engleski

Study of the interaction of dipeptidyl peptidase 3 and SH2 domain-containing protein 3C

Dipeptidyl peptidase 3 (DPP3) is a zinc metallopeptidase that cleaves dipeptides from unsubstituted amino-termini of 3 to 10 residues long peptides in vitro. Its physiological role is still unconfirmed, however, it is presumed that it has a role in the final stages of protein turnover in cells and in the regulation of blood pressure and pain. These proposed functions are related to its peptidase activity, while it also has a role in the NRF2-KEAP1 oxidative stress response signaling pathway through its interaction with KEAP1 protein. SH2 domain- containing protein 3C (SH2D3C) was identified as a putative interactor of DPP3 in SILAC-MS screening of HEK293T cells stably overexpressing HA-tagged DPP3. SH2D3C is one of three members of the NSP family of proteins which contain both, SH2 domain and a domain similar to guanine nucleotide exchange factor domains of Ras family GTPases (Ras GEF-like domain). SH2D3C interacts with the phosphorylated cytoplasmic domain of small Ras family GTPases, EphB2, R-Ras, and Rap1A, but does not exhibit GTP exchange activity in vitro. It is involved in the regulation of cell adhesion and migration, tissue organization, and the regulation of the immune response. Therefore, the interaction of DPP3 and SH2D3C may be a link between these processes and the response to oxidative stress. The interaction between DPP3 and SH2D3C-isoforms 2 and 3 and C- terminal Ras GEF-like domain was confirmed by GST- DPP3 pulldown, indicating that it binds DPP3 through its C-terminal domain. Molecular docking revealed two representative modes of binding, one in the interdomain cleft of DPP3 and another resembling KEAP1-DPP3 binding. The interaction was confirmed by bimolecular fluorescence complementation (BiFC) in NIH 3T3 cells and detected in the cytoplasm and membrane ruffles. The interaction was also confirmed by co- immunoprecipitation of endogenous proteins in HeLa and SH-SY5Y cells, and overexpression of SH2D3C isoform 3 was also found to downregulate mRNA expression of NQO1 and NRF2. Further experiments to determine the role of DPP3- SH2D3C interaction are in progress.

DPP3, SH2D3C, NRF2-KEAP1 pathway

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