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Molecular and Cellular Biochemistry

, Volume 409, Issue 1–2, pp 13–22 | Cite as

Novel protein–protein interactions of TPPII, p53, and SIRT7

  • Jarmila Nahálková
Article

Abstract

Novel protein–protein interactions of TPPII, SIRT7, and p53 were detected by co-immunoprecipitation using both HeLa cell lysates and the cytoplasmic fraction prepared by fractionation of mouse liver tissue. The interactions were further verified in vivo by in situ proximity ligation assay (PLA) within control HEK293 cells transformed with empty vector, highactTPPII HEK293 cells over-expressing murine TPPII displaying high specific enzymatic activity and in lowactTPPII HEK293 cells over-expressing human TPPII having low specific activity of the enzyme. Besides an abundant cytoplasmic localization of TPPII-p53 interaction signal, the nuclear interactions were also demonstrated. The cytoplasmic interactions were likewise detected between TPPII and SIRT7 in control HEK293 and lowactTPPII HEK293 cells. The interactions of SIRT7 with p53 were confirmed in three HEK293 cell transformants as well. The cytoplasmic occurrence of SIRT7 protein was demonstrated by immunofluorescence, when both nucleolar and cytoplasmic signals were identified within HEK293 cells and primary human fibroblasts. The unique cytoplasmic localization of SIRT7 protein was discussed based on an epitope specificity of N-terminus specific SIRT7 antibodies utilized in the present study compared with C-terminus specific antibodies previously used for nuclear detection of SIRT7 by other authors. The epitope sequence of N-terminal antibodies is occurring in all three splicing variants of SIRT7 compared to the epitope of C-terminal antibody, which is specific exclusively to the splicing variant 1. The cytoplasmic localization of p53 detected by immunofluorescence supported the results from its interactions with TPPII and SIRT7 observed by in situ PLA within model cells. Novel interactions of TPPII, p53, and SIRT7 presented in this study might contribute to the knowledge of the regulatory effects of these proteins on apoptotic pathways and to the understanding mechanisms of aging and lifespan regulation.

Keywords

TPPII SIRT7 p53 Apoptosis Cellular senescence Aging 

Notes

Acknowledgments

I would like to thank Prof. Birgitta Tomkinson who provided HEK293 transformants and a space in her laboratory. The primary human fibroblasts were kindly provided by “Parkinson Institute Biobank” (http://www.parkinsonbiobank.com) of the Telethon Genetic Biobank Network supported by TELETHON Italy (Project No. GTB07001) and by “Fondazione Grigioni per il Morbo di Parkinson. Mouse liver tissue was provided by Dr. Maria Ringvall from animals, which were sacrificed for other kind of an experimental work covered by her own ethical approval. The experimental work with human fibroblasts was part of the project approved by Regional Ethical Committee (EPN) in Uppsala, Sweden under Registration Number 2014/044. The experimental research was financially supported by Försäkringskassan, O. E. och Edla Johanssons vetenskapliga stiftelse and Stiftelsen Sigurd och Elsa Goljes Minne. The funding sources did not have any role in the experimental design, analysis, and data interpretation or in making publishing decisions.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Medical Biochemistry and Microbiology (IMBIM), BMCUppsala UniversityUppsalaSweden

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