOBEC3 cytidine deaminases: an innate defensive network opposing exogenous retroviruses and endogenous retroelements. Annu Rev Immunol 26:317–353. doi: 10.1146/annurev.immunol.26.021607.090350 CrossRefPubMedGoogle Scholar
  6. 6.
    Vasudevan AA, Smits SH, Hoppner A, Häussinger D, Koenig BW, Münk C (2013) Structural features of antiviral DNA cytidine deaminases. Biol Chem 394(11):1357–1370. doi: 10.1515/hsz-2013-0165 CrossRefPubMedGoogle Scholar
  7. 7.
    Yu Q, Konig R, Pillai S, Chiles K, Kearney M, Palmer S et al (2004) Single-strand specificity of APOBEC3G accounts for minus-strand deamination of the HIV genome. Nat Struct Mol Biol 11(5):435–442. doi: 10.1038/nsmb758 CrossRefPubMedGoogle Scholar
  8. 8.
    Burns MB, Lackey L, Carpenter MA, Rathore A, Land AM, Leonard B et al (2013) APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494(7437):366–370. doi: 10.1038/nature11881 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Henderson S, Fenton T (2015) APOBEC3 genes: retroviral restriction factors to cancer drivers. Trends Mol Med 21(5):274–284. doi: 10.1016/j.molmed.2015.02.007 CrossRefPubMedGoogle Scholar
  10. 10.
    Burns MB, Leonard B, Harris RS (2015) APOBEC3B: pathological consequences of an innate immune DNA mutator. Biom J 38(2):102–110. doi: 10.4103/2319-4170.148904 Google Scholar
  11. 11.
    Chelico L, Pham P, Calabrese P, Goodman MF (2006) APOBEC3G DNA deaminase acts processively 3′ –> 5′ on single-stranded DNA. Nat Struct Mol Biol 13(5):392–399. doi: 10.1038/nsmb1086 CrossRefPubMedGoogle Scholar
  12. 12.
    Nowarski R, Britan-Rosich E, Shiloach T, Kotler M (2008) Hypermutation by intersegmental transfer of APOBEC3G cytidine deaminase. Nat Struct Mol Biol 15(10):1059–1066. doi: 10.1038/nsmb.1495 CrossRefPubMedGoogle Scholar
  13. 13.
    Sheehy AM, Gaddis NC, Malim MH (2003) The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif. Nat Med 9(11):1404–1407. doi: 10.1038/nm945 CrossRefPubMedGoogle Scholar
  14. 14.
    Yu X, Yu Y, Liu B, Luo K, Kong W, Mao P et al (2003) Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex. Science 302(5647):1056–1060. doi: 10.1126/science.1089591 CrossRefPubMedGoogle Scholar
  15. 15.
    Roberts SA, Sterling J, Thompson C, Harris S, Mav D, Shah R et al (2012) Clustered mutations in yeast and in human cancers can arise from damaged long single-strand DNA regions. Mol Cell 46(4):424–435. doi: 10.1016/j.molcel.2012.03.030 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Nik-Zainal S, Alexandrov LB, Wedge DC, Van Loo P, Greenman CD, Raine K et al (2012) Mutational processes molding the genomes of 21 breast cancers. Cell 149(5):979–993. doi: 10.1016/j.cell.2012.04.024 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Roberts SA, Lawrence MS, Klimczak LJ, Grimm SA, Fargo D, Stojanov P et al (2013) An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers. Nat Genet 45(9):970–976. doi: 10.1038/ng.2702 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Cancer Genome Atlas Research N (2014) Comprehensive molecular characterization of urothelial bladder carcinoma. Nature 507(7492):315–322. doi: 10.1038/nature12965 CrossRefGoogle Scholar
  19. 19.
    Hedegaard J, Lamy P, Nordentoft I, Algaba F, Hoyer S, Ulhoi BP et al (2016) Comprehensive transcriptional analysis of early-stage urothelial carcinoma. Cancer Cell 30(1):27–42. doi: 10.1016/j.ccell.2016.05.004 CrossRefPubMedGoogle Scholar
  20. 20.
    Lamy P, Nordentoft I, Birkenkamp-Demtroder K, Thomsen MB, Villesen P, Vang S et al (2016) Paired exome analysis reveals clonal evolution and potential therapeutic targets in urothelial carcinoma. Cancer Res 76(19):5894–5906. doi: 10.1158/0008-5472.CAN-16-0436 CrossRefPubMedGoogle Scholar
  21. 21.
    Chan K, Roberts SA, Klimczak LJ, Sterling JF, Saini N, Malc EP et al (2015) An APOBEC3A hypermutation signature is distinguishable from the signature of background mutagenesis by APOBEC3B in human cancers. Nat Genet 47(9):1067–1072. doi: 10.1038/ng.3378 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Long J, Delahanty RJ, Li G, Gao YT, Lu W, Cai Q et al (2013) A common deletion in the APOBEC3 genes and breast cancer risk. J Natl Cancer Inst 105(8):573–579. doi: 10.1093/jnci/djt018 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Xuan D, Li G, Cai Q, Deming-Halverson S, Shrubsole MJ, Shu XO et al (2013) APOBEC3 deletion polymorphism is associated with breast cancer risk among women of European ancestry. Carcinogenesis 34(10):2240–2243. doi: 10.1093/carcin/bgt185 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Caval V, Suspene R, Shapira M, Vartanian JP, Wain-Hobson S (2014) A prevalent cancer susceptibility APOBEC3A hybrid allele bearing APOBEC3B 3'UTR enhances chromosomal DNA damage. Nat Commun 5:5129. doi: 10.1038/ncomms6129 CrossRefPubMedGoogle Scholar
  25. 25.
    Nik-Zainal S, Wedge DC, Alexandrov LB, Petljak M, Butler AP, Bolli N et al (2014) Association of a germline copy number polymorphism of APOBEC3A and APOBEC3B with burden of putative APOBEC-dependent mutations in breast cancer. Nat Genet 46(5):487–491. doi: 10.1038/ng.2955 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Starrett GJ, Luengas EM, McCann JL, Ebrahimi D, Temiz NA, Love RP et al (2016) The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis. Nat Commun 7:12918. doi: 10.1038/ncomms12918 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Jaguva Vasudevan AA, Perkovic M, Bulliard Y, Cichutek K, Trono D, Häussinger D, Münk C (2013) Prototype foamy virus bet impairs the dimerization and cytosolic solubility of human APOBEC3G. J Virol 87(16):9030–9040. doi: 10.1128/JVI.03385-12 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Marino D, Perkovic M, Hain A, Jaguva Vasudevan AA, Hofmann H, Hanschmann KM et al (2016) APOBEC4 enhances the replication of HIV-1. PLoS One 11(6):e0155422. doi: 10.1371/journal.pone.0155422 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Fogg MJ, Pearl LH, Connolly BA (2002) Structural basis for uracil recognition by archaeal family B DNA polymerases. Nat Struct Biol 9(12):922–927. doi: 10.1038/nsb867 CrossRefPubMedGoogle Scholar
  30. 30.
    Firbank SJ, Wardle J, Heslop P, Lewis RJ, Connolly BA (2008) Uracil recognition in archaeal DNA polymerases captured by X-ray crystallography. J Mol Biol 381(3):529–539. doi: 10.1016/j.jmb.2008.06.004 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Ananda Ayyappan Jaguva Vasudevan
    • 1
    • 2
  • Wolfgang Goering
    • 2
    • 3
  • Dieter Häussinger
    • 1
  • Carsten Münk
    • 1
    Email author
  1. 1.Clinic for Gastroenterology, Hepatology, and Infectiology, Medical FacultyHeinrich Heine University DüsseldorfDüsseldorfGermany
  2. 2.Department of Urology, Medical FacultyHeinrich Heine University DüsseldorfDüsseldorfGermany
  3. 3.Institute of Pathology, Medical FacultyHeinrich Heine University DüsseldorfDüsseldorfGermany

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