Nuclear cytoplasmic trafficking of proteins is a major response of human fibroblasts to oxidative stress

Abstract

We have used a subcellular spatial razor approach based on LC-MS/MS-based proteomics with SILAC isotope labeling to determine changes in protein abundances in the nuclear and cytoplasmic compartments of human IMR90 fibroblasts subjected to mild oxidative stress. We show that response to mild tert-butyl hydrogen peroxide treatment includes redistribution between the nucleus and cytoplasm of numerous proteins not previously associated with oxidative stress. The 121 proteins with the most significant changes encompass proteins with known functions in a wide variety of subcellular locations and of cellular functional processes (transcription, signal transduction, autophagy, iron metabolism, TCA cycle, ATP synthesis) and are consistent with functional networks that are spatially dispersed across the cell. Both nuclear respiratory factor 2 and the proline regulatory axis appear to contribute to the cellular metabolic response. Proteins involved in iron metabolism or with iron/heme as a cofactor as well as mitochondrial proteins are prominent in the response. Evidence suggesting that nuclear import/export and vesicle-mediated protein transport contribute to the cellular response was obtained. We suggest that measurements of global changes in total cellular protein abundances need to be complemented with measurements of the dynamic subcellular spatial redistribution of proteins to obtain comprehensive pictures of cellular function.

Keywords: DNA replication; SILAC; mass spectrometry; oxidative stress; peroxide; quantitative proteomics.

Figure 1

Characterization of the cellular response to oxidative stress and the subcellular fractionation. (A) Western blotting of NRF2 and KEAP1 of the nuclear (N) and cytoplasmic (C) fractions for untreated (U) or THP-treated (Ox) cells showing cellular response to oxidative stress. (B) Flow cytometry determination of the cellular distribution over the cell cycle.

Figure 2

Distribution over the nucleus (N) and cytoplasm (C) for all identified proteins (detected) or for the set of proteins (121-OxS set) selected as showing the most significant changes in compartmentalized abundance (S_c, S_n), total abundance (S_t), or distribution between the nucleus and cytoplasm (S_n/S_c).

Figure 3

Analysis of the enrichment/purity of the nuclear fraction. Table: The number of quantified proteins in the nuclear fraction that are annotated to other subcellular locations and the number showing appreciable change in their nuclear fraction (S_n/S_t = f_s/f_u). (A) Left: log₂(f_s/f_u) as a function of the average number of ratio counts over the nucleus and total data sets for proteins with GO annotation to mitochondria and nucleus (red, 143 proteins) or to mitochondria but not nucleus (blue, 218 proteins). Right: number of proteins versus log₂(f_s/f_u). (B) Log₂(f_s/f_u) for selected high abundance proteins with at least 100 SILAC ratio counts, including at least 50 in the nucleus, that have GO annotation to multiple subcellular locations. The current GO cellular component annotations for these proteins are given in parentheses according to the letter code in the table.

Figure 4

Subcellular characterization of the 87 proteins of the 121-OxS set for which S_n, S_c, and S_t were all measured. (A) Plot of changes in nucleocytoplasmic distribution (S_n/S_c) versus changes in total abundance (S_t). The red bounding box corresponds to 1.5-fold changes in total abundance and 0.57 > S_n/S_c > 1.74 (|log₂(S_n/S_c)| > 0.9). A few proteins appear just inside the bounds because the combinations of basal distribution, changes in total abundance, and changes in distribution can lead to individual compartmental abundances (S_n or S_c) being selected as significant. (B) 3D spatial razor plot for the 87 proteins. The dotted green lines correspond to the bound |log₂(S_n/S_c)| = 0.8. (C) Same as panel B for selected, labeled proteins or protein complexes. (See the text.) In panels B and C changes in total abundance S_t (perpendicular to the page) are color-coded according to the scale at the right.

Figure 5

Visual summary of the most significant changes in subcellular abundance (S_n or S_c) and in nucleocytoplasmic distribution (S_n/S_c) for the proteins of the 121-OxS set quantified in both the nuclear and cytoplasmic compartments. A selection of some of the GO biological process terms annotated to the proteins (see text) is indicated.

Figure 6

Network analysis for the 240-OxS and 121-OxS data sets. (A) STRING interaction network for the 240-OxS set. Nodes corresponding to the 121-OxS set are colored in red, nodes suggested by STRING and quantified in the MS data but not included in the 121-OxS set are colored in yellow, and nodes suggested by STRING but not quantified in the MS data are colored in gray. Node sizes are mapped to the betweenness centrality values, while edge colors and sizes are mapped to edge betweenness values, as calculated by the Network Analysis plugin for the Cytoscape software. Betweenness centrality is a measure of a node’s centrality and importance in a network. Edge betweenness is a topological measure defined as a normalized number of shortest paths between two nodes. (B) Thumbnails of the interaction network: nodes colored in blue indicate significant changes in S_n, S_c, S_t, or S_n/S_c of proteins that are included in the 121-OxS set.

Figure 7

Three selected processes/networks. These dense networks are highly interrelated and were extensively quantified in the MS measurements (yellow nodes) but only a small subset of the proteins in each network (red nodes) was included in the most significant changes.

Figure 8

Analysis of proteins annotated to vesicular trafficking. Left: log₂(S_n/S_t) = log₂(f_s/f_u) as a function of the average number of ratio counts over the nucleus and total data sets for proteins annotated to “vesicle-mediated transport” or to “cytoplasmic vesicle”. Proteins with (red, 70 proteins) or without (blue, 181 proteins) annotation to nucleus are indicated. Right: number of proteins versus log₂(f_s/f_u). Table: Data for 28 proteins of the 121-OxS set.