The Experts below are selected from a list of 17934 Experts worldwide ranked by ideXlab platform
Athar H. Chishti - One of the best experts on this subject based on the ideXlab platform.
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Calpain-1 ablation partially rescues disease-associated hallmarks in models of Machado-Joseph disease
Human molecular genetics, 2020Co-Authors: Jonasz J. Weber, Athar H. Chishti, Eva Haas, Yacine Maringer, Stefan Hauser, Nicolas Casadei, Olaf Riess, Jeannette Hübener-schmidAbstract:Proteolytic fragmentation of polyglutamine-expanded ataxin-3 is a concomitant and modifier of the molecular pathogenesis of Machado-Joseph disease (MJD), the most common autosomal dominant cerebellar ataxia. Calpains, a group of calcium-dependent cysteine proteases, are important mediators of ataxin-3 cleavage and implicated in multiple neurodegenerative conditions. Pharmacologic and genetic approaches lowering calpain activity showed beneficial effects on molecular and behavioural disease characteristics in MJD model organisms. However, specifically targeting one of the calpain isoforms by genetic means has not yet been evaluated as a potential therapeutic strategy. In our study, we tested whether calpains are overactivated in the MJD context and if reduction or ablation of calpain-1 expression ameliorates the disease-associated phenotype in MJD cells and mice. In all analysed MJD models, we detected an elevated calpain activity at baseline. Lowering or removal of calpain-1 in cells or mice counteracted calpain system overactivation and led to reduced cleavage of ataxin-3 without affecting its aggregation. Moreover, calpain-1 knockout in YAC84Q mice alleviated excessive fragmentation of important synaptic proteins. Despite worsening some motor characteristics, YAC84Q mice showed a rescue of body weight loss and extended survival upon calpain-1 knockout. Together, our findings emphasize the general potential of calpains as a therapeutic target in MJD and other neurodegenerative diseases.
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Calpain-2 Compensation Promotes Angiotensin II-Induced Ascending and Abdominal Aortic Aneurysms in Calpain-1 Deficient Mice
PloS one, 2013Co-Authors: Venkateswaran Subramanian, Athar H. Chishti, Jessica J. Moorleghen, Deborah A. Howatt, Anju Balakrishnan, Haruhito A. UchidaAbstract:Background and Objective Recently, we demonstrated that angiotensin II (AngII)-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA) in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2) in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development. Methodology/Results To investigate the relative contribution of calpain-1 and -2 in development of AngII-induced AAs, male LDLr −/− mice that were either calpain-1 +/+ or −/− were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min) for 4 weeks. Calpain-1 deficiency had no significant effect on body weight or blood pressure during AngII infusion. Moreover, calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AAs. Interestingly, AngII infusion induced increased expression of Calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410, a calpain inhibitor, along with AngII-infusion significantly attenuated AngII-induced ascending and abdominal AA formation in both calpain-1 +/+ and −/− mice as compared to vehicle administered mice. Furthermore, BDA-410 administration attenuated AngII-induced aortic medial hypertrophy and macrophage accumulation. Western blot and immunostaining analyses revealed BDA-410 administration attenuated AngII-induced C-terminal fragmentation of filamin A, an actin binding cytoskeletal protein in aorta. Conclusion Calpain-2 compensates for loss of calpain-1, and both calpain isoforms are involved in AngII-induced aortic aneurysm formation in mice.
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Abstract 516: Calpain-2 Compensation Promotes Angiotensin II-induced Ascending and Abdominal Aortic Aneurysms in Calpain-1 Deficient Mice
Arteriosclerosis Thrombosis and Vascular Biology, 2013Co-Authors: Venkateswaran Subramanian, Athar H. Chishti, Jessica J. Moorleghen, Deborah A. Howatt, Anju Balakrishnan, Haruhito A. UchidaAbstract:Background and Objective We demonstrated that angiotensin II (AngII)-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA) in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2) in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development. Methods and Results Male LDLr-/- mice that were either calpain-1 +/+ or -/- were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min) for 4 weeks. Calpain-1 deficiency had no effect on body weight or blood pressure during AngII infusion. Intimal areas of ascending aorta and maximum external width of the suprarenal abdominal aorta were measured by en face. Calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AA development (P = NS). Interestingly, AngII infusion induced increased expression of Calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410 along with AngII infusion significantly attenuated AngII-induced ascending (Vehicle - +/+: 15.0 ± 1.0, -/-: 14.9 ± 0.8; BDA: +/+: 11.5 ± 0.6, -/-: 12.1 ± 0.6 mm 2 ; P Conclusion Calpain-2 compensates for loss of calpain-1, and promotes AngII-induced ascending and abdominal AAs in calpain-1 deficient mice.
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targeted gene inactivation reveals a functional role of calpain 1 in platelet spreading
Journal of Thrombosis and Haemostasis, 2012Co-Authors: S. M. Kuchay, Adam J. Wieschhaus, Marina Marinkovic, Ira M. Herman, Athar H. ChishtiAbstract:To cite this article: Kuchay SM, Wieschhaus AJ, Marinkovic M, Herman IM, Chishti AH. Targeted gene inactivation reveals a functional role of Calpain-1 in platelet spreading. J Thromb Haemost 2012; 10: 1120–32. Summary. Background: Calpains are implicated in a wide range of cellular functions including the maintenance of hemostasis via the regulation of cytoskeletal modifications in platelets. Objectives: Determine the functional role of calpain isoforms in platelet spreading. Methods and Results: Platelets from calpain-1 )/) mice show enhanced spreading on collagenand fibrinogen-coated surfaces as revealed by immunofluorescence, differential interference contrast (DIC) and scanning electron microscopy. The treatment of mouse platelets with MDL, a cell permeable inhibitor of calpains 1/2, resulted in increased spreading. The PTP1B-mediated enhanced tyrosine dephosphorylation in calpain-1 )/) platelets did not fully account for the enhanced spreading as platelets from the double knockout mice lacking calpain-1 and PTP1B showed only a partial rescue of the spreading phenotype. In nonadherent platelets, proteolysis and GTPase activity of RhoA and Rac1 were indistinguishable between the wild-type (WT) and calpain-1 )/) platelets. In contrast, the ECM-adherent calpain-1 )/) platelets showed higher Rac1 activity at the beginning of spreading, whereas RhoA was more active at later time points. The ECM-adherent calpain-1 )/) platelets showed an elevated level of RhoA protein but not Rac1 and Cdc42. Proteolysis of recombinant RhoA, but not Rac1 and Cdc42, indicates that RhoA is a calpain-1 substrate in vitro. Conclusions: Potentiation of the platelet spreading phenotype in calpain-1 )/) mice suggests a novel role of calpain-1 in hemostasis, and may explain the normal bleeding time observed in the calpain-1 )/) mice.
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Targeted gene inactivation reveals a functional role of calpain‐1 in platelet spreading
Journal of Thrombosis and Haemostasis, 2012Co-Authors: S. M. Kuchay, Adam J. Wieschhaus, Marina Marinkovic, Ira M. Herman, Athar H. ChishtiAbstract:To cite this article: Kuchay SM, Wieschhaus AJ, Marinkovic M, Herman IM, Chishti AH. Targeted gene inactivation reveals a functional role of Calpain-1 in platelet spreading. J Thromb Haemost 2012; 10: 1120–32. Summary. Background: Calpains are implicated in a wide range of cellular functions including the maintenance of hemostasis via the regulation of cytoskeletal modifications in platelets. Objectives: Determine the functional role of calpain isoforms in platelet spreading. Methods and Results: Platelets from calpain-1 )/) mice show enhanced spreading on collagenand fibrinogen-coated surfaces as revealed by immunofluorescence, differential interference contrast (DIC) and scanning electron microscopy. The treatment of mouse platelets with MDL, a cell permeable inhibitor of calpains 1/2, resulted in increased spreading. The PTP1B-mediated enhanced tyrosine dephosphorylation in calpain-1 )/) platelets did not fully account for the enhanced spreading as platelets from the double knockout mice lacking calpain-1 and PTP1B showed only a partial rescue of the spreading phenotype. In nonadherent platelets, proteolysis and GTPase activity of RhoA and Rac1 were indistinguishable between the wild-type (WT) and calpain-1 )/) platelets. In contrast, the ECM-adherent calpain-1 )/) platelets showed higher Rac1 activity at the beginning of spreading, whereas RhoA was more active at later time points. The ECM-adherent calpain-1 )/) platelets showed an elevated level of RhoA protein but not Rac1 and Cdc42. Proteolysis of recombinant RhoA, but not Rac1 and Cdc42, indicates that RhoA is a calpain-1 substrate in vitro. Conclusions: Potentiation of the platelet spreading phenotype in calpain-1 )/) mice suggests a novel role of calpain-1 in hemostasis, and may explain the normal bleeding time observed in the calpain-1 )/) mice.
Michel Baudry - One of the best experts on this subject based on the ideXlab platform.
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Calpain-1 and Calpain-2 in the Brain: New Evidence for a Critical Role of Calpain-2 in Neuronal Death
Cells, 2020Co-Authors: Yubin Wang, Yan Liu, Michel BaudryAbstract:Calpains are a family of soluble calcium-dependent proteases that are involved in multiple regulatory pathways. Our laboratory has focused on the understanding of the functions of two ubiquitous calpain isoforms, calpain-1 and Calpain-2, in the brain. Results obtained over the last 30 years led to the remarkable conclusion that these two calpain isoforms exhibit opposite functions in the brain. Calpain-1 activation is required for certain forms of synaptic plasticity and corresponding types of learning and memory, while Calpain-2 activation limits the extent of plasticity and learning. Calpain-1 is neuroprotective both during postnatal development and in adulthood, while Calpain-2 is neurodegenerative. Several key protein targets participating in these opposite functions have been identified and linked to known pathways involved in synaptic plasticity and neuroprotection/neurodegeneration. We have proposed the hypothesis that the existence of different PDZ (PSD-95, DLG and ZO-1) binding domains in the C-terminal of calpain-1 and Calpain-2 is responsible for their association with different signaling pathways and thereby their different functions. Results with Calpain-2 knock-out mice or with mice treated with a selective Calpain-2 inhibitor indicate that Calpain-2 is a potential therapeutic target in various forms of neurodegeneration, including traumatic brain injury and repeated concussions.
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Calpain-1 and Calpain-2 in the Brain: Dr. Jekill and Mr Hyde?
Current neuropharmacology, 2019Co-Authors: Michel BaudryAbstract:While the calpain system has now been discovered for over 50 years, there is still a paucity of information regarding the organization and functions of the signaling pathways regulated by these proteases, although calpains play critical roles in many cell functions. Moreover, calpain overactivation has been shown to be involved in numerous diseases. Among the 15 calpain isoforms identified, calpain-1 (aka µ-calpain) and Calpain-2 (aka m-calpain) are ubiquitously distributed in most tissues and organs, including the brain. We have recently proposed that calpain-1 and calpain- 2 play opposite functions in the brain, with calpain-1 activation being required for triggering synaptic plasticity and neuroprotection (Dr. Jekill), and Calpain-2 limiting the extent of plasticity and being neurodegenerative (Mr. Hyde). Calpain-mediated cleavage has been observed in cytoskeleton proteins, membrane-associated proteins, receptors/channels, scaffolding/anchoring proteins, and protein kinases and phosphatases. This review will focus on the signaling pathways related to local protein synthesis, cytoskeleton regulation and neuronal survival/death regulated by calpain-1 and Calpain-2, in an attempt to explain the origin of the opposite functions of these 2 calpain isoforms. This will be followed by a discussion of the potential therapeutic applications of selective regulators of these 2 calpain isoforms.
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a molecular dynamics study of michaelis complex for designing selective transition state analog inhibitors for cysteine protease calpain 2
Biophysical Journal, 2016Co-Authors: Payal Chatterjee, Michel Baudry, Abdelaziz Alsamarah, David Kent, Li Qian, David Wych, Christine N Pham, Alla Avetisyan, Steven Standley, Yun LuoAbstract:Calpains constitute a family of intracellular cysteine proteases, which catalyze the cleavage of target proteins in response to Ca2+ signaling. We recently discovered that, calpain-1 induces long-term potentiation, while Calpain-2 limits the extent of potentiation and also is involved in neurodegeneration by NMDA activation. There is therefore a strong rationale to develop Calpain-2 selective inhibitors, as such compounds could enhance learning and memory and offer neuroprotection. Among several reversible inhibitors that have been developed to target calpains, peptidyl α-ketoamide analogs carry the most stable electrophilic warhead. Although several cocrystal structures of calpain-1 and α-ketoamide are available, it is not clear how to design Calpain-2 specific inhibitors based on the calpain-1 model, as the protease core is highly conserved. Structural alignment reveals that the major difference between calpain-1 and Calpain-2 is located at the flexible gating loop. Molecular dynamics simulation of ligand-protease Michaelis complex is therefore necessary to capture the difference in the noncovalent binding interaction between the inhibitor and two isoforms and thus to guide the structure-based design. The Michaelis complex model of Calpain-2 with a ketoamide compound was built from a cocrystal structure of a potent calpain-1 inhibitor. Effects of catalytic triad protonation states on the stability of the complex were investigated. The model was validated using existing Calpain-2 inhibitors. Based on the simulation results, we modified the prime side (C-terminal) of the α-ketoamide peptidomimetics to favor Calpain-2 over calpain-1. The dynamic of the calpain gating loop was explored using long-time scale simulation. The results show that molecular dynamics simulation of Michaelis complex can be a critical tool to explore the molecular bases of calpain activation and to design Calpain-2 selective inhibitors for clinical therapeutics.
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Calpain-1 and Calpain-2: The Yin and Yang of Synaptic Plasticity and Neurodegeneration
Trends in neurosciences, 2016Co-Authors: Michel BaudryAbstract:Many signaling pathways participate in both synaptic plasticity and neuronal degeneration. While calpains participate in these phenomena, very few studies have evaluated the respective roles of the two major calpain isoforms in the brain, calpain-1 and Calpain-2. We review recent studies indicating that calpain-1 and Calpain-2 exhibit opposite functions in both synaptic plasticity and neurodegeneration. Calpain-1 activation is required for the induction of long-term potentiation (LTP) and is generally neuroprotective, while Calpain-2 activation limits the extent of potentiation and is neurodegenerative. This duality of functions is related to their associations with different PDZ-binding proteins, resulting in differential subcellular localization, and offers new therapeutic opportunities for a number of indications in which these proteases have previously been implicated.
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Regulation of Calpain-2 in Neurons: Implications for Synaptic Plasticity
Molecular Neurobiology, 2010Co-Authors: Sohila Zadran, Michel BaudryAbstract:The family of calcium-dependent neutral proteases, calpains, was discovered more than 30 years ago, but their functional roles in the nervous system under physiological or pathological conditions still remain unclear. Although calpain was proposed to participate in synaptic plasticity and in learning and memory in the early 1980s, the precise mechanism regarding its activation, its target(s) and the functional consequences of its activation have remained controversial. A major issue has been the identification of roles of the two major calpain isoforms present in the brain, calpain-1 and Calpain-2, and the calcium requirement for their activation, which exceeds levels that could be reached intracellularly under conditions leading to changes in synaptic efficacy. In this review, we discussed the features of calpains that make them ideally suited to link certain patterns of presynaptic activity to the structural modifications of dendritic spines that could underlie synaptic plasticity and learning and memory. We then summarize recent findings that provide critical answers to the various questions raised by the initial hypothesis, and that further support the idea that, in brain, Calpain-2 plays critical roles in developmental and adult synaptic plasticity.
Haruhito A. Uchida - One of the best experts on this subject based on the ideXlab platform.
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Inducible Depletion of Calpain-2 Mitigates Abdominal Aortic Aneurysm in Mice
2020Co-Authors: Latha Muniappan, Jessica J. Moorleghen, Deborah A. Howatt, Anju Balakrishnan, Michihiro Okuyama, Aida Javidan, Devi Thiagarajan, Weihua Jiang, Lihua Yang, Haruhito A. UchidaAbstract:BACKGROUND: Cytoskeletal structural proteins maintain cell structural integrity by bridging extracellular matrix (ECM) with contractile filaments. During AAA development, (i) aortic medial degeneration is associated with loss of smooth muscle cell (SMC) integrity, and (ii) fibrogenic mesenchymal cells (FMSCs) mediates ECM remodeling. Calpains cleave cytoskeletal proteins that maintain cell structural integrity. Pharmacological inhibition of calpains exert beneficial effects on Angiotensin II (AngII)-induced AAAs in low density receptor deficient (LDLR-/-) mice. OBJECTIVES: To evaluate the functional contribution of FMSCs-derived Calpain-2 on (i) cytoskeletal structural protein and ECM alterations, and (ii) AAA progression. METHODS: Calpain-2 protein, and cytoskeletal protein (e.g. filamin or talin) fragmentation in human and mice AAA tissues were assessed by immunohistochemical and western blot analyses. LDLR-/- mice that were either inducible-whole body or FMSC-specific Calpain-2 deficient were fed a fat-enriched diet and infused with AngII for 4 weeks. The association of cytoskeletal protein to ECM was evaluated using aortic SMCs, in vitro. In addition, the effect of Calpain-2 deficiency on the stability of established AAA was examined. RESULTS: Calpain-2 protein, and filamin/talin fragmentation are significantly elevated in AAAs. Ubiquitous or FMSC-specific depletion of Calpain-2 suppressed AngII-induced AAAs, filamin/talin fragmentation and promoted ECM protein, collagen. Calpain-2 silencing in SMCs reduced AngII-induced filamin/talin fragmentation. In addition, silencing of filamin or talin in SMCs significantly reduced collagen protein. Furthermore, Calpain-2 deficiency suppressed established AAA rupture. CONCLUSION: Calpain-2 activation promotes cytoskeletal structural protein fragmentation and ECM degradation of experimental AAA aortas. Treatment with Calpain-2 specific inhibitor may facilitate the clinical management of AAA.
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Calpain-2 Compensation Promotes Angiotensin II-Induced Ascending and Abdominal Aortic Aneurysms in Calpain-1 Deficient Mice
PloS one, 2013Co-Authors: Venkateswaran Subramanian, Athar H. Chishti, Jessica J. Moorleghen, Deborah A. Howatt, Anju Balakrishnan, Haruhito A. UchidaAbstract:Background and Objective Recently, we demonstrated that angiotensin II (AngII)-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA) in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2) in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development. Methodology/Results To investigate the relative contribution of calpain-1 and -2 in development of AngII-induced AAs, male LDLr −/− mice that were either calpain-1 +/+ or −/− were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min) for 4 weeks. Calpain-1 deficiency had no significant effect on body weight or blood pressure during AngII infusion. Moreover, calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AAs. Interestingly, AngII infusion induced increased expression of Calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410, a calpain inhibitor, along with AngII-infusion significantly attenuated AngII-induced ascending and abdominal AA formation in both calpain-1 +/+ and −/− mice as compared to vehicle administered mice. Furthermore, BDA-410 administration attenuated AngII-induced aortic medial hypertrophy and macrophage accumulation. Western blot and immunostaining analyses revealed BDA-410 administration attenuated AngII-induced C-terminal fragmentation of filamin A, an actin binding cytoskeletal protein in aorta. Conclusion Calpain-2 compensates for loss of calpain-1, and both calpain isoforms are involved in AngII-induced aortic aneurysm formation in mice.
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Abstract 516: Calpain-2 Compensation Promotes Angiotensin II-induced Ascending and Abdominal Aortic Aneurysms in Calpain-1 Deficient Mice
Arteriosclerosis Thrombosis and Vascular Biology, 2013Co-Authors: Venkateswaran Subramanian, Athar H. Chishti, Jessica J. Moorleghen, Deborah A. Howatt, Anju Balakrishnan, Haruhito A. UchidaAbstract:Background and Objective We demonstrated that angiotensin II (AngII)-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA) in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2) in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development. Methods and Results Male LDLr-/- mice that were either calpain-1 +/+ or -/- were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min) for 4 weeks. Calpain-1 deficiency had no effect on body weight or blood pressure during AngII infusion. Intimal areas of ascending aorta and maximum external width of the suprarenal abdominal aorta were measured by en face. Calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AA development (P = NS). Interestingly, AngII infusion induced increased expression of Calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410 along with AngII infusion significantly attenuated AngII-induced ascending (Vehicle - +/+: 15.0 ± 1.0, -/-: 14.9 ± 0.8; BDA: +/+: 11.5 ± 0.6, -/-: 12.1 ± 0.6 mm 2 ; P Conclusion Calpain-2 compensates for loss of calpain-1, and promotes AngII-induced ascending and abdominal AAs in calpain-1 deficient mice.
Darrel E. Goll - One of the best experts on this subject based on the ideXlab platform.
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isolation and characterization of μ calpain m calpain and calpastatin from postmortem muscle i initial steps
Journal of Animal Science, 2007Co-Authors: J P Camou, John A. Marchello, S W Mares, R Vazquez, Michael D Taylor, Valery F Thompson, Darrel E. GollAbstract:Evidence has indicated that -calpain, m-calpain, and calpastatin have important roles in the proteolytic degradation that results in postmortem ten- derization.Simpleassaysofthese3proteinsatdifferent times postmortem, however, has shown that calpas- tatin and -calpain both rapidly lose their activity dur- ing postmortem storage, so that proteolytic activity of -calpain is nearly zero after 3 d postmortem, even when assayed at pH 7.5 and 25°C, and ability of calpas- tatin to inhibit the calpains is 30% or less of its ability when assayed at death. m-Calpain, however, retains much of its proteolytic activity during postmortem stor- age, but the Ca 2+ requirement of m-calpain is much higher than that reported to exist in postmortem mus- cle. Consequently, it is unclear how the calpain system functions in postmortem muscle. To clarify this issue, we have initiated attempts to purify the 2 calpains and calpastatin from bovine semitendinosus muscle after 11-13 d postmortem. The known properties of the cal- pains and calpastatin in postmortem muscle have im- portant effects on approaches that can be used to purify them. A hexyl-TSK hydrophobic interaction column is a critical first step in separating calpastatin from the
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interaction of calpastatin with calpain a review
Biological Chemistry, 2004Co-Authors: Amanda Wendt, Valery F Thompson, Darrel E. GollAbstract:Calpastatin is a multiheaded inhibitor capable of inhibiting more than one calpain molecule. Each inhibitory domain of calpastatin has three subdomains, A, B, and C; A binds to domain IV and C binds to domain VI of the calpains. Crystallographic evidence shows that binding of C to domain VI involves hydrophobic interactions at a site near the first EF-hand in domain VI. Sequence homology suggests that binding of A to calpain domain IV also involves hydrophobic interactions near the EF1-hand of domain IV. Neither subdomain A nor C have inhibitory activity without subdomain B, but both increase the inhibitory activity of B. Subdomain B peptides have no inhibitory activity unless they contain at least 13 amino acids, and inhibitory activity increases with the number of amino acid residues, suggesting that inhibition requires interaction over a large area of the calpain molecule. Although subdomain B inhibition kinetically is competitive in nature, subdomain B does not seem to interact with the active site of the calpains directly, but may bind to domain III of the calpains and act to block access to the active site. It is possible that subdomain B binds to calpain only after it has been activated by Ca 2 + .
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is calpain activity regulated by membranes and autolysis or by calcium and calpastatin
BioEssays, 1992Co-Authors: Darrel E. Goll, Valery F Thompson, Richard G Taylor, Teresa ZalewskaAbstract:Abstract Although the Ca(2+)-dependent proteinase (calpain) system has been found in every vertebrate cell that has been examined for its presence and has been detected in Drosophila and parasites, the physiological function(s) of this system remains unclear. Calpain activity has been associated with cleavages that alter regulation of various enzyme activities, with remodeling or disassembly of the cell cytoskeleton, and with cleavages of hormone receptors. The mechanism regulating activity of the calpain system in vivo also is unknown. It has been proposed that binding of the calpains to phospholipid in a cell membrane lowers the Ca2+ concentration, [Ca2+], required for the calpains to autolyze, and that autolysis converts an inactive proenzyme into an active protease. Recent studies, however, show that the calpains bind to specific proteins and not to phospholipids, and that binding to cell membranes does not affect the [Ca2+] required for autolysis. It seems likely that calpain activity is regulated by binding of Ca2+ to specific sites on the calpain molecule, with binding to each site eliciting a response (proteolytic activity, calpastatin binding, etc.) specific for that site. Regulation must also involve an, as yet, undiscovered mechanism that increases the affinity of the Ca(2+)-binding sites for Ca2+.
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studies of the alpha actinin actin interaction in the z disk by using calpain
Journal of Biological Chemistry, 1991Co-Authors: Darrel E. Goll, William R Dayton, I Singh, R M RobsonAbstract:Abstract Both mu- and m-calpain (the micro- and millimolar Ca(2+)-requiring Ca(2+)-dependent proteinases) can completely remove Z-disks from skeletal muscle myofibrils and leave a space devoid of filaments in the Z-disk area. alpha-Actinin, a principal protein component of Z-disks, is removed from myofibrils by the calpains, and a 100-kDa polypeptide that comigrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the alpha-actinin subunit is released into the supernatant. Purified calpain does not degrade purified actin or purified alpha-actinin as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by N- and C-terminal amino acid analysis of calpain-treated and untreated alpha-actinin and actin. The 100-kDa polypeptide released from myofibrils by calpain elutes identically with native alpha-actinin off DEAE-cellulose and hydroxyapatite columns and, after purification, binds to pure F-actin in the same manner that untreated, native alpha-actinin binds. Calpain-released alpha-actinin also accelerates the rate of superprecipitation of reconstituted actomyosin, a sensitive property characteristic of native alpha-actinin. Consequently, the calpains release alpha-actinin from the Z-disk of myofibrils without degrading it or without altering its ability to bind to actin. These results indicate that alpha-actinin does not simply cross-link thin filaments across the Z-disk but that at least one additional protein (or perhaps an altered actin or alpha-actinin) is involved in the alpha-actinin/actin interaction in Z-disks.
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Studies of the alpha-actinin/actin interaction in the Z-disk by using calpain.
The Journal of biological chemistry, 1991Co-Authors: Darrel E. Goll, William R Dayton, I Singh, R M RobsonAbstract:Abstract Both mu- and m-calpain (the micro- and millimolar Ca(2+)-requiring Ca(2+)-dependent proteinases) can completely remove Z-disks from skeletal muscle myofibrils and leave a space devoid of filaments in the Z-disk area. alpha-Actinin, a principal protein component of Z-disks, is removed from myofibrils by the calpains, and a 100-kDa polypeptide that comigrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the alpha-actinin subunit is released into the supernatant. Purified calpain does not degrade purified actin or purified alpha-actinin as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by N- and C-terminal amino acid analysis of calpain-treated and untreated alpha-actinin and actin. The 100-kDa polypeptide released from myofibrils by calpain elutes identically with native alpha-actinin off DEAE-cellulose and hydroxyapatite columns and, after purification, binds to pure F-actin in the same manner that untreated, native alpha-actinin binds. Calpain-released alpha-actinin also accelerates the rate of superprecipitation of reconstituted actomyosin, a sensitive property characteristic of native alpha-actinin. Consequently, the calpains release alpha-actinin from the Z-disk of myofibrils without degrading it or without altering its ability to bind to actin. These results indicate that alpha-actinin does not simply cross-link thin filaments across the Z-disk but that at least one additional protein (or perhaps an altered actin or alpha-actinin) is involved in the alpha-actinin/actin interaction in Z-disks.
R M Robson - One of the best experts on this subject based on the ideXlab platform.
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studies of the alpha actinin actin interaction in the z disk by using calpain
Journal of Biological Chemistry, 1991Co-Authors: Darrel E. Goll, William R Dayton, I Singh, R M RobsonAbstract:Abstract Both mu- and m-calpain (the micro- and millimolar Ca(2+)-requiring Ca(2+)-dependent proteinases) can completely remove Z-disks from skeletal muscle myofibrils and leave a space devoid of filaments in the Z-disk area. alpha-Actinin, a principal protein component of Z-disks, is removed from myofibrils by the calpains, and a 100-kDa polypeptide that comigrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the alpha-actinin subunit is released into the supernatant. Purified calpain does not degrade purified actin or purified alpha-actinin as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by N- and C-terminal amino acid analysis of calpain-treated and untreated alpha-actinin and actin. The 100-kDa polypeptide released from myofibrils by calpain elutes identically with native alpha-actinin off DEAE-cellulose and hydroxyapatite columns and, after purification, binds to pure F-actin in the same manner that untreated, native alpha-actinin binds. Calpain-released alpha-actinin also accelerates the rate of superprecipitation of reconstituted actomyosin, a sensitive property characteristic of native alpha-actinin. Consequently, the calpains release alpha-actinin from the Z-disk of myofibrils without degrading it or without altering its ability to bind to actin. These results indicate that alpha-actinin does not simply cross-link thin filaments across the Z-disk but that at least one additional protein (or perhaps an altered actin or alpha-actinin) is involved in the alpha-actinin/actin interaction in Z-disks.
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Studies of the alpha-actinin/actin interaction in the Z-disk by using calpain.
The Journal of biological chemistry, 1991Co-Authors: Darrel E. Goll, William R Dayton, I Singh, R M RobsonAbstract:Abstract Both mu- and m-calpain (the micro- and millimolar Ca(2+)-requiring Ca(2+)-dependent proteinases) can completely remove Z-disks from skeletal muscle myofibrils and leave a space devoid of filaments in the Z-disk area. alpha-Actinin, a principal protein component of Z-disks, is removed from myofibrils by the calpains, and a 100-kDa polypeptide that comigrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the alpha-actinin subunit is released into the supernatant. Purified calpain does not degrade purified actin or purified alpha-actinin as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by N- and C-terminal amino acid analysis of calpain-treated and untreated alpha-actinin and actin. The 100-kDa polypeptide released from myofibrils by calpain elutes identically with native alpha-actinin off DEAE-cellulose and hydroxyapatite columns and, after purification, binds to pure F-actin in the same manner that untreated, native alpha-actinin binds. Calpain-released alpha-actinin also accelerates the rate of superprecipitation of reconstituted actomyosin, a sensitive property characteristic of native alpha-actinin. Consequently, the calpains release alpha-actinin from the Z-disk of myofibrils without degrading it or without altering its ability to bind to actin. These results indicate that alpha-actinin does not simply cross-link thin filaments across the Z-disk but that at least one additional protein (or perhaps an altered actin or alpha-actinin) is involved in the alpha-actinin/actin interaction in Z-disks.
