Spatial resolution of calpain-catalyzed proteolysis in focal cerebral ischemia

Kambe, Atsushi; Yokota, Masayuki; Saido, Takaomi C.; Satokata, Ichiro; Fujikawa, Hirokazu; Tabuchi, Sadaharu; Kamitani, Hideki; Watanabe, Takashi

doi:10.1016/j.brainres.2005.01.080

Cited by 14 publications

(14 citation statements)

References 38 publications

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“…In the central nervous system, the activation of calpain has been implicated in the process of neuronal damage due to global or focal cerebral ischemia (21,22). The NMDA receptor function is downregulated through calpain-cleavage of its NR2 subunits induced by prolonged glutamate stimulation (23).…”

Section: Discussionmentioning

confidence: 99%

“…Calpain, as a mediator in Ca 2+ signal transduction, plays an important role in the basic cell life processes such as cell differentiation, cell division and growth (See review, 9). In the central nervous system, the activation of calpain has been implicated in the process of neuronal damage due to global or focal cerebral ischemia (21,22). The NMDA receptor function is downregulated through calpain-cleavage of its NR2 subunits induced by prolonged glutamate stimulation (23).…”

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confidence: 99%

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Role of μ-calpain in proteolytic cleavage of brain l-glutamic acid decarboxylase

Sha

Jin

et al. 2008

Brain Research

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Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for γ-aminobutyric acid (GABA) biosynthesis. Previously, we reported the presence of truncated forms of GAD in vivo and in vitro. In addition, an unidentified endogenous protease responsible for proteolytic cleavage of full-length GAD (fGAD) to its truncated form (tGAD) was also observed. In this communication, we report that μ-calpain is a good candidate for conversion of fGAD 67 to tGAD 67 . This conclusion is based on the following observations: 1. Purified recombinant GAD 67 is cleaved by μ-calpain at specific sites; 2. In brain synaptosomal preparation, GAD 67 is cleaved to its truncated form by an endogenous protease which is inhibited by specific calpain inhibitors; 3. In μ-calpain knockout mice, the level of tGAD in the brain is greatly reduced compared with the wild type; 4. when μ-calpain gene is silenced by siRNA, the level of tGAD is also markedly reduced compared to the control group; 5. μ-calpain is activated by neuronal stimulation and Ca 2+ -influx. The physiological significance of calpain in regulation of GABA synthesis and GABAergic neurotransmission is also discussed. KeywordsCalpain; GABA synthesis; GAD; Proteolytic CleavageIn the central nervous system (CNS), γ-aminobutyric acid (GABA) is synthesized by a single enzymatic reaction catalyzed by L-glutamic decarboxylase (EC 4.1.1.15; GAD) (1,2). Mammalian species express two isoforms of GAD, namely, GAD 65 and GAD 67 , referring to GAD with a molecular weight of 65 kDa and 67 kDa, respectively. Previously, we reported **Address correspondence and reprint requests to Dr. Jang-Yen Wu, Florida Atlantic University, Department of Biomedical Science, 777 Glades Road, Boca Raton, FL 33431-0991, U.S. ; Email: jwu@fau.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the presence of the truncated GAD (tGAD) derived from proteolytic cleavage of the full-length (fGAD) in vivo as well as in vitro (3,4). The presence of smaller forms of GAD was also observed from other laboratories (5-8). However, no information was reported regarding the identity of the endogenous proteases responsible for GAD cleavage and their physiological significance. Recently, we found that recombinant human brain GAD 65 is specifically cleaved at arginine 69 from the N-terminal of fGAD 65 to produce tGAD 65 , which is ∼2-3 fold more active than fGAD 65 (3). In addition, GAD 67 was found to be cleaved at two specific sites, one at arginine 70 and another at arginine 90, to produ...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Role of μ-calpain in proteolytic cleavage of brain l-glutamic acid decarboxylase

Sha

Jin

et al. 2008

Brain Research

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“…At the molecular level, increases in intracellular calcium have been shown to promote Tau hyperphosphorylation (33). Given that ␣4 cleavage is mediated by calpains, which can be hyperactivated in response to elevated calcium flux (34), it is enticing to speculate that the hyperphosphorylation of Tau could result from up-regulated ␣4 cleavage and a loss of PP2Ac (1) in cells. Previous studies have suggested that ␣4 facilitates MID1-dependent polyubiquitination of PP2Ac (2A) and subsequent proteasomal degradation; however, our findings challenge this paradigm and support a model in which MID1 serves as the E3 ligase for ␣4 (2B), leading to a conformational change in ␣4 whereby the UIM of ␣4 binds in cis to the covalently attached ubiquitin (Ub; 3).…”

Section: Discussionmentioning

confidence: 99%

Monoubiquitination Promotes Calpain Cleavage of the Protein Phosphatase 2A (PP2A) Regulatory Subunit α4, Altering PP2A Stability and Microtubule-associated Protein Phosphorylation

Watkins

Wang

Mazalouskas

et al. 2012

Journal of Biological Chemistry

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Background: ␣4 binds to the PP2A catalytic subunit and the microtubule-associated E3 ligase MID1. Results: MID1-dependent monoubiquitination promotes calpain-mediated cleavage of ␣4, altering its phosphatase regulatory function. Conclusion: Defects in this regulatory process may underlie the MAP hypophosphorylation and hyperphosphorylation seen in Opitz syndrome and Alzheimer disease. Significance: Pharmacological agents that interfere with ␣4 monoubiquitination or cleavage are potential therapeutics to treat Alzheimer disease.

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“…The major calpain isoforms, -and m-calpains (also known as calpains 1 and 2), cleave a wide variety of proteins in the cell, including the cytoskeletal proteins ␣-spectrin, integrin subunit ␤3 and filamin A, the CDK5 regulator p35 (1), and the mitochondrial membrane permeability factor Bax (2), supporting their involvement in a wide variety of cellular processes ranging from cell motility (3) and cell cycling (4) to cell death by apoptosis and necrosis (5). When a cell loses its ability to control calcium levels following an ischemic episode or acute neural/cerebral injury, calpains are constitutively activated and contribute to furthering cell damage by degrading nontarget proteins such as other cytoskeletal components, resulting in apoptosis or necrosis and irreversible tissue damage (6). In a similar manner but on a different time scale, calpain overactivation also contributes to the development of cataracts and Alzheimer disease.…”

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confidence: 99%

Development of Calpain-specific Inactivators by Screening of Positional Scanning Epoxide Libraries

Cuerrier

Moldoveanu

Campbell

et al. 2007

Journal of Biological Chemistry

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Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)-or (R,R)-epoxide stereochemistries, and incorporate both natural and nonnatural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m-and -calpain rapidly (k 2 /K i ‫؍‬ 131,000 and 16,500 M ؊1 s ؊1 , respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat -calpain inactivated by the epoxide compounds WR ␥-cyano-␣-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR ␥-cyano-␣-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.

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Spatial resolution of calpain-catalyzed proteolysis in focal cerebral ischemia

Cited by 14 publications

References 38 publications

Role of μ-calpain in proteolytic cleavage of brain l-glutamic acid decarboxylase

Role of μ-calpain in proteolytic cleavage of brain l-glutamic acid decarboxylase

Monoubiquitination Promotes Calpain Cleavage of the Protein Phosphatase 2A (PP2A) Regulatory Subunit α4, Altering PP2A Stability and Microtubule-associated Protein Phosphorylation

Development of Calpain-specific Inactivators by Screening of Positional Scanning Epoxide Libraries

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