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Weng T, Zhang X, He J, Yang Y, Li C. Bioinformatics-based analysis of the relationship between plasminogen regulatory genes and photoaging. J Cosmet Dermatol 2024; 23:2270-2278. [PMID: 38634239 DOI: 10.1111/jocd.16266] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2023] [Revised: 02/08/2024] [Accepted: 02/23/2024] [Indexed: 04/19/2024]
Abstract
BACKGROUND Ultraviolet radiation causes skin photoaging by producing a variety of enzymes, which impact both skin health and hinder beauty. Currently, the early diagnosis and treatment of photoaging remain a challenge. Bioinformatics analysis has strong advantages in exploring core genes and the biological pathways of photoaging. AIMS To screen and validate key risk genes associated with plasminogen in photoaging and to identify potential target genes for photoaging. METHODS Two human transcriptome datasets were obtained by searching the Gene Expression Omnibus (GEO) database, and the mRNAs in the GSE131789 dataset were differentially analyzed, and then the weighted gene co-expression network analysis (WGCNA) was performed to find out the strongest correlations. Template genes, interaction analysis of differentially expressed genes (DEGs), modular genes with the most WGCNA correlations, and genecard database genes related to plasminogen were performed, and further Kyoto genes and Genome Encyclopedia (KEGG) pathway analysis. Two different algorithms, least absolute shrinkage and selection operator (LASSO) and support vector machines-recursive feature elimination (SVM-RFE), were used to find key genes. Then the data set (GSE206495) was validated and analyzed. Real-time PCR was performed to validate the expression of key genes through in vitro cellular experiments. RESULTS IFI6, IFI44L, HRSP12, and BMP4 were screened from datasets as key genes for photoaging and further analysis showed that these genes have significant diagnostic value for photoaging. CONCLUSION IFI6, IFI44L, HRSP12, and BMP4 play a key role in the pathogenesis of photoaging, and serve as promising potential predictive biomarkers for photoaging.
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Affiliation(s)
- Tengyu Weng
- Department of Dermatology, First Medical Center of PLA General Hospital, Beijing, China
| | - Xiaoning Zhang
- Department of Dermatology, First Medical Center of PLA General Hospital, Beijing, China
| | - Juan He
- Department of Dermatology, First Medical Center of PLA General Hospital, Beijing, China
| | - Yi Yang
- Department of Dermatology, Third Medical Center of PLA General Hospital, Beijing, China
| | - Chengxin Li
- Department of Dermatology, First Medical Center of PLA General Hospital, Beijing, China
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Fulton RL, Downs DM. Modulators of a robust and efficient metabolism: Perspective and insights from the Rid superfamily of proteins. Adv Microb Physiol 2023; 83:117-179. [PMID: 37507158 PMCID: PMC10642521 DOI: 10.1016/bs.ampbs.2023.04.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/30/2023]
Abstract
Metabolism is an integrated network of biochemical pathways that assemble to generate the robust, responsive physiologies of microorganisms. Despite decades of fundamental studies on metabolic processes and pathways, our understanding of the nuance and complexity of metabolism remains incomplete. The ability to predict and model metabolic network structure, and its influence on cellular fitness, is complicated by the persistence of genes of unknown function, even in the best-studied model organisms. This review describes the definition and continuing study of the Rid superfamily of proteins. These studies are presented with a perspective that illustrates how metabolic complexity can complicate the assignment of function to uncharacterized genes. The Rid superfamily of proteins has been divided into eight subfamilies, including the well-studied RidA subfamily. Aside from the RidA proteins, which are present in all domains of life and prevent metabolic stress, most members of the Rid superfamily have no demonstrated physiological role. Recent progress on functional assignment supports the hypothesis that, overall, proteins in the Rid superfamily modulate metabolic processes to ensure optimal organismal fitness.
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Affiliation(s)
- Ronnie L Fulton
- Department of Microbiology, University of Georgia, Athens, GA, United States
| | - Diana M Downs
- Department of Microbiology, University of Georgia, Athens, GA, United States.
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3
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Irons JL, Hodge-Hanson K, Downs DM. RidA Proteins Protect against Metabolic Damage by Reactive Intermediates. Microbiol Mol Biol Rev 2020; 84:e00024-20. [PMID: 32669283 PMCID: PMC7373157 DOI: 10.1128/mmbr.00024-20] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The Rid (YjgF/YER057c/UK114) protein superfamily was first defined by sequence homology with available protein sequences from bacteria, archaea, and eukaryotes (L. Parsons, N. Bonander, E. Eisenstein, M. Gilson, et al., Biochemistry 42:80-89, 2003, https://doi.org/10.1021/bi020541w). The archetypal subfamily, RidA (reactive intermediate deaminase A), is found in all domains of life, with the vast majority of free-living organisms carrying at least one RidA homolog. In over 2 decades, close to 100 reports have implicated Rid family members in cellular processes in prokaryotes, yeast, plants, and mammals. Functional roles have been proposed for Rid enzymes in amino acid biosynthesis, plant root development and nutrient acquisition, cellular respiration, and carcinogenesis. Despite the wealth of literature and over a dozen high-resolution structures of different RidA enzymes, their biochemical function remained elusive for decades. The function of the RidA protein was elucidated in a bacterial model system despite (i) a minimal phenotype of ridA mutants, (ii) the enzyme catalyzing a reaction believed to occur spontaneously, and (iii) confusing literature on the pleiotropic effects of RidA homologs in prokaryotes and eukaryotes. Subsequent work provided the physiological framework to support the RidA paradigm in Salmonella enterica by linking the phenotypes of mutants lacking ridA to the accumulation of the reactive metabolite 2-aminoacrylate (2AA), which damaged metabolic enzymes. Conservation of enamine/imine deaminase activity of RidA enzymes from all domains raises the likelihood that, despite the diverse phenotypes, the consequences when RidA is absent are due to accumulated 2AA (or a similar reactive enamine) and the diversity of metabolic phenotypes can be attributed to differences in metabolic network architecture. The discovery of the RidA paradigm in S. enterica laid a foundation for assessing the role of Rid enzymes in diverse organisms and contributed fundamental lessons on metabolic network evolution and diversity in microbes. This review describes the studies that defined the conserved function of RidA, the paradigm of enamine stress in S. enterica, and emerging studies that explore how this paradigm differs in other organisms. We focus primarily on the RidA subfamily, while remarking on our current understanding of the other Rid subfamilies. Finally, we describe the current status of the field and pose questions that will drive future studies on this widely conserved protein family to provide fundamental new metabolic information.
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Affiliation(s)
- Jessica L Irons
- Department of Microbiology, University of Georgia, Athens, Georgia, USA
| | | | - Diana M Downs
- Department of Microbiology, University of Georgia, Athens, Georgia, USA
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Dókus LE, Yousef M, Bánóczi Z. Modulators of calpain activity: inhibitors and activators as potential drugs. Expert Opin Drug Discov 2020; 15:471-486. [DOI: 10.1080/17460441.2020.1722638] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Affiliation(s)
- Levente Endre Dókus
- Department of Organic Chemistry, Institute of Chemistry, Eötvös Loránd University, Budapest, Hungary
| | - Mo’ath Yousef
- Department of Organic Chemistry, Institute of Chemistry, Eötvös Loránd University, Budapest, Hungary
| | - Zoltán Bánóczi
- Department of Organic Chemistry, Institute of Chemistry, Eötvös Loránd University, Budapest, Hungary
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5
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Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein. Viruses 2019; 11:v11121106. [PMID: 31795245 PMCID: PMC6950447 DOI: 10.3390/v11121106] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2019] [Revised: 11/26/2019] [Accepted: 11/26/2019] [Indexed: 12/25/2022] Open
Abstract
Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C’s cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.
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6
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Hong LTT, Hachiya T, Hase S, Shiwa Y, Yoshikawa H, Sakakibara Y, Nguyen SLT, Kimura K. Poly-γ-glutamic acid production of Bacillus subtilis (natto) in the absence of DegQ: A gain-of-function mutation in yabJ gene. J Biosci Bioeng 2019; 128:690-696. [PMID: 31272833 DOI: 10.1016/j.jbiosc.2019.05.014] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2019] [Revised: 05/21/2019] [Accepted: 05/27/2019] [Indexed: 02/06/2023]
Abstract
Poly-γ-glutamic acid (γPGA) production by Bacillus subtilis is regulated by the quorum sensing system where DegQ transmits the cell density signal to a DNA-binding protein DegU. A mutation suppressing the γPGA-negative phenotype of degQ gene knock-out mutant (ΔdegQ) was identified through whole genome sequencing. The mutation conferred an amino acid substitution of Ser103 to phenylalanine (S103F) in yabJ that belongs to the highly conserved YjgF/YER057c/UK114 family. Genetic experiments including LacZ-fusion assay of γPGA synthetic operon confirmed that the suppressor mutation (yabJS103F) was responsible for the recovery of γPGA production. The yabJ itself was not essential for the γPGA production and the mutant allele enabled γPGA production of the ΔdegQ strain even in the presence of wild type yabJ. Thus, yabJS103F was a dominant positive allele. degU-lacZ fusion gene was hyper-expressed in cells carrying the yabJS103F, but disruption of yabJ did not affect the transcription level of the degU-lacZ. These observations suggested that YabJ acquired a function to stimulate expression of degU by the S103F mutation which is involved in the regulation of γPGA synthesis.
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Affiliation(s)
- Le Thi Thu Hong
- Food Research Institute, National Agriculture and Food Research Organization (NFRI/NARO), Tsukuba, Ibaraki 305-8642, Japan
| | - Tsuyoshi Hachiya
- Department of Bioscience and Informatics, Keio University, Yokohama, Kanagawa 223-8522, Japan
| | - Sumitaka Hase
- Department of Bioscience and Informatics, Keio University, Yokohama, Kanagawa 223-8522, Japan
| | - Yuh Shiwa
- Department of Molecular Microbiology, Tokyo University of Agriculture, Setagaya, Tokyo 156-8502, Japan
| | - Hirofumi Yoshikawa
- Genome Research Center, NODAI Research Institute, Tokyo University of Agriculture, Setagaya, Tokyo 156-8502, Japan; Department of Bioscience, Tokyo University of Agriculture, Setagaya, Tokyo 156-8502, Japan
| | - Yasubumi Sakakibara
- Department of Bioscience and Informatics, Keio University, Yokohama, Kanagawa 223-8522, Japan
| | - Sy Le Thanh Nguyen
- Food Research Institute, National Agriculture and Food Research Organization (NFRI/NARO), Tsukuba, Ibaraki 305-8642, Japan
| | - Keitarou Kimura
- Food Research Institute, National Agriculture and Food Research Organization (NFRI/NARO), Tsukuba, Ibaraki 305-8642, Japan.
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Identification of a perchloric acid-soluble protein (PSP)-like ribonuclease from Trichomonas vaginalis. Parasitol Res 2018; 117:3639-3652. [PMID: 30191309 DOI: 10.1007/s00436-018-6065-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2018] [Accepted: 08/23/2018] [Indexed: 01/08/2023]
Abstract
A perchloric acid-soluble protein (PSP), named here tv-psp1, was identified in Trichomonas vaginalis. It is expressed under normal culture conditions according to expressed sequence tag (EST) analysis. On the other hand, Tv-PSP1 protein was identified by mass spectrometry with a 40% of identity to human PSP (p14.1). Polyclonal antibodies against recombinant Tv-PSP1 (rTv-PSP1) recognized a single band at 13.5 kDa in total protein parasite extract by SDS-PAGE and a high molecular weight band analyzed by native PAGE. Structural analysis of Tv-PSP1, using dynamic light scattering, size exclusion chromatography, and circular dichroism spectroscopy, showed a trimeric structure stable at 7 M urea with 38% α-helix and 14% β-sheet in solution and a molecular weight of 40.5 kD. Tv-PSP1 models were used to perform dynamic simulations over 100 ns suggesting a stable homotrimeric structure. Tv-PSP1 was located in the nucleus, cytoplasm, and hydrogenosomes of T. vaginalis, and the in silico analysis by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) showed interactions with RNA binding proteins. The preliminary results of RNA degradation analysis with the recombinant Tv-PSP1 showed RNA partial deterioration suggesting a possible putative ribonuclease function.
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8
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Zhang Y, Liu NM, Wang Y, Youn JY, Cai H. Endothelial cell calpain as a critical modulator of angiogenesis. Biochim Biophys Acta Mol Basis Dis 2017; 1863:1326-1335. [PMID: 28366876 DOI: 10.1016/j.bbadis.2017.03.021] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2016] [Revised: 03/04/2017] [Accepted: 03/28/2017] [Indexed: 12/13/2022]
Abstract
Calpains are a family of calcium-dependent non-lysosomal cysteine proteases. In particular, calpains residing in the endothelial cells play important roles in angiogenesis. It has been shown that calpain activity can be increased in endothelial cells by growth factors, primarily vascular endothelial growth factor (VEGF). VEGF/VEGFR2 induces calpain 2 dependent activation of PI3K/AMPK/Akt/eNOS pathway, and consequent nitric oxide production and physiological angiogenesis. Under pathological conditions such as tumor angiogenesis, endothelial calpains can be activated by hypoxia. This review focuses on the molecular regulatory mechanisms of calpain activation, and the newly identified mechanistic roles and downstream signaling events of calpains in physiological angiogenesis, and in the conditions of pathological tumor angiogenesis and diabetic wound healing, as well as retinopathy and atherosclerosis that are also associated with an increase in calpain activity. Further discussed include the differential strategies of modulating angiogenesis through manipulating calpain expression/activity in different pathological settings. Targeted limitation of angiogenesis in cancer and targeted promotion of angiogenesis in diabetic wound healing via modulations of calpains and calpain-dependent signaling mechanisms are of significant translational potential. Emerging strategies of tissue-specific targeting, environment-dependent targeting, and genome-targeted editing may turn out to be effective regimens for targeted manipulation of angiogenesis through calpain pathways, for differential treatments including both attenuation of tumor angiogenesis and potentiation of diabetic angiogenesis.
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Affiliation(s)
- Yixuan Zhang
- Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at University of California Los Angeles (UCLA), CA 90095, USA; Division of Cardiology, Department Medicine, Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California Los Angeles (UCLA), CA 90095, USA
| | - Norika Mengchia Liu
- Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at University of California Los Angeles (UCLA), CA 90095, USA; Division of Cardiology, Department Medicine, Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California Los Angeles (UCLA), CA 90095, USA
| | - Yongchen Wang
- Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at University of California Los Angeles (UCLA), CA 90095, USA; Division of Cardiology, Department Medicine, Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California Los Angeles (UCLA), CA 90095, USA
| | - Ji Youn Youn
- Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at University of California Los Angeles (UCLA), CA 90095, USA; Division of Cardiology, Department Medicine, Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California Los Angeles (UCLA), CA 90095, USA
| | - Hua Cai
- Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at University of California Los Angeles (UCLA), CA 90095, USA; Division of Cardiology, Department Medicine, Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California Los Angeles (UCLA), CA 90095, USA.
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9
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Müller A, Langklotz S, Lupilova N, Kuhlmann K, Bandow JE, Leichert LIO. Activation of RidA chaperone function by N-chlorination. Nat Commun 2014; 5:5804. [PMID: 25517874 PMCID: PMC4284807 DOI: 10.1038/ncomms6804] [Citation(s) in RCA: 61] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2014] [Accepted: 11/07/2014] [Indexed: 12/25/2022] Open
Abstract
Escherichia coli RidA is a member of a structurally conserved, yet functionally highly diverse protein family involved in translation inhibition (human), Hsp90-like chaperone activity (fruit fly) and enamine/imine deamination (Salmonella enterica). Here, we show that E. coli RidA modified with HOCl acts as a highly effective chaperone. Although activation of RidA is reversed by treatment with DTT, ascorbic acid, the thioredoxin system and glutathione, it is independent of cysteine modification. Instead, treatment with HOCl or chloramines decreases the amino group content of RidA by reversibly N-chlorinating positively charged residues. N-chlorination increases hydrophobicity of RidA and promotes binding to a wide spectrum of unfolded cytosolic proteins. Deletion of ridA results in an HOCl-sensitive phenotype. HOCl-mediated N-chlorination thus is a cysteine-independent post-translational modification that reversibly turns RidA into an effective chaperone holdase, which plays a crucial role in the protection of cytosolic proteins during oxidative stress. Hypochlorous acid generated by neutrophils acts as a potent antibacterial agent. Müller et al. now show that this oxidant directly activates a protective counter-response in E. coli by N-chlorinating the protein RidA and converting it into an effective protein chaperone.
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Affiliation(s)
- Alexandra Müller
- Institute of Biochemistry and Pathobiochemistry-Microbial Biochemistry, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany
| | - Sina Langklotz
- Biology of Microorganisms, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany
| | - Nataliya Lupilova
- Institute of Biochemistry and Pathobiochemistry-Microbial Biochemistry, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany
| | - Katja Kuhlmann
- Medizinisches Proteom-Center, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany
| | - Julia Elisabeth Bandow
- Biology of Microorganisms, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany
| | - Lars Ingo Ole Leichert
- Institute of Biochemistry and Pathobiochemistry-Microbial Biochemistry, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany
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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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Abstract
Calpain is a conserved family of calcium-dependent, cytosolic, neutral cysteine proteases. The best characterized members of the family are the ubiquitously expressed calpain 1 and calpain 2. They perform controlled proteolysis of their target proteins. The regulation of these enzymes includes autolysis, calcium, phosphorylation as a posttranslational modification, and binding of calpastatin, phospholipids or activator proteins, respectively. Calpain are implicated in many physiological and pathological processes. They have significant role in the cell proliferation, differentiation and migration in a variety of mammalian cell types, contributing to the development of angiogenesis, vascular remodeling, and cancer. Therefore the knowledge of the precise mechanism of calpain signaling could provide therapeutic approaches in these processes.
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Affiliation(s)
- Laszlo Kovacs
- Department of Pharmacology & Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA
| | - Yunchao Su
- Department of Pharmacology & Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA
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12
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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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Andersson KM, Meerupati T, Levander F, Friman E, Ahrén D, Tunlid A. Proteome of the nematode-trapping cells of the fungus Monacrosporium haptotylum. Appl Environ Microbiol 2013; 79:4993-5004. [PMID: 23770896 PMCID: PMC3754708 DOI: 10.1128/aem.01390-13] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2013] [Accepted: 06/07/2013] [Indexed: 01/08/2023] Open
Abstract
Many nematophagous fungi use morphological structures called traps to capture nematodes by adhesion or mechanically. To better understand the cellular functions of adhesive traps, the trap cell proteome of the fungus Monacrosporium haptotylum was characterized. The trap of M. haptotylum consists of a unicellular structure called a knob that develops at the apex of a hypha. Proteins extracted from knobs and mycelia were analyzed using SDS-PAGE and liquid chromatography-tandem mass spectrometry (LC-MS-MS). The peptide sequences were matched against predicted gene models from the recently sequenced M. haptotylum genome. In total, 336 proteins were identified, with 54 expressed at significantly higher levels in the knobs than in the mycelia. The upregulated knob proteins included peptidases, small secreted proteins with unknown functions, and putative cell surface adhesins containing carbohydrate-binding domains, including the WSC domain. Phylogenetic analysis showed that all upregulated WSC domain proteins belonged to a large, expanded cluster of paralogs in M. haptotylum. Several peptidases and homologs of experimentally verified proteins in other pathogenic fungi were also upregulated in the knob proteome. Complementary profiling of gene expression at the transcriptome level showed poor correlation between the upregulation of knob proteins and their corresponding transcripts. We propose that the traps of M. haptotylum contain many of the proteins needed in the early stages of infection and that the trap cells can tightly control the translation and degradation of these proteins to minimize the cost of protein synthesis.
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Affiliation(s)
| | | | - Fredrik Levander
- Bioinformatics Infrastructure for Life Sciences, Department of Immunotechnology, Lund University, Lund, Sweden
| | - Eva Friman
- Microbial Ecology, Department of Biology, Lund University, Lund, Sweden
| | - Dag Ahrén
- Microbial Ecology, Department of Biology, Lund University, Lund, Sweden
- Bioinformatics Infrastructure for Life Sciences, Department of Biology, Lund University, Lund, Sweden
| | - Anders Tunlid
- Microbial Ecology, Department of Biology, Lund University, Lund, Sweden
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Lindemann C, Lupilova N, Müller A, Warscheid B, Meyer HE, Kuhlmann K, Eisenacher M, Leichert LI. Redox proteomics uncovers peroxynitrite-sensitive proteins that help Escherichia coli to overcome nitrosative stress. J Biol Chem 2013; 288:19698-714. [PMID: 23696645 DOI: 10.1074/jbc.m113.457556] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Peroxynitrite is a highly reactive chemical species with antibacterial properties that are synthesized in immune cells. In a proteomic approach, we identified specific target proteins of peroxynitrite-induced modifications in Escherichia coli. Although peroxynitrite caused a fairly indiscriminate nitration of tyrosine residues, reversible modifications of protein thiols were highly specific. We used a quantitative redox proteomic method based on isotope-coded affinity tag chemistry and identified four proteins consistently thiol-modified in cells treated with peroxynitrite as follows: AsnB, FrmA, MaeB, and RidA. All four were required for peroxynitrite stress tolerance in vivo. Three of the identified proteins were modified at highly conserved cysteines, and MaeB and FrmA are known to be directly involved in the oxidative and nitrosative stress response in E. coli. In in vitro studies, we could show that the activity of RidA, a recently discovered enamine/imine deaminase, is regulated in a specific manner by the modification of its single conserved cysteine. Mutation of this cysteine 107 to serine generated a constitutively active protein that was not susceptible to peroxynitrite.
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Affiliation(s)
- Claudia Lindemann
- Medical Proteome Center, Ruhr University Bochum, Universitätsstrasse 150, 44780 Bochum, Germany
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Abstract
Calpains are a family of complex multi-domain intracellular enzymes that share a calcium-dependent cysteine protease core. These are not degradative enzymes, but instead carry out limited cleavage of target proteins in response to calcium signalling. Selective cutting of cytoskeletal proteins to facilitate cell migration is one such function. The two most abundant and extensively studied members of this family in mammals, calpains 1 and 2, are heterodimers of an isoform-specific 80 kDa large subunit and a common 28 kDa small subunit. Structures of calpain-2, both Ca2+-free and bound to calpastatin in the activated Ca2+-bound state, have provided a wealth of information about the enzyme's structure-function relationships and activation. The main association between the subunits is the pairing of their C-terminal penta-EF-hand domains through extensive intimate hydrophobic contacts. A lesser contact is made between the N-terminal anchor helix of the large subunit and the penta-EF-hand domain of the small subunit. Up to ten Ca2+ ions are co-operatively bound during activation. The anchor helix is released and individual domains change their positions relative to each other to properly align the active site. Because calpains 1 and 2 require ~30 and ~350 μM Ca2+ ions for half-maximal activation respectively, it has long been argued that autoproteolysis, subunit dissociation, post-translational modifications or auxiliary proteins are needed to activate the enzymes in the cell, where Ca2+ levels are in the nanomolar range. In the absence of robust support for these mechanisms, it is possible that under normal conditions calpains are transiently activated by high Ca2+ concentrations in the microenvironment of a Ca2+ influx, and then return to an inactive state ready for reactivation.
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De Franceschi L, Franco RS, Bertoldi M, Brugnara C, Matté A, Siciliano A, Wieschhaus AJ, Chishti AH, Joiner CH. Pharmacological inhibition of calpain-1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease. FASEB J 2012; 27:750-9. [PMID: 23085996 DOI: 10.1096/fj.12-217836] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Sickle cell disease (SCD) is a globally distributed hereditary red blood cell (RBC) disorder. One of the hallmarks of SCD is the presence of circulating dense RBCs, which are important in SCD-related clinical manifestations. In human dense sickle cells, we found reduced calpastatin activity and protein expression compared to either healthy RBCs or unfractionated sickle cells, suggesting an imbalance between activator and inhibitor of calpain-1 in favor of activator in dense sickle cells. Calpain-1 is a nonlysosomal cysteine proteinase that modulates multiple cell functions through the selective cleavage of proteins. To investigate the relevance of this observation in vivo, we evaluated the effects of the orally active inhibitor of calpain-1, BDA-410 (30 mg/kg/d), on RBCs from SAD mice, a mouse model for SCD. In SAD mice, BDA-410 improved RBC morphology, reduced RBC density (D(20); from 1106 ± 0.001 to 1100 ± 0.001 g/ml; P<0.05) and increased RBC-K(+) content (from 364 ± 10 to 429 ± 12.3 mmol/kg Hb; P<0.05), markedly reduced the activity of the Ca(2+)-activated K(+)channel (Gardos channel), and decreased membrane association of peroxiredoxin-2. The inhibitory effect of calphostin C, a specific inhibitor of protein kinase C (PKC), on the Gardos channel was eliminated after BDA-410 treatment, which suggests that calpain-1 inhibition affects the PKC-dependent fraction of the Gardos channel. BDA-410 prevented hypoxia-induced RBC dehydration and K(+) loss in SAD mice. These data suggest a potential role of BDA-410 as a novel therapeutic agent for treatment of SCD.
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17
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Pu YG, Jiang YL, Ye XD, Ma XX, Guo PC, Lian FM, Teng YB, Chen Y, Zhou CZ. Crystal structures and putative interface of Saccharomyces cerevisiae mitochondrial matrix proteins Mmf1 and Mam33. J Struct Biol 2011; 175:469-74. [PMID: 21600990 DOI: 10.1016/j.jsb.2011.05.005] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2011] [Revised: 04/22/2011] [Accepted: 05/04/2011] [Indexed: 10/18/2022]
Abstract
The yeast Saccharomyces cerevisiae mitochondrial matrix factor Mmf1, a member in the YER057c/Yigf/Uk114 family, participates in isoleucine biosynthesis and mitochondria maintenance. Mmf1 physically interacts with another mitochondrial matrix protein Mam33, which is involved in the sorting of cytochrome b₂ to the intermembrane space as well as mitochondrial ribosomal protein synthesis. To elucidate the structural basis for their interaction, we determined the crystal structures of Mmf1 and Mam33 at 1.74 and 2.10 Å, respectively. Both Mmf1 and Mam33 adopt a trimeric structure: each subunit of Mmf1 displays a chorismate mutase fold with a six-stranded β-sheet flanked by two α-helices on one side, whereas a subunit of Mam33 consists of a twisted six-stranded β-sheet surrounded by five α-helices. Biochemical assays combined with structure-based computational simulation enable us to model a putative complex of Mmf1-Mam33, which consists of one Mam33 trimer and two tandem Mmf1 trimers in a head-to-tail manner. The two interfaces between the ring-like trimers are mainly composed of electrostatic interactions mediated by complementary negatively and positively charged patches. These results provided the structural insights into the putative function of Mmf1 during mitochondrial protein synthesis via Mam33, a protein binding to mitochondrial ribosomal proteins.
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Affiliation(s)
- You-Guang Pu
- Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, People's Republic of China
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18
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Abstract
The calpains are a conserved family of cysteine proteinases that catalyse the controlled proteolysis of many specific substrates. Calpain activity is implicated in several fundamental physiological processes, including cytoskeletal remodelling, cellular signalling, apoptosis and cell survival. Calpain expression is altered during tumorigenesis, and the proteolysis of numerous substrates, such as inhibitors of nuclear factor-κB (IκB), focal adhesion proteins (including, focal adhesion kinase and talin) and proto-oncogenes (for example, MYC), has been implicated in tumour pathogenesis. Recent evidence indicates that the increased expression of certain family members might influence the response to cancer therapies, providing justification for the development of novel calpain inhibitors.
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Affiliation(s)
- Sarah J Storr
- University of Nottingham, School of Molecular Medical Sciences, Nottingham NG5 1PB, UK
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19
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Bukowska A, Lendeckel U, Bode-Böger SM, Goette A. Physiologic and Pathophysiologic Role of Calpain: Implications for the Occurrence of Atrial Fibrillation. Cardiovasc Ther 2010; 30:e115-27. [DOI: 10.1111/j.1755-5922.2010.00245.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
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20
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Lambrecht JA, Browne BA, Downs DM. Members of the YjgF/YER057c/UK114 family of proteins inhibit phosphoribosylamine synthesis in vitro. J Biol Chem 2010; 285:34401-7. [PMID: 20817725 DOI: 10.1074/jbc.m110.160515] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The YjgF/YER057c/UK114 family of proteins is highly conserved across all three domains of life and currently lacks a consensus biochemical function. Analysis of Salmonella enterica strains lacking yjgF has led to a working model in which YjgF functions to remove potentially toxic secondary products of cellular enzymes. Strains lacking yjgF synthesize the thiamine precursor phosphoribosylamine (PRA) by a TrpD-dependent mechanism that is not present in wild-type strains. Here, PRA synthesis was reconstituted in vitro with anthranilate phosphoribosyltransferase (TrpD), threonine dehydratase (IlvA), threonine, and phosphoribosyl pyrophosphate. TrpD-dependent PRA formation in vitro was inhibited by S. enterica YjgF and the human homolog UK114. Thus, the work herein describes the first biochemical assay for diverse members of the highly conserved YjgF/YER057c/UK114 family of proteins and provides a means to dissect the cellular functions of these proteins.
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Affiliation(s)
- Jennifer A Lambrecht
- Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA
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21
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Shaikh S, Samanta K, Kar P, Roy S, Chakraborti T, Chakraborti S. m-Calpain-mediated cleavage of Na+/Ca2+ exchanger-1 in caveolae vesicles isolated from pulmonary artery smooth muscle. Mol Cell Biochem 2010; 341:167-80. [PMID: 20372982 DOI: 10.1007/s11010-010-0448-z] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2009] [Accepted: 03/17/2010] [Indexed: 01/30/2023]
Abstract
Using m-calpain antibody, we have identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of m-calpain in caveolae vesicles isolated from bovine pulmonary artery smooth muscle plasma membrane. In addition, 78, 35, and 18 kDa immunoreactive bands of m-calpain have also been detected. Casein zymogram studies also revealed the presence of m-calpain in the caveolae vesicles. We have also identified Na(+)/Ca(2+) exchanger-1 (NCX1) in the caveolae vesicles. Purification and N-terminal sequence analyses of these two proteins confirmed their identities as m-calpain and NCX1, respectively. We further sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of NCX1 in the caveolae vesicles. Treatment of the caveolae vesicles with the calcium ionophore, A23187 (1 microM) in presence of CaCl(2) (1 mM) appears to cleave NCX1 (120 kDa) to an 82 kDa fragment as revealed by immunoblot study using NCX1 monoclonal antibody; while pretreatment with the calpain inhibitors, calpeptin or MDL28170; or the Ca(2+) chelator, BAPTA-AM did not cause a discernible change in the NCX protein profile. In vitro cleavage of the purified NCX1 by the purified m-calpain supports this finding. The cleavage of NCX1 by m-calpain in the caveolae vesicles may be interpreted as an important mechanism of Ca(2+) overload, which could arise due to inhibition of Ca(2+) efflux by the forward-mode NCX and that could lead to sustained Ca(2+) overload in the smooth muscle leading to pulmonary hypertension.
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Affiliation(s)
- Soni Shaikh
- Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, 741235 West Bengal, India
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22
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Thakur KG, Praveena T, Gopal B. Mycobacterium tuberculosis Rv2704 is a member of the YjgF/YER057c/UK114 family. Proteins 2010; 78:773-8. [PMID: 19899170 PMCID: PMC3068300 DOI: 10.1002/prot.22623] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Affiliation(s)
- Krishan Gopal Thakur
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
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23
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Hailfinger S, Rebeaud F, Thome M. Adapter and enzymatic functions of proteases in T-cell activation. Immunol Rev 2009; 232:334-47. [DOI: 10.1111/j.1600-065x.2009.00830.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
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24
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Ma H, Nakajima E, Shih M, Azuma M, Shearer TR. Expression of calpain small subunit 2 in mammalian tissues. Curr Eye Res 2009; 29:337-47. [PMID: 15590481 DOI: 10.1080/02713680490516242] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
PURPOSE The purpose of the current experiments was to more closely define the distribution and the function of calpain small subunit 2 (css2). Css2 is a newly discovered regulatory protein for the calcium activated proteases, mu- and m-calpains. METHODS Tissues from rat, monkey, and man of various ages were used to determine expression patterns of css2 by relative quantitative RT-PCR using 18S rRNA as an endogenous standard. Recombinant css2 and the 80 kDa catalytic subunit of m-calpain (80 kDa/css2) were co-expressed in Escherichia coli. Casein zymography was used to measure the enzymatic activity of 80 kDa/css2 proteins. Lens alpha-crystallin and beta B1-crystallin were used as substrates to determine proteolysis by 80 kDa/css2. Computer-based homology modeling was used to predict interactions between the traditional small subunit (css1) or css2 with the 80 kDa catalytic subunit. RESULTS Css2 appears to be a functional equivalent of css1 in vitro in that the calcium-dependent proteolytic activity of 80 kDa/css2 was similar to recombinant m-calpain (80 kDa/css1). In rat and human lens, css2 transcripts increased with age, whereas css1 transcripts decreased with age. Human beta B1-crystallin and rat alpha A-crystallin were cleaved similarly by 80 kDa/css2 and 80 kDa/css1. Interestingly, alpha A-insert crystallin was not hydrolyzed when css2 was substituted for css1 in the calpain dimer, suggesting that css2 may perform different functions from css1 in terms of proteolysis of lens crystallins during maturational growth of the lens. Css2 may also assist in the proper folding of the 80 kDa subunit and regulate protease activity in the absence of calcium. CONCLUSIONS The wide distribution of css2 transcripts in rat and monkey suggested that css2 is a second, widely distributed (rather than tissue-specific) calpain small subunit, in addition to the long-recognized css1. Further studies at the protein level will indicate if css2 has unique functions apart from css1.
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Affiliation(s)
- H Ma
- Department of Integrative Biosciences, Oregon Health & Science University, Portland, Oregon 97239, USA.
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25
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Chong CL, Huang SF, Hu CP, Chen YL, Chou HY, Chau GY, Shew JY, Tsai YL, Chen CT, Chang C, Chen ML. Decreased expression of UK114 is related to the differentiation status of human hepatocellular carcinoma. Cancer Epidemiol Biomarkers Prev 2008; 17:535-42. [PMID: 18349270 DOI: 10.1158/1055-9965.epi-07-0506] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Previous studies have identified that the expression of UK114 is tissue specific and the protein has been found to be most abundant in liver and kidney. However, the expression of UK114 in human hepatocellular carcinoma and its relationship to differentiation and transformation of hepatocellular carcinoma have not been studied. In this study, the expression of UK114 in human hepatocellular carcinoma was examined by Northern and Western blot analyses. We found that UK114 was significantly down-regulated in most of hepatocellular carcinoma tissues compared with adjacent nontumor tissues (72.7%) at both mRNA and protein levels. We looked into the possibility that this decreased expression of UK114 in the hepatocellular carcinoma tissues may play a role in the differentiation or tumorigenicity of hepatocellular carcinoma. Immunohistochemical staining showed that the reduced expression of UK114 in hepatocellular carcinoma tissues was correlated with the tumor differentiation status as graded by the Edmondson-Steiner classification. On the other hand, overexpression of UK114 was not able to suppress the proliferation of human hepatoma cells and tumorigenicity in nude mice. These results suggest that UK114 does not seem to act as a tumor suppressor gene; however, it may useful as a biomarker that will assist in the grading of the differentiation status of hepatocellular carcinoma samples.
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Affiliation(s)
- Chin-Liew Chong
- Division of Molecular and Genomic Medicine, National Health Research Institutes, 35, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, [corrected] Republic of China
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26
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YjgF is required for isoleucine biosynthesis when Salmonella enterica is grown on pyruvate medium. J Bacteriol 2008; 190:3057-62. [PMID: 18296521 DOI: 10.1128/jb.01700-07] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The YjgF/YER057c/UK114 family of proteins is conserved across the three domains of life, yet no biochemical function has been clearly defined for any member of this family. In Salmonella enterica, a deletion of yjgF results in a requirement for isoleucine when the mutant strain is grown in glucose-serine or pyruvate medium. Feedback inhibition of IlvA is required for the curative effect of isoleucine on glucose-serine medium. On pyruvate medium, yjgF mutants are unable to synthesize enough isoleucine for growth. From this study, we conclude that the isoleucine requirement of a yjgF mutant on pyruvate is a consequence of the decreased transaminase B (IlvE) activity that has previously been characterized in these mutants.
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27
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Samanta K, Kar P, Ghosh B, Chakraborti T, Chakraborti S. Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum. Biochim Biophys Acta Gen Subj 2007; 1770:1297-307. [PMID: 17656025 DOI: 10.1016/j.bbagen.2007.06.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2007] [Revised: 06/18/2007] [Accepted: 06/22/2007] [Indexed: 11/25/2022]
Abstract
Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.
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Affiliation(s)
- Krishna Samanta
- Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India
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28
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Salem M, Nath J, Killefer J. Cloning of the Calpain Regulatory Subunit cDNA from Fish Reveals a Divergent Domain-V. Anim Biotechnol 2007; 15:145-57. [PMID: 15595700 DOI: 10.1081/labt-200036706] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Calpains are Ca2+-dependent intracellular cysteine proteases, including the ubiquitously expressed micro- and m-calpains. Both are heterodimers, consisting of a distinct catalytic subunit and a common regulatory subunit. We describe cloning and sequencing of the calpain small (regulatory) subunit (cpns) cDNA from rainbow trout. This represents the first fish and lower vertebrate full cDNA of cpns. The rainbow trout cpns cDNA was used to retrieve the zebra fish and Japanese flounder homologues. We present evidence that fish cpns, unlike the conventional mammalian predominant isoform, cpnsl, is lacking the glycine-rich region of domain V. Because the glycine-rich region is known to play a role in membrane targeting, this divergent cpns suggests potentially different functional and activation mechanisms of the fish calpain system. A phylogenetic tree for the cpns gene superfamily has been constructed and the evolution of cpns considered.
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Affiliation(s)
- M Salem
- Animal Sciences, West Virginia University, Morgantown, West Virginia, USA
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29
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Abstract
Calpains, particularly conventional dimeric calpains, have claimed to be involved in the cell degeneration processes that characterize numerous disease conditions linked to dysfunctions of cellular Ca2+ homeostasis. The evidence supporting their involvement has traditionally been indirect and circumstantial, but recent work has added more solid evidence supporting the role of ubiquitous dimeric calpains in the process of neurodegeneration. The only disease condition in which a calpain defect has been conclusively involved concerns an atypical monomeric calpain: the muscle specific calpain-3, also known as p94. Inactivating defects in its gene cause a muscular dystrophy termed LGMD-2A. The molecular mechanism by which the absence of the proteolytic activity of calpain-3 causes the dystrophic process is unknown. Another atypical calpain, which has been characterized recently as a Ca2(+)-dependent protease, calpain 10, appears To be involved in the etiology of type 2 diabetes. The involvement has been inferred essentially from genetic evidence. Also in the case of type 2 diabetes the molecular mechanisms that could link the disease to calpain 10 are unknown.
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Affiliation(s)
- I Bertipaglia
- Department of Biochemistry, University of Padova, Italy
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30
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Del Bello B, Moretti D, Gamberucci A, Maellaro E. Cross-talk between calpain and caspase-3/-7 in cisplatin-induced apoptosis of melanoma cells: a major role of calpain inhibition in cell death protection and p53 status. Oncogene 2006; 26:2717-26. [PMID: 17130844 DOI: 10.1038/sj.onc.1210079] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
The contribution of different proteolytic systems, in particular calpains and effector caspases, in apoptotic cell death is still controversial. In this paper, we show that during cisplatin-induced apoptosis of human metastatic melanoma cells, calpain activation, as measured in intact cells by two different fluorescent substrates, is an early event, taking place well before caspase-3/-7 activation, and progressively increasing during 48 h of treatment. Such activation appears to be independent from any intracellular calcium imbalance; in fact, an increase of cytosolic calcium along with emptying of the reticular stores occur only at very late stages, uniquely in frankly apoptotic, detached cells. Calpain activation proves to be an early and crucial event in the apoptotic machinery, as demonstrated by the significant protection of cell death in samples co-treated with the calpain inhibitors, MDL 28170, calpeptin and PD 150606, where a variable but significant reduction of both caspase-3/-7 activity and cell detachment is observed. Consistently, such a protective effect can be at least partially due to the impairment of cisplatin-induced p53 activation, occurring early in committed, preapoptotic cells. Furthermore, in late apoptotic cells, calpain activity is also responsible for the formation of a novel p53 proteolytic fragment (approximately 26 kDa), whose function is so far to be elucidated.
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Affiliation(s)
- B Del Bello
- Department of Physiopathology and Experimental Medicine, University of Siena, Siena, Italy
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31
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Saez ME, Ramirez-Lorca R, Moron FJ, Ruiz A. The therapeutic potential of the calpain family: new aspects. Drug Discov Today 2006; 11:917-23. [PMID: 16997142 DOI: 10.1016/j.drudis.2006.08.009] [Citation(s) in RCA: 95] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2006] [Revised: 07/20/2006] [Accepted: 08/14/2006] [Indexed: 12/30/2022]
Abstract
The calpain family is a group of cysteine proteases unique in their dependency on calcium to attain functionally active forms. Calpains are involved in a wide range of cellular calcium-regulated functions, including signal transduction, cell proliferation and differentiation, and apoptosis. Moreover, altered calpain activity has been observed in several human diseases. Specific calpain inhibitors hold promise for the treatment of neuromuscular and neurodegenerative diseases in which calpains have been shown to be upregulated (e.g. Parkinson's disease and Duchenne muscular dystrophy). Conversely, calpain activators could be a useful approach for those diseases where reduced calpain activity has been observed, such as type 2 diabetes or metabolic syndrome.
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Affiliation(s)
- Maria E Saez
- Department of Structural Genomics, Neocodex, Centro de Negocios Charles Darwin s/n, Isla de la Cartuja, 41092-Sevilla, Spain.
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Simonovic M, Zhang Z, Cianci CD, Steitz TA, Morrow JS. Structure of the calmodulin alphaII-spectrin complex provides insight into the regulation of cell plasticity. J Biol Chem 2006; 281:34333-40. [PMID: 16945920 DOI: 10.1074/jbc.m604613200] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
AlphaII-spectrin is a major cortical cytoskeletal protein contributing to membrane organization and integrity. The Ca2+-activated binding of calmodulin to an unstructured insert in the 11th repeat unit of alphaII-spectrin enhances the susceptibility of spectrin to calpain cleavage but abolishes its sensitivity to several caspases and to at least one bacterially derived pathologic protease. Other regulatory inputs including phosphorylation by c-Src also modulate the proteolytic susceptibility of alphaII-spectrin. These pathways, acting through spectrin, appear to control membrane plasticity and integrity in several cell types. To provide a structural basis for understanding these crucial biological events, we have solved the crystal structure of a complex between bovine calmodulin and the calmodulin-binding domain of human alphaII-spectrin (Protein Data Bank ID code 2FOT). The structure revealed that the entire calmodulin-spectrin-binding interface is hydrophobic in nature. The spectrin domain is also unique in folding into an amphiphilic helix once positioned within the calmodulin-binding groove. The structure of this complex provides insight into the mechanisms by which calmodulin, calpain, caspase, and tyrosine phosphorylation act on spectrin to regulate essential cellular processes.
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Affiliation(s)
- Miljan Simonovic
- Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520, USA
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33
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Murphy RM, Verburg E, Lamb GD. Ca2+ activation of diffusible and bound pools of mu-calpain in rat skeletal muscle. J Physiol 2006; 576:595-612. [PMID: 16857710 PMCID: PMC1890353 DOI: 10.1113/jphysiol.2006.114090] [Citation(s) in RCA: 97] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Skeletal muscle fibres contain ubiquitous and muscle-specific calcium-dependent proteases known as calpains. During normal activity, intracellular [Ca(2+)] in muscle fibres increases to high levels ( approximately 2-20 microm), and it is not apparent how this can be reconciled with the activation properties of the calpains. Calpains evidently do not cause widespread proteolytic damage within muscle fibres under normal circumstances, but do have a role in necrosis in dystrophic muscle fibres. In this study, we examined the in situ localization and regulation of calpains in muscle fibres in order to identify how they are attuned to normal function. The sarcolemma of individual muscle fibres of the rat was removed by microdissection (fibre 'skinning') in order to determine the compartmentalization and diffusibility of the two most Ca(2+)-sensitive calpains, mu-calpain and calpain-3, and to permit precise manipulation of cytoplasmic [Ca(2+)] under physiological in situ conditions. Passive force production in stretched fibres, which indicates the patency of the important elastic structural protein titin, was used as a sensitive assay of the amount of diffusible proteolytic activity in individual fibre segments and in muscle homogenates at set [Ca(2+)]. All calpain-3 is bound tightly within a fibre, whereas most mu-calpain ( approximately 0.2 microm) is initially freely diffusible in the cytoplasm at resting [Ca(2+)] but binds within seconds at high [Ca(2+)]. [Ca(2+)] has to be raised to >/= 2 microm for >/= 1 min to initiate detectable autolysis of mu-calpain and to activate appreciable proteolytic activity. If the [Ca(2+)] is raised sufficiently for long enough to initiate substantial autolysis of mu-calpain, the Ca(2+) sensitivity of the proteolytic activity is greatly increased, and it remains active even at 300 nm Ca(2+), with activity only ceasing if the [Ca(2+)] is decreased to approximately 50 nm Ca(2+), close to the normal resting [Ca(2+)]. These findings on the Ca(2+)- and time-dependent binding, autolytic and proteolytic properties of mu-calpain under physiological conditions demonstrate how it is precisely attuned to avoid uncontrolled proteolytic activity under normal circumstances, and indicate why it could lead to substantial proteolytic damage if resting or localized [Ca(2+)] is elevated, as is likely to occur after eccentric contraction and in dystrophic muscle.
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Affiliation(s)
- Robyn M Murphy
- Department of Zoology, La Trobe University, Bundoora campus, Melbourne, Victoria 3086, Australia
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34
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Pozdniakovaite N, Popendikyte V. DNA methylation differences in human p14.5 gene promoter region in normal and proliferating cells. Dev Growth Differ 2006; 47:493-9. [PMID: 16179076 DOI: 10.1111/j.1440-169x.2005.00824.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Methylation status of cytosines and its changes during cell proliferation was analyzed in the 5'-flanking region of the human p14.5 gene, which encodes a member of the YER057c/YIL051c/YjgF protein family. We describe evidence of dramatic DNA methylation differences revealed in the study, and present detailed mapping of methylated cytosines (metC) at the 5'-flanking region of the p14.5 gene in several human normal tissues and tumor cells lines. DNA methylation profiles demonstrated aberrant distribution of metC positions with the different degree of methylation along all analyzed 5'-flanking regions of the p14.5 gene in cancer cells. We investigated DNA methylation changes in p14.5 5'-flanking region during cell differentiation by using DNA samples of freshly isolated monocytes and macrophages. According to our data, cellular differentiation processes from monocytes to macrophages are related to the elevated degree of DNA methylation of the p14.5 gene at the putative binding motifs for several transcription factors. The present findings indicate that some cytosines in the promoter region may have some significance in the degree of expression of the p14.5 gene during cell proliferation and cancerogenesis.
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Fernández-Montalván A, Assfalg-Machleidt I, Pfeiler D, Fritz H, Jochum M, Machleidt W. μ-Calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation. Biol Chem 2006; 387:617-27. [PMID: 16740134 DOI: 10.1515/bc.2006.079] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Mu- and m-calpain are cysteine proteases requiring micro- and millimolar Ca2+ concentrations for their activation in vitro. Among other mechanisms, interaction of calpains with membrane phospholipids has been proposed to facilitate their activation by nanomolar [Ca2+] in living cells. Here the interaction of non-autolysing, C115A active-site mutated heterodimeric human mu-calpain with phospholipid bilayers was studied in vitro using protein-to-lipid fluorescence resonance energy transfer and surface plasmon resonance. Binding to liposomes was Ca2+-dependent, but not selective for specific phospholipid head groups. [Ca2+]0.5 for association with lipid bilayers was not lower than that required for the exposure of hydrophobic surface (detected by TNS fluorescence) or for enzyme activity in the absence of lipids. Deletion of domain V reduced the lipid affinity of the isolated small subunit (600-fold) and of the heterodimer (10- to 15-fold), thus confirming the proposed role of domain V for membrane binding. Unexpectedly, mutations in the acidic loop of the 'C2-like' domain III, a putative Ca2+ and phospholipid-binding site, did not affect lipid affinity. Taken together, these results support the hypothesis that in vitro membrane binding of mu-calpain is due to the exposed hydrophobic surface of the active conformation and does not reduce the Ca2+ requirement for activation.
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Affiliation(s)
- Amaury Fernández-Montalván
- Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgische Klinik, Ludwig-Maximilians-Universität München, Nussbaumstr. 20, D-80336 München, Germany
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Murphy RM, Snow RJ, Lamb GD. μ-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans. Am J Physiol Cell Physiol 2006; 290:C116-22. [PMID: 16107503 DOI: 10.1152/ajpcell.00291.2005] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
μ-calpain and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that μ-calpain becomes proteolytically active in the presence of 2–200 μM Ca2+. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca2+concentration levels ([Ca2+]i; ∼1 μM) than the levels at which μ-calpain activation occurs. We have demonstrated the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca2+] levels (0, 2.5, 10, and 25 μM). Autolysis of calpain-3 was found to occur across a [Ca2+] range similar to that for μ-calpain, and both calpains displayed a seemingly higher Ca2+sensitivity in human than in rat muscle homogenates, with ∼15% autolysis observed after 1-min exposure to 2.5 μM Ca2+in human muscle and almost none after 1- to 2-min exposure to the same [Ca2+]ilevel in rat muscle. During muscle activity, [Ca2+]imay transiently peak in the range found to autolyze μ-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s “all-out” cycling, n = 8; and 70% V̇o2 peakuntil fatigue, n = 3) on the amount of autolyzed μ-calpain or calpain-3 in human muscle. No significant autolysis of μ-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca2+]ithat occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.
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Affiliation(s)
- Robyn M Murphy
- Dept. of Zoology, La Trobe Univ., Victoria 3086, Australia.
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Abstract
The calpain family of proteases has been implicated in cellular processes such as apoptosis, proliferation and cell migration. Calpains are involved in several key aspects of migration, including: adhesion and spreading; detachment of the rear; integrin- and growth-factor-mediated signaling; and membrane protrusion. Our understanding of how calpains are activated and regulated during cell migration has increased as studies have identified roles for calcium and phospholipid binding, autolysis, phosphorylation and inhibition by calpastatin in the modulation of calpain activity. Knockout and knockdown approaches have also contributed significantly to our knowledge of calpain biology, particularly with respect to the specific functions of different calpain isoforms. The mechanisms by which calpain-mediated proteolysis of individual substrates contributes to cell motility have begun to be addressed, and these efforts have revealed roles for proteolysis of specific substrates in integrin activation, adhesion complex turnover and membrane protrusion dynamics. Understanding these mechanisms should provide avenues for novel therapeutic strategies to treat pathological processes such as tumor metastasis and chronic inflammatory disease.
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Affiliation(s)
- Santos J Franco
- Program in Cellular and Molecular Biology, University of Wisconsin, Madison, WI 53705, USA
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Costelli P, Reffo P, Penna F, Autelli R, Bonelli G, Baccino FM. Ca(2+)-dependent proteolysis in muscle wasting. Int J Biochem Cell Biol 2005; 37:2134-46. [PMID: 15893952 DOI: 10.1016/j.biocel.2005.03.010] [Citation(s) in RCA: 113] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2004] [Revised: 02/24/2005] [Accepted: 03/11/2005] [Indexed: 11/25/2022]
Abstract
Skeletal muscle wasting is a prominent feature of cachexia, a complex systemic syndrome that frequently complicates chronic diseases such as inflammatory and autoimmune disorders, cancer and AIDS. Muscle wasting may also develop as a manifestation of primary or neurogenic muscular disorders. It is now generally accepted that muscle depletion mainly arises from increased protein catabolism. The ubiquitin-proteasome system is believed to be the major proteolytic machinery in charge of such protein breakdown, yet there is evidence suggesting that Ca(2+)-dependent system, lysosomes and, in some conditions at least, even caspases are involved as well. The role of Ca(2+)-dependent proteolysis in skeletal muscle wasting is reviewed in the present paper. This system relies on the activity of calpains, a family of Ca(2+)-dependent cysteine proteases, whose regulation is complex and not completely elucidated. Modulations of Ca(2+)-dependent proteolysis have been associated with muscle protein depletion in various pathological contexts and particularly with muscle dystrophies. Calpains can only perform a limited proteolysis of their substrates, however they may play a critical role in initiating the breakdown of myofibrillar protein, by releasing molecules that become suitable for further degradation by proteasomes. Some evidence would also support a role for lysosomes and caspases in muscle wasting. Thus it cannot be excluded that different intracellular proteolytic systems may coordinately concur in shifting muscle protein turnover towards excess catabolism. Many different signals have been proposed as potentially involved in triggering the enhanced protein breakdown that underlies muscle wasting. How they are transduced to initiate the hypercatabolic response and to activate the proteolytic pathways remains largely unknown, however.
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Affiliation(s)
- Paola Costelli
- Dipartimento di Medicina e Oncologia Sperimentale, Università di Torino, Corso Raffaello 30, 10125 Torino, Italy.
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Mistiniene E, Pozdniakovaite N, Popendikyte V, Naktinis V. Structure-based ligand binding sites of protein p14.5, a member of protein family YER057c/YIL051c/YjgF. Int J Biol Macromol 2005; 37:61-8. [PMID: 16198412 DOI: 10.1016/j.ijbiomac.2005.08.008] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2005] [Revised: 08/23/2005] [Accepted: 08/23/2005] [Indexed: 11/22/2022]
Abstract
Seventeen mutants with one, two or three amino acids substitutions of human protein p14.5, homologue to well-known tumor antigen from goat liver UK114 and a member of proteins YER057c/YIL051c/YjgF family, have been used for structure-functional relation studies and ligand binding analysis using cross-linking by triacryloyl-hexahydro-s-triazine (TAT), size exclusion chromatography, free fatty acid and 8-anilino-1-naphthalenesulfonic acid (ANS) binding assays. Amino acids having the most significant impact on the ligand binding activity have been determined: R107, N93, Y21 and F89. Arginine 107 has been identified as the most accessible amino acid in the cleft. Trimeric structure of protein p14.5 has been confirmed as being essential for stoichiometric small ligand binding activity and oligomeric structure of p14. Ligand binding activity may be related with the biological functions of these proteins, which still are not understood well.
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Affiliation(s)
- Edita Mistiniene
- Institute of Biotechnology, V.A. Graiciuno 8, LT-02241, Vilnius 28, Lithuania
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40
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Kanouchi H, Matsumoto M, Taga M, Yamada K, Oka T, Toné S, Minatogawa Y. Nuclear transfer of perchloric acid-soluble protein by endoplasmic reticulum stressors. Protein Sci 2005; 14:2344-9. [PMID: 16081652 PMCID: PMC2253487 DOI: 10.1110/ps.051481105] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Perchloric acid-soluble protein (PSP) is highly conserved during evolution from bacteria to mammals. Although PSP has been recognized as an inhibitor of translation and proliferation in vitro, its precise biological role has not yet been elucidated. Since we previously found similar distributions for PSP and the endoplasmic reticulum (ER) and Golgi complex, the intracellular distribution of PSP was analyzed in more detail. Immunofluorescence studies indicated that PSP co-localized with the ER and Golgi complex, since the distribution pattern of PSP was well matched to both of these organelles. An immunoelectron microscopic study revealed PSP was located not only in the cytosol but also on the surface of the outer ER membrane. Since PSP was present on the ER, we speculated that it may be associated with ER function. Therefore, we analyzed whether or not the ER stress response, which is one of the ER functions, affected PSP expression. The results showed that various ER stressors (thapsigargin, A23187, tunicamycin, brefeldin A, and cisplatin) provoked a dramatic change in the localization of PSP from outside of the nucleus to inside the nucleus within 3 h. Moreover, the ER stressors induced PSP expression. These results suggest that PSP is involved in the cellular response to ER stressors, and that the change in localization of PSP from the ER to the nucleus may be associated with ER stress responses.
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Affiliation(s)
- Hiroaki Kanouchi
- Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki-city, Okayama, 701-0192, Japan.
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Nicola C, Timoshenko AV, Dixon SJ, Lala PK, Chakraborty C. EP1 receptor-mediated migration of the first trimester human extravillous trophoblast: the role of intracellular calcium and calpain. J Clin Endocrinol Metab 2005; 90:4736-46. [PMID: 15886234 DOI: 10.1210/jc.2005-0413] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
CONTEXT The root cause of preeclampsia in the human lies in the placenta, where a subpopulation of cytotrophoblast cells called extravillous trophoblasts (EVT), known to be involved in the invasion of the uterine endometrium and utero-placental arteries, become less invasive, resulting in poor perfusion of maternal blood into placenta. OBJECTIVES Because EVT migrate into the prostaglandin (PG) E2-rich decidua, we tested the roles of PGE2 and PGE2-mediated signaling in EVT migration, using our well-characterized EVT line HTR-8/Svneo as well as first trimester villus explants in culture. DESIGN mRNA expression of different PGE2 receptors (EPs) in HTR-8/Svneo cells was studied using RT-PCR. To characterize the functional significance of EP receptors in EVT, different EP receptor agonists and antagonists were used in our migration assay systems and in the measurements of intracellular concentration of Ca2+ ([Ca2+]i) and calpain activity. RESULTS Exogenous PGE2 stimulated EVT migration both in vitro and in the villus explant cultures. Although EVT expressed mRNA for all EP receptors (EP 1-4), a functional predominance of EP1 and EP4 was demonstrated in migration assays using specific EP agonists and antagonists. EP1-receptor-mediated signaling events such as activation of phospholipase C and elevation of cytosolic free [Ca2+]i were confirmed by the following findings: 1) exogenous PGE2 or an EP1 agonist, but not an EP4 agonist, increased [Ca2+]i, which could be blocked with an EP1 antagonist as well as BAPTA and thapsigargin; 2) phospholipase C inhibitor U73122, BAPTA, and thapsigargin inhibited PGE2-mediated migratory response of EVT; and 3) PGE2-mediated EVT migration was shown to be dependent on a class of Ca2+-dependent proteases called calpains, known to be involved in cell detachment from substratum during migratory responses. The presence of PGE2 stimulated calpain activity, whereas two calpain inhibitors, calpastatin and N-Ac-Leu-Leu-methioninal (ALLM), blocked EVT migration. CONCLUSION PGE2 stimulates EVT migration by signaling through EP1 receptors, increasing [Ca2+]i, and activating calpain.
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Affiliation(s)
- Catalin Nicola
- Department of Anatomy and Cell Biology, University of Western Ontario, London, Canada N6A 5C1
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42
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Farkas A, Nardai G, Csermely P, Tompa P, Friedrich P. DUK114, the Drosophila orthologue of bovine brain calpain activator protein, is a molecular chaperone. Biochem J 2005; 383:165-70. [PMID: 15250825 PMCID: PMC1134055 DOI: 10.1042/bj20040668] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2004] [Revised: 07/06/2004] [Accepted: 07/13/2004] [Indexed: 11/17/2022]
Abstract
UK114, the goat liver tumour antigen, is a member of a widely distributed family of conserved low-molecular-mass proteins (YER057c/YjgF/UK114), the function of which is ill understood. To the various orthologues diverse functions have been ascribed, such as translation inhibition, regulation of purine repressor or calpain activation. Owing to a limited sequence similarity to Hsp90 (heat-shock protein 90), they have also been proposed to be molecular chaperones; however, this has never been tested. In the present paper, we report the cloning and characterization of the Drosophila orthologue, DUK114. In brief, DUK114 had no effect that would have qualified it as a calpain activator. In contrast, it proved to be a very potent molecular chaperone in in vitro assays. In a heat-aggregation test, it significantly decelerated the formation of citrate synthase aggregates. In a reverse assay, the recovery of the enzyme from urea- and heat-induced denatured states was accelerated almost 3-fold. On a molar basis, the chaperone activity of the 15-kDa DUK114 is comparable with that of Hsp90, the almost 6-times-larger archetypal molecular chaperone. In similar assays, DUK114 was ineffective with Drosophila calpain A or calpain B. To test for its chaperone activity in vivo, DUK114 was transfected into Schneider (S2) cells; after heat shock, the number of viable non-transfected cells started to increase after a lag time; in the presence of DUK114, cell proliferation started at once. Our work is the first experimental evidence that DUK114, and possibly other members of this family, are molecular chaperones.
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Affiliation(s)
- Attila Farkas
- *Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary
| | - Gábor Nardai
- †Department of Medical Chemistry, Semmelweis University, Budapest, Hungary
| | - Peter Csermely
- †Department of Medical Chemistry, Semmelweis University, Budapest, Hungary
| | - Peter Tompa
- *Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary
| | - Peter Friedrich
- *Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary
- To whom correspondence should be addressed (email )
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43
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Zimowska M, Constantin B, Papy-Garcia D, Raymond G, Cognard C, Caruelle JP, Moraczewski J, Martelly I. Novel glycosaminoglycan mimetic (RGTA, RGD120) contributes to enhance skeletal muscle satellite cell fusion by increasing intracellular Ca2+ and calpain activity. J Cell Physiol 2005; 205:237-45. [PMID: 15887234 DOI: 10.1002/jcp.20403] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Glycosaminoglycans (GAG) are classes of molecules that play an important role in cellular processes. The use of GAG mimetics called regenerating agent (RGTA) represents a tool to investigate the effect of GAG moiety on cellular behavior. A first member of the RGTA family (RG1192), a dextran polymers with defined amounts of sulfate, carboxymethyl, as well as hydrophobic groups (benzylamide), was shown to stimulate skeletal muscle repair after damage and myoblast differentiation. To obtain a comprehensive insight into the mechanism of action of GAG mimetics, we investigated the effect on myoblast differentiation of a novel RGTA, named RGD120, which was devoid of hydrophobic substitution and had ionic charge similar to heparin. Myoblasts isolated from adult rat skeletal muscles and grown in primary cultures were used in this study. We found that chronic treatment with RGD120 increased the growth of adult myoblasts and induced their precocious fusion into myotubes in vitro. It also partially overcame the inhibitory effect of the calpain inhibitor N-acetyl-leu-leu-norleucinal (ALLN) on these events. Western blot and zymography analyses revealed that milli calpain was slightly increased by RGD120 chronic treatment. In addition, using fluorescent probes (Indo-1 and Boc-leu-met-MAC), we demonstrated that RGD120 added to prefusing myoblast cultures accelerates myoblast fusion into myotubes, induced an increase of cytosolic free calcium concentration, and concomitantly an increase of intracellular calpain protease activity. Altogether, these results suggested that the efficiency of RGD120 in stimulating myogenesis might be in part explained through its effect on calcium mobilization as well as on the calpain amount and activity.
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MESH Headings
- Animals
- Blotting, Western
- Calcium/metabolism
- Calpain/analysis
- Calpain/metabolism
- Cell Differentiation
- Cell Extracts/pharmacology
- Cell Fusion
- Cells, Cultured
- Dose-Response Relationship, Drug
- Fluorescence
- Glycosaminoglycans/chemistry
- Glycosaminoglycans/pharmacology
- Immunohistochemistry
- Male
- Molecular Mimicry
- Muscle, Skeletal/cytology
- Myoblasts, Skeletal/cytology
- Myoblasts, Skeletal/drug effects
- Myoblasts, Skeletal/physiology
- Rats
- Rats, Wistar
- Satellite Cells, Skeletal Muscle/cytology
- Spectrometry, Fluorescence
- Time Factors
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Affiliation(s)
- M Zimowska
- Department of Cytology, Faculty of Biology, Warsaw University, Poland
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Pozdniakovaite N, Popendikyte V. Identification of differentially expressed genes in yeast Saccharomyces cerevisiae cells with inactivated Mmf1p and Hmf1p, members of proteins family YERO57c/YJGF. Dev Growth Differ 2004; 46:545-54. [PMID: 15610144 DOI: 10.1111/j.1440-169x.2004.00771.x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
We used differential display analysis of mRNA to investigate the differences between gene expression in wild-type (wt) yeast Saccharomyces cerevisiae cells and mutated ones with disrupted activity of genes MMF1 and HMF1, members of the YERO57c/YJGF family. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to determine the differences in the degree of expression of 14 specific transcripts in normal and mutated yeast cells. Obtained data demonstrate that disruption of genes encoding proteins Mmf1p, Hmf1p (or both of them) result in the correlative variation of expression level of the target 12 genes both in the cells with changed phenotype (mmf1 and mmf1 hmf1) and in the cells retaining w.t. shape and growth rate (wt cells, hmf1). Metabolic processes and cellular pathways have been indicated for Mmf1p and Hmf1p based on the different profiles of the expression of 14 genes in mmf1, hmf1 yeast S. cerevisiae cells.
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45
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Friedrich P. The intriguing Ca2+ requirement of calpain activation. Biochem Biophys Res Commun 2004; 323:1131-3. [PMID: 15451413 DOI: 10.1016/j.bbrc.2004.08.194] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2004] [Indexed: 10/26/2022]
Abstract
Mammalian ubiquitous micro- and m-calpains, as well as their Drosophila homologs, Calpain A and Calpain B, are Ca(2+)-activated cytoplasmic proteases that act by limited proteolysis of target proteins. Calpains are thought to be part of many cellular signaling pathways. These enzymes, however, require such high Ca(2+) concentration for half-maximal activation in vitro, [Ca(2+)](0.5), that hardly ever occurs in intact cells. This major dilemma has pervaded the literature on calpains for decades. In this paper several considerations are put forward that challenge the orthodox view and envisage mechanisms that may govern calpain action in vivo. The "unphysiologically" high Ca(2+) demand for activation may turn out to be an evolutionarily adjusted safety device.
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Affiliation(s)
- Peter Friedrich
- Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 7, H-1518 Budapest, Hungary.
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46
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Friedrich P, Tompa P, Farkas A. The calpain-system of Drosophila melanogaster: coming of age. Bioessays 2004; 26:1088-96. [PMID: 15382138 DOI: 10.1002/bies.20106] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Drosophila melanogaster is one of the most popular and powerful model organisms that help our understanding of mammalian (human) life processes at the molecular level. Calpains are Ca(2+)-activated cytoplasmic proteases thought to play multiple roles in intracellular signal processing by limited proteolysis of target substrate proteins, thereby changing their function. The calpain superfamily consists of 14 genes in mammals, but only 4 genes in Drosophila. One may assume that the calpain system, i.e. recognizing calpain-dependent life processes and identifying the substrates cleaved while exerting their functions, would prove easier to solve in Drosophila than in mammals. Recently, major progress has been made in characterizing Drosophila Calpain A, Calpain B and Calpain C. The fourth member, Calpain D (or SOL), was analyzed earlier. At this juncture, it seems justifiable to summarize our knowledge about the Drosophila enzymes, in comparison to the ubiquitous mammalian ones, as regards structure-function relations, mode of activation by Ca(2+) and other factors, inhibition, potential targeting, expression pattern in vivo, etc. Equipped with all this information, we may now embark on the genetic modification of family members, interpreting mutant phenotypes in terms of the cell biology of calpains.
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Affiliation(s)
- Peter Friedrich
- Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary.
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47
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Glading A, Bodnar RJ, Reynolds IJ, Shiraha H, Satish L, Potter DA, Blair HC, Wells A. Epidermal growth factor activates m-calpain (calpain II), at least in part, by extracellular signal-regulated kinase-mediated phosphorylation. Mol Cell Biol 2004; 24:2499-512. [PMID: 14993287 PMCID: PMC355832 DOI: 10.1128/mcb.24.6.2499-2512.2004] [Citation(s) in RCA: 220] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
How m-calpain is activated in cells has challenged investigators because in vitro activation requires near-millimolar calcium. Previously, we demonstrated that m-calpain activation by growth factors requires extracellular signal-regulated kinase (ERK); this enables tail deadhesion and allows productive motility. We now show that ERK directly phosphorylates and activates m-calpain both in vitro and in vivo. We identified serine 50 as required for epidermal growth factor (EGF)-induced calpain activation in vitro and in vivo. Replacing the serine with alanine limits activation by EGF and subsequent cell deadhesion and motility. A construct with the serine converted to glutamic acid displays constitutive activity in vivo; expression of an estrogen receptor fusion construct produces a tamoxifen-sensitive enzyme. Interestingly, EGF-induced m-calpain activation occurs in the absence of increased intracellular calcium levels; EGF triggers calpain even in the presence of intracellular calcium chelators and in calcium-free media. These data provide evidence that m-calpain can be activated through the ERK cascade via direct phosphorylation and that this activation may occur in the absence of cytosolic calcium fluxes.
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Affiliation(s)
- A Glading
- Departments of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA
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48
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Baliova M, Betz H, Jursky F. Calpain-mediated proteolytic cleavage of the neuronal glycine transporter, GlyT2. J Neurochem 2003; 88:227-32. [PMID: 14675166 DOI: 10.1046/j.1471-4159.2003.02192.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The glycine transporter 2 (GlyT2) belongs to the family of Na+/CL--dependent plasma membrane transporters and is localized on the presynaptic terminals of glycinergic neurons. GlyT2 differs from other family members by its extended N-terminal cytoplasmic region. We report that activation of a Ca2+-dependent protease, most likely calpain, in spinal cord synaptosomes or cultured spinal cord neurons, results in partial proteolysis of GlyT2. Regions sensitive to calpain cleavage in vivo are located in the N-terminal and, to a lesser extent, C-terminal regions of the transporter protein. Incubation of a GlyT2 N-terminal fusion protein with spinal cord extract in the presence of calcium followed by protein sequence analysis localized the major N-terminal cleavage site after methionine 156, with a second cleavage site being situated after glycine 164. Interestingly, the size of the N-terminally truncated GlyT2 protein (70 kDa) is similar to that of most other transporter family members, and truncated GlyT2 displayed full transport activity upon expression in HEK293 cells. Our data suggest that Ca2+-triggered proteolysis may contribute to the regulation of GlyT2 trafficking and/or function in the neuronal plasma membrane.
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Affiliation(s)
- Martina Baliova
- Department of Neurobiology, Institute of Molecular Biology, SAS, Bratislava, Slovakia
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49
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De Tullio R, Stifanese R, Salamino F, Pontremoli S, Melloni E. Characterization of a new p94-like calpain form in human lymphocytes. Biochem J 2003; 375:689-96. [PMID: 12882647 PMCID: PMC1223710 DOI: 10.1042/bj20030706] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2003] [Revised: 07/17/2003] [Accepted: 07/25/2003] [Indexed: 11/17/2022]
Abstract
Human circulating PBMC (peripheral blood mononuclear cells) contain three calpain isoforms distinguishable on the basis of their chromatographic properties. Two of these proteases belong to the ubiquitous calpain subfamily, corresponding to the classical mu- and m-calpain forms. The third, which shows peculiar activating and regulatory properties, is an alternatively spliced calpain 3 (p94) form. This new calpain differs from calpain 3 in that it has lost IS1 insertion and exon 15, a lysine-rich sequence regarded as a nuclear translocation signal. PBMC p94-calpain undergoes activation and inactivation without the accumulation of a low-Ca2+-requiring form that is typical of the classical activation processes of mu- and m-calpain. Furthermore, it differs from the ubiquitous forms in that it displays a lower sensitivity to calpastatin. On the basis of these selective properties, it can be postulated that PBMC p94-calpain can be activated in response to specific stimuli that are not effective on the other calpain isoenzymes. The enzyme is preferentially expressed in B- and T-lymphocytes, whereas it is poorly expressed in natural killer cells and almost undetectable in polymorphonuclear cells. This distribution might reflect the specific function of this protease, which is preferentially present in cells devoted to the production of the humoral, rather than to the cellular, immune response.
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Affiliation(s)
- Roberta De Tullio
- Department of Experimental Medicine, Biochemistry Section, and Excellence Center for Biomedical Research, University of Genova Viale Benedetto XV, 1, 16132 Genova, Italy
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Leinala EK, Arthur JSC, Grochulski P, Davies PL, Elce JS, Jia Z. A second binding site revealed by C-terminal truncation of calpain small subunit, a penta-EF-hand protein. Proteins 2003; 53:649-55. [PMID: 14579356 DOI: 10.1002/prot.10453] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
The subunits in calpain and in the related penta-EF-hand (PEF) proteins are bound through contacts between the unpaired EF-hand 5 from each subunit. To study subunit binding further, a tetra-EF-hand 18 kDa N- and C-terminally truncated form of the calpain small subunit was prepared (18k). This protein does not combine with the calpain large subunit to form active calpain, but forms homodimers in solution, as shown by ultracentrifugation. The X-ray structure of the 18k protein in the presence of cadmium was solved to a resolution of 2.0 A. The structure of the monomer is almost identical to the known structure of the calpain small subunit, but the 18k protein forms an oligomer in the crystal by the use of two binding sites. One of these sites is an artefact arising from the C-terminal truncation, but the other is a naturally occurring site that is fully exposed to water in intact purified calpain. The characteristics of this site suggest that it may be important in binding other protein modulators involved in the regulation of calpain and of PEF proteins.
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Affiliation(s)
- E K Leinala
- Department of Biochemistry, Queen's University, Kingston, Ontario, Canada
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