Bioinformatic Analysis of Differential Protein Expression in Calu-3 Cells Exposed to Carbon Nanotubes

Abstract

Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 hexposure to 10 μg/mL and 100 ng/mL of two common carbon nanoparticles, single- and multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study the differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformaticanalysis of proteins identified in LFQMS. Interestingly, after exposure to ahigh concentration (10 μg/mL; 0.4 μg/cm²) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm²) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins in common to both nanotubes occurred within the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The majority of the protein changes represent a decrease in amount suggesting a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure.

Keywords: airway epithelia; barrier epithelia; label-free quantitative mass spectrometry; protein interaction networks.

Figure 1

Changes in protein abundance in response to carbon nanotube exposure. The bars depict the number of proteins whose expression was increased (above 0) or decreased (below 0) over controls in response to 24 h exposure to carbon nanotubes at the concentrations listed on the figure. Actual numbers of proteins whose abundance are altered are also shown on the graph. Protein expression data filtered by ANOVA p < 0.01 and Pairwise Multiple Comparison p < 0.05 of different CNTs (SWCNT, MWCNT) and concentrations (10 μg/mL, 100 ng/mL).

Figure 2

Volcano plots of all proteins identified and quantified, illustrating their magnitude, significance, and direction of differential expression observed after 24 h exposure to CNTs (SWCNT, MWCNT) at two different concentrations, 10 μg/mL, 100 ng/mL. The horizontal red line in each graph signifies Pairwise Multiple Comparisons p-value < 0.05 compared to proteins in control cultures grown in parallel.

Figure 3

Venn diagram illustrating common identities of the proteins altered in response to 24 h CNT exposure. Overlaps of identical significantly altered protein expressions after exposure to two different concentrations (10 μg/mL, 100 ng/mL) of either SWCNT or MWCNT are indicated by the numbers inside each of the different compartments.

Figure 4

Overlap of the changes in protein abundance in common Biological Functions after 24 h exposure to a low concentration (100 ng/mL) of MWCNT or SWCNT. The numbers of proteins that are altered in response to low dose carbon nanotube exposure within defined biological functions are listed. The Venn diagrams show the number of proteins with shared identity altered in response to both MWCNT and SWCNT.

Figure 5

Identity and interaction of proteins within functional network that are altered by 24 h carbon nanotube exposure. STRING database was used to illustrate the interactions of the proteins that were altered by exposure to a low dose of both SWCNT and MWCNT. The strength of the interactions is indicated by the thickness of the connecting lines. Proteins that have no connecting lines have been identified as altered by both SWCNT and MWCNT exposure but are not part of a network that connects them to any of the other altered proteins. (A) Proteins within functional network of “Cell Death”. Proteins in the left circle are component of adherens junctions, and proteins in the right circle are component of ribonucleoprotein complex. Hub proteins in the network: RPLP0, ribosomal protein large P0; RPS3A, ribosomal protein S3A; EEF1A1, eukaryotic translation elongation factor 1 alpha-like 7; TPT1, tumor protein translationally-controlled 1; HSP90AA1, heat shock protein 90 kDa alpha (cytosolic) class A member 1; STAT1, signal transducer and activator of transcription 1, 91 kDa; STIP1, stress-induced-phosphoprotein 1; CDH1, cadherin 1 type 1 E-cadherin (epithelial); JUP, junction plakoglobin; (B) Proteins within functional network of “Cell Survival”. Proteins in the upper circle are involved in the regulation of cell death, and proteins in the lower circle are located on the nucleoplasm. Hub proteins in the network: CDH1; STAT1; (C) Proteins within functional network of “Organization of Cytoplasm”. Proteins in the upper circle are component of actin cytoskeleton, and proteins in the lower circle are located on plasma membrane. Hub proteins in the network: CDH1; (D) Proteins within functional network of “Proliferation of Cells”. Proteins in the left circle are component of anchoring junctions, and proteins in the right circle are involved in the ATP catabolic process. Hub proteins in the network: JUP; EZR, ezrin; CDH1; LMNA, lamin A/C; HNRNPR, heterogeneous nuclear ribonucleoprotein R; GNB2L1, guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1; TPT1; STAT1; (E) Proteins within functional network of “Cell Movement”. Proteins in the upper circle are component of actin cytoskeleton, and proteins in the lower circle are involved in the regulation of cell death. Hub proteins in the network: HSP90AA1; CDH1; GNB2L1; (F) Proteins within functional network of “Viral Infection”. Proteins in the circle are located on the nucleoplasm. Hub proteins in the network: STAT1; HNRNPU, heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A); TPT1.