The expression of calpain 1 and calpain 2 in spermatogenic cells and spermatozoa of the mouse

There is some evidence suggesting that Ca2+is involved in processes that occur during the development and function of spermatozoa. Calcium-dependent proteins, such as calmodulin, are expressed during mammalian spermatogenesis further suggesting that Ca2+takes part in its regulation. However, the precise roles of Ca2+in spermatogenesis remain to be elucidated. Calpains are a family of Ca2+-dependent cysteine proteases whose members are expressed ubiquitously or in a tissue-specific manner. Calpain has been demonstrated to mediate specific Ca2+-dependent processes including cell fusion, mitosis and meiosis. We herein followed the expression pattern of calpain’s ubiquitous isoforms, 1 and 2, throughout spermatogenesis at the RNA and protein levels by RT-PCR and Western blotting analysis. Both RNA and protein studies revealed that these isoforms are expressed in all spermatogenic cells. The expression of calpain 1 levels is slightly higher in spermatocytes entering the meiotic phase. Both calpain isoforms are also expressed in mouse spermatozoa and are localized to the acrosomal cap. Inducing capacitated spermatozoa to undergo the acrosome reaction in the presence of a selective calpain inhibitor significantly reduced the acrosome reaction rate in a dose-dependent manner. Thus, calpain, a pluripotential protease with numerous substrates, may serve as an effector in more than one pathway in the complex process of spermatogenesis and in the events preceding fertilization, such as the acrosome reaction.

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The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Directly Binding the Calpain Small Subunit

Endocrinology ◽

10.1210/en.2004-1637 ◽

2005 ◽

Vol 146 (5) ◽

pp. 2336-2344 ◽

Cited By ~ 12

Author(s):

Masako Shimada ◽

Matthew J. Mahon ◽

Peter A. Greer ◽

Gino V. Segre

Keyword(s):

Parathyroid Hormone ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Small Subunit ◽

Hek293 Cells ◽

Calpain Inhibitor ◽

Dependent Manner ◽

Intact Cells ◽

Calcium Dependent

Abstract We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes ΔNt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of ΔNt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.

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Conventional calpains and programmed cell death.

Acta Biochimica Polonica ◽

10.18388/abp.2011_2238 ◽

2011 ◽

Vol 58 (3) ◽

Cited By ~ 32

Author(s):

Paulina Łopatniuk ◽

Jacek M Witkowski

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

System Development ◽

Cysteine Proteases ◽

Drug Induced ◽

Calcium Dependent ◽

Immune System Development ◽

Apoptotic Pathways ◽

Induced Apoptosis ◽

And Function

The evidence on the crucial role of a family of calcium-dependent cysteine proteases called calpains in programmed cell death is rich and still growing. However, understanding of the mechanisms of their functions in apoptosis is not full yet. Calpains have been implicated in both physiological and pathological cell death control, especially in various malignancies, but also in the immune system development and function. There is also growing evidence on calpain involvement in apoptosis execution in certain pathological conditions of the central nervous system, in cardiovascular diseases, etc. Understanding of the clinical significance of calpain activation pathways, after intense studies of the influence of calpain activity on drug-induced apoptosis, seems especially important lately, as calpains have become noticed as potential therapeutic targets. To allow pharmacological targeting of these enzymes, thorough knowledge of their patterns of activation and further interactions with already known apoptotic pathways is necessary. A comprehensive summary of both well established and recently obtained information in the field is an important step that may lead to future advances in the use of calpain-targeted agents in the clinic.

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Rab coupling protein is selectively degraded by calpain in a Ca2+-dependent manner

Biochemical Journal ◽

10.1042/bj20042116 ◽

2005 ◽

Vol 389 (1) ◽

pp. 223-231 ◽

Cited By ~ 15

Author(s):

Nicolas MARIE ◽

Andrew J. LINDSAY ◽

Mary W. McCAFFREY

Keyword(s):

Plasma Membrane ◽

Membrane Trafficking ◽

Calpain Inhibitor ◽

Dependent Manner ◽

Interacting Proteins ◽

Wild Type ◽

A431 Cells ◽

Calcium Dependent ◽

Coupling Protein ◽

Calpain Inhibitors

RCP (Rab coupling protein) belongs to the recently identified Rab11-FIPs (Rab11 family of interacting proteins). All the Rab-FIP members have the ability to bind Rab11 tightly via a Rab-binding domain located near their C-termini. RCP belongs to the class I Rab11-FIP subfamily, characterized by the presence of a conserved C2 domain near its N-terminus. The function of this protein in Rab11-dependent membrane trafficking remains to be fully understood. In the present study, we have identified three putative PEST (Pro, Glu, Ser/Thr-rich) sequences in RCP. PEST motifs play a role in targeting a protein for proteolytic degradation. We have demonstrated that RCP undergoes calcium-dependent degradation which can be prevented by specific calpain inhibitors. Using a mutant, lacking the three PEST sequences, RCPΔPEST, we demonstrated that they are necessary for the cleavage of RCP by calpains. When expressed in A431 cells, RCPΔPEST displays significantly greater localization to the plasma membrane, compared with the wild-type protein. Similarly, treatment with the calpain inhibitor, calpeptin, results in the redistribution of endogenous RCP to the periphery of the cell. We propose that once the Rab11/RCP-regulated cargo has been delivered from the endocytic recycling compartment to the plasma membrane, RCP is inactivated by calpain-mediated proteolysis.

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Sperm require beta-N-acetylglucosaminidase to penetrate through the egg zona pellucida

Development ◽

10.1242/dev.118.4.1279 ◽

1993 ◽

Vol 118 (4) ◽

pp. 1279-1289 ◽

Cited By ~ 6

Author(s):

D.J. Miller ◽

X. Gong ◽

B.D. Shur

Keyword(s):

Zona Pellucida ◽

Acrosome Reaction ◽

Competitive Inhibitor ◽

Dependent Manner ◽

Surface Receptors ◽

Acrosomal Exocytosis ◽

Sperm Penetration ◽

Glycosidase Inhibitor ◽

Beta Glucosidase ◽

And Function

Fertilization in the mouse is initiated by sperm beta 1,4-galactosyltransferase (GalTase) binding to terminal N-acetylglucosamine residues on the zona pellucida glycoprotein ZP3. Binding of ZP3 induces exocytosis of the sperm acrosome, whose contents are believed to digest a penetration slit in the zona matrix through which sperm reach the egg. As a consequence of acrosomal exocytosis, GalTase is redistributed to the lateral aspect of the sperm head, where its function remains unknown. In this location, GalTase could conceivably impede zona penetration by binding to N-acetylglucosamine residues exposed on zona pellucida glycoproteins. Therefore, in this study we investigated the presence and function of acrosomal glycosidases capable of removing the GalTase-binding site from zona pellucida glycoproteins. beta-N-acetylglucosaminidase was found at very high levels in sperm, being more than 20-fold higher than other glycosidases assayed. The specific isozymic variant was identified as beta-hexosaminidase B. beta-N-acetylglucosaminidase was localized to sperm acrosomes by biochemical and indirect immunofluorescence studies and was released during the acrosome reaction, as expected for an enzyme involved in zona penetration. To determine if, in fact, acrosomal beta-N-acetylglucosaminidase facilitated penetration through the zona, an assay was developed using eggs that were rendered incapable of triggering the block to polyspermy. A specific competitive inhibitor of beta-N-acetylglucosaminidase activity, PUGNAC, inhibited sperm penetration of the zona in a dose-dependent manner, whereas a closely related beta-glucosidase inhibitor, PUGLU, had no effect on zona penetration or on beta-N-acetylglucosaminidase activity. Neither glycosidase inhibitor affected sperm motility or induction of the acrosome reaction. These results demonstrate that beta-N-acetylglucosaminidase is found in sperm acrosomes and is released during the acrosome reaction, at which time it facilitates sperm penetration through the zona. These results also imply that sperm have developed mechanisms to prevent the formation of stable interactions between surface receptors and their zona pellucida ligands during penetration.

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Homocysteine-mediated activation and mitochondrial translocation of calpain regulates MMP-9 in MVEC

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00377.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. H2825-H2835 ◽

Cited By ~ 55

Author(s):

Karni S. Moshal ◽

Mahavir Singh ◽

Utpal Sen ◽

Dorothea Susanne E. Rosenberger ◽

Brooke Henderson ◽

...

Keyword(s):

Extracellular Matrix ◽

Oxidative Burst ◽

Cysteine Proteases ◽

Mitochondrial Pathway ◽

Extracellular Matrix Remodeling ◽

Matrix Remodeling ◽

Dependent Manner ◽

Mitochondrial Translocation ◽

Calcium Dependent

Hyperhomocysteinemia (HHcy) is associated with atherosclerosis, stroke, and dementia. Hcy causes extracellular matrix remodeling by the activation of matrix metalloproteinase-9 (MMP-9), in part, by inducing redox signaling and modulating the intracellular calcium dynamics. Calpains are the calcium-dependent cysteine proteases that are implicated in mitochondrial damage via oxidative burst. Mitochondrial abnormalities have been identified in HHcy. The mechanism of Hcy-induced extracellular matrix remodeling by MMP-9 activation via mitochondrial pathway is largely unknown. We report a novel role of calpains in mitochondrial-mediated MMP-9 activation by Hcy in cultured rat heart microvascular endothelial cells. Our observations suggested that calpain regulates Hcy-induced MMP-9 expression and activity. We showed that Hcy activates calpain-1, but not calpain-2, in a calcium-dependent manner. Interestingly, the enhanced calpain activity was not mirrored by the decreased levels of its endogenous inhibitor calpastatin. We presented evidence that Hcy induces the translocation of active calpain from cytosol to mitochondria, leading to MMP-9 activation, in part, by causing intramitochondrial oxidative burst. Furthermore, studies with pharmacological inhibitors of calpain (calpeptin and calpain-1 inhibitor), ERK (PD-98059) and the mitochondrial uncoupler FCCP suggested that calpain and ERK-1/2 are the major events within the Hcy/MMP-9 signal axis and that intramitochondrial oxidative stress regulates MMP-9 via ERK-1/2 signal cascade. Taken together, these findings determine the novel role of mitochondrial translocation of calpain-1 in MMP-9 activation during HHcy, in part, by increasing mitochondrial oxidative tress.

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Guizhi Fuling Wan, a Traditional Chinese Herbal Formula, Sensitizes Cisplatin-Resistant Human Ovarian Cancer Cells through Inactivation of the PI3K/AKT/mTOR Pathway

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/4651949 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 5

Author(s):

Li Han ◽

Xiaojuan Guo ◽

Hua Bian ◽

Lei Yang ◽

Zhong Chen ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cells ◽

Mtor Pathway ◽

Cell Counting ◽

Ovarian Cancer Cells ◽

Dependent Manner ◽

Bioactive Constituents ◽

Anticancer Efficacy ◽

Protein Levels ◽

And Function

The aim of the study was to explore the possible mechanisms that Guizhi Fuling Wan (GFW) enhances the sensitivity of the SKOV3/DDP ovarian cancer cells and the resistant xenograft tumours to cisplatin. Rat medicated sera containing GFW were prepared by administering GFW to rats, and the primary bioactive constituents of the sera were gallic acid, paeonol, and paeoniflorin analysed by HPLC/QqQ MS. Cell counting kit-8 analysis was shown that coincubation of the sera with cisplatin/paclitaxel enhanced significantly the cytotoxic effect of cisplatin or paclitaxel in SKOV3/DDP cells. The presence of the rat medicated sera containing GFW resulted in an increase in rhodamine 123 accumulation by flow cytometric assays and a decrease in the protein levels of P-gp, phosphorylation of AKT at Ser473, and mTOR in a dose-dependent manner in SKOV3/DDP cells by western blot analysis, but the sera had no effect on the protein levels of PI3K p110αand total AKT. The low dose of GFW enhanced the anticancer efficacy of cisplatin and paclitaxel treatment in resistant SKOV3/DDP xenograft tumours. GFW could sensitize cisplatin-resistant SKOV3/DDP cells by inhibiting the protein level and function of P-gp, which may be medicated through inactivation of the PI3K/AKT/mTOR pathway.

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Calpain 1 and 2 Are Required for RNA Replication of Echovirus 1

Journal of Virology ◽

10.1128/jvi.01375-07 ◽

2007 ◽

Vol 82 (3) ◽

pp. 1581-1590 ◽

Cited By ~ 29

Author(s):

Paula Upla ◽

Varpu Marjomäki ◽

Liisa Nissinen ◽

Camilla Nylund ◽

Matti Waris ◽

...

Keyword(s):

Cysteine Proteases ◽

Host Cells ◽

Calpain Inhibitor ◽

Calpain Activity ◽

Caveolin 1 ◽

Calcium Dependent ◽

Cytoplasmic Proteins ◽

Calpain 2 ◽

Calpain Inhibitors

ABSTRACT Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. Confocal microscopy and immunoelectron microscopy showed that the EV1/caveolin-1-positive vesicles also contain calpain 1 and 2. Our results indicate that calpains are not required for virus entry but that they are important at a later stage of infection. Calpain inhibitors blocked the production of EV1 particles after microinjection of EV1 RNA into the cells, and they effectively inhibited the synthesis of viral RNA in the host cells. Thus, both calpain 1 and calpain 2 are essential for the replication of EV1 RNA.

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Calpain Inhibitors Alter the Excitable Membrane Properties of Cultured Aplysia Neurons

Journal of Neurophysiology ◽

10.1152/jn.90487.2008 ◽

2008 ◽

Vol 100 (5) ◽

pp. 2784-2793 ◽

Cited By ~ 4

Author(s):

Arkady Khoutorsky ◽

Micha E. Spira

Keyword(s):

Calcium Influx ◽

Potassium Conductance ◽

Cysteine Proteases ◽

Excitable Membrane ◽

Membrane Properties ◽

Calpain Inhibitor ◽

Calcium Dependent ◽

Aplysia Neurons ◽

The One ◽

Calpain Inhibitors

The calpain superfamily of calcium-dependent papain-like cysteine proteases constitutes highly conserved proteases that function to posttranslationally modify substrates by partial proteolysis. Calpains are known to proteolyze >100 substrates that lack strong sequence homology. Consequently, the calpain superfamily has been implicated in playing a central role in diverse physiological and pathological processes. Investigation of the physiological functions of calpains, on the one hand, and the need to develop pharmacological reagents to inhibit calpain-mediated pathological processes, on the other hand, led to the development of numerous calpain inhibitors. Using cultured Aplysia neurons and voltage-clamp analysis, we report here that the calpain inhibitors calpeptin, MG132, and the calpain inhibitor XII inhibit voltage-gated potassium conductance and moderately reduce the sodium conductance. These consequently lead to spike broadening and increased calcium influx. Such alterations of the excitable membrane properties may alter the normal patterns of neuronal and muscle electrical activities and thus should be taken into account when evaluating the effects of calpain inhibitors as protective/therapeutic drugs and as research tools.

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Concentric zones of active RhoA and Cdc42 around single cell wounds

The Journal of Cell Biology ◽

10.1083/jcb.200411109 ◽

2005 ◽

Vol 168 (3) ◽

pp. 429-439 ◽

Cited By ~ 196

Author(s):

Hélène A. Benink ◽

William M. Bement

Keyword(s):

Single Cell ◽

Rho Gtpases ◽

Actin Filaments ◽

Xenopus Oocytes ◽

Dependent Manner ◽

Calcium Dependent ◽

Normal Zone ◽

And Function ◽

Distinct Features ◽

Dependent Processes

Rho GTPases control many cytoskeleton-dependent processes, but how they regulate spatially distinct features of cytoskeletal function within a single cell is poorly understood. Here, we studied active RhoA and Cdc42 in wounded Xenopus oocytes, which assemble and close a dynamic ring of actin filaments (F-actin) and myosin-2 around wound sites. RhoA and Cdc42 are rapidly activated around wound sites in a calcium-dependent manner and segregate into distinct, concentric zones around the wound, with active Cdc42 in the approximate middle of the F-actin array and active RhoA on the interior of the array. These zones form before F-actin accumulation, and then move in concert with the closing array. Microtubules and F-actin are required for normal zone organization and dynamics, as is crosstalk between RhoA and Cdc42. Each of the zones makes distinct contributions to the organization and function of the actomyosin wound array. We propose that similar rho activity zones control related processes such as cytokinesis.

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Structural and lipid-binding characterization of human annexin A13a reveals strong differences with its long A13b isoform

Biological Chemistry ◽

10.1515/hsz-2016-0242 ◽

2017 ◽

Vol 398 (3) ◽

pp. 359-371 ◽

Cited By ~ 3

Author(s):

Sara Fernández-Lizarbe ◽

Emilio Lecona ◽

Angélica Santiago-Gómez ◽

Nieves Olmo ◽

María Antonia Lizarbe ◽

...

Keyword(s):

Calcium Binding ◽

Structural Characteristics ◽

Colon Adenocarcinoma ◽

Human Colon ◽

Lipid Binding ◽

Tryptophan Residue ◽

Dependent Manner ◽

Calcium Dependent ◽

Protein Levels ◽

Acidic Phospholipids

Abstract Annexin A13 is the founder member of the vertebrate family of annexins, which are comprised of a tetrad of unique conserved domains responsible for calcium-dependent binding to membranes. Its expression is restricted to epithelial intestinal and kidney cells. Alternative splicing in the N-terminal region generates two isoforms, A13a and A13b, differing in a deletion of 41 residues in the former. We have confirmed the expression of both isoforms in human colon adenocarcinoma cells at the mRNA and protein levels. We have cloned, expressed, and purified human annexin A13a for the first time to analyze its structural characteristics. Its secondary structure and thermal stability differs greatly from the A13b isoform. The only tryptophan residue (Trp186) is buried in the protein core in the absence of calcium but is exposed to the solvent after calcium binding even though circular dichroism spectra are quite similar. Non-myristoylated annexin A13a binds in a calcium-dependent manner to acidic phospholipids but not to neutral or raft-like liposomes. Calcium requirements for binding to phosphatidylserine are around 6-fold lower than those required by the A13b isoform. This fact could account for the different subcellular localization of both annexins as binding to basolateral membranes seems to be calcium-dependent and myristoylation-independent.

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