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A Novel Interaction between Thyroid Receptor Interacting Protein 13 with Tetratricopeptide Repeat Domain 5 Regulates p53 Activity in Murine Inner Medullary Collecting Duct Cells
Author(s) -
Hama Taketsugu,
Pressly Jeffrey David,
O'Brien Shan,
Park Frank
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.747.2
Subject(s) - signal transduction , microbiology and biotechnology , dna damage , tetratricopeptide , gene knockdown , kidney , cancer research , receptor , dna repair , transcription factor , cytoprotection , biology , chemistry , medicine , endocrinology , cell culture , apoptosis , dna , gene , genetics
Renal epithelial cells adapt to biological stressors to maintain their normal biological function. Repair pathways are initiated within the damaged tubular epithelial cells and allow for recovery from the biological stress, whereas more severe injury may circumvent the recovery phase by activating programmed cell death pathways. Emerging evidence has uncovered that thyroid receptor interacting protein 13 (TRIP13) may play a crucial role in the repair process of genomic DNA following damage stimuli. Towards this end, our lab has recently identified increased production of Trip13 transcript levels in renal tubular epithelial cells following acute kidney injury, but there is a paucity of data regarding its mechanism of action in the kidney. In this study we investigated the relationship between TRIP13 and p53, a pivotal transcription factor that acts as a central hub for signal transduction pathways to direct a multifaceted signaling output following biological stress. To investigate the effect of TRIP13 in response to DNA damage, we initially exposed genetic knockdown of TRIP13 in murine inner medullary collecting duct cells to H 2 O 2 , which is known to induce double strand breaks. We demonstrated a significant reduction in cell numbers by 40–50% compared to IMCD‐Ctrl and also revealed increased levels of phospho‐p53 (Serine 15) and total p53 following H 2 O 2 insult compared to the IMCD‐Ctrl cells. To further investigate the mechanism by which TRIP13 induces p53 levels and/or activity, we pursued a yeast two‐hybrid screen approach to identify new protein‐protein interactions that could participate in this response. Our yeast two‐hybrid approach identified a protein, tetratricopeptide repeat domain 5 (TTC5), which is known to function as a stress‐inducible transcription p53 cofactor to augment p53 expression, stability and/or activity. Confirmation of a direct interaction between TRIP13 and TTC5 was demonstrated using co‐immunoprecipitation techniques. Moreover, fluorescent microscopy revealed that TRIP13 was largely localized to the cytoplasm with modest signals from the nucleus. In the presence of over‐expressed TTC5, TRIP13 was strongly detected in the nucleus. Western blotting using nuclear fractionation revealed that p53 induction was reduced in overexpression of EGFP‐TRIP13 and myc‐TTC5 compared to control conditions. Thus, our data suggests that TRIP13 interacts with TTC5 to reduce p53 activity following DNA damage. Support or Funding Information This study was funded by NIH RO1‐DK90123, and UTHSC seed grant program.