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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

Protein abundance in the aggregated fraction of the human brain proteome as extracted using detergent/acetate buffer together with ultracentrifugation/ERLIC fractionation (a). Embedded panel details the most abundant proteins detected in the aggregated fraction (b)
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Fig2: Protein abundance in the aggregated fraction of the human brain proteome as extracted using detergent/acetate buffer together with ultracentrifugation/ERLIC fractionation (a). Embedded panel details the most abundant proteins detected in the aggregated fraction (b)

Mentions: Protein aggregation into amyloid plaques is a pathologic hallmark of multiple neurodegenerative diseases, but therapies directed against amyloid proteins have shown little clinical benefit in human patients so far, suggesting a critical role for non-amyloid proteins in the formation of plaques. Having optimized our protocol for extraction of both soluble and aggregated proteins from post-mortem samples of human brain, we next conducted a proteome-wide analysis of amyloid structure, relative abundance and plaque composition in human brain tissues affected by dementia. To do this, we extracted both soluble and aggregated amyloidal proteins from brain using our UC-based method, subjected the aggregated proteins to further extraction and solubilization in formic acid (FA), and separated the deamidated peptides by ERLIC fractionation and structural analysis by LC-MS/MS. Using this approach, we identified a total of 5225 ± 248 brain tissue proteins, of which 4339 ± 104 individual proteins remained after application of a stringent false-discovery rate (FDR; cutoff value <1.0 %) and the characterized proteins have been identified with at least 2 unique peptides. Overlap analysis revealed that while 1697 proteins were present in both the soluble and aggregated fractions, 159 individual brain proteins were specific to the aggregated (pellet) fraction only (Fig. 1). We observed significant enrichment of S100A9, ferritin, mitochondrial creatine kinase (U-type), and hemoglobin subunits (α, β, ε and ζ) within the protein aggregate (Fig. 2a, b), as well as increased abundance of collagen, palmitoyl-protein thioesterase 1, laminin, coronin-1A, S100-B, S100-A8, Syntaxin-binding protein 2, cathepsin G, grancalcin, and syntaxin-binding protein 2 (Additional file 1: Table S1A–D). While protein S100A9 was present in both sample types, the levels detected in the pellet fraction (protein score 5083.50 ± 172.50, emPAI 300.74 ± 0.00) were far higher than those detected in the soluble fraction (1315.50 ± 119.50; 8.81 ± 0.00), suggesting substantial enrichment of S100A9 in brain amyloid aggregates (Fig. 2, Additional file 2: Table S2 and Additional file 1: Table S1C). Similarly, the iron-storing protein ferritin was also enriched in the pelleted protein aggregate (emPAI 450.45 ± 0.00) relative to the levels detected in the soluble fraction (15.20 ± 0.00; Fig. 2b). Also enriched in the pellet fraction were the hemoglobin subunits α, β, ε and ζ, consistent with reports that elevated hemoglobin levels are associated with increased risk of neurodegenerative disease and rapid cognitive decline (Additional file 2: Table S2 and Additional file 1: Table S1D). Conversely, a distinct subset of brain proteins was more abundant in the soluble fraction than in the pellet, including neuromodulin, glial fibrillary acidic protein, myelin basic protein isoform 4, synaptosomal associated protein 25, brain acid soluble protein 1, and components of β-tubulin (Fig. 3a and b). After further separation of the soluble fraction by UC-ERLIC, we were also able to detect several different isoforms of APBB2 and amyloid-like protein APLP2, as well as APBB1, APBB1IP and SAA4. These data indicated that multiple amyloid-like protein isoforms displayed only intermediate levels of aggregation and were therefore largely absent from the pellet fraction (Fig. 4).Fig. 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)

Protein abundance in the aggregated fraction of the human brain proteome as extracted using detergent/acetate buffer together with ultracentrifugation/ERLIC fractionation (a). Embedded panel details the most abundant proteins detected in the aggregated fraction (b)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Protein abundance in the aggregated fraction of the human brain proteome as extracted using detergent/acetate buffer together with ultracentrifugation/ERLIC fractionation (a). Embedded panel details the most abundant proteins detected in the aggregated fraction (b)
Mentions: Protein aggregation into amyloid plaques is a pathologic hallmark of multiple neurodegenerative diseases, but therapies directed against amyloid proteins have shown little clinical benefit in human patients so far, suggesting a critical role for non-amyloid proteins in the formation of plaques. Having optimized our protocol for extraction of both soluble and aggregated proteins from post-mortem samples of human brain, we next conducted a proteome-wide analysis of amyloid structure, relative abundance and plaque composition in human brain tissues affected by dementia. To do this, we extracted both soluble and aggregated amyloidal proteins from brain using our UC-based method, subjected the aggregated proteins to further extraction and solubilization in formic acid (FA), and separated the deamidated peptides by ERLIC fractionation and structural analysis by LC-MS/MS. Using this approach, we identified a total of 5225 ± 248 brain tissue proteins, of which 4339 ± 104 individual proteins remained after application of a stringent false-discovery rate (FDR; cutoff value <1.0 %) and the characterized proteins have been identified with at least 2 unique peptides. Overlap analysis revealed that while 1697 proteins were present in both the soluble and aggregated fractions, 159 individual brain proteins were specific to the aggregated (pellet) fraction only (Fig. 1). We observed significant enrichment of S100A9, ferritin, mitochondrial creatine kinase (U-type), and hemoglobin subunits (α, β, ε and ζ) within the protein aggregate (Fig. 2a, b), as well as increased abundance of collagen, palmitoyl-protein thioesterase 1, laminin, coronin-1A, S100-B, S100-A8, Syntaxin-binding protein 2, cathepsin G, grancalcin, and syntaxin-binding protein 2 (Additional file 1: Table S1A–D). While protein S100A9 was present in both sample types, the levels detected in the pellet fraction (protein score 5083.50 ± 172.50, emPAI 300.74 ± 0.00) were far higher than those detected in the soluble fraction (1315.50 ± 119.50; 8.81 ± 0.00), suggesting substantial enrichment of S100A9 in brain amyloid aggregates (Fig. 2, Additional file 2: Table S2 and Additional file 1: Table S1C). Similarly, the iron-storing protein ferritin was also enriched in the pelleted protein aggregate (emPAI 450.45 ± 0.00) relative to the levels detected in the soluble fraction (15.20 ± 0.00; Fig. 2b). Also enriched in the pellet fraction were the hemoglobin subunits α, β, ε and ζ, consistent with reports that elevated hemoglobin levels are associated with increased risk of neurodegenerative disease and rapid cognitive decline (Additional file 2: Table S2 and Additional file 1: Table S1D). Conversely, a distinct subset of brain proteins was more abundant in the soluble fraction than in the pellet, including neuromodulin, glial fibrillary acidic protein, myelin basic protein isoform 4, synaptosomal associated protein 25, brain acid soluble protein 1, and components of β-tubulin (Fig. 3a and b). After further separation of the soluble fraction by UC-ERLIC, we were also able to detect several different isoforms of APBB2 and amyloid-like protein APLP2, as well as APBB1, APBB1IP and SAA4. These data indicated that multiple amyloid-like protein isoforms displayed only intermediate levels of aggregation and were therefore largely absent from the pellet fraction (Fig. 4).Fig. 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