Proteome-wide lysine acetylation in cortical astrocytes and alterations that occur during infection with brain parasite Toxoplasma gondii

Abstract

Lysine acetylation is a reversible post-translational modification (PTM) that has been detected on thousands of proteins in nearly all cellular compartments. The role of this widespread PTM has yet to be fully elucidated, but can impact protein localization, interactions, activity, and stability. Here we present the first proteome-wide survey of lysine acetylation in cortical astrocytes, a subtype of glia that is a component of the blood-brain barrier and a key regulator of neuronal function and plasticity. We identified 529 lysine acetylation sites across 304 proteins found in multiple cellular compartments that largely function in RNA processing/transcription, metabolism, chromatin biology, and translation. Two hundred and seventy-seven of the acetylated lysines we identified on 186 proteins have not been reported previously in any other cell type. We also mapped an acetylome of astrocytes infected with the brain parasite, Toxoplasma gondii. It has been shown that infection with T. gondii modulates host cell gene expression, including several lysine acetyltransferase (KAT) and deacetylase (KDAC) genes, suggesting that the host acetylome may also be altered during infection. In the T. gondii-infected astrocytes, we identified 34 proteins exhibiting a level of acetylation >2-fold and 24 with a level of acetylation <2-fold relative to uninfected astrocytes. Our study documents the first acetylome map for cortical astrocytes, uncovers novel lysine acetylation sites, and demonstrates that T. gondii infection produces an altered acetylome.

Fig 1. Workflow for acetylome analysis of cortical astrocytes.

Protein lysates generated from infected and uninfected cortical astrocytes were subjected to proteolysis to generate a peptide suspension. Immunoaffinity purification with anti-acetyl-lysine antibody enriched for acetylated peptides (acetylation mark is denoted by red dot). Identification of acetylated peptides was achieved with mass spectrometry.

Fig 2. Global features of the cortical astrocyte acetylome.

Acetylated proteins (A) and residues (B) were sorted into functional groups based on GO analyses. Lysine acetylation is most prevalent on proteins involved in RNA processing and transcription, metabolism, and chromatin biology. C. The 277 novel acetyl-lysine sites identified in our study are present on proteins that encompass a wide range of cellular functions. D. Proteins detected as lysine-acetylated were grouped based on their respective cellular location.

Fig 3. Localization of acetylated tubulin in astrocytes.

The subcellular localization of α-tubulin acetylated at K40 (upper panels) and total α-tubulin (lower panels) through immunofluorescence of isolated cortical astrocytes (green). DAPI staining to highlight nuclei is indicated in blue. Scale bar = 20 μm.

Fig 4. Analysis of acetylated lysine sites.

A. Heat map of amino acid composition of acetylation sites in Rattus norvegicus astrocytes, displaying amino acids that are significantly enriched (green) or absent (red) relative to the general amino acid composition of the Rattus norvegicus proteome. B. Sequence motifs of astrocyte acetylation sites +/-7 amino acids from the targeted lysine residue. Motifs were compiled using all acetylated peptides or only those found in histones, non-histone proteins, or mitochondrial proteins.