Calpain Purification Through Calpastatin and Calcium: Strategy and Procedures.

This chapter describes the strategy and procedures for the calcium-mediated affinity purification of calpain. The affinity capture method exploits the reversible binding properties of calpain's intrinsically disordered protein (IDP) inhibitor, calpastatin. IDPs are easily produced in heterologous expression hosts and purified to homogeneity. Combining these properties with in vivo biotinylation leads to a simplified purification strategy whereby biotinylated human calpastatin domain 1 (hCSD1) can capture calpain efficiently from a complex biological mixture with only a single chromatographic step and in a considerably reduced time. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest in order to capture its binding partner in a calcium- and chelator-based protocol.

Single-Step Purification of Calpain-1, Calpain-2, and Calpastatin Using Anion-Exchange Chromatography.

Purification and separation of calpains and calpastatin are used to determine the individual activities of calpain-1 and calpain-2 and their inhibitor calpastatin. We discuss here a method to purify these enzymes using dialysis followed by separation using anion-exchange chromatography coupled with gradient elution. Swollen DEAE Sephacel is used as the column matrix in this method. Calpastatin and both domains of calpain are weakly basic molecules that effectively bind with the DEAE Sephacel and separate well using a stepwise, increasing gradient of NaCl to elute the proteins. Calpastatin binds most weakly with the column matrix, so it elutes first, followed by calpain-1 and, finally, calpain-2.

Bacterial Expression and Purification of Calpains.

The production of recombinant proteins has been a cornerstone of the study of protein structure and function. As an example, the expression and purification of recombinant rat calpain-2 in bacteria was essential for solving the first crystal structures of the calpains in both calcium-free and calcium-bound forms. Here we describe the production and purification of recombinant rat calpain-2 from Escherichia coli using anion-exchange, affinity, and size-exclusion chromatographies. The heterodimeric enzyme is produced from a stable two-plasmid system. The order in which the protocol is carried out has been optimized to reduce unnecessary concentration and dialysis steps. The typical yield of this multi-domain enzyme from 4 L of E. coli culture is about 20 mg. The production of whole structures for the other calpain family members has been fraught with difficulty. To circumvent this roadblock, a certain amount of structure-function information can be gleaned about these other calpain isoforms by expressing just their protease core. These "mini-calpains" have been useful for X-ray co-crystallography with calpain inhibitors.Here we also present a variation of the whole enzyme production and purification protocol optimized for the expression and purification of the calpain-1 and calpain-3 protease cores (mini-calpains).

FRET-Based Assays to Determine Calpain Activity.

Calpains are signaling proteases that have relatively little sequence specificity but some preferences for certain residues on either side of the scissile bond. As with most proteases, they mainly cut unstructured or extended regions of their target proteins. The tendency for concentrated calpain to rapidly autoproteolyze when activated by calcium complicates the kinetic assessment of calpain activity. As calpain autoproteolyzes, the amount of fully active enzyme continuously decreases until all of the calpain molecules have been cut and their activity reduced to a tiny fraction of the starting rate. To accurately measure calpain kinetics, only the initial rate of substrate hydrolysis, where autoproteolysis is minimal, can be used. To accomplish this, a method for rapid, quantifiable determination of substrate cleavage is required. Many of the existing assays are lacking in their sensitivity to accurately quantify calpain activity within this timeframe. However, the FRET peptide substrates developed by Cuerrier et al. have been shown to have sufficiently high affinity between substrate and enzyme to accurately measure the initial enzyme reaction velocity at substrate concentrations above the Km value. With a suitably sensitive fluorimeter, sufficient data can be obtained to evaluate calpain kinetics and inhibition. Here we describe a facile, reliable calpain assay based on the continuous monitoring of FRET fluorescence from the highly sensitive calpain-specific substrate, (EDANS)-EPLFAERK-(DABCYL). We illustrate some difficulties associated with determining kinetic constants of whole calpains that are simultaneously undergoing autoproteolysis and how the assay can be used to help characterize calpain-specific inhibitors. We also present a variation of this fluorescence-based assay for high-throughput screening using the calpain protease core and a fluorescence plate reader.

Casein Zymography for Analysis of Calpain-1 and Calpain-2 Activity.

Casein zymography is used to detect calpain activity in cell or tissue lysates. In this technique, lysates are loaded into a polyacrylamide gel containing casein, and the enzymes within the lysates are electrophoretically separated. The casein embedded in the gel acts as a substrate for calpains in the lysates, and its degradation reflects the activity of these enzymes. Calpain degradation of the casein is visualized as distinct bands that are devoid of dye when stained with Coomassie Brilliant Blue G-250. We describe here how calpains can be extracted from muscle tissue and assayed for activity using this technique. This technique is also generally applicable to lysates from other types of cells or tissues.

Immunohistochemical Localization of Calpains in the Amphibian Xenopus laevis.

Though histochemical techniques have been used for decades, they are still very important in basic research. They make it possible to work on fixed tissues and provide a large amount of information in a relatively short time and at a low cost. Here we describe methods for indirect immunohistochemistry and immunofluorescence on sections of tadpoles and tissues of adult amphibians belonging to the species Xenopus laevis. The objective is to localize calpains within tissues in order to understand their involvement in cellular processes.

Use of the ß-Glucuronidase (GUS) Reporter System to Localize Promoter Activities of the Endogenous Plant Calpain DEFECTIVE KERNEL1 (DEK1).

The DEFECTIVE KERNEL1 (DEK1) gene encodes the unique plant calpain protein, which is fundamental for cell specification, development, and growth of land plants. The ß-glucuronidase (GUS) reporter gene system has been used to characterize and localize gene expression over several decades. When cloning a promoter upstream of the uidA/GUS reporter gene, you visualize when the promoter is activating expression by monitoring enzymatic activity of GUS, by detecting ß-glucuronidase cleavage products. Here we describe a protocol for monitoring the DEK1 promoter activity in different tissues in Arabidopsis by GUS staining.

Insertion sequence 1 from calpain-3 is functional in calpain-2 as an internal propeptide.

Calpains are intracellular, calcium-activated cysteine proteases. Calpain-3 is abundant in skeletal muscle, where its mutation-induced loss of function causes limb-girdle muscular dystrophy type 2A. Unlike the small subunit-containing calpain-1 and -2, the calpain-3 isoform homodimerizes through pairing of its C-terminal penta-EF-hand domain. It also has two unique insertion sequences (ISs) not found in the other calpains: IS1 within calpain-3's protease core and IS2 just prior to the penta-EF-hand domain. Production of either native or recombinant full-length calpain-3 to characterize the function of these ISs is challenging. Therefore, here we used recombinant rat calpain-2 as a stable surrogate and inserted IS1 into its equivalent position in the protease core. As it does in calpain-3, IS1 occupied the catalytic cleft and restricted the enzyme's access to substrate and inhibitors. Following activation by Ca2+, IS1 was rapidly cleaved by intramolecular autolysis, permitting the enzyme to freely accept substrate and inhibitors. The surrogate remained functional until extensive intermolecular autoproteolysis inactivated the enzyme, as is typical of calpain-2. Although the small-molecule inhibitors E-64 and leupeptin limited intermolecular autolysis of the surrogate, they did not block the initial intramolecular cleavage of IS1, establishing its role as a propeptide. Surprisingly, the large-molecule calpain inhibitor, calpastatin, completely blocked enzyme activity, even with IS1 intact. We suggest that calpastatin is large enough to oust IS1 from the catalytic cleft and take its place. We propose an explanation for why calpastatin can inhibit calpain-2 bearing the IS1 insertion but cannot inhibit WT calpain-3.

In vivo biotinylated calpastatin improves the affinity purification of human m-calpain.

Recently we established a novel affinity purification method for calpain by exploiting the specific and reversible binding properties of its intrinsically disordered protein inhibitor, calpastatin. The immobilization strategy relied on the strength and specificity of the biotin - streptavidin interaction. Here, we report an improved and optimized method that even enables the general applicability of in vivo biotinylated (intrinsically disordered) proteins in any affinity capture strategy. Since in vitro chemical biotinylation is only accomplished with reagents that lack exact site specificity, it can not only cause sample heterogeneity but it can also hamper the functionality of the biotinylated molecules. Therefore, we have developed a recombinant expression protocol to produce in vivo biotinylated human calpastatin domain 1 (hCSD1) in Escherichia coli. We have experimentally verified that the biotinylated polypeptide tag is compatible with the intrinsically disordered state of hCSD1 and that it does not influence the functional properties of this intrinsically disordered protein (IDP). The in vivo biotinylated hCSD1 was then used without the need of any prepurification step prior to the affinity capturing of its substrate, human m-calpain. This leads to a simplified purification strategy that allows capturing the calpain efficiently from a complex biological mixture with only a single chromatogaphic step and in a considerably reduced timeframe. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest, and its practical implementation will showcase the power to exploit the properties of IDPs in affinity capture strategies.

Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin.

Calpains are calcium-activated proteases that have biomedical and biotechnological potential. Their activity is tightly regulated by their endogenous inhibitor, calpastatin that binds to the enzyme only in the presence of calcium. Conventional approaches to purify calpain comprise multiple chromatographic steps, and are labor-intensive, leading to low yields. Here we report a new purification procedure for the human m-calpain based on its reversible calcium-mediated interaction with the intrinsically disordered calpastatin. We exploit the specific binding properties of human calpastatin domain 1 (hCSD1) to physically capture human m-calpain from a complex biological mixture. The dissociation of the complex is mediated by chelating calcium, upon which heterodimeric calpain elutes while hCSD1 remains immobilized onto the stationary phase. This novel affinity-based purification was compared to the conventional multistep purification strategy and we find that it is robust, it yields a homogeneous preparation, it can be scaled up easily and it rests on a non-disruptive step that maintains close to physiological conditions that allow further biophysical and functional studies.

Partial autolysis of µ/m-calpain during post mortem aging of chicken muscle.

The objective of this study was to investigate changes occurring in µ/m-calpain in post mortem chicken muscles and to determine the origin of the unknown bands found in calpain casein zymography. The unknown bands were reported with slightly greater mobility compared to conventional µ/m-calpain bands in casein zymography. Identification of these bands was accomplished using native polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry and with protein phosphatase treatment. Results showed that the unknown bands were corresponding to µ/m-calpain, and dephosphorylation by protein phosphatase did not change their appearance. The calpain samples were then incubated with various concentrations of Ca2+ to determine the relationship between changes in µ/m-calpain and the appearance of the unknown bands. The products of µ/m-calpain partial autolysis were found to be consistent with the appearance of the unknown bands. Therefore, the appearance of these bands did not result from phosphorylation of µ/m-calpain as previously hypothesized, but from partial autolysis of µ/m-calpain. Also their presence suggests that µ/m-calpain undergoes partial autolysis during aging which may play certain roles in meat quality improvement.

Efficient expression and purification of recombinant human µ-calpain using an Escherichia coli expression system.

Calpains comprise a superfamily of Ca(2+) -regulated cysteine proteases that are indispensable for the regulation of various cellular functions. Of these, the mammalian µ- and m-calpains are the best characterized isoforms. They are ubiquitously expressed and form heterodimers consisting of a distinct 80-kDa catalytic subunit (CAPN1 for µ-calpain and CAPN2 for m-calpain) and a common 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for structural and functional studies; however, no low-cost, simple and efficient bacterial expression system for µ-calpain has been available, because the protein forms aggregates. Here, we established an efficient method for producing active recombinant human µ-calpain using an Escherichia coli expression system. This was achieved by co-expressing CAPN1 and CAPNS1 lacking the N-terminal Gly-rich domain (CAPNS1ΔGR) in the SoluBL21 strain. From 1 L of E. coli culture, over 2 and 6 mg, respectively, of µ-calpain and its active-site mutant µ-calpain:C115S (CAPN1:C115S+CAPNS1ΔGR) were purified by two successive column chromatographies. Compared to the native enzyme, the purified µ-calpain showed almost identical properties, demonstrating its suitability for use in structural and functional studies. This is the first report of the bacterial expression and the simple and efficient purification of active recombinant µ-calpain.

Identification of active Plasmodium falciparum calpain to establish screening system for Pf-calpain-based drug development.

BACKGROUND: With the increasing resistance of malaria parasites to available drugs, there is an urgent demand to develop new anti-malarial drugs. Calpain inhibitor, ALLN, is proposed to inhibit parasite proliferation by suppressing haemoglobin degradation. This provides Plasmodium calpain as a potential target for drug development. Pf-calpain, a cysteine protease of Plasmodium falciparum, belongs to calpain-7 family, which is an atypical calpain not harboring Ca2+-binding regulatory motifs. In this present study, in order to establish the screening system for Pf-calpain specific inhibitors, the active form of Pf-calpain was first identified. METHODS: Recombinant Pf-calpain including catalytic subdomain IIa (rPfcal-IIa) was heterologously expressed and purified. Enzymatic activity was determined by both fluorogenic substrate assay and gelatin zymography. Molecular homology modeling was carried out to address the activation mode of Pf-calpain in the aspect of structural moiety. RESULTS: Based on the measurement of enzymatic activity and protease inhibitor assay, it was found that the active form of Pf-calpain only contains the catalytic subdomain IIa, suggesting that Pf-calpain may function as a monomeric form. The sequence prediction indicates that the catalytic subdomain IIa contains all amino acid residues necessary for catalytic triad (Cys-His-Asn) formation. Molecular modeling suggests that the Pf-calpain subdomain IIa makes an active site, holding the catalytic triad residues in their appropriate orientation for catalysis. The mutation analysis further supports that those amino acid residues are functional and have enzymatic activity. CONCLUSION: The identified active form of Pf-calpain could be utilized to establish high-throughput screening system for Pf-calpain inhibitors. Due to its unique monomeric structural property, Pf-calpain could be served as a novel anti-malarial drug target, which has a high specificity for malaria parasite. In addition, the monomeric form of enzyme may contribute to relatively simple synthesis of selective inhibitors.

Expression and purification of recombinant calpain-derived N-terminal peptides from glycine transporter GlyT2.

Glycine transporter GlyT2 contains an extended N-terminal domain which is about three times longer than the N-termini of its closest family members. We previously found that this domain could be separated from the transporter by proteolysis with calpain resulting in the generation of at least two GlyT2N derived peptides. In this work we analyzed the properties of these peptides using bio-informatics, by expressing them in mammalian cell lines and by overexpressing them in bacteria. When expressed in mammalian cell lines, these peptides show widespread localization in the cytoplasm. Their unusually high number of alanine, proline and glycine residues suggests that they posses significant disorder and conformational flexibility, which is supported by their thermal resistivity. Making use of these phenomena, we developed a simple purification method for obtaining pure recombinant GlyT2N derived calpain fragments without using an affinity tag. This procedure can be used to obtain peptide fragments in large amounts for structural, interaction studies or for determining their potential biological activity.

Impacto do tamponamento do CA2+ nuclear na expressão gênica associada à radioresistência em carcinoma de células escamosas de cabeça e pescoço

O Carcinoma de células escamosas de cabeça e pescoço acomete mucosas do trato aerodigestivo superior, incluindo cavidade oral, laringe e faringe e é caracterizado por elevada agressividade e radioresistência, apresentando, muitas vezes, recidivas após o tratamento. Em trabalho anterior, foi demonstrado pelo nosso grupo que o tamponamento do Ca2+ nuclear é capaz de reduzir a taxa proliferação do carcinoma de células escamosas de cabeça e pescoço(células A431), bem como a sobrevida após a irradiação. Neste trabalho, investigamos quais genes estariam envolvidos na radiosensibilização observada mediante tamponamento do Ca2+nuclear, utilizando o protocolo RaSH (rapid subtraction hybridization), que permitiu a identificação de genes diferencialmente expressos nas células apenas irradiadas (RX) e nas células irradiadas em que o Ca2+ nuclear fora tamponado (RX/Ca2+T). Foram sequenciados 91 clones, dos quais 8 mostraram elevada similaridade (93-100%) com sequências conhecidas depositadas no genebank (NCBI). Entre os clones selecionados, 3 foram escolhidos, sendo a Calpaína e a NLK selecionadas como mais expressas na condição RX/Ca2+T e a Kinesina,selecionada na condição RX. Estas proteínas são envolvidas em vias de sinalização críticas ao desenvolvimento tumoral, com base na análise por Blastn. Como a Kinesina foi identificada como superexpressa na condição RX, nós investigamos se seu silenciamento era capaz de produzir efeitos similares aos induzidos pelo tamponamento do Ca2+ nuclear. O silenciamento da Kinesina com siRNA promoveu a redução da formação de colônias e da sobrevida, em associação (90±2%) ou não (60±2%) com RX. Estes dados mostram que a Kinesina é um potencial alvo molecular a ser utilizado na radiosensibilização de carcinoma epidermóide de cabeça e pescoço.

Impacto do tamponamento do CA2+ nuclear na expressão gênica associada à radioresistência em carcinoma de células escamosas de cabeça e pescoço / Impact of nuclear Ca2 + buffering in gene expression associated with radioresistência in squamous cell carcinoma of head and neck

O Carcinoma de células escamosas de cabeça e pescoço acomete mucosas do trato aerodigestivo superior, incluindo cavidade oral, laringe e faringe e é caracterizado por elevada agressividade e radioresistência, apresentando, muitas vezes, recidivas após o tratamento. Em trabalho anterior, foi demonstrado pelo nosso grupo que o tamponamento do Ca2+ nuclear é capaz de reduzir a taxa proliferação do carcinoma de células escamosas de cabeça e pescoço(células A431), bem como a sobrevida após a irradiação. Neste trabalho, investigamos quais genes estariam envolvidos na radiosensibilização observada mediante tamponamento do Ca2+nuclear, utilizando o protocolo RaSH (rapid subtraction hybridization), que permitiu a identificação de genes diferencialmente expressos nas células apenas irradiadas (RX) e nas células irradiadas em que o Ca2+ nuclear fora tamponado (RX/Ca2+T). Foram sequenciados 91 clones, dos quais 8 mostraram elevada similaridade (93-100%) com sequências conhecidas depositadas no genebank (NCBI). Entre os clones selecionados, 3 foram escolhidos, sendo a Calpaína e a NLK selecionadas como mais expressas na condição RX/Ca2+T e a Kinesina,selecionada na condição RX. Estas proteínas são envolvidas em vias de sinalização críticas ao desenvolvimento tumoral, com base na análise por Blastn. Como a Kinesina foi identificada como superexpressa na condição RX, nós investigamos se seu silenciamento era capaz de produzir efeitos similares aos induzidos pelo tamponamento do Ca2+ nuclear. O silenciamento da Kinesina com siRNA promoveu a redução da formação de colônias e da sobrevida, em associação (90±2%) ou não (60±2%) com RX. Estes dados mostram que a Kinesina é um potencial alvo molecular a ser utilizado na radiosensibilização de carcinoma epidermóide de cabeça e pescoço...

The effect of temperature on the activity of µ- and m-calpain and calpastatin during post-mortem storage of porcine longissimus muscle.

The experiment was conducted to determine the effect of temperature during post-mortem muscle storage on the activity of the calpain system, the myofibril fragmentation and the free calcium concentration. Porcine longissimus muscle were incubated from 2h post-mortem at temperatures of 2, 15, 25 and 30 °C and sampling times were at 2, 6, 24, 48 and 120 h post-mortem. After 120 h at 30 °C the free calcium concentration increased to 530 µM from 440 µM at 2 °C. Incubation at temperatures higher than 2 °C resulted in the appearance of autolyzed m-calpain activity and a decrease of native m-calpain activity. Native m-calpain decreased more slowly than native µ-calpain, and the autolysis process started later. Myofibril fragmentation increased with storage time and incubation temperature, while calpastatin activity decreased. The study showed that high temperature incubation not only rapidly activated µ-calpain but at higher temperatures and later time points also m-calpain.

Efficient expression and purification of recombinant human m-calpain using an Escherichia coli expression system at low temperature.

Calpain belongs to the superfamily of Ca(2+)-regulated cysteine proteases, which are indispensable to the regulation of various cellular functions. Of the 15 mammalian calpain isoforms, µ- and m-calpains are the best characterized. Both µ- and m-calpain are ubiquitously expressed and exist as heterodimers, containing a distinct 80-kDa catalytic subunit (CAPN1 and CAPN2, respectively) and the common, 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for use in structural and physiological studies, however Escherichia coli systems have proven incompatible with large-scale preparation of calpain, with the exception of rat m-calpain. Here, we have established a highly efficient method to purify active recombinant human m-calpain using an E. coli expression system at low temperature (22°C). This was achieved by co-expressing CAPN2 with a C-terminal histidine-tag, and CAPNS1, lacking the first Gly-repeated region at the N-terminal. After three sequential passes through a chromatographic column, ~5 mg of human m-calpain was homogenously purified from 1 l of E. coli culture. Proteins were stable for several months. This is the first report of efficient, large-scale purification of recombinant human m-calpain using an E. coli expression system.

m-Calpain-mediated cleavage of Na+/Ca2+ exchanger-1 in caveolae vesicles isolated from pulmonary artery smooth muscle.

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.

A novel Ca2+-activated protease from germinating Vigna radiata seeds and its role in storage protein mobilization.

Calcium (Ca(2+))-dependent/activated proteases make decisive cleavages in proteins, affecting their further degradation/activation. Few such Ca(2+)-dependent proteases have been reported from plants, and none during germination-related events. Seeds are woken up from their quiescent state upon imbibition of water. The subsequent process of germination is strongly influenced by hormones (mainly gibberellins) and light, with both resulting in change in intracellular Ca(2+). We have investigated the effect of Ca(2+) on protease activity in extracts prepared from dry Vigna radiata (L.) Wilczec seeds and cotyledons 4, 24, 48 and 72h post-imbibition. Ca(2+)-activated protease activity is present at a very low level in dry seeds, rises with imbibition and peaks 24h post-imbibition. Subsequently, the activity rapidly declines, even as total protease activity continues to rise. Calcium activation of proteolysis was reversed by ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 1,10, phenanthroline, chlorpromazine and by beta-mercaptoethanol in a concentration-dependent manner. Protease activity was also inhibited by para chloro mercuribenzoate (pCMB) and l-trans-epoxysuccinyl-leucylamido(4-guanidino) butane (E 64), while phenyl methyl sulfonyl fluoride (PMSF) and pepstatin did not effect Ca(2+) activation. The protease could be separated from the calmodulin fraction by size-exclusion chromatography, while retaining its ability for Ca(2+) activation, excluding the possibility of activation through calmodulin-based pathways. The presence of a Ca(2+)-activated protease in the cotyledons suggests its role in a predetermined program of germination involving elevation of cytosolic Ca(2+) levels during germination. This protease could be an important enzyme interfacing cytoplasmic signaling events and initiation of storage protein mobilization during seed germination.