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Protein Citrullination: A Proposed Mechanism for Pathology in Traumatic Brain Injury.

Lazarus RC, Buonora JE, Flora MN, Freedy JG, Holstein GR, Martinelli GP, Jacobowitz DM, Mueller GP - Front Neurol (2015)

Bottom Line: The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved.This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia.Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins.

View Article: PubMed Central - PubMed

Affiliation: Program in Neuroscience, Uniformed Services University of the Health Sciences , Bethesda, MD , USA.

ABSTRACT
Protein citrullination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS), and prion disease. Citrullination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citrullination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved. Immunofluorescence revealed that controlled cortical impact in rats profoundly up--regulated protein citrullination in the cerebral cortex, external capsule, and hippocampus. This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia. Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins. Proteins most notably affected were those also reported to be citrullinated in other disorders, including prion disease and MS. In vivo findings were extended in an in vitro model of simulated TBI employing normal human astrocytes. Pharmacologically induced calcium excitotoxicity was shown to activate the citrullination and breakdown of glial fibrillary acidic protein, producing a novel candidate TBI biomarker and potential target for autoimmune recognition. In summary, these findings demonstrate that the effects of TBI on protein citrullination are selective with respect to brain region, cell type, and proteins modified, and may contribute to a role for autoimmune dysfunction in chronic pathology following TBI.

No MeSH data available.


Related in: MedlinePlus

Identification of proteins that are citrullinated in response to CCI. Extracts of control (C) and injured (I) cerebral cortex were fractionated by fluid-phase isoelectric focusing into defined pH ranges (shown at top) and then further resolved according to molecular weight using one-dimensional gel electrophoresis. Proteins were then transferred to nitrocellulose membranes and probed for protein-bound citrulline (see “Materials and Methods”) (right panel, “Western blot”). Gels run in parallel were visualized with Coomassie (left panel). Sixteen features showing increased citrullination in response to CCI (black numbered boxes, right panel) were mapped to corresponding Coomassie features (red numbered boxes, left panel) and identified by peptide mass finger printing and tandem mass spectrometry. Proteins identified are listed in the lower panel. Analysis of the mass spectra data set with a variable modification search for the citrullination of arginine residues revealed several proteins with sites of citrullination on arginine residues with Mascot score corresponding to p < 0.05 (Cit Pep). Several of these proteins (Prev Report) are also citrullinated in other pathologies, including ALDOA, ALDOC, GFAP, PRDX1, COF1, ENOA, ENOG, MBP, and tubulin (beta) in prion disease (20, 21); GFAP and MBP in Alzheimer’s disease and multiple sclerosis (16); ENOA in rheumatoid arthritis (22); and GRP78 in Type I diabetes (23). Images are representative of six independent experiments. A total of n = 4 CCI and n = 4 control animals were examined.
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Figure 8: Identification of proteins that are citrullinated in response to CCI. Extracts of control (C) and injured (I) cerebral cortex were fractionated by fluid-phase isoelectric focusing into defined pH ranges (shown at top) and then further resolved according to molecular weight using one-dimensional gel electrophoresis. Proteins were then transferred to nitrocellulose membranes and probed for protein-bound citrulline (see “Materials and Methods”) (right panel, “Western blot”). Gels run in parallel were visualized with Coomassie (left panel). Sixteen features showing increased citrullination in response to CCI (black numbered boxes, right panel) were mapped to corresponding Coomassie features (red numbered boxes, left panel) and identified by peptide mass finger printing and tandem mass spectrometry. Proteins identified are listed in the lower panel. Analysis of the mass spectra data set with a variable modification search for the citrullination of arginine residues revealed several proteins with sites of citrullination on arginine residues with Mascot score corresponding to p < 0.05 (Cit Pep). Several of these proteins (Prev Report) are also citrullinated in other pathologies, including ALDOA, ALDOC, GFAP, PRDX1, COF1, ENOA, ENOG, MBP, and tubulin (beta) in prion disease (20, 21); GFAP and MBP in Alzheimer’s disease and multiple sclerosis (16); ENOA in rheumatoid arthritis (22); and GRP78 in Type I diabetes (23). Images are representative of six independent experiments. A total of n = 4 CCI and n = 4 control animals were examined.

Mentions: Proteomic analysis revealed that the effects of CCI on protein citrullination were specific to a discrete subset of proteins making up the entire brain proteome, and further, that the proteins involved are primarily associated with cytoskeletal structure and metabolic processes (Figure 8). Shown in the upper left panel are the proteomes of control and injured cerebral cortex fractionated by fluid-phase isoelectric focusing. Each pair of lanes, control (C) and CCI (I), show the proteins present in the four different pI partitions. As visualized by Commassie staining, CCI did not appreciably affect the general pattern of protein staining across the four pI fractions. In contrast, the pattern of protein citrullination was dramatically impacted by CCI (upper right panel). Consistent with immunohistochemistry findings (Figures 2 and 3), little protein citrullination was observed in control cortex (C), whereas CCI (I) resulted in the intense labeling of a distinctive subset of the fractionated proteins. The immunoreactive signals of the western blot were mapped to Commassie features of the protein gel, and proteins were identified by peptide mass fingerprinting and tandem mass spectrometry. The proteins identified are presented in the lower panel of Figure 8. These proteins are functionally grouped as cytoskeletal components (including dynamin-1, GFAP, and several forms of tubulin); those involved in metabolic processes (including peroxiredoxin-1, dihydropyrimidinase-related protein 2, and creatine kinase B-type); and proteins involved in cell–cell signaling and synaptic transmission (synapsin-2, syntaxin-binding protein 1, and amphyiphysin). It should be noted that these functional groups of affected proteins – cytoskeletal components, metabolic proteins, and proteins involved in cell–cell signaling – may have been preferentially identified as citrullinated due to their relatively high abundance. These abundant protein types may be more easily identifiable through proteomic analyses than less abundant proteins, which may also be citrullinated but not easily identified. In this regard, however, only a small proportion of the very high abundance proteins were identified as being citrullinated, indicating a notable degree of specificity in the citrullination response.


Protein Citrullination: A Proposed Mechanism for Pathology in Traumatic Brain Injury.

Lazarus RC, Buonora JE, Flora MN, Freedy JG, Holstein GR, Martinelli GP, Jacobowitz DM, Mueller GP - Front Neurol (2015)

Identification of proteins that are citrullinated in response to CCI. Extracts of control (C) and injured (I) cerebral cortex were fractionated by fluid-phase isoelectric focusing into defined pH ranges (shown at top) and then further resolved according to molecular weight using one-dimensional gel electrophoresis. Proteins were then transferred to nitrocellulose membranes and probed for protein-bound citrulline (see “Materials and Methods”) (right panel, “Western blot”). Gels run in parallel were visualized with Coomassie (left panel). Sixteen features showing increased citrullination in response to CCI (black numbered boxes, right panel) were mapped to corresponding Coomassie features (red numbered boxes, left panel) and identified by peptide mass finger printing and tandem mass spectrometry. Proteins identified are listed in the lower panel. Analysis of the mass spectra data set with a variable modification search for the citrullination of arginine residues revealed several proteins with sites of citrullination on arginine residues with Mascot score corresponding to p < 0.05 (Cit Pep). Several of these proteins (Prev Report) are also citrullinated in other pathologies, including ALDOA, ALDOC, GFAP, PRDX1, COF1, ENOA, ENOG, MBP, and tubulin (beta) in prion disease (20, 21); GFAP and MBP in Alzheimer’s disease and multiple sclerosis (16); ENOA in rheumatoid arthritis (22); and GRP78 in Type I diabetes (23). Images are representative of six independent experiments. A total of n = 4 CCI and n = 4 control animals were examined.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4585288&req=5

Figure 8: Identification of proteins that are citrullinated in response to CCI. Extracts of control (C) and injured (I) cerebral cortex were fractionated by fluid-phase isoelectric focusing into defined pH ranges (shown at top) and then further resolved according to molecular weight using one-dimensional gel electrophoresis. Proteins were then transferred to nitrocellulose membranes and probed for protein-bound citrulline (see “Materials and Methods”) (right panel, “Western blot”). Gels run in parallel were visualized with Coomassie (left panel). Sixteen features showing increased citrullination in response to CCI (black numbered boxes, right panel) were mapped to corresponding Coomassie features (red numbered boxes, left panel) and identified by peptide mass finger printing and tandem mass spectrometry. Proteins identified are listed in the lower panel. Analysis of the mass spectra data set with a variable modification search for the citrullination of arginine residues revealed several proteins with sites of citrullination on arginine residues with Mascot score corresponding to p < 0.05 (Cit Pep). Several of these proteins (Prev Report) are also citrullinated in other pathologies, including ALDOA, ALDOC, GFAP, PRDX1, COF1, ENOA, ENOG, MBP, and tubulin (beta) in prion disease (20, 21); GFAP and MBP in Alzheimer’s disease and multiple sclerosis (16); ENOA in rheumatoid arthritis (22); and GRP78 in Type I diabetes (23). Images are representative of six independent experiments. A total of n = 4 CCI and n = 4 control animals were examined.
Mentions: Proteomic analysis revealed that the effects of CCI on protein citrullination were specific to a discrete subset of proteins making up the entire brain proteome, and further, that the proteins involved are primarily associated with cytoskeletal structure and metabolic processes (Figure 8). Shown in the upper left panel are the proteomes of control and injured cerebral cortex fractionated by fluid-phase isoelectric focusing. Each pair of lanes, control (C) and CCI (I), show the proteins present in the four different pI partitions. As visualized by Commassie staining, CCI did not appreciably affect the general pattern of protein staining across the four pI fractions. In contrast, the pattern of protein citrullination was dramatically impacted by CCI (upper right panel). Consistent with immunohistochemistry findings (Figures 2 and 3), little protein citrullination was observed in control cortex (C), whereas CCI (I) resulted in the intense labeling of a distinctive subset of the fractionated proteins. The immunoreactive signals of the western blot were mapped to Commassie features of the protein gel, and proteins were identified by peptide mass fingerprinting and tandem mass spectrometry. The proteins identified are presented in the lower panel of Figure 8. These proteins are functionally grouped as cytoskeletal components (including dynamin-1, GFAP, and several forms of tubulin); those involved in metabolic processes (including peroxiredoxin-1, dihydropyrimidinase-related protein 2, and creatine kinase B-type); and proteins involved in cell–cell signaling and synaptic transmission (synapsin-2, syntaxin-binding protein 1, and amphyiphysin). It should be noted that these functional groups of affected proteins – cytoskeletal components, metabolic proteins, and proteins involved in cell–cell signaling – may have been preferentially identified as citrullinated due to their relatively high abundance. These abundant protein types may be more easily identifiable through proteomic analyses than less abundant proteins, which may also be citrullinated but not easily identified. In this regard, however, only a small proportion of the very high abundance proteins were identified as being citrullinated, indicating a notable degree of specificity in the citrullination response.

Bottom Line: The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved.This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia.Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins.

View Article: PubMed Central - PubMed

Affiliation: Program in Neuroscience, Uniformed Services University of the Health Sciences , Bethesda, MD , USA.

ABSTRACT
Protein citrullination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS), and prion disease. Citrullination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citrullination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved. Immunofluorescence revealed that controlled cortical impact in rats profoundly up--regulated protein citrullination in the cerebral cortex, external capsule, and hippocampus. This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia. Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins. Proteins most notably affected were those also reported to be citrullinated in other disorders, including prion disease and MS. In vivo findings were extended in an in vitro model of simulated TBI employing normal human astrocytes. Pharmacologically induced calcium excitotoxicity was shown to activate the citrullination and breakdown of glial fibrillary acidic protein, producing a novel candidate TBI biomarker and potential target for autoimmune recognition. In summary, these findings demonstrate that the effects of TBI on protein citrullination are selective with respect to brain region, cell type, and proteins modified, and may contribute to a role for autoimmune dysfunction in chronic pathology following TBI.

No MeSH data available.


Related in: MedlinePlus