Earlier, the biological effect of tripeptidylpeptidase II (TPPII) in cells was often associated only with a protein degradation, since the enzyme functions downstream of the ubiquitin-proteasome system and provides together with other amino- and exopeptidases free amino acid pool for biosynthesis. However, several previous functional studies including cancer cells, knockout mouse models and specific TPPII substrates suggested, that the enzyme plays additional cellular functions. In cancer cells, TPPII was shown to be required for their survival and a previous experimental work also confirmed its regulatory effects on apoptosis, the cell cycle and DNA damage responses. It also regulated the cellular level of important neuropeptides like cholecystokinin and somatostatin and the deficiency in experimental animals was manifested by a shortened lifespan, early ageing and immunosenescence. Since the detail molecular mechanisms how TPPII controls these cellular events are not known yet, here protein interaction partners were analysed with the aim to identify corresponding pathways. Both conventional co-IP and very sensitive method in situ Proximity Ligation Assay (PLA) were exploited for the identification. In situ PLA is an established method for the detection of single molecule interactions based on the principle, that only the specifically bound probes localized in very close proximity (< 16 nm) can form circular DNA. The formed circular template can be further amplified by the rolling-circle amplification and be detected by fluorescent markers within cells. The used methods identified a novel protein-protein interactions with p53 and SIRT7, which are important targets for development of anti-tumour therapeutics, neuroprotective treatments and for improvement of the symptoms of diseases of ageing. TPPII was also implicated in the anti-cancer drug development, since inhibitors of the enzymatic activity were proposed as potential tumor-suppressing agents. One of the TPPII interacting proteins SIRT7 belongs to the sirtuin family, which is currently under detailed investigation due to neuroprotective effects of several members. Another interacting protein p53 is the key protein, which regulation represents one of the main challenges for both cancer research and for the treatment of neurodegenerative diseases. Recently, neuroprotective strategies towards inactivation of p53 and/or decreasing of its expression in neurons were very seriously considered. Its transcription is activated by binding to MYBBP1A, which interaction with TPPII was very recently discovered (Nahálková & Tomkinson, 2014). Reference: Nahálková, J., & Tomkinson, B. (2014). Arch Biochem Biophys, 564, 128–35.
Mol Cell Biochem. 2015 Jul 14.
Department of Medical Biochemistry and Microbiology (IMBIM), BMC, Uppsala University, Box 582, 751 23, Uppsala, Sweden, [email protected]
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.Go To Mol Cell Biochem.