Transient Receptor Potential Channel A1 (TRPA1) Regulates Sulfur Mustard-Induced Expression of Heat Shock 70 kDa Protein 6 (HSPA6) In Vitro

Abstract

The chemosensory transient receptor potential ankyrin 1 (TRPA1) ion channel perceives different sensory stimuli. It also interacts with reactive exogenous compounds including the chemical warfare agent sulfur mustard (SM). Activation of TRPA1 by SM results in elevation of intracellular calcium levels but the cellular consequences are not understood so far. In the present study we analyzed SM-induced and TRPA1-mediated effects in human TRPA1-overexpressing HEK cells (HEKA1) and human lung epithelial cells (A549) that endogenously exhibit TRPA1. The specific TRPA1 inhibitor AP18 was used to distinguish between SM-induced and TRPA1-mediated or TRPA1-independent effects. Cells were exposed to 600 µM SM and proteome changes were investigated 24 h afterwards by 2D gel electrophoresis. Protein spots with differential staining levels were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano liquid chromatography electrospray ionization tandem mass spectrometry. Results were verified by RT-qPCR experiments in both HEKA1 or A549 cells. Heat shock 70 kDa protein 6 (HSPA6) was identified as an SM-induced and TRPA1-mediated protein. AP18 pre-treatment diminished the up-regulation. RT-qPCR measurements verified these results and further revealed a time-dependent regulation. Our results demonstrate that SM-mediated activation of TRPA1 influences the protein expression and confirm the important role of TRPA1 ion channels in the molecular toxicology of SM.

Identification of creatine kinase and alpha‐1 antitrypsin as protein targets of alkylation by sulfur mustard

“…This technology was applied to a human cell culture (HEK293 cells) and plasma as matrices often used in SM research 15,23,30 . The HEK293 cells are a common and robust cell line easy in handling and maintaining with a high reproducibility 31 .…”

“…The HEK293 cells are a common and robust cell line easy in handling and maintaining with a high reproducibility 31 . In addition, the effects of SM on these cells were investigated in several studies before 23,32 . Accordingly, proteins derived from lysates of cell culture exposed to SM and proteins derived from plasma exposed to CEES were labeled with the IR800‐maleimide dye and compared with non‐exposed blanks labeled with the IR680‐maleimide dye.…”

“…Therefore, the identification of proteins modified by SM and similar poisons represents a pivotal challenge to understand the toxicity of alkylating agents. Diverse proteomics technologies were used for the targeted and nontargeted analysis of biological systems exposed to SM 17,22–26 . In general, in many proteomics studies, diverse techniques were applied that allow relative quantification of proteins to figure out the influence of any factor 27,28 .…”

Evidence of sulfur mustard poisoning by detection of the albumin‐derived dipeptide biomarker C(‐HETE)P after nicotinylation

“…[14][15][16]19 Following proteolysis of the HETE-HSA-adduct found at C 34 with pepsin the alkylated dodecapeptide LQQC 34 (-HETE) PFEDHVKL is produced, 1 with proteinase K the alkylated tripeptide C(-HETE)PF is generated 1,14 and pronase-catalyzed hydrolysis yields the alkylated dipeptide C(-HETE)P. 15,19 These peptides represent wellestablished and internationally accepted long-term biomarkers of exposure to SM detectable for up to four weeks after exposure using liquid chromatography-electrospray ionization tandem-mass spectrometry (LC-ESI MS/MS) techniques. 1,8,14 Nevertheless, the current incidents of poisoning with chemical warfare agents-also including the highly toxic organophosphorus nerve agents 11,20,21 -demand the search for potential novel biomarker molecules 17,[22][23][24][25][26][27][28][29] as well as the continuous optimization of already established methods. 16,[30][31][32] Therefore, we addressed the precolumn derivatization of C(-HETE)P to produce a derivative suited as an additional biomarker of exposure.…”

Transient Receptor Potential (TRP) Channels in Health and Disease

“…The authors of this manuscript used a proteomic approach to identify differentially regulated proteins in TRPA1-expressing HEK293 and A549 cells after SM treatment. The selective TRPA1 inhibitor AP18 was used to distinguish the TRPA1-mediated effect from unspecific effects [15].…”