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Regulation of L-glutamic acid decarboxylase by post-translational modifications

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Date Issued:
2005
Summary:
In the central nervous system (CNS), the rate-limiting step in GABA synthesis is the reaction catalyzed by L-glutamic acid decarboxylase (GAD). Alternations in the level of GABA or GAD have been linked to various neurological disorders. Mammalian species express two isoforms of GAD, namely, GAD65 and GAD67, referring to GAD with a molecular weight of 65 kDa and 67 kDa, respectively. Numerous studies have been done to elucidate the mechanisms that control the regulation of GAD at the level of gene expression, protein synthesis, saturation of co-factor, pyridoxal 5'-phosphate (PLP), and post-translational modification. Our previous studies had demonstrated the presence of the truncated form of human brain L-glutamic decarboxylase 65 (tGAD65) in vivo as well as in vitro and found that tGAD65 was more active than the full-length GAD65 (fGAD65). In addition, the recombinant human brain GAD67 has been found to be specifically cleaved at two specific sites, one at arginine 70 and another at arginine 90, to produce two truncated forms of GAD 67 (tGAD67). It seems that the formation of tGAD is catalyzed by specific proteases instead of a random degradation. Furthermore, it has been found that GAD65 is regulated by the Ca2+-free form of calmodulin (apoCaM). My research focus is to elucidate the regulation of GABA biosynthesis through regulation of its synthesizing enzyme, especially GAD67, by protein phosphorylation, proteolytic cleavage and apoCaM. Experiments presented here have been conducted to demonstrate the molecular cloning, expression, and purification of human brain tGAD67. The purified protein was further characterized by kinetic studies and phosphorylation studies. Truncated forms of hGAD67 were much less active than the full-length form. Both truncated enzymes are also phosphorylated by protein kinase A (PKA) as is full-length hGAD67. A deletion of 1-70 aa from the N-terminal results in additional protein kinase C (PKC) phosphorylation. Several phosphopeptides and possible phosphorylation sites are suggested by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis. Furthermore, evidence of mu-calpain, not m-calpain, as the protease responsible for GAD cleavage in vivo as well as in vitro is presented. In addition, evidence on the effect of ApoCaM on GAD67 activity, phosphporylation and proteolytic cleavage by mu-calpain is discussed. Finally, an overall model of GAD regulation by a variety of mechanisms including protein phosphorylation, mu-calpain proteolytic cleavage and apoCaM is proposed.
Title: Regulation of L-glutamic acid decarboxylase by post-translational modifications.
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Name(s): Sha, Di
Florida Atlantic University, Degree Grantor
Charles E. Schmidt College of Science
Center for Complex Systems and Brain Sciences
Type of Resource: text
Genre: Electronic Thesis Or Dissertation
Issuance: monographic
Date Issued: 2005
Publisher: Florida Atlantic University
Place of Publication: Boca Raton, Fla.
Physical Form: application/pdf
Extent: 238 p.
Language(s): English
Summary: In the central nervous system (CNS), the rate-limiting step in GABA synthesis is the reaction catalyzed by L-glutamic acid decarboxylase (GAD). Alternations in the level of GABA or GAD have been linked to various neurological disorders. Mammalian species express two isoforms of GAD, namely, GAD65 and GAD67, referring to GAD with a molecular weight of 65 kDa and 67 kDa, respectively. Numerous studies have been done to elucidate the mechanisms that control the regulation of GAD at the level of gene expression, protein synthesis, saturation of co-factor, pyridoxal 5'-phosphate (PLP), and post-translational modification. Our previous studies had demonstrated the presence of the truncated form of human brain L-glutamic decarboxylase 65 (tGAD65) in vivo as well as in vitro and found that tGAD65 was more active than the full-length GAD65 (fGAD65). In addition, the recombinant human brain GAD67 has been found to be specifically cleaved at two specific sites, one at arginine 70 and another at arginine 90, to produce two truncated forms of GAD 67 (tGAD67). It seems that the formation of tGAD is catalyzed by specific proteases instead of a random degradation. Furthermore, it has been found that GAD65 is regulated by the Ca2+-free form of calmodulin (apoCaM). My research focus is to elucidate the regulation of GABA biosynthesis through regulation of its synthesizing enzyme, especially GAD67, by protein phosphorylation, proteolytic cleavage and apoCaM. Experiments presented here have been conducted to demonstrate the molecular cloning, expression, and purification of human brain tGAD67. The purified protein was further characterized by kinetic studies and phosphorylation studies. Truncated forms of hGAD67 were much less active than the full-length form. Both truncated enzymes are also phosphorylated by protein kinase A (PKA) as is full-length hGAD67. A deletion of 1-70 aa from the N-terminal results in additional protein kinase C (PKC) phosphorylation. Several phosphopeptides and possible phosphorylation sites are suggested by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis. Furthermore, evidence of mu-calpain, not m-calpain, as the protease responsible for GAD cleavage in vivo as well as in vitro is presented. In addition, evidence on the effect of ApoCaM on GAD67 activity, phosphporylation and proteolytic cleavage by mu-calpain is discussed. Finally, an overall model of GAD regulation by a variety of mechanisms including protein phosphorylation, mu-calpain proteolytic cleavage and apoCaM is proposed.
Identifier: 9780542379024 (isbn), 12170 (digitool), FADT12170 (IID), fau:9077 (fedora)
Collection: FAU Electronic Theses and Dissertations Collection
Note(s): Adviser: Jang-Yen Wu.
Thesis (Ph.D.)--Florida Atlantic University, 2005.
Subject(s): Biology, Neuroscience
Chemistry, Biochemistry
Held by: Florida Atlantic University Libraries
Persistent Link to This Record: http://purl.flvc.org/fcla/dt/12170
Sublocation: Digital Library
Use and Reproduction: Copyright © is held by the author, with permission granted to Florida Atlantic University to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Use and Reproduction: http://rightsstatements.org/vocab/InC/1.0/
Host Institution: FAU
Is Part of Series: Florida Atlantic University Digital Library Collections.