20. June 2012 · Comments Off · Categories: Neuropathy · Tags: , , , , ,
Title Abnormal Protein Expression and Modification in the Spinal Cord with ALS
Abstract

Amyotrophic lateral sclerosis (ALS) is progressive fatal neurodegenerative disease in which motor neurons selectively degenerate. The incidence of ALS is reported to be average 1.89 per 100.000/year, which leads to death usually form respiratory failure within 2 to 5 years. ALS is characterized pathologically by the loss of motor neurons in motor cortex, brainstem and spinal cord. However, the exact mechanisms of this disease are still being unraveled.Recently, the study on mechanism of ALS used to focus on the mutations in the Copper-Zinc superoxide dismutase (SOD1) gene, abnormal expression and modification of TDP-43, changes reflecting free radical damage, the over stimulation of glutamate receptors and neurofilamnet aggregation. But the exact molecular pathway causing motor neuron degenerative in ALS is not yet known. Some changes cross in ALS subtypes, and it become to be complicated understanding. Proteomics is the large-scale study of proteins, particularly their expression and posttranslational modification, which become more and more important skills for research. In this present study, we used this method to explore the underlying mechanisms of ALS, and in order to find out the biomarker for early diagnosis and therapies.Objective: To investigate the difference of protein expression and modification in different area of the nervous system. To address the pathophysiological mechanisms underlying the development of ALS in this project.Method:①The quantification and separation of protein can be used by One-dimensional gel electrophoresis and Two-dimensional gel electrophoresis, which can also purify protein.②The sequence and posttranslational modification was identified by LC-tandem mass spectrometry (LC-MS/MS).③The significant and modify protein can be confirmed by Western blot and 2D Western blotResult:①To identify the difference in protein expression and modification by 2D gel using postmortem tissues, we didn’t find significant difference of protein expression except spinal cord. These proteins are modified with different charges.②Using LC-MS/MS, the most significant difference is GFAP, which is up-regulated in spinal cord of ALS. We also found some others are different in protein expression, such as: NFL, Vimentin and so on. To confirm that these filament protein are expressed differently, we analyzed the protein expression by western blotting. We find the NFL is decreased, the Vementin is up-regulated. In addition, some of them were degraded into fragments.③Increased GFAP were also confirmed by western bloting, which is consisted with the result of 2D gel. When the lysate were dialysis against with PBS, the degraded fragment of GFAP were more in ALS than NON-ALS.④Using LC-MS/MS, The oxidation sites were more in degraded fragment GFAP from high molecular weight to low molecular weight. The methionine is these peptides are highly oxidated. By using the antibody against common acetylated protein, we find protein acetylation is different in ALS and NON-ALS. Using LC-MS/MS, we first identified three acetylated protein. That is Tublin, GFAP and UBS3A.Conclusion: Our research suggested that filament protein play important role in pathology of ALS. The differently protein expression and modification offer futher insight into the protential mechanisms of ALS.

Category Neuropathy
Keywords Acetyaltion, ALS, Filament protein, GFAP, oxidation, Proteomics,
FileType PDF
Pages 147
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