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Identification of ROS‐induced Poly(ADP‐ribose)‐Associated Proteins by LC‐MS/MS
Author(s) -
IslasRobles Argel,
Munoz Frances,
Lau Serrine,
Monks Terrence
Publication year - 2017
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.31.1_supplement.819.12
Subject(s) - poly adp ribose polymerase , chemistry , dna damage , oxidative stress , microbiology and biotechnology , glutathione , programmed cell death , intracellular , biochemistry , biology , apoptosis , polymerase , dna , enzyme
Hydroquinone occurs ubiquitously found in the environment, is metabolized via oxidation and glutathione conjugation to 2,3,5‐tris‐(glutathion‐S‐yl)hydroquinone (TGHQ). TGHQ is nephrotoxic, nephrocarcinogenic and generates ROS, which cause DNA strand breaks, hyperactivation of poly(ADP‐ribose) polymerase‐1 (PARP‐1), increase in intracellular calcium concentration ([iCa 2+ ]), and ultimately cell death. PARP‐1 is an abundant nuclear protein involved in DNA repair, transcriptional regulation, intracellular trafficking and is an epigenetic mediator. PARP‐1 catalyzes the attachment on target proteins of a posttranscriptional modification, consisting of multi branched ADP‐ribose polymers (PAR). We have shown that ROS stress promotes PARP‐1 hyperactivation and elevations in [iCa 2+ ] which are reciprocally coupled, resulting in renal tubule cell death. The molecular mechanism of this interaction is unclear. Using LC‐MS/MS we have identified proteins associated with PAR that have a potential role in ROS mediated toxicity. Kidney proximal tubule cells (HK‐2) were treated with 400 μM TGHQ creating a ROS cell stress environment leading to PARP‐1 hyperactivation. PAR‐associated proteins were immunoprecipitated using an antibody against PAR, followed by subsequent separation by SDS PAGE. Tryptic peptides were obtained from the protein Gel sections and analyzed by LC‐MS/MS. Scaffold was used to validate MS/MS based peptides, protein identification, and perform the relative abundance analysis by spectral counts. Protein‐protein interaction modeling and gene ontology (GO) analysis were performed in Network Analyst. Coomassie stained protein gels showed differentially PARylated proteins between treated and control samples, the former displaying far more abundant bands. The spectral counting analysis allowed us to obtain fold‐changes in protein abundance relative to control. 361 PAR‐associated proteins were confidently identified as modified by TGHQ treatment. From this cohort, 276 proteins have increased PAR association while 85 are decreased. Protein network analysis revealed 2 subnetworks on the PAR‐increased proteins data set, the functions of which, are related to RNA metabolism and cellular organization. Cellular compartmentalization functions were prominently identified on the PAR‐decreased proteins. 13 proteins from the whole cohort contain GO annotations related to calcium. Importantly, Neuroblast Differentiation‐Associated Protein (AHNAK), Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) and General Transcription Factor II‐I (GTF2I) are directly involved in modulation of [iCa 2+ ]. Currently we are investigating the possible role of these PAR‐associated proteins in ROS induced cell death.