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Title: Quantitative Proteomic Profiling of Low Dose Ionizing Radiation Effects in a Human Skin Model

Abstract

To assess molecular responses to low doses of radiation that may be encountered during medical diagnostic procedures, nuclear accidents, or terrorist acts, a quantitative global proteomic approach was used to identify protein alterations in a reconstituted human skin tissue treated with 10 cGy of ionizing radiation. Subcellular fractionation was employed to remove highly abundant structural proteins and provide insight on radiation induced alterations in protein abundance and localization. In addition, peptides were post-fractionated using high resolution 2-dimensional liquid chromatography to increase the dynamic range of detection of protein abundance and translocation changes. Quantitative data was obtained by labeling peptides with 8-plex isobaric iTRAQ tags. A total of 207 proteins were detected with statistically significant alterations in abundance and/or subcellular localization compared to sham irradiated tissues. Bioinformatics analysis of the data indicated that the top canonical pathways affected by low dose radiation are related to cellular metabolism. Among the proteins showing alterations in abundance, localization and proteolytic processing was the skin barrier protein filaggrin which is consistent with our previous observation that ionizing radiation alters profilaggrin processing with potential effects on skin barrier functions. In addition, a large number of proteases and protease regulators were affected by low dose radiation exposuremore » indicating that altered proteolytic activity may be a hallmark of low dose radiation exposure. While several studies have demonstrated altered transcriptional regulation occurs following low dose radiation exposures, the data presented here indicates post-transcriptional regulation of protein abundance, localization, and proteolytic processing play an important role in regulating radiation responses in complex human tissues.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1171288
Report Number(s):
PNNL-SA-102010
40691; 24390; KP1602020; 400403309
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Proteomes, 2(3):382-398
Country of Publication:
United States
Language:
English
Subject:
Environmental Molecular Sciences Laboratory

Citation Formats

Hengel, Shawna, Aldrich, Joshua T., Waters, Katrina M., Pasa-Tolic, Ljiljana, and Stenoien, David L. Quantitative Proteomic Profiling of Low Dose Ionizing Radiation Effects in a Human Skin Model. United States: N. p., 2014. Web. doi:10.3390/proteomes2030382.
Hengel, Shawna, Aldrich, Joshua T., Waters, Katrina M., Pasa-Tolic, Ljiljana, & Stenoien, David L. Quantitative Proteomic Profiling of Low Dose Ionizing Radiation Effects in a Human Skin Model. United States. doi:10.3390/proteomes2030382.
Hengel, Shawna, Aldrich, Joshua T., Waters, Katrina M., Pasa-Tolic, Ljiljana, and Stenoien, David L. Tue . "Quantitative Proteomic Profiling of Low Dose Ionizing Radiation Effects in a Human Skin Model". United States. doi:10.3390/proteomes2030382.
@article{osti_1171288,
title = {Quantitative Proteomic Profiling of Low Dose Ionizing Radiation Effects in a Human Skin Model},
author = {Hengel, Shawna and Aldrich, Joshua T. and Waters, Katrina M. and Pasa-Tolic, Ljiljana and Stenoien, David L.},
abstractNote = {To assess molecular responses to low doses of radiation that may be encountered during medical diagnostic procedures, nuclear accidents, or terrorist acts, a quantitative global proteomic approach was used to identify protein alterations in a reconstituted human skin tissue treated with 10 cGy of ionizing radiation. Subcellular fractionation was employed to remove highly abundant structural proteins and provide insight on radiation induced alterations in protein abundance and localization. In addition, peptides were post-fractionated using high resolution 2-dimensional liquid chromatography to increase the dynamic range of detection of protein abundance and translocation changes. Quantitative data was obtained by labeling peptides with 8-plex isobaric iTRAQ tags. A total of 207 proteins were detected with statistically significant alterations in abundance and/or subcellular localization compared to sham irradiated tissues. Bioinformatics analysis of the data indicated that the top canonical pathways affected by low dose radiation are related to cellular metabolism. Among the proteins showing alterations in abundance, localization and proteolytic processing was the skin barrier protein filaggrin which is consistent with our previous observation that ionizing radiation alters profilaggrin processing with potential effects on skin barrier functions. In addition, a large number of proteases and protease regulators were affected by low dose radiation exposure indicating that altered proteolytic activity may be a hallmark of low dose radiation exposure. While several studies have demonstrated altered transcriptional regulation occurs following low dose radiation exposures, the data presented here indicates post-transcriptional regulation of protein abundance, localization, and proteolytic processing play an important role in regulating radiation responses in complex human tissues.},
doi = {10.3390/proteomes2030382},
journal = {Proteomes, 2(3):382-398},
number = ,
volume = ,
place = {United States},
year = {Tue Jul 29 00:00:00 EDT 2014},
month = {Tue Jul 29 00:00:00 EDT 2014}
}
  • Outside the protection of earth’s atmosphere, astronauts are exposed to low doses of high linear energy transfer (LET) radiation. Future NASA plans for deep space missions or a permanent settlement on the moon are limited by the health risks associated with space radiation exposures. There is a paucity of direct epidemiological data for low dose exposures to space radiation-relevant high LET ions. Health risk models are used to estimate the risk for such exposures, though these models are based on high dose experiments. There is increasing evidence, however, that low and high dose exposures result in different signaling events atmore » the molecular level, and may involve different response mechanisms. Further, despite their low abundance, high LET particles have been identified as the major contributor to health risk during manned space flight. The human skin is exposed in every external radiation scenario, making it an ideal epithelial tissue model in which to study radiation induced effects. Here, we exposed an in vitro three dimensional (3-D) human organotypic skin tissue model to low doses of high LET oxygen (O), silicon (Si) and iron (Fe) ions. We measured proliferation and differentiation profiles in the skin tissue and examined the integrity of the skin’s barrier function. We discuss the role of secondary particles in changing the proportion of cells receiving a radiation dose, emphasizing the possible impact on radiation-induced health issues in astronauts.« less
  • Background: High doses of ionizing radiation result in biological damage, however the precise relationships between long term health effects, including cancer, and low dose exposures remain poorly understood and are currently extrapolated using high dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose dependent responses to radiation. Principle Findings: We have identified 6845 unique phosphopeptides (2566 phosphoproteins) from control and irradiated (2 and 50 cGy) primary human skin fibroblasts one hour post-exposure. Dual statistical analyses based on spectral counts and peakmore » intensities identified 287 phosphopeptides (from 231 proteins) and 244 phosphopeptides (from 182 proteins) that varied significantly following exposure to 2 and 50 cGy respectively. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatics analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role of MAP kinase and protein kinase A (PKA) signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. Conlcusions: Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provides a basis for the systems level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at different radiation doses and elucidate the impact of low dose radiation exposure on human health.« less
  • Our objective here was to perform a quantitative phosphoproteomic study on a reconstituted human skin tissue to identify low and high dose ionizing radiation dependent signaling in a complex 3-dimensional setting. Application of an isobaric labeling strategy using sham and 3 radiation doses (3, 10, 200 cGy) resulted in the identification of 1113 unique phosphopeptides. Statistical analyses identified 151 phosphopeptides showing significant changes in response to radiation and radiation dose. Proteins responsible for maintaining skin structural integrity including keratins and desmosomal proteins (desmoglein, desmoplakin, plakophilin 1 and 2,) had altered phosphorylation levels following exposure to both low and high dosesmore » of radiation. A phosphorylation site present in multiple copies in the linker regions of human profilaggrin underwent the largest fold change. Increased phosphorylation of these sites coincided with altered profilaggrin processing suggesting a role for linker phosphorylation in human profilaggrin regulation. These studies demonstrate that the reconstituted human skin system undergoes a coordinated response to ionizing radiation involving multiple layers of the stratified epithelium that serve to maintain skin barrier functions and minimize the damaging consequences of radiation exposure.« less
  • Understanding how human organs respond to ionizing radiation (IR) at a systems biology level and identifying biomarkers for IR exposure at low doses can help provide a scientific basis for establishing radiation protection standards. Little is known regarding the physiological responses to low dose IR at the metabolite level, which represents the end-point of biochemical processes inside cells. Using a full thickness human skin tissue model and GC-MS-based metabolomics analysis, we examined the metabolic perturbations at three time points (3, 24 and 48 hr) after exposure to 3, 10 and 200 cGy of X-rays. PLS-DA score plots revealed dose- andmore » time-dependent clustering between sham and irradiated groups. Importantly, a comparable number of metabolites were detected to have significant change 48 hr after exposure to 3 and 10 cGy of irradiation, when compared with the high dose of 200 cGy. Biochemical pathway analysis showed perturbations to DNA/RNA damage and repair, lipid and energy metabolisms, even at low doses of IR.« less