Inhibition of P-glycoprotein-mediated multidrug efflux by aminomethylene and ketomethylene analogs of reversins

Several aminomethylene analogs and a ketomethylene analog of reversins were synthesized in order to evaluate their ability to inhibit P-glycoprotein-mediated drug efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein. These analogs retained good activity compared to cyclosporin A and the original reversins.     

The tandem chain extension aldol reaction used for synthesis of ketomethylene tripeptidomimetics targeting hPEPT1

The rationale for targeting the human di-/tripeptide transporter hPEPT1 for oral drug delivery has been well established by several drug and prodrug cases. The aim of this study was to synthesize novel ketomethylene modified tripeptidomimetics and to investigate their binding affinity for hPEPT1. Three related tripeptidomimetics of the structure H-Phe-ψ[COCH₂]-Ser(Bz)-X_aa-OH were synthesized applying the tandem chain extension aldol reaction, where amino acid derived β-keto imides were stereoselectively converted to α-substituted γ-keto imides. In addition, three corresponding tripeptides, composed of amide bonds, were synthesized for comparison of binding affinities. The six investigated compounds were all defined as high affinity ligands (K_i-values <0.5 mM) for hPEPT1 by measuring the concentration dependent inhibition of apical [¹⁴C]Gly-Sar uptake in Caco-2 cells. Consequently, the ketomethylene replacement for the natural amide bond and α-side chain modifications appears to offer a promising strategy to modify tripeptidic structures while maintaining a high affinity for hPEPT1.     

Synthesis of ketomethylene amino pseudopeptide analogues via reductive amination of glyoxals derived from alpha-amino acids

The reductive amination of an amino acid derived glyoxal, with the free amino group of a protected amino acid or oligopeptide fragment, has been developed as a simple and efficient method for the preparation of ketomethylene amino pseudo-oligopeptide isosteres Aa psi(COCH2NH)Aa. Trichlorosilane-DMF is the reagent of choice for the reduction.     

Comparison of calpain I and calpain II from carp muscle

1. The content of calpain II is 3.4 times more than that of calpain I when estimated by the elution profiles from a column of DEAE-cellulose. 2. Calpain I required 1 mM Ca2+ and calpain II required 5 mM Ca2+ to show the full activities. These data demonstrated that Ca2+-sensitivities of both calpains were lower than those of mammalian calpains, respectively. 3. The optimum caseinolytic activity was pH 7.2 for calpain I and pH 7.5 for calpain II. 4. The molecular weight of calpain I was estimated to be 110 k and that of calpain II to be 120 k by gel filtration. 5. Calpain I was much more heat-stable than calpain II around 50-60 degrees C. 6. Both calpains were sensitive to calpastatin, an endogenous inhibitor for calpain.     

Loss of calpain 10 causes mitochondrial dysfunction during chronic hyperglycemia

We showed that renal calpain 10, a mitochondrial and cytosolic Ca(2+)-regulated cysteine protease, is specifically decreased in kidneys of diabetic rats and mice, and is associated with diabetic nephropathy. The goals of this study were to examine renal calpain 10 and mitochondrial dysfunction in streptozotocin-induced hyperglycemic rats and determine the effects of siRNA-mediated knock down of renal calpain 10 on mitochondrial function. Four weeks after streptozotocin injection, calpain 10 protein and mRNA were decreased and calpain 10 substrates accumulated. We detected increased state 2 respiration in isolated renal mitochondria and increased markers of mitochondrial fission and mitophagy. All changes were prevented by daily insulin injection. Compared to scrambled siRNA, calpain 10 siRNA resulted in a marked decrease in renal calpain 10 at 2, 5 and 7 days. In concert with the loss of renal calpain 10, calpain 10 substrates accumulated, mitochondrial fusion decreased, mitochondrial fission and mitophagy increased. In summary, insulin-sensitive hyperglycemia induced loss of renal calpain 10 is correlated with renal mitochondrial dysfunction, fission and mitophagy, and specific depletion of renal calpain 10 produces similar mitochondrial defects. These results provide evidence that diabetes-induced renal mitochondrial dysfunction and renal injury may directly result from the loss of renal calpain 10.     

Interaction of human calpains I and II with high molecular weight and low molecular weight kininogens and their heavy chain: mechanism of interaction and the role of divalent cations

Calpain I prepared from human erythrocytes was half-maximally and maximally activated at 23 and 35 microM calcium ion, and two preparations of calpain II from human liver and kidney were half-maximally activated at 340 and 220 microM calcium ion and maximally activated at 900 microM calcium ion, respectively. High molecular weight (HMW) and low molecular weight (LMW) kininogens isolated from human plasma and the heavy chain prepared from these proteins inhibited calpain I as well as calpain II. The molar ratios of calpains to HMW kininogen to give complete inhibition of calpains were 1.4 for calpain I and 2.0 for calpain II, and those of calpains to heavy chain were 0.40-0.66 for calpain I and 0.85 for calpain II. LMW kininogen did not completely inhibit the calpains even with an excess amount of kininogen. The apparent binding ratio of calpain to HMW kininogen estimated from the disc gel electrophoretic analysis, however, was found to be 2:1, whereas those of calpain to LMW kininogen and of calpain to heavy chain were found to be 1:1. Calpains and kininogens failed to form complexes in the absence of calcium ion. In the presence of calcium ion, however, they formed the complexes, which were dissociable by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The minimum concentrations of calcium ion required to induce complex formation between calpain I and kininogens and calpain II and kininogens were 70 and 100 microM, respectively. Some other divalent cations such as Mn2+, Sr2+, and Ba2+ were also able to induce the complex formation between calpains and kininogens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic alpha,beta-unsaturated esters

The proteolytic processing of polyproteins by the 3CL protease of severe acute respiratory syndrome coronavirus is essential for the viral propagation. A series of tripeptide alpha,beta-unsaturated esters and ketomethylene isosteres, including AG7088, are synthesized and assayed to target the 3CL protease. Though AG7088 is inactive (IC50 > 100 microM), the ketomethylene isosteres and tripeptide alpha,beta-unsaturated esters containing both P1 and P2 phenylalanine residues show modest inhibitory activity (IC50 = 11-39 microM). The Phe-Phe dipeptide inhibitors 18a-e are designed on the basis of computer modeling of the enzyme-inhibitor complex. The most potent inhibitor 18c with an inhibition constant of 0.52 microM is obtained by condensation of the Phe-Phe dipeptide alpha,beta-unsaturated ester with 4-(dimethylamino)cinnamic acid. The cell-based assays also indicate that 18c is a nontoxic anti-SARS agent with an EC50 value of 0.18 microM.     

Detection and some properties of calpain II (high-Ca2+-requiring form of calpain) in carp (Cyprinus carpio) erythrocytes

In order to examine the existence of calpain I, a low (micromolar)-Ca2+-requiring form of calpain, in fish tissues, carp erythrocytes were chosen as the experimental material, since only calpain I is known to exist in mammalian erythrocytes. By DEAE-cellulose chromatography, calpain and calpastatin (specific inhibitor for calpain) were separated from carp erythrocyte hemolysate. Carp erythrocyte calpain is classified as calpain II, a high (millimolar)-Ca2+-requiring form of calpain, from the result of Ca2+-requirement for the activity.     

Mitochondrial calpain 10 activity and expression in the kidney of multiple species

Calpains, Ca²⁺-activated cysteine proteases, have been implicated in the progression of multiple disease states. We recently identified calpain 10 as a mitochondrial calpain that is involved in Ca²⁺-induced mitochondrial dysfunction. The goals of this study were to characterize the expression and activity of renal mitochondrial calpain 10 in rabbit, mouse, and rat. Using shRNA technology and immunoblot analysis three previously postulated splice variants of calpain 10 were identified (50, 56, and 75 kDa). SLLVY-AMC zymography and immunoblot analysis was used to directly link calpeptin-sensitive calpain activity to calpain 10 splice variants. Rabbit, mouse, and rat kidney mitochondria contained 75 kDa (calpain 10a), 56 kDa (calpain 10c or 10d), and 50 kDa (calpain 10e) splice variants. Interestingly, zymography yielded distinct bands of calpain activity containing multiple calpain 10 splice variants in all species. These results provide evidence that several previously postulated splice variants of calpain 10 are localized to the mitochondria in kidneys of rabbits, rats, and mice.     

Activation of calpain I and calpain II: a comparative study using terbium as a fluorescent probe for calcium-binding sites

The present study demonstrates the activation of calpain I and calpain II by micromolar levels of terbium and has utilized the enhancement in the fluorescence of protein-bound terbium to study and compare the calcium binding sites of the two enzymes. Calpain I and calpain II were isolated from bovine erythrocytes and brain, respectively. While the rates of activation of calpain I by terbium and calcium are comparable, the rate of activation of calpain II was much greater in the presence of terbium than in the presence of calcium. Binding of terbium ions to calpains was monitored by the enhanced terbium fluorescence and by the changes in the intrinsic protein fluorescence of calpains. Stoichiometric titrations indicated that calpain I and calpain II bound four and six molar equivalents of terbium ion, respectively. During the titration, the intrinsic protein fluorescence of calpain II was successively quenched whereas that of calpain I showed an abrupt drop just prior to the saturation. The association constants (Ka) increased from 10(5) to 10(7) M-1 for calpain I and from 10(4) to 10(6) M-1 for calpain II with addition of increasing molar equivalents of terbium. Titration of enzymatic activities with calcium showed that the activation of calpain I required fewer molar equivalents of metal ions than were necessary for the activation of calpain II, in agreement with stoichiometric titration with terbium.     

Early activation and redistribution of calpain activity in skeletal muscle during hindlimb unweighting and reweighting

The aims of this study were the following: (i) to determine whether activation of the Ca2+-activated protease, calpain, is an early event during hindlimb unweighting (HU) in skeletal muscle; and (ii) to assess whether calpain activity is greater during reweighting compared with HU alone. Rats were exposed to 12, 24, and 72 h, or 9 d of HU, followed by reweighting for 0, 12, or 24 h. Calpain activities were assayed for total, soluble, and particulate fractions. Total calpain activity was increased in the soleus at all HU time points, whereas activities were elevated in the gastrocnemius only after 9 d of HU. With reweighting, calpain activity remained elevated at all time points for both muscles. In general, reweighting the gastrocnemius increased its calpain activity more than during HU only, whereas reweighting the soleus did not produce additional increases in its calpain activity. The increases in calpain activity were associated with a proportional increase in activity of the particulate (membrane- and protein-associated) fraction. The results suggest that calpain activation is an early event during HU in the soleus, and that the increases in calpain activity in both muscles are associated with a redistribution of activity from cytosolic to particulate fractions.     

Characteristics of mitochondrial calpains

Calpains are considered to be cytoplasmic enzymes, although several studies have shown that calpain-like protease activities also exist in mitochondria. We partially purified mitochondrial calpain from swine liver mitochondria and characterized. Only one type of mitochondrial calpain was detected by the column chromatographies. The mitochondrial calpain was stained with anti-mu-calpain and calpain small subunit antibodies. The susceptibility of mitochondrial calpain to calpain inhibitors and the optimum pH differ from those of cytosolic mu- and m-calpains. The Ca(2+)-dependency of mitochondrial calpain was similar to that of cytosolic mu-calpain. Therefore, we named the protease mitochondrial mu-like calpain. In zymogram analysis, two types of caseinolytic enzymes existed in mitochondria and showed different mobilities from cytosolic mu- and m-calpains. The upper major band was stained with anti-mu-calpain and calpain small subunit antibodies (mitochondrial calpain I, mitochondrial mu-like calpain). The lower band was stained only with anti-calpain small subunit antibody (mitochondrial calpain II, unknown mitochondrial calpain). Calpastatin was not detected in mitochondrial compartments. The mitochondrial calpain processed apoptosis-inducing factor (AIF) to truncated AIF (tAIF), releasing tAIF into the intermembrane space. These results indicate that mitochondrial calpain, which differs from mu- and m-calpains, seems to be a ubiquitous calpain and may play a role in mitochondrial apoptotic signalling.     

Glutamate Neurotoxicity Is Independent of Calpain I Inhibition in Primary Cultures of Cerebellar Granule Cells

Glutamate-induced neurotoxicity and calpain activity were studied in primary cultures of rat cerebellar granule neurons and glial cells. Calpain activation, as monitored by quantitative immunoblotting of spectrin, required micromolar concentrations of Ca2+ in neuronal homogenates (calpain I) and millimolar Ca2+ concentrations in glial homogenates (calpain II). Glutamate-induced toxicity and calpain activation were observed in neuronal, but not in glial, cultures. In neurons, calpain I activation by glutamate was dose-dependent and persisted after withdrawal of neurotoxic doses of glutamate. Natural (GM1) and semisynthetic (LIGA4) gangliosides or the glutamate receptor blocker MK-801 prevented calpain I activation and delayed neuronal death elicited by glutamate. GM1 and LIGA4 had no effect on calpain I activity in neuronal homogenates, however. Furthermore, two calpain I inhibitors (leupeptin and N-acetyl-Leu-Leu-norleucinal) prevented glutamate-induced spectrin degradation, but failed to affect glutamate neurotoxicity. These results thus suggest that glutamate-induced neurotoxicity is independent of calpain I activation.     

Investigation of the structural basis of the interaction of calpain II with phospholipid and with carbohydrate.

Two forms of pig kidney calpain II were isolated, both of which appeared to contain an intact 80 kDa large subunit, but which showed specific proteolytic degradation at the N-terminal end of the 30 kDa small subunit. The structure of each of these molecules was investigated by amino acid sequence analysis. The forms corresponded to molecules with small subunits starting at residue 38 (degraded calpain A) and at residue 62 (degraded calpain B) of the complete sequence. These molecules were tested for their ability to interact with phosphatidylinositol and with carbohydrate (agarose gel-filtration media). Calpain and degraded calpain A, but not degraded calpain B, would interact with phosphatidylinositol. Thus the sequence (G)17TAMRILG (residues 38-61) is essential for the interaction. Neither calpain nor the degraded forms of the enzyme showed specific interaction with carbohydrate.     

Identification of both calpains I and II in nucleated chicken erythrocytes

Chicken erythrocytes were found to contain two species of calpains which differ in elution profile from DEAE-cellulose and in Ca2+ requirement. After partial purification, one of them was half-maximally activated by 10 microM Ca2+ and the other by 180 microM Ca2+. The low- and high-Ca2+-requiring proteases cross-reacted only with the respective monospecific antibodies for mammalian calpain I and calpain II, respectively. Approximately 5 times more calpain I than calpain II is present in chicken erythrocytes. By immunoelectrophoretic blot analysis, both calpains I and II from chicken erythrocytes were proved to be heterodimers composed of 76 and 28 kDa, and 80 and 28 kDa subunits, respectively. Our present finding that the heavy subunit of calpain I is smaller than that of calpain II is noteworthy, since the opposite is known to be true of various mammalian calpains. An immunological study has revealed that the calpain I newly found in chicken erythrocytes is not derived from calpain II. Thus, the co-existence of calpains I and II in one animal species also holds in chickens, contrary to the previously advocated notion that chickens have only one type of calpain.     

Changes in contents of calpain and calpastatin in rat liver during growth

Variation of calpain I, calpain II, and calpastatin in rat liver during growth from 0 to 14 weeks was studied by chromatographic fractionation of the liver cytosol and enzyme assays on the eluted fractions. When compared in terms of units per g wet liver, high-Ca2+-requiring calpain II always exceeded low-Ca2+-requiring calpain I in male and female rats. The level of calpain II in neonatal (0 week) rat liver was 1.9-2.9 times higher than that for the adults (7 to 14 weeks). The contents of calpastatin, calpain-specific inhibitor protein, were were always higher than those of calpain II in adult rat liver, but the difference was much less, or sometimes even reversed, in neonatal and young (1 and 2 weeks) animals. In general, the variation was more pronounced in female than in male rats.     

Cloning and molecular characterization of calpain, a calcium-activated neutral proteinase, from different strains of Schistosoma japonicum

cDNA coding for calpain of Schistosoma japonicum were cloned and sequenced, and serological basis of host responses to calpain were analyzed. cDNA of calpain from S. japonicum of two different isolates, Yamanashi strain (Sj-J) and Hunan strain (Sj-C), were 2, 468 bp and 2, 465 bp in length, including the same number (2, 274) of open reading frame. Nucleotide sequence and amino acid sequence between the two calpains are 99.1% and 98.8% identity, respectively. Sj-J and Sj-C calpains were considered to be translated as a preproenzyme, and a 746-amino acid mature enzyme contains eight motifs without a signal peptide at the N-terminal based on the deduced amino acid sequences. mRNA for calpain were detectable in different developmental stages, however, sera obtained from mice immunized with recombinant calpain showed enhanced binding to cercarial antigen. Human sera from S. japonicum-infected individuals recognized the large subunit of schistosomal calpain, and light-infected sera showed stronger reactivities to the recombinant calpain than moderate/high infection cases. When we tested synthetic peptides, there were four common human B cell epitopes in schistosomal calpain, all of which are shared with S. mansoni. Together with these results, calpain of S. japonicum seems to be not only a vaccine candidate, but also a target antigen for immunodiagnosis of human schistosomiasis.     

Characterization of calpain I-binding proteins in human erythrocyte plasma membrane

The calpain-binding components on the plasma membrane were characterized using calpain I. 125I-labeled calpain was bound to inside-out membrane vesicles from human erythrocyte in a Ca2(+)-dependent manner, but not to right-side-out membrane vesicles. The maximum binding was observed at more than 5 microM Ca2+. The binding amount of calpain to the inside-out membrane vesicles was decreased when the vesicles were pretreated with 100 micrograms/ml of trypsin, chymotrypsin, elastase, or pronase P for 30 min at 37 degrees C, although the binding to the vesicles pretreated with 200 micrograms/ml of phospholipase A2 or C was not affected. Calpain-binding proteins in the membrane were analyzed by using a modified immunoblotting for calpain. Immunostained bands of 240, 220, 89, 72, 52, and 36 kDa were detected as the calpain-binding proteins in the native membrane. All of these bands had disappeared in trypsin-treated membrane. The disappearance of bands was dose-dependent with respect to trypsin and paralleled the reduction of binding amount of calpain to the trypsinized membrane. In calpain-treated membrane, the 240 and 36 kDa bands were retained in the blotting, though the other bands disappeared dose-dependently with respect to calpain. These results suggested that the significant component in the inner surface of plasma membrane for binding of calpain was proteinaceous and the calpain-binding proteins could be classified into two species, i.e. substrates of calpain (220, 89, 72, and 52 kDa protein) and non-substrates (240 and 36 kDa protein).     

Distinct regulatory functions of calpain 1 and 2 during neural stem cell self-renewal and differentiation

Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC) self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, β-III Tubulin and glial fibrillary acidic protein (GFAP). Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.     

Schistosoma japonicum: localization of calpain in the penetration glands and secretions of cercariae

A monoclonal antibody was generated against the large subunit of Schistosoma japonicum calpain to study the localization and possible function of the molecule in vivo. Mice were immunized with recombinant S. japonicum calpain and polyclonal antisera and a monoclonal antibody specific to schistosome calpain was obtained. In immunohistochemistry, a monoclonal antibody against S. japonicum calpain, KG-2E11, bound weakly to calpain expressed at the surface of adult worm tegument, however, it bound strongly to the cercarial secretions ("footprints") of S. japonicum, emitted from the penetration glands. The present study indicates that calpain is multifunctional as it is expressed at various locations in different developmental stages. Calpain-based vaccines could thus possibly induce protective immunity against cercariae and the following early developing stages.