Determination of the role of PAR‐associated proteins in ROS‐mediated cell death
Authors: Islas‐Robles Argel , Lau Serrine S. , Monks Terrence J. 
Published: June 2021

2,3,5‐Tris‐(glutathion‐S‐yl)hydroquinone (TGHQ) is a nephrotoxic and nephrocarcinogenic metabolite of hydroquinone. TGHQ can redox cycle, generating ROS which cause DNA strand breaks, hyperactivation of poly(ADP‐ribose) polymerase‐1 (PARP‐1), increases in intracellular calcium concentration (i[Ca2+]), and cell death. PARP‐1 is a nuclear protein involved in DNA repair, transcriptional regulation and intracellular trafficking, and catalyzes the attachment of a posttranscriptional modification, consisting of multi branched ADP‐ribose polymers (PAR) on target proteins. ROS stress promotes PARP‐1 hyperactivation and elevations in i[Ca2+] which are reciprocally coupled, resulting in cell death. The molecular mechanism of this interaction is unclear. The aim of the present study was to identify TGHQ‐induced PAR‐associated proteins and their potential role in cell death. Human kidney proximal tubule cells (HK‐2) were treated with 400 μM TGHQ to induce ROS‐stress leading to PARP‐1 hyperactivation. PAR‐associated proteins were immunoprecipitated, followed by subsequent separation by SDS PAGE and proteomic analysis. A relative protein abundance analysis by spectral counts allowed us to obtain fold‐changes in protein abundance relative to control. 361 PAR‐associated proteins were significantly identified as modified by TGHQ treatment. From this cohort, 276 proteins showed an increased PAR association while 85 were decreased. Additionally, 13 targets had Gene Ontology annotations related to calcium. Of these, neuroblast differentiation‐associated protein (AHNAK), calcium homeostasis endoplasmic reticulum protein (CHERP) and general transcription factor II‐I (TFII‐I) are directly involved in modulation of i[Ca2+]. TFII‐I is a transcription factor that regulates i[Ca2+] by sequestering phospholipase C (PLC) in the cytosol. Under basal conditions free PLC binds and promotes the translocation of the transient receptor potential channel (TRPC3) to the plasma membrane leading to increases in i[Ca2+]. TFII‐I increased PAR‐association might therefore lead to calcium influx via TRPC3. Using co‐immunoprecipitation and immunodetection against TFII‐I, we confirmed increased TFII‐I PAR‐association. The effect of a TRPC3 inhibitor on TGHQ‐dependent modulation of i[Ca2+] was determined by live imaging of HK‐2 cells with the calcium sensitive dye fluo‐8 AM. Inhibition of TRPC3 promoted a delayed but higher increase in i[Ca2+]. Cellular compartmentalization of TFII‐I under TGHQ treatment was determined by cellular fractionation. TGHQ induced TFII‐I translocation from the nucleus to the cytosol as well as decreased tyrosine phosphorylation, which might have a functional effect on the transactivation potential of TFII‐I. In summary, LC‐MS/MS identified proteins with altered PARylation under ROS stress. TFII‐I is a novel PAR interacting protein which participates in signaling pathways with potential importance in TGHQ‐mediated cell death. Currently we are investigating the transcriptional and DNA repair effects of PAR‐associated TFII‐I that might be relevant to the mechanism of ROS‐dependent toxicity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.