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Branched-chain alpha-keto acids accumulating in maple syrup urine disease induce reorganization of phosphorylated GFAP in C6-glioma cells.
Metab Brain Dis. 2005 Sep; 20(3):205-17.MB

Abstract

In this study we investigate the effects of the branched-chain keto acids (BCKA) alpha-ketoisocaproic (KIC), alpha-ketoisovaleric (KIV), and alpha-keto-beta-methylvaleric (KMV) acids, metabolites accumulating in maple syrup urine disease (MSUD), on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and cytoskeletal reorganization in C6-glioma cells. We observed that after 3 h treatment with KIC, KIV, or KMV cells showed retracted cytoplasm with bipolar processes containing packed GFAP filaments as revealed by immunocytochemistry. Western Blot analysis by anti-GFAP monoclonal antibody demonstrated that BCKA were not able to alter GFAP immunocontent in total cell homogenate, but the immunocontent as well as the in vitro (32)P incorporation into GFAP recovered into the high salt Triton-insoluble cytoskeletal fraction were significantly increased. Western Blot using monoclonal antiphosphoserine antibody showed that BCKA induced increased immunocontent of phosphoserine-containing amino acids in several proteins in total cell homogenate. In addition, the immunocontent of phosphoserine-containing amino acids was also greatly increased in GFAP recovered in the high-salt Triton insoluble cytoskeletal fraction, corresponding to the polymerized intermedite filament (IF) proteins present in the cell. In conclusion, our results indicate that KIC, KIV, or KMV increased the serine/threonine in vitro phosphorylation of GFAP leading to increased Triton-insoluble GFAP immunocontent and cytoskeletal reorganization. Considering IF networks can be regulated by phosphorylation of polypeptide subunits leading to reorganization of the IF filamentous structure, we could suppose that GFAP hyperphosphorylation and disorganization of cellular structure could be involved in the brain damage characteristic of MSUD patients.

Authors+Show Affiliations

Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16167198

Citation

Funchal, Cláudia, et al. "Branched-chain Alpha-keto Acids Accumulating in Maple Syrup Urine Disease Induce Reorganization of Phosphorylated GFAP in C6-glioma Cells." Metabolic Brain Disease, vol. 20, no. 3, 2005, pp. 205-17.
Funchal C, Dos Santos AQ, Jacques-Silva MC, et al. Branched-chain alpha-keto acids accumulating in maple syrup urine disease induce reorganization of phosphorylated GFAP in C6-glioma cells. Metab Brain Dis. 2005;20(3):205-17.
Funchal, C., Dos Santos, A. Q., Jacques-Silva, M. C., Zamoner, A., Gottfried, C., Wajner, M., & Pessoa-Pureur, R. (2005). Branched-chain alpha-keto acids accumulating in maple syrup urine disease induce reorganization of phosphorylated GFAP in C6-glioma cells. Metabolic Brain Disease, 20(3), 205-17.
Funchal C, et al. Branched-chain Alpha-keto Acids Accumulating in Maple Syrup Urine Disease Induce Reorganization of Phosphorylated GFAP in C6-glioma Cells. Metab Brain Dis. 2005;20(3):205-17. PubMed PMID: 16167198.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Branched-chain alpha-keto acids accumulating in maple syrup urine disease induce reorganization of phosphorylated GFAP in C6-glioma cells. AU - Funchal,Cláudia, AU - Dos Santos,André Quincozes, AU - Jacques-Silva,Maria Caroline, AU - Zamoner,Ariane, AU - Gottfried,Carmem, AU - Wajner,Moacir, AU - Pessoa-Pureur,Regina, PY - 2005/04/25/received PY - 2005/06/07/accepted PY - 2005/9/17/pubmed PY - 2005/11/11/medline PY - 2005/9/17/entrez SP - 205 EP - 17 JF - Metabolic brain disease JO - Metab Brain Dis VL - 20 IS - 3 N2 - In this study we investigate the effects of the branched-chain keto acids (BCKA) alpha-ketoisocaproic (KIC), alpha-ketoisovaleric (KIV), and alpha-keto-beta-methylvaleric (KMV) acids, metabolites accumulating in maple syrup urine disease (MSUD), on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and cytoskeletal reorganization in C6-glioma cells. We observed that after 3 h treatment with KIC, KIV, or KMV cells showed retracted cytoplasm with bipolar processes containing packed GFAP filaments as revealed by immunocytochemistry. Western Blot analysis by anti-GFAP monoclonal antibody demonstrated that BCKA were not able to alter GFAP immunocontent in total cell homogenate, but the immunocontent as well as the in vitro (32)P incorporation into GFAP recovered into the high salt Triton-insoluble cytoskeletal fraction were significantly increased. Western Blot using monoclonal antiphosphoserine antibody showed that BCKA induced increased immunocontent of phosphoserine-containing amino acids in several proteins in total cell homogenate. In addition, the immunocontent of phosphoserine-containing amino acids was also greatly increased in GFAP recovered in the high-salt Triton insoluble cytoskeletal fraction, corresponding to the polymerized intermedite filament (IF) proteins present in the cell. In conclusion, our results indicate that KIC, KIV, or KMV increased the serine/threonine in vitro phosphorylation of GFAP leading to increased Triton-insoluble GFAP immunocontent and cytoskeletal reorganization. Considering IF networks can be regulated by phosphorylation of polypeptide subunits leading to reorganization of the IF filamentous structure, we could suppose that GFAP hyperphosphorylation and disorganization of cellular structure could be involved in the brain damage characteristic of MSUD patients. SN - 0885-7490 UR - https://www.unboundmedicine.com/medline/citation/16167198/Branched_chain_alpha_keto_acids_accumulating_in_maple_syrup_urine_disease_induce_reorganization_of_phosphorylated_GFAP_in_C6_glioma_cells_ L2 - https://doi.org/10.1007/s11011-005-7208-x DB - PRIME DP - Unbound Medicine ER -