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Park J, Choi H, Kim B, Chae U, Lee DG, Lee SR, Lee S, Lee HS, Lee DS. Peroxiredoxin 5 (Prx5) decreases LPS-induced microglial activation through regulation of Ca 2+/calcineurin-Drp1-dependent mitochondrial fission. Free Radic Biol Med 2016; 99:392-404. [PMID: 27585948 DOI: 10.1016/j.freeradbiomed.2016.08.030] [Citation(s) in RCA: 57] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/15/2016] [Revised: 07/24/2016] [Accepted: 08/28/2016] [Indexed: 12/30/2022]
Abstract
Microglial activation is a hallmark of neurodegenerative diseases. ROS activates microglia by regulating transcription factors to express pro-inflammatory genes and is associated with disruption of Ca2+ homeostasis through thiol redox modulation. Recently, we reported that Prx5 can regulate activation of microglia cells by governing ROS. In addition, LPS leads to excessive mitochondrial fission, and regulation of mitochondrial dynamics involved in a pro-inflammatory response is important for the maintenance of microglial activation. However, the precise relationship among these signals and the role of Prx5 in mitochondrial dynamics and microglial activation is still unknown. In this study, we demonstrated that Ca2+/calcineurin-dependent de-phosphorylation of Drp1 induces mitochondrial fission and regulates mitochondrial ROS production, which influences the expression of pro-inflammatory mediators in LPS-induced microglia cells. Moreover, it is likely that cytosolic and Nox-derived ROS were upstream of mitochondrial fission and mitochondrial ROS generation in activated microglia cells. Prx5 regulates LPS-induced mitochondrial fission through modulation of Ca2+/calcineurin-dependent Drp1 de-phosphorylation by eliminating Nox-derived and cytosolic ROS. Therefore, we suggest that mitochondrial dynamics may be essential for understanding pro-inflammatory responses and that Prx5 may be used as a new therapeutic target to prevent neuroinflammation and neurodegenerative diseases.
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Affiliation(s)
- Junghyung Park
- School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea; College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea
| | - Hoonsung Choi
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Jeollabuk-do, Republic of Korea
| | - Bokyung Kim
- School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea; College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea
| | - Unbin Chae
- School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea; College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea
| | - Dong Gil Lee
- School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea; College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea
| | - Sang-Rae Lee
- National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Chungcheongbuk-do, Republic of Korea
| | - Seunghoon Lee
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Jeollabuk-do, Republic of Korea
| | - Hyun-Shik Lee
- School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea; College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea
| | - Dong-Seok Lee
- School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea; College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea.
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Neuhof C, Neuhof H. Calpain system and its involvement in myocardial ischemia and reperfusion injury. World J Cardiol 2014; 6:638-652. [PMID: 25068024 PMCID: PMC4110612 DOI: 10.4330/wjc.v6.i7.638] [Citation(s) in RCA: 61] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/26/2013] [Revised: 01/26/2014] [Accepted: 05/29/2014] [Indexed: 02/06/2023] Open
Abstract
Calpains are ubiquitous non-lysosomal Ca2+-dependent cysteine proteases also present in myocardial cytosol and mitochondria. Numerous experimental studies reveal an essential role of the calpain system in myocardial injury during ischemia, reperfusion and postischemic structural remodelling. The increasing Ca2+-content and Ca2+-overload in myocardial cytosol and mitochondria during ischemia and reperfusion causes an activation of calpains. Upon activation they are able to injure the contractile apparatus and impair the energy production by cleaving structural and functional proteins of myocytes and mitochondria. Besides their causal involvement in acute myocardial dysfunction they are also involved in structural remodelling after myocardial infarction by the generation and release of proapoptotic factors from mitochondria. Calpain inhibition can prevent or attenuate myocardial injury during ischemia, reperfusion, and in later stages of myocardial infarction.
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3
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Calpain-1 inhibitors for selective treatment of rheumatoid arthritis: what is the future? Future Med Chem 2013; 5:2057-74. [DOI: 10.4155/fmc.13.172] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Effective small-molecule treatment of inflammatory diseases remains an unmet need in medicine. Current treatments are either limited in effectiveness or invasive. The latest biologics prevent influx of inflammatory cells to damaged tissue. Calpain-1 is a calcium-activated cysteine protease that plays an important role in neutrophil motility. It is, therefore, a potential target for intervention in inflammatory disease. Many inhibitors of calpains have been developed but most are unselective and so unsuitable for drug use. However, recent series of α-mercaptoacrylate inhibitors target regulatory domains of calpain-1 and are much more specific. These compounds are effective in impairing the cell spreading mechanism of neutrophils in vitro and raise the possibility of treating rheumatoid arthritis with a pill; however, challenges still remain. Improved bioavailability is needed and solution of their precise mode of action should prompt the development of specific calpain-1 screens for novel classes of inhibitors.
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Abstract
INTRODUCTION Calpains represent a family of neutral, calcium-dependent proteases, which modify the function of their target proteins by partial truncation. These proteases have been implicated in numerous cell functions, including cell division, proliferation, migration, and death. In the CNS, where µ-calpain and m-calpain are the main calpain isoforms, their activation has been linked to synaptic plasticity as well as to neurodegeneration. This review will focus on the role of calpains in synaptic plasticity and discuss the possibility of developing methods to manipulate calpain activity for therapeutic purposes. AREAS COVERED This review covers the literature showing how calpains are implicated in synaptic plasticity and in a number of conditions associated with learning impairment. The possibility of developing new drugs targeting these enzymes for treating these conditions is discussed. EXPERT OPINION As evidence accumulates that calpain activation participates in neurodegeneration and cancer, there is interest in developing therapeutic approaches using direct or indirect calpain inhibition. In particular, a peptide derived from the calpain truncation site of mGluR1α was shown to decrease neurodegeneration following neonatal hypoxia/ischemia. More selective approaches need to be developed to target calpain or some of its substrates for therapeutic indications associated with deregulation of synaptic plasticity.
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Affiliation(s)
- Michel Baudry
- Western University of Health Sciences, Graduate College of Biomedical Sciences, Basic Medical Sciences, COMP , 309 E 2nd St, Pomona, CA 91766, USA.
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Baumgärtel K, Mansuy IM. Neural functions of calcineurin in synaptic plasticity and memory. Learn Mem 2012; 19:375-84. [PMID: 22904368 DOI: 10.1101/lm.027201.112] [Citation(s) in RCA: 131] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Major brain functions depend on neuronal processes that favor the plasticity of neuronal circuits while at the same time maintaining their stability. The mechanisms that regulate brain plasticity are complex and engage multiple cascades of molecular components that modulate synaptic efficacy. Protein kinases (PKs) and phosphatases (PPs) are among the most important of these components that act as positive and negative regulators of neuronal signaling and plasticity, respectively. In these cascades, the PP protein phosphatase 2B or calcineurin (CaN) is of particular interest because it is the only Ca(2+)-activated PP in the brain and a major regulator of key proteins essential for synaptic transmission and neuronal excitability. This review describes the primary properties of CaN and illustrates its functions and modes of action by focusing on several representative targets, in particular glutamate receptors, striatal enriched protein phosphatase (STEP), and neuromodulin (GAP43), and their functional significance for synaptic plasticity and memory.
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Affiliation(s)
- Karsten Baumgärtel
- Dorris Neuroscience Center, Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037-1000, USA
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6
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Powers SK, Smuder AJ, Criswell DS. Mechanistic links between oxidative stress and disuse muscle atrophy. Antioxid Redox Signal 2011; 15:2519-28. [PMID: 21457104 PMCID: PMC3208252 DOI: 10.1089/ars.2011.3973] [Citation(s) in RCA: 146] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Long periods of skeletal muscle inactivity promote a loss of muscle protein resulting in fiber atrophy. This disuse-induced muscle atrophy results from decreased protein synthesis and increased protein degradation. Recent studies have increased our insight into this complicated process, and evidence indicates that disturbed redox signaling is an important regulator of cell signaling pathways that control both protein synthesis and proteolysis in skeletal muscle. The objective of this review is to outline the role that reactive oxygen species play in the regulation of inactivity-induced skeletal muscle atrophy. Specifically, this report will provide an overview of experimental models used to investigate disuse muscle atrophy and will also highlight the intracellular sources of reactive oxygen species and reactive nitrogen species in inactive skeletal muscle. We then will provide a detailed discussion of the evidence that links oxidants to the cell signaling pathways that control both protein synthesis and degradation. Finally, by presenting unresolved issues related to oxidative stress and muscle atrophy, we hope that this review will serve as a stimulus for new research in this exciting field.
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Affiliation(s)
- Scott K Powers
- Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, 32611, USA.
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Nejatbakhsh N, Feng ZP. Calcium binding protein-mediated regulation of voltage-gated calcium channels linked to human diseases. Acta Pharmacol Sin 2011; 32:741-8. [PMID: 21642945 DOI: 10.1038/aps.2011.64] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Calcium ion entry through voltage-gated calcium channels is essential for cellular signalling in a wide variety of cells and multiple physiological processes. Perturbations of voltage-gated calcium channel function can lead to pathophysiological consequences. Calcium binding proteins serve as calcium sensors and regulate the calcium channel properties via feedback mechanisms. This review highlights the current evidences of calcium binding protein-mediated channel regulation in human diseases.
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Abdul HM, Baig I, LeVine H, Guttmann RP, Norris CM. Proteolysis of calcineurin is increased in human hippocampus during mild cognitive impairment and is stimulated by oligomeric Abeta in primary cell culture. Aging Cell 2011; 10:103-13. [PMID: 20969723 PMCID: PMC3021581 DOI: 10.1111/j.1474-9726.2010.00645.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Recent reports demonstrate that the activation and interaction of the protease calpain (CP) and the protein phosphatase calcineurin (CN) are elevated in the late stages of Alzheimer's disease (AD). However, the extent to which CPs and CN interact during earlier stages of disease progression remains unknown. Here, we investigated CP and CN protein levels in cytosolic, nuclear, and membrane fractions prepared from human postmortem hippocampal tissue from aged non-demented subjects, and subjects diagnosed with mild cognitive impairment (MCI). The results revealed a parallel increase in CP I and the 48 kDa CN-Aα (ΔCN-Aα48) proteolytic fragment in cytosolic fractions during MCI. In primary rat hippocampal cultures, CP-dependent proteolysis and activation of CN was stimulated by application of oligomeric Aβ((1-42)) peptides. Deleterious effects of Aβ on neuronal morphology were reduced by blockade of either CP or CN. NMDA-type glutamate receptors, which help regulate cognition and neuronal viability, and are modulated by CPs and CN, were also investigated in human hippocampus. Relative to controls, MCI subjects showed significantly greater proteolytic levels of the NR2B subunit. Within subjects, the extent of NR2B proteolysis was strongly correlated with the generation of ΔCN-Aα48 in the cytosol. A similar proteolytic pattern for NR2B was also observed in primary rat hippocampal cultures treated with oligomeric Aβ and prevented by inhibition of CP or CN. Together, the results demonstrate that the activation and interaction of CPs and CN are increased early in cognitive decline associated with AD and may help drive other pathologic processes during disease progression.
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Affiliation(s)
- Hafiz Mohmmad Abdul
- The Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536
| | - Irfan Baig
- The Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536
- Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536
| | - Harry LeVine
- The Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536
- Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536
| | - Rodney P Guttmann
- The Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536
- Graduate Center for Gerontology, University of Kentucky, Lexington, KY 40536
| | - Christopher M. Norris
- The Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536
- Department of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, KY40536
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Potential role of calcineurin in pathogenic conditions. Mol Cell Biochem 2009; 338:133-41. [DOI: 10.1007/s11010-009-0346-4] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2009] [Accepted: 11/19/2009] [Indexed: 12/14/2022]
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Xifró X, Giralt A, Saavedra A, García-Martínez JM, Díaz-Hernández M, Lucas JJ, Alberch J, Pérez-Navarro E. Reduced calcineurin protein levels and activity in exon-1 mouse models of Huntington's disease: role in excitotoxicity. Neurobiol Dis 2009; 36:461-9. [PMID: 19733666 DOI: 10.1016/j.nbd.2009.08.012] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2009] [Revised: 08/03/2009] [Accepted: 08/28/2009] [Indexed: 11/28/2022] Open
Abstract
Calcineurin is a serine/threonine phosphatase involved in the regulation of glutamate receptors signaling. Here, we analyzed whether the regulation of calcineurin protein levels and activity modulates the susceptibility of striatal neurons to excitotoxicity in R6/1 and R6/1:BDNF+/- mouse models of Huntington's disease. We show that calcineurin inhibition in wild-type mice drastically reduced quinolinic acid-induced striatal cell death. Moreover, calcineurin A and B were differentially regulated during disease progression with a specific reduction of calcineurin A protein levels and calcineurin activity at the onset of the disease in R6/1:BDNF+/- mice. Analysis of the conditional mouse model Tet/HD94 showed that mutant huntingtin specifically controls calcineurin A protein levels. Finally, calcineurin activation induced by intrastriatal quinolinic acid injection in R6/1 mouse was lower than in wild-type mice. Therefore, reduction of calcineurin activity by alteration of calcineurin A expression participates in the pathophysiology of Huntington's disease and contributes to the excitotoxic resistance observed in exon-1 mouse models.
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Affiliation(s)
- Xavier Xifró
- Departament de Biologia Cel.lular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, IDIBAPS, Casanova 143, E-08036 Barcelona, Spain
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Aharonovitz O, Livne AA, Granot Y. 42 kDa Protein as a Substrate for Protein Phosphatase (s) in Intact Human Blood Platelets. Platelets 2009; 6:17-23. [DOI: 10.3109/09537109509013257] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Vosler PS, Brennan CS, Chen J. Calpain-mediated signaling mechanisms in neuronal injury and neurodegeneration. Mol Neurobiol 2008; 38:78-100. [PMID: 18686046 PMCID: PMC2726710 DOI: 10.1007/s12035-008-8036-x] [Citation(s) in RCA: 284] [Impact Index Per Article: 16.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2008] [Accepted: 07/17/2008] [Indexed: 12/19/2022]
Abstract
Calpain is a ubiquitous calcium-sensitive protease that is essential for normal physiologic neuronal function. However, alterations in calcium homeostasis lead to persistent, pathologic activation of calpain in a number of neurodegenerative diseases. Pathologic activation of calpain results in the cleavage of a number of neuronal substrates that negatively affect neuronal structure and function, leading to inhibition of essential neuronal survival mechanisms. In this review, we examine the mechanistic underpinnings of calcium dysregulation resulting in calpain activation in the acute neurodegenerative diseases such as cerebral ischemia and in the chronic neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, prion-related encephalopathy, and amylotrophic lateral sclerosis. The premise of this paper is that analysis of the signaling and transcriptional consequences of calpain-mediated cleavage of its various substrates for any neurodegenerative disease can be extrapolated to all of the neurodegenerative diseases vulnerable to calcium dysregulation.
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Affiliation(s)
- P S Vosler
- Department of Neurology, University of Pittsburgh School of Medicine, S-507, Biomedical Science Tower, Pittsburgh, PA 15213, USA
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Yuen PW, KW Wang K. Section Review: Central & Peripheral Nervous Systems: Therapeutic potential of calpain inhibitors in neurodegenerative disorders. Expert Opin Investig Drugs 2008. [DOI: 10.1517/13543784.5.10.1291] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
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Kuchay SM, Kim N, Grunz EA, Fay WP, Chishti AH. Double knockouts reveal that protein tyrosine phosphatase 1B is a physiological target of calpain-1 in platelets. Mol Cell Biol 2007; 27:6038-52. [PMID: 17576811 PMCID: PMC1952154 DOI: 10.1128/mcb.00522-07] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. Gene targeting was used to evaluate the physiological function of mouse calpain-1 and establish that its inactivation results in reduced platelet aggregation and clot retraction potentially by causing dephosphorylation of platelet proteins. Here, we report that calpain-1 null (Capn1-/-) platelets accumulate protein tyrosine phosphatase 1B (PTP1B), which correlates with enhanced tyrosine phosphatase activity and dephosphorylation of multiple substrates. Treatment of Capn1-/- platelets with bis(N,N-dimethylhydroxamido)hydroxooxovanadate, an inhibitor of tyrosine phosphatases, corrected the aggregation defect and recovered impaired clot retraction. More importantly, platelet aggregation, clot retraction, and tyrosine dephosphorylation defects were rescued in the double knockout mice lacking both calpain-1 and PTP1B. Further evaluation of mutant mice by the ferric chloride-induced arterial injury model suggests that the Capn1-/- mice are relatively resistant to thrombosis in vivo. Together, our results demonstrate that PTP1B is a physiological target of calpain-1 and suggest that a similar mechanism may regulate calpain-1-mediated tyrosine dephosphorylation in other cells.
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Affiliation(s)
- Shafi M Kuchay
- Department of Pharmacology, UIC Cancer Center, University of Illinois at Chicago, 909 South Wolcott Avenue, Room 5097, Chicago, IL 60612-3725, USA
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Abstract
PURPOSE OF REVIEW There is considerable interest in understanding the function and mechanism of calpains in platelet aggregation, spreading, and granular secretion pathways. Recent insights from the calpain-1 knockout platelets suggest a pivotal role of these cysteine proteases in the regulation of outside-in signaling, aggregation, and clot retraction. RECENT FINDINGS The calpain-1 knockout mouse provided direct evidence for the role of calpain-1 in platelet aggregation and clot retraction. Reduced tyrosine phosphorylation of platelet proteins correlated with reduced platelet aggregation and clot retraction. Future investigations of the mechanism of platelet defects in calpain-1 null mice may unveil the physiological functions of this important and elusive protease in mammalian cells. SUMMARY This review focuses on the role of calpains in platelets with a particular emphasis on recent findings in calpain-1 null platelets. Previous studies used synthetic inhibitors to study the role of calpains in platelet function yielding useful information about the identification of calpain substrates. The development of calpain-1 null mice demonstrated that calpain-1 plays an important function in the regulation of platelet aggregation and clot retraction. Since the combined deletion of calpain-1 and calpain-2 genes results in embryonic lethality, the calpain-1 null mouse remains the only experimental model available to study the physiological role of calpains in mammalian cells.
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Affiliation(s)
- Shafi M Kuchay
- Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA
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Abstract
Skeletal muscle inactivity is associated with a loss of muscle protein and reduced force-generating capacity. This disuse-induced muscle atrophy results from both increased proteolysis and decreased protein synthesis. Investigations of the cell signaling pathways that regulate disuse muscle atrophy have increased our understanding of this complex process. Emerging evidence implicates oxidative stress as a key regulator of cell signaling pathways, leading to increased proteolysis and muscle atrophy during periods of prolonged disuse. This review will discuss the role of reactive oxygen species in the regulation of inactivity-induced skeletal muscle atrophy. The specific objectives of this article are to provide an overview of muscle proteases, outline intracellular sources of reactive oxygen species, and summarize the evidence that connects oxidative stress to signaling pathways contributing to disuse muscle atrophy. Moreover, this review will also discuss the specific role that oxidative stress plays in signaling pathways responsible for muscle proteolysis and myonuclear apoptosis and highlight gaps in our knowledge of disuse muscle atrophy. By presenting unresolved issues and suggesting topics for future research, it is hoped that this review will serve as a stimulus for the expansion of knowledge in this exciting field.
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Affiliation(s)
- Scott K Powers
- Department of Applied Physiology and Kinesiology, PO Box 118205, University of Florida, Gainesville, FL 32611, USA.
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Hernández-Espinosa D, Morton AJ. Calcineurin inhibitors cause an acceleration of the neurological phenotype in a mouse transgenic for the human Huntington's disease mutation. Brain Res Bull 2006; 69:669-79. [PMID: 16716837 DOI: 10.1016/j.brainresbull.2006.03.013] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2006] [Accepted: 03/23/2006] [Indexed: 10/24/2022]
Abstract
Calcineurin (CaN) is a Ca(2+)- and calmodulin-dependent protein serine-threonine phosphatase that is thought to play an important role in the neuronal response to changes in the intracellular Ca(2+) concentration. CaN has been implicated in numerous physiological processes including learning and memory. Decreases in CaN expression are thought to be responsible for some of the pathological features seen in brain ischemia, Down's syndrome and Alzheimer's disease. In this study, we examined the possibility of CaN playing a role in the progressive neurological phenotype of the R6/2 mouse of Huntington's disease. We studied the effects of the CaN inhibitors cyclosporin A and FK506 on the progressive neurological phenotype in the R6/2 mouse. We found that an immunosuppressive dose of both drugs dramatically accelerated the main features of the neurological phenotype in R6/2 mice. This was unlikely to be due solely to the immunosuppressive action of these drugs, since treatment with cyclophosphamide, an immunosuppressant drug with a mechanism of action that is not mediated via CaN, did not have deleterious effects on the R6/2 mouse. If anything, cyclophosphamide improved the neurological symptoms in the R6/2 mice. Together, our data suggest a central role for CaN in the deleterious phenotype of the R6/2 mouse. Treatments aimed at preventing the loss of CaN or stimulating its function may be beneficial in the treatment of HD.
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Affiliation(s)
- David Hernández-Espinosa
- Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, United Kingdom
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Kurz JE, Parsons JT, Rana A, Gibson CJ, Hamm RJ, Churn SB. A Significant Increase in Both Basal and Maximal Calcineurin Activity following Fluid Percussion Injury in the Rat. J Neurotrauma 2005; 22:476-90. [PMID: 15853464 DOI: 10.1089/neu.2005.22.476] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Calcineurin, a neuronally enriched, calcium-stimulated phosphatase, is an important modulator of many neuronal processes, including several that are physiologically related to the pathology of traumatic brain injury. This study examined the effects of moderate, central fluid percussion injury on the activity of this important neuronal enzyme. Animals were sacrificed at several time-points postinjury and cortical, hippocampal, and cerebellar homogenates were assayed for calcineurin activity by dephosphorylation of p-nitrophenol phosphate. A significant brain injury-dependent increase was observed in both hippocampal and cortical homogenates under both basal and maximally-stimulated reaction conditions. This increase persisted 2-3 weeks post-injury. Brain injury did not alter substrate affinity, but did induce a significant increase in the apparent maximal dephosphorylation rate. Unlike the other brain regions, no change in calcineurin activity was observed in the cerebellum following brain injury. No brain region tested displayed a significant change in calcineurin enzyme levels as determined by Western blot, demonstrating that increased enzyme synthesis was not responsible for the observed increase in activity. The data support the conclusion that fluid percussion injury results in increased calcineurin activity in the rat forebrain. This increased activity has broad physiological implications, possibly resulting in altered cellular excitability or a greater likelihood of neuronal cell death.
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Affiliation(s)
- Jonathan E Kurz
- Department of Neurology, Medical College of Virginia/Virginia Commonwealth University, Richmond, VA 23298, USA
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Xiang B, Liu P, Jiang G, Zou K, Yi F, Yang S, Wei Q. The catalytically active domain in the A subunit of calcineurin. Biol Chem 2004; 384:1429-34. [PMID: 14669985 DOI: 10.1515/bc.2003.158] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Calcineurin (CaN) is a heterodimer composed of a catalytic subunit A (CaNA) and a regulatory subunit B (CaNB). We report here an active truncated mutation of the rat CaNAdelta that contains only the catalytic domain (residues 1-347, also known as a/CaNA). The p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were higher than that of CaNA. Both p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were unaffected by CaM and the B-subunit; the B-subunit and CaM have relatively little effect on p-nitrophenyl phosphatase activity and a crucial effect on protein phosphatase activity of CaNA. Mn2+ and Ni2+ ions effeciently activated CaNA. The Km of a/CaNA was about 16 mM, and the k(cat) of a/CaNA was 10.03 s(-1) using pNPP as substrate. With RII peptide as a substrate, the Km of a/CaNA was about 21 microM and the k(cat) of a/CaNA was 0.51 s(-1). The optimum reaction temperature was about 45 degrees C, and the optimum reaction pH was about 7.2. Our results indicate that a/CaNA is the catalytic core of CaNA, and CaN and the B-subunit binding domain itself might play roles in the negative regulation of the phosphatase activity of CaN. The results provide the basis for future studies on the catalytic domain of CaN.
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Affiliation(s)
- Benqiong Xiang
- Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing 100875, PR China
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Kumar R, Musiyenko A, Oldenburg A, Adams B, Barik S. Post-translational generation of constitutively active cores from larger phosphatases in the malaria parasite, Plasmodium falciparum: implications for proteomics. BMC Mol Biol 2004; 5:6. [PMID: 15230980 PMCID: PMC459218 DOI: 10.1186/1471-2199-5-6] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2004] [Accepted: 07/01/2004] [Indexed: 11/16/2022] Open
Abstract
Background Although the complete genome sequences of a large number of organisms have been determined, the exact proteomes need to be characterized. More specifically, the extent to which post-translational processes such as proteolysis affect the synthesized proteins has remained unappreciated. We examined this issue in selected protein phosphatases of the protease-rich malaria parasite, Plasmodium falciparum. Results P. falciparum encodes a number of Ser/Thr protein phosphatases (PP) whose catalytic subunits are composed of a catalytic core and accessory domains essential for regulation of the catalytic activity. Two examples of such regulatory domains are found in the Ca+2-regulated phosphatases, PP7 and PP2B (calcineurin). The EF-hand domains of PP7 and the calmodulin-binding domain of PP2B are essential for stimulation of the phosphatase activity by Ca+2. We present biochemical evidence that P. falciparum generates these full-length phosphatases as well as their catalytic cores, most likely as intermediates of a proteolytic degradation pathway. While the full-length phosphatases are activated by Ca+2, the processed cores are constitutively active and either less responsive or unresponsive to Ca+2. The processing is extremely rapid, specific, and occurs in vivo. Conclusions Post-translational cleavage efficiently degrades complex full-length phosphatases in P. falciparum. In the course of such degradation, enzymatically active catalytic cores are produced as relatively stable intermediates. The universality of such proteolysis in other phosphatases or other multi-domain proteins and its potential impact on the overall proteome of a cell merits further investigation.
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Affiliation(s)
- Rajinder Kumar
- Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, 307 University Blvd., Mobile, Alabama, USA 36688-0002
| | - Alla Musiyenko
- Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, 307 University Blvd., Mobile, Alabama, USA 36688-0002
| | - Anja Oldenburg
- Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, 307 University Blvd., Mobile, Alabama, USA 36688-0002
| | - Brian Adams
- Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, 307 University Blvd., Mobile, Alabama, USA 36688-0002
| | - Sailen Barik
- Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, 307 University Blvd., Mobile, Alabama, USA 36688-0002
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22
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Wu HY, Tomizawa K, Oda Y, Wei FY, Lu YF, Matsushita M, Li ST, Moriwaki A, Matsui H. Critical Role of Calpain-mediated Cleavage of Calcineurin in Excitotoxic Neurodegeneration. J Biol Chem 2004; 279:4929-40. [PMID: 14627704 DOI: 10.1074/jbc.m309767200] [Citation(s) in RCA: 183] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Calcineurin and calpain, a Ca2+/calmodulin-dependent protein phosphatase and a Ca2+-dependent cysteine protease, respectively, mediate neuronal cell death through independent cascades. Here, we report that during neuroexcitotoxicity, calcineurin A (CnA) is directly cleaved by calpain in vitro and in vivo, resulting in the enzyme being converted to an active form. Mass spectrometry identified three cleavage sites in CnA, two of which were constitutively active forms. Overexpression of the cleaved CnA induced caspase activity and neuronal cell death. Calpain inhibitors and membrane-permeable calpastatin peptides not only blocked the cleavage of CnA, but also protected against excitotoxic neuronal cell death in vitro and in vivo. These results indicate that CnA is a crucial target for calpain, and the calpain-mediated activation of CnA triggers excitotoxic neurodegeneration. This study established a molecular link between calpain and calcineurin, thereby demonstrating a new mechanism for proteolytical regulation of calcineurin by calpain in response to certain pathological states.
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Affiliation(s)
- Hai-Yan Wu
- Department of Physiology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata, Okayama, 700-8558 Japan
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23
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Abstract
Calpains are a family of calcium-dependent cysteine proteases under complex cellular regulation. By making selective limited proteolytic cleavages, they modulate the activity of enzymes, including key signaling molecules, and induce specific cytoskeletal rearrangements, accounting for their roles in cell motility, signal transduction, vesicular trafficking and structural stabilization. Calpain activation has been implicated in various aging phenomena and diseases of late life, including cataract formation, erythrocyte senescence, diabetes mellitus type 2, hypertension, arthritis, and neurodegenerative disorders. The early and pervasive involvement of calpains in Alzheimer's disease potentially influences the development of beta-amyloid and tau disturbances and their consequences for neurodegeneration and neuronal cell loss.
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Affiliation(s)
- Ralph A Nixon
- Center for Dementia Research, Nathan Kline Institute, Orangeburg, NY 10962, USA.
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24
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Abstract
The calpain system originally comprised three molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. Both mu- and m-calpain are heterodimers containing an identical 28-kDa subunit and an 80-kDa subunit that shares 55-65% sequence homology between the two proteases. The crystallographic structure of m-calpain reveals six "domains" in the 80-kDa subunit: 1). a 19-amino acid NH2-terminal sequence; 2). and 3). two domains that constitute the active site, IIa and IIb; 4). domain III; 5). an 18-amino acid extended sequence linking domain III to domain IV; and 6). domain IV, which resembles the penta EF-hand family of polypeptides. The single calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85 kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear, although all bind to three different places on the calpain molecule; binding to at least two of the sites is Ca2+ dependent. Since 1989, cDNA cloning has identified 12 additional mRNAs in mammals that encode polypeptides homologous to domains IIa and IIb of the 80-kDa subunit of mu- and m-calpain, and calpain-like mRNAs have been identified in other organisms. The molecules encoded by these mRNAs have not been isolated, so little is known about their properties. How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke, and brain trauma.
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Affiliation(s)
- Darrell E Goll
- Muscle Biology Group, University of Arizona, Tucson, AZ 85721, USA.
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25
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Kang S, Kassam N, Gauthier ML, O'Day DH. Post-mortem changes in calmodulin binding proteins in muscle and lung. Forensic Sci Int 2003; 131:140-7. [PMID: 12590053 DOI: 10.1016/s0379-0738(02)00426-7] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Estimation of post-mortem interval (PMI) remains an elusive issue in forensic investigations. In this study, we examined the possible use of calmodulin (CaM) binding proteins (CaMBPs) as indicators of PMI. Whole CaMBP populations from homogenized rat lung and rat skeletal muscle removed at 0, 24, 48 and 96 h post-mortem at 21 degrees C were detected by the calmodulin binding overlay technique (CaMBOT) using 35S-VU1-CaM and visualized by autoradiography. CaMBOT showed that, in both tissues, the CaMBP population remained relatively stable for up to 96 h post-mortem with the exception of a single approximately 200 kDa CaMBP that increased in 24 h post-mortem samples then showed decreasing amounts at subsequent times. Immunoblot analysis of the specific CaMBPs, Ca(2+)/CaM-dependent kinase II (CaMKII), calcineurin A (CNA), myristoylated alanine-rich C-kinase substrate (MARCKS) and inducible nitric oxide synthase (iNOS) were done on lung tissue samples. CaMKII levels did not change appreciably over the 96 h PMI examined. In contrast to iNOS levels, which varied from sample to sample, CNA and MARCKS showed predictable patterns of change: the level of MARCKS decreased steadily in the 0-96 h post-mortem lung samples while CNA underwent a shift in mobility on SDS-PAGE by 24 h post-mortem before slowly decreasing in amount. The stability of CaMKII levels over 96 h was also seen in skeletal muscle tissue while CNA showed variable levels at 0, 48 and 96 h with the presence of the rapidly migrating band at 24 h. These patterns of change in CaMBPs provide some insight into the post-mortem changes in calmodulin-mediated signaling components in lung and skeletal muscle and support the further study of CNA and CaMKII as potential markers for estimating short- and long-term PMIs.
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Affiliation(s)
- Susey Kang
- Department of Zoology, University of Toronto at Mississauga, Mississauga, Ont, Canada L5L 1C6
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26
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Abstract
Reactive oxygen and nitrogen species can be used as a messengers in normal cell functions. However, at oxidative stress levels they can disrupt normal physiological pathways and cause cell death. Such a switch is largely mediated through Ca(2+) signaling. Oxidative stress causes Ca(2+) influx into the cytoplasm from the extracellular environment and from the endoplasmic reticulum or sarcoplasmic reticulum (ER/SR) through the cell membrane and the ER/SR channels, respectively. Rising Ca(2+) concentration in the cytoplasm causes Ca(2+) influx into mitochondria and nuclei. In mitochondria Ca(2+) accelerates and disrupts normal metabolism leading to cell death. In nuclei Ca(2+) modulates gene transcription and nucleases that control cell apoptosis. Both in nuclei and cytoplasm Ca(2+) can regulate phosphorylation/dephosphorylation of proteins and can modulate signal transduction pathways as a result. Since oxidative stress is associated with many diseases and the aging process, understanding how oxidants alter Ca(2+) signaling can help to understand process of aging and disease, and may lead to new strategies for their prevention.
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Affiliation(s)
- Gennady Ermak
- Ethel Percy Andrus Gerontology Center, and Division of Molecular Biology, University of Southern California, Rm 306, 3715 McClintock Avenue, Los Angeles, CA 90089-0191, USA
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27
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Kurz JE, Sheets D, Parsons JT, Rana A, Delorenzo RJ, Churn SB. A significant increase in both basal and maximal calcineurin activity in the rat pilocarpine model of status epilepticus. J Neurochem 2001; 78:304-15. [PMID: 11461966 DOI: 10.1046/j.1471-4159.2001.00426.x] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
This study focused on the effects of status epilepticus on the activity of calcineurin, a neuronally enriched, calcium-dependent phosphatase. Calcineurin is an important modulator of many neuronal processes, including learning and memory, induction of apoptosis, receptor function and neuronal excitability. Therefore, a status epilepticus-induced alteration of the activity of this important phosphatase would have significant physiological implications. Status epilepticus was induced by pilocarpine injection and allowed to continue for 60 min. Brain region homogenates were then assayed for calcineurin activity by dephosphorylation of p-nitrophenol phosphate. A significant status epilepticus-dependent increase in both basal and Mn(2+)-dependent calcineurin activity was observed in homogenates isolated from the cortex and hippocampus, but not the cerebellum. This increase was resistant to 150 nM okadaic acid, but sensitive to 50 microM okadaic acid. The increase in basal activity was also resistant to 100 microM sodium orthovanadate. Both maximal dephosphorylation rate and substrate affinity were increased following status epilepticus. However, the increase in calcineurin activity was not found to be due to an increase in calcineurin enzyme levels. Finally, increase in calcineurin activity was found to be NMDA-receptor activation dependent. The data demonstrate that status epilepticus resulted in a significant increase in both basal and maximal calcineurin activity.
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Affiliation(s)
- J E Kurz
- Department of Biology, Virginia Commonwealth University, Richmond, Virginia, USA
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28
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Abstract
A systematic study on postmortem changes of brain proteins has not been performed so far and information is limited to basic principles of specific or nonspecific proteolysis or proteolysis of individual proteins. We studied protein level alterations in rat brain of animals kept at 23 degrees C for several postmortem times up to 72 h. Brain tissue protein extracts were analyzed by two-dimensional electrophoresis and the proteins with different levels were identified by matrix-assisted laser desorption ionization mass spectrometry. The changes observed mainly concerned structural proteins and enzymes. The levels of dihydropyrimidinase-related protein-2 decreased within 6 h and two new spots were detected representing shorter forms of the protein. Most of the other alterations appeared about 48 h postmortem. The most significant were reduced levels of neurofilament, alpha-internexin, synaptosomal-associated protein 25, glial fibrillary acidic protein, heat shock proteins, and dynamin-1; increased levels of 14-3-3 proteins and spectrin; and generation of shorter forms of certain proteins, such as tubulins, actin, and serum albumin. The results may be useful in neuropathology and brain protein studies.
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Affiliation(s)
- M Fountoulakis
- Pharmaceutical Research, Genomics Technologies, F. Hoffmann-La Roche, Ltd., Basel, Switzerland
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29
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Abstract
Calcineurin is a eukaryotic Ca(2+)- and calmodulin-dependent serine/threonine protein phosphatase. It is a heterodimeric protein consisting of a catalytic subunit calcineurin A, which contains an active site dinuclear metal center, and a tightly associated, myristoylated, Ca(2+)-binding subunit, calcineurin B. The primary sequence of both subunits and heterodimeric quaternary structure is highly conserved from yeast to mammals. As a serine/threonine protein phosphatase, calcineurin participates in a number of cellular processes and Ca(2+)-dependent signal transduction pathways. Calcineurin is potently inhibited by immunosuppressant drugs, cyclosporin A and FK506, in the presence of their respective cytoplasmic immunophilin proteins, cyclophilin and FK506-binding protein. Many studies have used these immunosuppressant drugs and/or modern genetic techniques to disrupt calcineurin in model organisms such as yeast, filamentous fungi, plants, vertebrates, and mammals to explore its biological function. Recent advances regarding calcineurin structure include the determination of its three-dimensional structure. In addition, biochemical and spectroscopic studies are beginning to unravel aspects of the mechanism of phosphate ester hydrolysis including the importance of the dinuclear metal ion cofactor and metal ion redox chemistry, studies which may lead to new calcineurin inhibitors. This review provides a comprehensive examination of the biological roles of calcineurin and reviews aspects related to its structure and catalytic mechanism.
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Affiliation(s)
- F Rusnak
- Section of Hematology Research and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.
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30
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Krebs EG, Graves JD. Interactions between protein kinases and proteases in cellular signaling and regulation. ADVANCES IN ENZYME REGULATION 2000; 40:441-70. [PMID: 10828362 DOI: 10.1016/s0065-2571(99)00030-8] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Affiliation(s)
- E G Krebs
- Department of Pharmacology, University of Washington Medical Center, Seattle, WA 98195, USA
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31
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Thompson VF, Saldaña S, Cong J, Goll DE. A BODIPY fluorescent microplate assay for measuring activity of calpains and other proteases. Anal Biochem 2000; 279:170-8. [PMID: 10706786 DOI: 10.1006/abio.1999.4475] [Citation(s) in RCA: 85] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The use of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL) labeled casein in autoquenching assays of proteolytic activity has been recently described, and we have adapted this assay to measurement of calpain activity. BODIPY-FL coupled to casein at a ratio of 8 mol of BODIPY-FL/mol of casein or higher produces a BODIPY-FL-casein substrate that can be used in an autoquenching assay of calpain proteolytic activity. This assay has a number of advantages for measuring calpain activity. (1) The procedure does not require precipitation and removal of undegraded protein, so it is much faster than other procedures that require a precipitation step, and it can be used directly in kinetic assays of proteolytic activity. (2) The BODIPY-FL-casein assay is easily adapted to a microtiter plate format, so it can be used to screen large numbers of samples. (3) Casein is an inexpensive and readily available protein substrate that more closely mimics the natural substrates of endoproteinases, such as the calpains, than synthetic peptide substrates do. Casein has K(m) values for micro- and m-calpain that are similar to those of other substrates such as fodrin or MAP2 that may be "natural" substrates for the calpains, and there is no reason to believe that calpain hydrolysis of casein is inherently different from hydrolysis of fodrin or MAP2, which are much less accessible as substrates for protease assays. (4) The BODIPY-FL-casein assay is capable of detecting 10 ng ( approximately 5 nM) of calpain and is nearly as sensitive as the most sensitive calpain assay reported thus far. (5) The BODIPY-FL-casein assay is as reproducible as the FITC-casein assay, whose reproducibility is comparable to or better than the reproducibility of other methods used to assay calpain activity. The BODIPY-FL-casein assay is a general assay for proteolytic activity and can be used with any protease that cleaves casein.
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Affiliation(s)
- V F Thompson
- Muscle Biology Group, University of Arizona, Tucson, Arizona, 85721, USA.
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32
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Morioka M, Hamada J, Ushio Y, Miyamoto E. Potential role of calcineurin for brain ischemia and traumatic injury. Prog Neurobiol 1999; 58:1-30. [PMID: 10321795 DOI: 10.1016/s0301-0082(98)00073-2] [Citation(s) in RCA: 97] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/16/2022]
Abstract
Calcineurin belongs to the family of Ca2+/calmodulin-dependent protein phosphatase, protein phosphatase 2B. Calcineurin is the only protein phosphatase which is regulated by a second messenger, Ca2+. Furthermore, calcineurin is highly localized in the central nervous system, especially in those neurons vulnerable to ischemic and traumatic insults. For these reasons, calcineurin is considered to play important roles in neuron-specific functions. Recently, on the basis of the finding that FK506 and cyclosporin A serve as calcineurin-specific inhibitors, this enzyme has become the subject of much study. It is clear that calcineurin is involved in many neuronal (or non-neuronal) functions such as neurotransmitter release, regulation of receptor functions, signal transduction systems, neurite outgrowth, gene expression and neuronal cell death. In this review, we describe the calcineurin functions, functions of the substrates, and the pathogenesis of traumatic and ischemic insults, and we discuss the potential role of calcineurin. There are many similarities in traumatic and ischemic pathogenesis of the brain in which the release of excessive glutamate is followed by an intracellular Ca2+ increase. However, the intracellular cascade which leads to neuronal cell death after the release of excess Ca2+ is unclear. Although calcineurin is thought to be a key toxic enzyme on the basis of studies using immunosuppressants (FK506 or cyclosporin A), many of the functions of the substrates for calcineurin protect against neuronal cell death. We concluded that calcineurin is a bi-directional enzyme for neuronal cell death, having protective and toxic actions, and the balance of the bi-directional effects may be important in ischemic and traumatic pathogenesis.
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Affiliation(s)
- M Morioka
- Department of Neurosurgery, Kumamoto University School of Medicine, Japan.
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33
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Hewitt KE, Lesiuk HJ, Tauskela JS, Morley P, Durkin JP. Selective coupling of mu-calpain activation with the NMDA receptor is independent of translocation and autolysis in primary cortical neurons. J Neurosci Res 1998; 54:223-32. [PMID: 9788281 DOI: 10.1002/(sici)1097-4547(19981015)54:2<223::aid-jnr10>3.0.co;2-5] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Excessive mu-calpain activation has been linked to several cellular pathologies including excitotoxicity and ischemia. In erythrocytes and other non-central nervous system (CNS) cells, calpain activation is thought to occur following a Ca2+-induced translocation of inactive cytosolic enzyme to membranes and subsequent autolysis. In the present report, we show that transiently exposing primary rat cortical neurons to lethal (50 microM) N-methyl-D-aspartic acid (NMDA) caused protracted calpain activation, measured as increased spectrin hydrolysis, but this was independent of translocation or autolysis of the protease. An anti-mu-calpain antibody showed that calpain was largely membrane associated in cortical neurons, and, consequently, neither translocation nor autolysis of the protease was observed following ionomycin or lethal NMDA treatment. By contrast, in rat erythrocytes, calpain was largely cytosolic and underwent rapid translocation and autolysis in response to ionomycin. Calpain-mediated spectrin hydrolysis was specifically coupled to Ca2+ entry through the NMDA receptor because nonspecific Ca2+ influx via ionomycin or KCl-mediated depolarization failed to activate the enzyme. Thus, calpain appears selectively linked to glutamate receptors in cortical neurons and regulated by mechanisms distinct from that occurring in many non-CNS cells. The data suggest that intracellular signals coupled to the NMDA receptor are responsible for activating calpain already associated with cellular membranes in cortical cells.
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Affiliation(s)
- K E Hewitt
- Cellular Neurobiology Group, Institute for Biological Sciences, National Research Council of Canada, University of Ottawa, Ontario
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34
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Kakkar R, Raju RV, Sharma RK. In vitro generation of an active calmodulin-independent phosphodiesterase from brain calmodulin-dependent phosphodiesterase (PDE1A2) by m-calpain. Arch Biochem Biophys 1998; 358:320-8. [PMID: 9784246 DOI: 10.1006/abbi.1998.0858] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
In the present study we have shown that bovine brain 60-kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme (CaMPDE - PDE1A2) is proteolyzed by a Ca2+-dependent cysteine protease, m-calpain. The proteolysis of PDE1A2 by m-calpain results in its conversion to a totally calmodulin (CaM)-independent form accompanied by degradation of PDE1A2 into a 45-kDa catalytic fragment and a 15-kDa fragment. The activity of PDE1A2 is unaffected by the presence or absence of CaM during cleavage, suggesting that the interaction between CaM and PDE1A2 does not alter substrate recognition by calpain. Furthermore, we provide evidence, based on the studies of CaM overlay and phosphorylation, that the cleavage site is not present either in the CaM-binding domain or phosphorylation site. N-terminal sequence analysis of the 45-kDa fragment indicated that cleavage occurs between residues 126Gln and 127Ala, and eliminates the CaM-dependent activity of carboxy termini PDE1A2. The present findings suggest that limited proteolysis in the brain through calpains could be an alternate mechanism for activating CaMPDE(s) and for regulating intracellular levels of cAMP.
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Affiliation(s)
- R Kakkar
- College of Medicine, University of Saskatchewan, Saskatoon, S7N 4H4, Canada
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35
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Abstract
A family of p34Cdc2 related protein kinases, the PITSLRE kinases, is generated by alternative splicing and promoter utilization from three duplicated and tandemly linked genes on human chromosome 1p36.3, which is frequently deleted during the late stages of tumorigenesis. PITSLRE mRNA, protein, and enzyme activity are induced during Fas receptor- and glucocorticoid-mediated apoptosis of human T cells. Several PITSLRE isoforms are specific targets of proteolysis during apoptosis, generating an enzymatically active 50 kDa isoform. Inhibition of this protease activity blocks PITSLRE processing and enzyme activation, as well as apoptosis. Thus, PITSLRE kinases may be integral downstream components of apoptotic signal transduction pathway(s). Furthermore, PITSLRE genes, and their products, are physically altered in human neuroblastoma tumors, suggesting that they may be tumor suppressors.
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Affiliation(s)
- J M Lahti
- Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
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36
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Kissmehl R, Treptau T, Kottwitz B, Plattner H. Occurrence of a para-nitrophenyl phosphate-phosphatase with calcineurin-like characteristics in Paramecium tetraurelia. Arch Biochem Biophys 1997; 344:260-70. [PMID: 9264538 DOI: 10.1006/abbi.1997.0208] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Using para-nitrophenyl phosphate (pNPP) as a substrate for enzymatic activity, we sought to identify CaN in Paramecium. We isolated three different pNPP-phosphatases from the soluble fraction of Paramecium cells by anion-exchange and affinity column chromatographies. One, pNPP-phosphatase Peak I, is very similar to mammalian CaN. Divalent cation dependency, inhibition by calmodulin (CaM) antagonists (trifluoperazine, calmidazolium), and insensitivity to various phosphatase inhibitors (heparin, okadaic acid, sodium vanadate, etc.) show similarity to mammalian CaN rather than to any other Paramecium pNPP-hydrolyzing enzymes tested. Polyclonal antibodies against bovine brain CaN recognizing subunits A (61 or 58 kDa) and B (17 kDa) of brain CaN cross-reacted with a 63-kDa protein in fractions containing Peak IpNPP-phosphatase activity and coeluted calmodulin. Overlay assays using biotinylated brain calmodulin indicated Ca2+-dependent CaM-binding by the 63-kDa protein. A Ca2+-binding protein with the same electrophoretic mobility as CaN B (17 kDa) was also present, though in other fractions from DEAE-cellulose chromatography. This finding strongly suggests that, in the absence of Ca2+, both subunits, A and B, were separated either before or during chromatographic processing. Our data support the existence of both subunits of a CaN-like phosphatase in Paramecium cells.
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Affiliation(s)
- R Kissmehl
- Faculty of Biology, University of Konstanz, Germany.
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37
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Lin GD, Chattopadhyay D, Maki M, Wang KK, Carson M, Jin L, Yuen PW, Takano E, Hatanaka M, DeLucas LJ, Narayana SV. Crystal structure of calcium bound domain VI of calpain at 1.9 A resolution and its role in enzyme assembly, regulation, and inhibitor binding. NATURE STRUCTURAL BIOLOGY 1997; 4:539-47. [PMID: 9228946 DOI: 10.1038/nsb0797-539] [Citation(s) in RCA: 138] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The three dimensional structure of calcium-bound domain VI of porcine calpain has been determined to 1.9 A resolution. The crystal structure reveals five EF-hands, one more than previously suggested. There are two EF-hand pairs, one pair (EF1-EF2) displays an 'open' conformation and the other (EF3-EF4) a 'closed' conformation. Unusually, a calcium atom is found at the C-terminal end of the calcium binding loop of EF4. With two additional residues in the calcium binding loop, the fifth EF-hand (EF5) is in a 'closed' conformation. EF5 pairs up with the corresponding fifth EF-hand of a non-crystallographically related molecule. Considering the EF5's role in a homodimer formation of domain VI, we suggest a model for the assembly of heterodimeric calpain. The crystal structure of a Ca2+ bound domain VI-inhibitor (PD150606) complex has been refined to 2.1 A resolution. A possible mode for calpain inhibition is discussed.
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Affiliation(s)
- G D Lin
- Center for Macromolecular Crystallography, University of Alabama at Birmingham 35294, USA
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38
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39
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Lnenicka GA, Hong SJ. Activity-dependent changes in voltage-dependent calcium currents and transmitter release. Mol Neurobiol 1997; 14:37-66. [PMID: 9170100 DOI: 10.1007/bf02740620] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Voltage-dependent Ca2+ channels are important in the regulation of neuronal structure and function, and as a result, they have received considerable attention. Recent studies have begun to characterize the diversity of their properties and the relationship of this diversity to their various cellular functions. In particular, Ca2+ channels play a prominent role in depolarization-secretion coupling, where the release of neurotransmitter is very sensitive to changes in voltage-dependent Ca2+ currents. An important feature of Ca2+ channels is their regulation by electrical activity. Depolarization can selectively modulate the properties of Ca2+ channel types, thus shaping the response of the neuron to future electrical activity. In this article, we examine the diversity of Ca2+ channels found in vertebrate and invertebrate neurons, and their short- and long-term regulation by membrane potential and Ca2+ influx. Additionally, we consider the extent to which this activity-dependent regulation of Ca2+ currents contributes to the development and plasticity of transmitter releasing properties. In the studies of long-term regulation, we focus on crustacean motoneurons where activity levels, Ca2+ channel properties, and transmitter releasing properties can be followed in identified neurons.
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Affiliation(s)
- G A Lnenicka
- Department of Biological Sciences, State University of New York, Albany 12222, USA
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40
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Guttmann RP, Elce JS, Bell PD, Isbell JC, Johnson GV. Oxidation inhibits substrate proteolysis by calpain I but not autolysis. J Biol Chem 1997; 272:2005-12. [PMID: 8999893 DOI: 10.1074/jbc.272.3.2005] [Citation(s) in RCA: 79] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
In this study, the effects of oxidation on calpain I autolysis and calpain-mediated proteolysis were examined. Calpain I was incubated with increasing concentrations of free calcium in the presence or absence of oxidant, and autolytic conversion of both the 80- and 30-kDa subunits was measured by immunoblotting utilizing monoclonal antibodies which recognize both autolyzed and non-autolyzed forms of each subunit, respectively. Autolytic conversion of the 80-kDa subunit of calpain I was not detected until free calcium concentration was greater than 40 microM, whereas autolysis of the 30-kDa subunit did not occur until the free calcium concentration was greater than 100 microM. In addition, autolytic conversion of either the 80- or 30-kDa subunit was not inhibited by the presence of oxidant. Calpain I activity was measured using the fluorescent peptide N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4- methylcoumarin or the microtubule-associated protein tau as substrate. Calpain I was found to have proteolytic activity at free calcium concentrations below that required for autolysis. Calpain I activity was strongly inhibited by oxidant at all calcium concentrations studied, suggesting that proteolytic activity of both the non-autolyzed 80-kDa and autolyzed 76-kDa forms was susceptible to oxidation. Interestingly, whereas oxidation did not inhibit autolytic conversion, the presence of high substrate concentrations did result in a significant reduction of autolysis without altering calpain proteolytic activity. Calpain I activity that had been inhibited by the presence of oxidant was recovered immediately by addition of the reducing agent dithiothreitol.
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Affiliation(s)
- R P Guttmann
- Department of Psychiatry, University of Alabama at Birmingham, Birmingham, Alabama 35294-0017, USA
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41
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YAQOOB MUHAMMAD, EDELSTEIN CHARLESL, SCHRIER ROBERTW. Identification of the novel calcium mediated cellular events in the pathogenesis of hypoxia-induced proximal tubular injury. Nephrology (Carlton) 1996. [DOI: 10.1111/j.1440-1797.1996.tb00133.x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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42
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Wang KK, Yuen PW. Development and therapeutic potential of calpain inhibitors. ADVANCES IN PHARMACOLOGY (SAN DIEGO, CALIF.) 1996; 37:117-52. [PMID: 8891101 DOI: 10.1016/s1054-3589(08)60949-7] [Citation(s) in RCA: 75] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Affiliation(s)
- K K Wang
- Department of Neuroscience Therapeutics Parke-Davis Pharmaceutical Research Warner-Lambert Company, Ann Arbor, Michigan 48105, USA
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43
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Abel K, Mieskes G, Walter U. Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets. FEBS Lett 1995; 370:184-8. [PMID: 7656973 DOI: 10.1016/0014-5793(95)00817-s] [Citation(s) in RCA: 48] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP-dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function.
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Affiliation(s)
- K Abel
- Medizinische Universitätsklinik, Klinische Biochemie und Pathobiochemie, Würzburg, Germany
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44
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Abstract
Many short-lived proteins which are devoid of proteolytic activity contain PEST sequences which are segments along the polypeptide chain that are rich in proline (P), glutamate (E), serine (S) and threonine (T). These designated PEST sequences are believed to be putative intramolecular signals for rapid proteolytic degradation. Calmodulin is a ubiquitous, 17 kDa, acidic Ca(2+)-binding protein which plays an important role in the regulation of many physiological processes through its interaction with a wide range of calmodulin-binding proteins. Several calmodulin-binding proteins are known to contain PEST sequences and are susceptible to proteolysis by endogenous neutral proteases such as calpain I and calpain II. In this report, we discuss the functions of PEST sequences in calmodulin-binding proteins and assess the correlation between calmodulin-binding proteins and PEST sequences.
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Affiliation(s)
- J A Barnes
- Department of Biochemistry, Faculty of Medical Sciences, University of The West Indies, St. Augustine, Trinidad and Tobago, West Indies
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45
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Murphy CT, Westwick J. Role of type 1 and type 2A phosphatases in signal transduction of platelet-activating-factor-stimulated rabbit platelets. Biochem J 1994; 301 ( Pt 2):531-7. [PMID: 8042999 PMCID: PMC1137113 DOI: 10.1042/bj3010531] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Calyculin A, the potent inhibitor of type 1 (PP1) and type 2A (PP2A) phosphatases, has been employed in order to investigate the role of endogenously activated PP1/PP2A in the signal-transduction pathway of platelet-activating-factor (PAF)-stimulated platelets. Calyculin A alone caused an increase in protein phosphorylation in unstimulated platelets, with the detection of a number of newly phosphorylated proteins, whereas in PAF-stimulated platelets phosphorylation of the major substrates of protein kinase C and myosin light-chain kinase were no longer transient, but phosphorylation was sustained. PP1/PP2A appear to play a role in Ca2+ homoeostasis, as inhibition of PP1/PP2A caused an inhibition of Ca2+ mobilization and Ca2+ influx through the plasma membrane in PAF-stimulated platelets. The effect of calyculin A on Ca2+ mobilization correlated with the observed inhibition of the production of the signal molecule Ins(1,4,5)P3. The release reaction (which is a Ca(2+)-dependent event) was also inhibited by calyculin A. The results are discussed in relation to the possible role of protein kinase C in mediating the events leading to the effects observed with calyculin A.
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Affiliation(s)
- C T Murphy
- School of Pharmacy and Pharmacology, University of Bath, Avon, U.K
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46
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Gilchrist JS, Czubryt MP, Pierce GN. Calcium and calcium-binding proteins in the nucleus. Mol Cell Biochem 1994; 135:79-88. [PMID: 7816059 DOI: 10.1007/bf00925963] [Citation(s) in RCA: 48] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Calcium has long been known to play a role as a key cytoplasmic second messenger, but until relatively recently its possible involvement in nuclear signal transduction and the regulation of nuclear events has not been extensively studied. Evidence revealing the presence of transmembrane nuclear Ca2+ gradients and a variety of intranuclear Ca2+ binding proteins has fueled renewed interest in this key ion and its involvement in cell-cycle timing and division, gene expression, and protein activation. This review will offer an overview of the current state of knowledge and theory regarding calcium orchestration of nuclear functions and events and discuss possible future directions in this field of study.
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Affiliation(s)
- J S Gilchrist
- Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada
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47
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Ishii H, Suzuki Y, Horie S, Nakagawa M, Kazama M. Participation of calpain I activation in the ATP release reaction of platelets stimulated with thrombin. BIOCHIMICA ET BIOPHYSICA ACTA 1992; 1175:37-43. [PMID: 1482695 DOI: 10.1016/0167-4889(92)90007-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Proteolytic activation of calpain (calcium-dependent neutral protease) I in thrombin-stimulated platelets was determined by following the production of the 76- and 78-kDa forms from the 80-kDa subunit of calpain I as measured by immunoblotting using monospecific antibody to human calpain I, and the correlation between the extents of calpain I activation and ATP release was investigated. When platelets were stimulated with thrombin in the range from 0.01 to 0.5 U/ml, the maximal 60% activation of calpain I was achieved within 15 s after the stimulation, and ATP release began after the maximal activation had been reached. The extent of ATP release decreased in parallel with the decrease in activation ratio of calpain I on treatment of platelets with EGTA or EST, a membrane-permeable inhibitor of calpain. Although pretreatment of platelets with EST did not affect the thrombin-dependent elevation of the cytosolic Ca2+ concentration, both the inhibition of calpain I activation and the reduction of ATP release were observed as a function of EST concentration. These results suggest that calpain I participates in one of the processes leading to the ATP release reaction of platelets stimulated with thrombin.
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Affiliation(s)
- H Ishii
- Department of Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan
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48
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Kuboki M, Ishii H, Horie S, Kazama M. Procalpain I in cytoplasm is translocated onto plasma and granule membranes during platelet stimulation with thrombin and then activated on the membranes. Biochem Biophys Res Commun 1992; 185:1122-7. [PMID: 1627133 DOI: 10.1016/0006-291x(92)91742-9] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Thrombin stimulation of platelets resulted in changes in the subcellular localization of calpain I, with a concomitant alteration of its molecular weight as measured by immunoblotting. Calpain I in resting platelets was distributed as procalpain I, an 80 kDa form which does not exhibit the enzyme activity, and 83% of the total antigen was localized in the cytosol fraction. When platelets were stimulated with thrombin, the total content of calpain I antigen was not significantly changed as compared with that of the resting platelets, though a decrease in the cytosolic distribution of 80 kDa form (from 83% to 47% of the total antigen) was observed with concomitant appearance of the active 76 kDa and intermediate 78 kDa forms of calpain I and increase in the 80 kDa form in the granule and membrane fractions. These results indicated that calpain I was translocated from the cytosol to both the plasma and granule membranes as procalpain I and then activated on the membranes during platelet stimulation with thrombin.
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Affiliation(s)
- M Kuboki
- Department of Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan
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49
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Ishii H, Suzuki Y, Kuboki M, Morikawa M, Inoue M, Kazama M. Activation of calpain I in thrombin-stimulated platelets is regulated by the initial elevation of the cytosolic Ca2+ concentration. Biochem J 1992; 284 ( Pt 3):755-60. [PMID: 1622393 PMCID: PMC1132603 DOI: 10.1042/bj2840755] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The source and concentration of Ca2+ required to activate calpain I were investigated in thrombin-stimulated platelets. The concentration of cytosolic free Ca2+ ([Ca2+]i) was measured in platelets containing fura-2-AM, and exhibited a biphasic response after stimulation with 0.05, 0.1 or 0.5 NIH units of thrombin/ml. An initial transient elevation, which was predominantly dependent upon Ca2+ released from the internal stores into the cytosol, peaked at 15 s after stimulation, and a secondary sustained elevation, which was due to Ca2+ influx, was observed following the initial elevation. Calpain I was present at about 540 ng/10(8) unstimulated platelets, as measured by immunoblotting using rabbit anti-(human calpain I) IgG. Calpain I was activated 10 s after thrombin stimulation, as determined by the appearance of the 78 kDa and 76 kDa forms on immunoblots. The activation ratio of calpain I was calculated as the amount of the 78 + 76 kDa forms as a percentage of the total (80 + 78 + 76 kDa), and was influenced by the extent of the initial transient [Ca2+]i elevation after stimulation. An initial increase in [Ca2+]i of 300 nM was required to achieve the maximal activation (60%) of calpain I, and half-maximal activation occurred at 160 nM- Ca2+]i. These results suggest that the activation of calpain I in platelets is regulated by the initial elevation in Ca2+]i after thrombin stimulation, and does not necessarily require a Ca2+ influx.
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Affiliation(s)
- H Ishii
- Department of Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan
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50
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Morioka M, Fukunaga K, Yasugawa S, Nagahiro S, Ushio Y, Miyamoto E. Regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II and calcineurin in the hippocampus of rat brain after transient forebrain ischemia. J Neurochem 1992; 58:1798-809. [PMID: 1313854 DOI: 10.1111/j.1471-4159.1992.tb10056.x] [Citation(s) in RCA: 56] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
We have investigated regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and calcineurin (Ca2+/calmodulin-dependent protein phosphatase) after transient forebrain ischemia. Immunoreactivity and enzyme activity of CaM kinase II decreased in regions CA1 and CA3, and in the dentate gyrus, of the hippocampus early (6-12 h) after ischemia, but the decrease in immunoreactivity gradually recovered over time, except in the CA1 region. Furthermore, the increase in Ca2+/calmodulin-independent activity was detected up to 3 days after ischemia in all regions tested, suggesting that the concentration of intracellular Ca2+ increased. In contrast to CaM kinase II, as immunohistochemistry and regional immunoblot analysis revealed, calcineurin was preserved in the CA1 region until 1.5 days and then lost with the increase in morphological degeneration of neurons. Immunoblot analysis confirmed the findings of the immunohistochemistry. These results suggest that there is a difference between CaM kinase II and calcineurin in regional and temporal loss after ischemia and that imbalance of Ca2+/calmodulin-dependent protein phosphorylation-dephosphorylation may occur.
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Affiliation(s)
- M Morioka
- Department of Neurosurgery, Kumamoto University Medical School, Japan
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