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Dementia-linked amyloidosis is associated with brain protein deamidation as revealed by proteomic profiling of human brain tissues.

Adav SS, Gallart-Palau X, Tan KH, Lim SK, Tam JP, Sze SK - Mol Brain (2016)

Bottom Line: Proteomics profiling of both soluble and aggregated amyloidal plaque demonstrated significant enrichment and deamidation of S100A9, ferritin, hemoglobin subunits, creatine kinase and collagen protein among the aggregated brain proteins.The multiple deamidated residues of S100A9 predicts introduction of negative charge that alter Ca(++) binding, suggesting increased capacity to form pathological aggregates in the brain.UC-coupled proteomics revealed that brain amyloidal plaques are enriched in deamidated proteins, and suggested that altered charge state and calcium-binding capacity of S100A9 may enhance protein aggregation and promote neurodegeneration in the human brain.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Division of Structural Biology and Biochemistry, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore. SSAdav@ntu.edu.sg.

ABSTRACT

Background: Aggregation of malformed proteins is a key feature of many neurodegenerative diseases, but the mechanisms that drive proteinopathy in the brain are poorly understood. We aimed to characterize aggregated proteins in human brain tissues affected by dementia.

Results: To characterize amyloidal plaque purified from post-mortem brain tissue of dementia patient, we applied ultracentrifugation-electrostatic repulsion hydrophilic interaction chromatography (UC-ERLIC) coupled mass spectrometry-based proteomics technologies. Proteomics profiling of both soluble and aggregated amyloidal plaque demonstrated significant enrichment and deamidation of S100A9, ferritin, hemoglobin subunits, creatine kinase and collagen protein among the aggregated brain proteins. Amyloidal plaques were enriched in the deamidated variant of protein S100A9, and structural analysis indicated that both the low- and high-affinity calcium binding motifs of S100A9 were deamidated exclusively in the aggregated fraction, suggesting altered charge state and function of this protein in brain tissues affected by dementia. The multiple deamidated residues of S100A9 predicts introduction of negative charge that alter Ca(++) binding, suggesting increased capacity to form pathological aggregates in the brain.

Conclusion: UC-coupled proteomics revealed that brain amyloidal plaques are enriched in deamidated proteins, and suggested that altered charge state and calcium-binding capacity of S100A9 may enhance protein aggregation and promote neurodegeneration in the human brain.

No MeSH data available.


Related in: MedlinePlus

Structural model of Protein S100A9 (RCSB Protein Data Bank accession code: 1XK4) showing deamidation sites. EF hand calcium binding motifs are shown in yellow and deamidation sites (area of modification) are highlighted in magenta and blue. Helices are shown in green while Ca ions are shown as red spheres. Lower panel shows only EF hands and area of modification with Ca ions in green
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Fig5: Structural model of Protein S100A9 (RCSB Protein Data Bank accession code: 1XK4) showing deamidation sites. EF hand calcium binding motifs are shown in yellow and deamidation sites (area of modification) are highlighted in magenta and blue. Helices are shown in green while Ca ions are shown as red spheres. Lower panel shows only EF hands and area of modification with Ca ions in green

Mentions: Many specific deamidation sites were exclusively identified in the aggregated proteins, including modifications of S100A9, ferritin, hemoglobin subunits, S100-A8, S100-B, collagens, mitochondrial creatine kinase (U-type), β-tubulin and laminin. The deamidation sites of protein S100A9 that were commonly identified in Mascot and Maxqunat were listed in Table 1. These specific structural modifications may have functional consequences that influence the progression of neurodegenerative diseases. For example, the inflammation-associated calcium binding protein S100A9 incorporates two EF-hand motifs that exhibit differential affinity for Ca++ binding, and deamidation of both motifs was detected exclusively in the aggregated fraction (Fig. 5). In contrast, only low levels of S100A9 were detected in the soluble fraction and the protein was not deamidated in this sample. These data suggest that deamidation and aggregation of brain proteins that regulate inflammatory processes may contribute to the pathology of human neurodegeneration. While these data suggested that protein deamidation and aggregation are key features of brain plaque formation, we also detected the restorative enzyme PIMT (ProteinL-isoaspartate O-methyltransferase) within the pellet aggregate, perhaps indicating a failed response to repair disrupted protein functions. In vitro incubation of protein samples at various different temperatures (10, 22 and 37 °C) for variable duration (1, 3 or 7 days) in different buffers like AAB (pH 6), ABB (pH 8) and TEAB (pH 8.5) revealed precipitation of protein except acetate buffer (pH 6). As alkaline condition is known to induce protein deamidation, this observation may suggest that deamidation causes aggregation of proteins.Table 1


Dementia-linked amyloidosis is associated with brain protein deamidation as revealed by proteomic profiling of human brain tissues.

Adav SS, Gallart-Palau X, Tan KH, Lim SK, Tam JP, Sze SK - Mol Brain (2016)

Structural model of Protein S100A9 (RCSB Protein Data Bank accession code: 1XK4) showing deamidation sites. EF hand calcium binding motifs are shown in yellow and deamidation sites (area of modification) are highlighted in magenta and blue. Helices are shown in green while Ca ions are shown as red spheres. Lower panel shows only EF hands and area of modification with Ca ions in green
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4759965&req=5

Fig5: Structural model of Protein S100A9 (RCSB Protein Data Bank accession code: 1XK4) showing deamidation sites. EF hand calcium binding motifs are shown in yellow and deamidation sites (area of modification) are highlighted in magenta and blue. Helices are shown in green while Ca ions are shown as red spheres. Lower panel shows only EF hands and area of modification with Ca ions in green
Mentions: Many specific deamidation sites were exclusively identified in the aggregated proteins, including modifications of S100A9, ferritin, hemoglobin subunits, S100-A8, S100-B, collagens, mitochondrial creatine kinase (U-type), β-tubulin and laminin. The deamidation sites of protein S100A9 that were commonly identified in Mascot and Maxqunat were listed in Table 1. These specific structural modifications may have functional consequences that influence the progression of neurodegenerative diseases. For example, the inflammation-associated calcium binding protein S100A9 incorporates two EF-hand motifs that exhibit differential affinity for Ca++ binding, and deamidation of both motifs was detected exclusively in the aggregated fraction (Fig. 5). In contrast, only low levels of S100A9 were detected in the soluble fraction and the protein was not deamidated in this sample. These data suggest that deamidation and aggregation of brain proteins that regulate inflammatory processes may contribute to the pathology of human neurodegeneration. While these data suggested that protein deamidation and aggregation are key features of brain plaque formation, we also detected the restorative enzyme PIMT (ProteinL-isoaspartate O-methyltransferase) within the pellet aggregate, perhaps indicating a failed response to repair disrupted protein functions. In vitro incubation of protein samples at various different temperatures (10, 22 and 37 °C) for variable duration (1, 3 or 7 days) in different buffers like AAB (pH 6), ABB (pH 8) and TEAB (pH 8.5) revealed precipitation of protein except acetate buffer (pH 6). As alkaline condition is known to induce protein deamidation, this observation may suggest that deamidation causes aggregation of proteins.Table 1

Bottom Line: Proteomics profiling of both soluble and aggregated amyloidal plaque demonstrated significant enrichment and deamidation of S100A9, ferritin, hemoglobin subunits, creatine kinase and collagen protein among the aggregated brain proteins.The multiple deamidated residues of S100A9 predicts introduction of negative charge that alter Ca(++) binding, suggesting increased capacity to form pathological aggregates in the brain.UC-coupled proteomics revealed that brain amyloidal plaques are enriched in deamidated proteins, and suggested that altered charge state and calcium-binding capacity of S100A9 may enhance protein aggregation and promote neurodegeneration in the human brain.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Division of Structural Biology and Biochemistry, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore. SSAdav@ntu.edu.sg.

ABSTRACT

Background: Aggregation of malformed proteins is a key feature of many neurodegenerative diseases, but the mechanisms that drive proteinopathy in the brain are poorly understood. We aimed to characterize aggregated proteins in human brain tissues affected by dementia.

Results: To characterize amyloidal plaque purified from post-mortem brain tissue of dementia patient, we applied ultracentrifugation-electrostatic repulsion hydrophilic interaction chromatography (UC-ERLIC) coupled mass spectrometry-based proteomics technologies. Proteomics profiling of both soluble and aggregated amyloidal plaque demonstrated significant enrichment and deamidation of S100A9, ferritin, hemoglobin subunits, creatine kinase and collagen protein among the aggregated brain proteins. Amyloidal plaques were enriched in the deamidated variant of protein S100A9, and structural analysis indicated that both the low- and high-affinity calcium binding motifs of S100A9 were deamidated exclusively in the aggregated fraction, suggesting altered charge state and function of this protein in brain tissues affected by dementia. The multiple deamidated residues of S100A9 predicts introduction of negative charge that alter Ca(++) binding, suggesting increased capacity to form pathological aggregates in the brain.

Conclusion: UC-coupled proteomics revealed that brain amyloidal plaques are enriched in deamidated proteins, and suggested that altered charge state and calcium-binding capacity of S100A9 may enhance protein aggregation and promote neurodegeneration in the human brain.

No MeSH data available.


Related in: MedlinePlus