Proteomic analysis reveals differentially regulated protein acetylation in human amyotrophic lateral sclerosis spinal cord.

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease that primarily affects motor neurons in the brain and spinal cord. Histone deacetylase (HDAC) inhibitors have neuroprotective effects potentially useful for the treatment of neurodegenerative diseases including ALS; however, the molecular mechanisms underlying their potential efficacy is not well understood. Here we report that protein acetylation in urea-soluble proteins is differently regulated in post-mortem ALS spinal cord. Two-dimensional electrophoresis (2-DE) analysis reveals several protein clusters with similar molecular weight but different charge status. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identifies glial fibrillary acidic protein (GFAP) as the dominant component in the protein clusters. Further analysis indicates six heavily acetylated lysine residues at positions 89, 153, 189, 218, 259 and 331 of GFAP. Immunoprecipitation followed by Western blotting confirms that the larger form of GFAP fragments are acetylated and upregulated in ALS spinal cord. Further studies demonstrate that acetylation of the proteins additional to GFAP is differently regulated, suggesting that acetylation and/or deacetylation play an important role in pathogenesis of ALS

Identification of Protein Spots on the 2D gel by LC-MS/MS^a.

a<p>The differentially expressed protein spots on the 2-D gels of ALS were excised and proteins identified by LC-MS/MS.</p>b<p>The length of identified peptide fragments divided by the length of the protein.</p>c<p>Mascot algorithm score; the minimum score is required to have a statistic significance (p<0.05).</p

Identification of the Acetylated Proteins in ALS Spinal Cord by Immunoprecipitation and LC-MS/MS^a.

a<p>The proteins that were immunoprecipitated with the antibody against acetylated lysine were resolved on SDS-PAGE and stained with Rubby-RED. The protein bands corresponding to the Western blots using the same antibody were excised and identified LC-MS/MS.</p

The acetylated lysine residues in GFAP.

<p>(<b>A</b>) The GFAP sequence with six identified positions for lysine acetylation. Bold sequence, the identified tryptic peptide; underlined K, the acetylated lysine residue. (<b>B</b>) A schematic diagram of human GFAP structure with four α-helical coiled-coil domains (CC1a, CC1b, CC2a, CC2b) and the positions for acetylation (arrows). The acetylated lysines fell into the highly conserved coiled-coil domains.</p

Immunoprecipitation followed by Western blotting confirms the up-regulation of the acetylated GFAP in ALS.

<p>The GFAP fragments in the soluble fractions from three individual ALS and non-ALS spinal cord samples were immunoprecipitated with the anti-GFAP antibody, and analysed by Western blotting using the antibody against acetyl-lysine (the right panel). Western blotting with β-actin was used as a control for the inputted proteins (the left panel). (<b>B</b>) Quantification of the acetylated GFAP. The relative acetylated GFAP to β-actin were compared between each pair of ALS and non-ALS samples (*ALS/non-ALS = 2.1±0.2, p<0.003, n = 3).</p

Acetylated Lysine and Peptides of GFAP Identified by LC-MS/MS^a.

a<p>Acetylation was confirmed by MS and MS/MS of the peptide.</p>b<p>Numbering according Genbank accession # P14136; underline indicates the acetylated lysine.</p>c<p>Monoisotopic mass of the neutral peptide.</p>d<p>Mascot algorithm score of each acetylated peptide.</p

Differentially regulated protein acetylation in ALS and non-ALS spinal cords by Western blotting and immunoprecipitation.

<p>(<b>A</b>) Western blotting analysis of total acetylated proteins. The urea-soluble proteins from ALS and non-ALS spinal cords were resolved on SDS-PAGE and followed by Western blotting using the antibody against acetyl-lysine. Arrows indicate bands found in ALS spinal cords, while arrowheads indicate the bands found in non-ALS counterparts. (<b>B</b>) Immunoprecipitation of the acetylated proteins. The soluble protein fractions were immunoprecipitated with the antibody against acetyl-lysine, resolved by SDS-PAGE and stained with Sypro Ruby. The protein bands labelled with U0, U1, … U8 were recovered, digested with trypsin and identified with LC-MS/MS. The proteins that were identified by LC-MS/MS are indicated to the left.</p

Comparison of urea-soluble proteins from ALS and non-ALS spinal cords by 2D SDS-PAGE.

<p>(<b>A</b>) Urea-soluble whole tissue lysates were prepared from pooled ALS or non-ALS spinal cords in the RIPA lysis buffer containing 8 M of urea. The first dimension was 18-cm immobilized pH gradient isoelectric focusing (IEF) from pI = 3–11; the second dimension was 10% SDS-PAGE. The gels were stained with Sypro Rubby. High quality spots marked with a–i and a’–i’ were randomly selected as references to normalize the differences between different gels. (<b>B</b>) Differentially expressed protein clusters between ALS and non-ALS spinal cords. The cluster A, B and S from ALS and A’, B’ and S’ from non-ALS were excised from the 2-D gels and subjected to LC-MS/MS protein identification. (<b>C</b>) Western blotting analysis of the protein clusters with anti-GFAP antibody. The urea-soluble whole tissue lysates were resolved by mini-2D SDS-PAGE, transferred to the PVDF membrane and detected with the antibody against GFAP.</p

Western Blotting analysis of the GFAP fragments in the soluble and insoluble protein fractions of ALS and non-ALS spinal cords.

<p>(<b>A, B</b>) Western Blotting analysis of the GFAP fragments in the insoluble (A) and soluble (B) fractions. The urea-soluble proteins were dialyzed against PBS and centrifuged. The pellet (insoluble fraction) and the supernatant (soluble fraction) were analyzed by Western blotting and detected with the anti-GFAP antibody. GFAP-a and GFAP-b, the larger forms of GFAP; GFAP-s, the degraded GFAP fragments; β-actin, internal control. (<b>C</b>) Quantitation of two larger forms of GFAP fragments. The expression levels of GFAP-a and GFAP-b relative to β-actin were calculated. The large forms of GFAP are preferably found in the insoluble fractions (* p<0.05, n = 4).</p