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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

Specificity of anti-protein citrulline mAb 6B3 detection for western blotting. Displayed on the left are two Coomassie-stained profiles showing the protein composition of native fibrinogen (Fib) and citrullinated fibrinogen (C-Fib). The western blot (right) shows three immunoblots in which citrullinated fibrinogen was probed with active mAb 6B3 (Active/C-Fib; left lane); citrullinated fibrinogen was probed with immunoneutralized mAb 6B3 (Neutralized/C-Fib; middle lane); and native fibrinogen was probed with active mAb 6B3 (Active/Fib; right lane).
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Figure 7: Specificity of anti-protein citrulline mAb 6B3 detection for western blotting. Displayed on the left are two Coomassie-stained profiles showing the protein composition of native fibrinogen (Fib) and citrullinated fibrinogen (C-Fib). The western blot (right) shows three immunoblots in which citrullinated fibrinogen was probed with active mAb 6B3 (Active/C-Fib; left lane); citrullinated fibrinogen was probed with immunoneutralized mAb 6B3 (Neutralized/C-Fib; middle lane); and native fibrinogen was probed with active mAb 6B3 (Active/Fib; right lane).

Mentions: Figure 7 demonstrates the specificity of the 6B3 antibody for detecting citrullinated proteins in a western blot format. Shown on the left is the Coomassie staining for human fibrinogen (Fib) and the same preparation of fibrinogen that was enzymatically citrullinated by reaction with PAD4 (C-Fib). The protein staining shows that the characteristic profile of purified human fibrinogen is modestly affected by reaction with PAD4. Presented on the right are immunoblots showing the reactivity of mAb 6B3 with the citrullinated fibrinogen preparation (Active/C-Fib) and the elimination of this reactivity by preabsorption of 6B3 with citrullinated fibrinogen prior to blotting (Neutralized/C-Fib). There was no reactivity of mAb 6B3 with fibrinogen that had not been citrullinated by PAD4 treatment (Active/Fib).


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)

Specificity of anti-protein citrulline mAb 6B3 detection for western blotting. Displayed on the left are two Coomassie-stained profiles showing the protein composition of native fibrinogen (Fib) and citrullinated fibrinogen (C-Fib). The western blot (right) shows three immunoblots in which citrullinated fibrinogen was probed with active mAb 6B3 (Active/C-Fib; left lane); citrullinated fibrinogen was probed with immunoneutralized mAb 6B3 (Neutralized/C-Fib; middle lane); and native fibrinogen was probed with active mAb 6B3 (Active/Fib; right lane).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Specificity of anti-protein citrulline mAb 6B3 detection for western blotting. Displayed on the left are two Coomassie-stained profiles showing the protein composition of native fibrinogen (Fib) and citrullinated fibrinogen (C-Fib). The western blot (right) shows three immunoblots in which citrullinated fibrinogen was probed with active mAb 6B3 (Active/C-Fib; left lane); citrullinated fibrinogen was probed with immunoneutralized mAb 6B3 (Neutralized/C-Fib; middle lane); and native fibrinogen was probed with active mAb 6B3 (Active/Fib; right lane).
Mentions: Figure 7 demonstrates the specificity of the 6B3 antibody for detecting citrullinated proteins in a western blot format. Shown on the left is the Coomassie staining for human fibrinogen (Fib) and the same preparation of fibrinogen that was enzymatically citrullinated by reaction with PAD4 (C-Fib). The protein staining shows that the characteristic profile of purified human fibrinogen is modestly affected by reaction with PAD4. Presented on the right are immunoblots showing the reactivity of mAb 6B3 with the citrullinated fibrinogen preparation (Active/C-Fib) and the elimination of this reactivity by preabsorption of 6B3 with citrullinated fibrinogen prior to blotting (Neutralized/C-Fib). There was no reactivity of mAb 6B3 with fibrinogen that had not been citrullinated by PAD4 treatment (Active/Fib).

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