Calcium-dependent Regulator Protein Research Articles

Fluorometric Measurement of Calmodulin-Dependent Peptide-Protein Interactions Using Dansylated Calmodulin.

The assessment of peptide-protein interactions is a pivotal aspect of studying the functionality and mechanisms of various bioactive peptides. In this context, it is essential to employ methods that meet specific criteria, including sensitivity, biocompatibility, versatility, simplicity, and the ability to offer real-time monitoring. In cellular contexts, only a few proteins naturally possess inherent fluorescence, specifically those containing aromatic amino acids, particularly tryptophan. Nonetheless, by covalently attaching fluorescent markers, almost all proteins can be modified for monitoring purposes. Among the early extrinsic fluorescent probes designed for this task, dansyl chloride (DNSC) is a notable option due to its versatile nature and reliable performance. DNSC has been the primary choice as a fluorogenic derivatizing reagent for analyzing amino acids in proteins and peptides for an extended period of time. In our work, we have effectively utilized the distinctive properties of dansylated-calmodulin (D-CaM) for monitoring the interaction dynamics between proteins and peptides, particularly in the context of their association with calmodulin (CaM), a calcium-dependent regulatory protein. This technique not only enables us to scrutinize the affinity of diverse ligands but also sheds light on the intricate role played by calcium in these interactions. Key features • Dynamic fluorescence and real-time monitoring: dansyl-modified CaM enables sensitive, real-time fluorescence, providing valuable insights into the dynamics of molecular interactions and ligand binding. • Selective interaction and stable fluorescent adducts: DNSC selectively interacts with primary amino groups, ensuring specific detection and forming stable fluorescent sulfonamide adducts. • Versatility in research and ease of identification: D-CaM is a versatile tool in biological research, facilitating identification, precise quantification, and drug assessment for therapeutic development. • Sensitivity to surrounding alterations: D-CaM exhibits sensitivity to its surroundings, particularly ligand-induced changes, offering subtle insights into molecular interactions and environmental influences.

Unnatural enzyme activation by a metal-responsive regulatory protein.

As a result of calcium ion binding, the calcium-dependent regulatory protein calmodulin (CaM) undergoes a conformational change, enabling it to bind to and activate a variety of enzymes. However, the detoxification enzyme glutathione S-transferase (GST) is notably not among the enzymes activated by CaM. In this study, we demonstrate the feasibility of establishing, in vitro, an artificial regulatory link between CaM and GST using bifunctional chemical transducer (CT) molecules possessing binders for CaM and GST. We show that the CTs convert the constitutively active GST into a triggerable enzyme whose activity is unnaturally regulated by the CaM conformational state and consequently, by the level of calcium ions. The ability to reconfigure the regulatory function of CaM demonstrates a novel mode by which CTs could be employed to mediate artificial protein crosstalk, as well as a new means to achieve artificial control of enzyme activity by modulating the coordination of metal ions. Within this study, we also investigated the impact of covalent interaction between the CTs and the enzyme target. This investigation offers further insights into the mechanisms governing the function of CTs and the possibility of rendering them isoform specific.

The structure analysis of human MRP14, a calcium-dependent regulator protein in inflammatory process

The structure analysis of human MRP14, a calcium-dependent regulator protein in inflammatory process

Specific Proteins of the Organ of Corti

The mammalian organ of Corti has achieved a degree of perfection unequaled in other hair cell systems. Although cellular metabolism requires the coordinated action of thousands of proteins, the physical processes underlying auditory transduction in the OC are undoubtedly mediated by a much smaller subset of these. OCP1, OCP2, and CBP-15-identified by 2D-PAGE-are apparently members of this elite class. OCP1 and OCP2 are restricted to the supporting cells of the organ of Corti and adjacent epithelia. Their distribution closely parallels the boundaries of the epithelial gap junction system, implying a role in cochlear potassium and pH homeostasis. CBP-15 was recently shown to be identical to oncomodulin, the mammalian /?-parvalbumin, heretofore documented only in the placenta and neoplasms. Expression of this small calcium-binding protein in the OC is restricted to the outer hair cells, where it may function as a calcium-dependent regulatory protein.

Expression of a calmodulin inhibitor peptide in progenitor alveolar type II cells disrupts lung development.

Calmodulin (CaM) is a major intracellular Ca2+ mediator protein involved in cell growth and differentiation. To evaluate calmodulin function in lung, it was necessary to construct a gene that encodes a high-affinity calmodulin binding peptide, since chemically synthesized calmodulin inhibitors lack binding and targeting specificity. This calmodulin inhibitor peptide gene was targeted to type II epithelial cells in transgenic mice using the human surfactant protein C promoter. Neutralization of calmodulin function in progenitor type II epithelial pneumocytes alters epithelial cell growth and differentiation, which prevents branching morphogenesis of the bronchial tree. Newborn transgenic animals have undeveloped lungs. This study indicates that type II lung epithelial cells require functional CaM for proliferation and development. The targeting of specific inhibitor peptides to a single lung cell type is an approach to evaluate the role of calmodulin, the ubiquitous calcium-dependent regulator protein, in lung development and disease.

Activity-dependent short-term enhancement of intercellular coupling

It was reported previously that repeated brief tetanization of the posterior eight nerve can produce long-term homosynaptic potentiations of the electrotonic and chemical components of the mixed EPSP evoked in the Mauthner cell lateral dendrite by a single stimulus to the nerve. We show here that the same stimulus paradigm can lead, alternatively, to short-term enhancements of both excitatory responses. These transient modifications last for approximately 3 min, with a time course similar to post-tetanic potentiation at chemical synapses. However, a different stimulus pattern that transiently increases the presynaptic calcium concentration, paired-nerve stimuli, does not have any significant effect on electrotonic transmission, whereas it facilitates the chemically mediated EPSP. On the other hand, induction of the short-lasting potentiation of coupling, which depended on the discontinuous or burst-like property of the tetanizing paradigm, required NMDA-receptor activation and was blocked by postsynaptic intradendritic injections of the calcium chelator bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid. The ineffectiveness of presynaptic calcium in potentiating electrotonic coupling likely reflects the involvement of a calcium-dependent regulatory protein in the postsynaptic cell and suggests that hemichannels on the two sides of a gap junction plaque can be modified independently. NMDA-mediated modulation of gap junctions could be widespread, because both types of channels coexist during development and in several mammalian adult central nervous system structures such as hippocampus.

Spore swelling inDictyosteliumis a dynamic process mediated by calmodulin

Spore swelling is a necessary prelude to the emergence of amoebae during spore germination in Dictyostelium discoideum. Previous work has shown that the initiation of this event requires the activity of the calcium-dependent regulatory protein calmodulin. In this study, the use of trifluoperazine, an inhibitor of calmodulin function, has shown that calmodulin activity is required throughout the swelling phase. When fully swollen spores were treated with trifluoperazine they rapidly returned to the same size and shape observed prior to swelling. These data suggest that spore swelling in D. discoideum is a dynamic process which is mediated by calmodulin.

Role of calmodulin as an endogenous initiatory factor in compensatory lung growth after pneumonectomy

Compensatory lung growth after pneumonectomy is a well established phenomenon in young humans and experimental animals. To date, the cellular initiating and/or regulatory factor(s) responsible for this growth response have yet to be established. We have studied changes in lung content and activity of calmodulin, a calcium-dependent regulatory protein in relation with lung mass and DNA content during postpneumonectomy compensatory lung growth in 4-week-old rats. We observed that after left pneumonectomy, right lung calmodulin content (measured by radioimmunoassay) and calmodulin activity (measured by cyclic nucleotide phosphodiesterase activation) were increased at days 1 and 2 after surgery in the pneumonectomized rats, preceding the increase of lung mass and DNA content which started at day 3. Treatment of the pneumonectomized rats with a highly specific calmodulin antagonist, trifluoperazine, immediately after the surgery, resulted in diminished lung calmodulin activity, sparing the calmodulin content, and in concomitant reduction of lung mass and DNA content to values intermediate between those of controls and the pneumonectomized animals. Based on these findings, we conclude that calmodulin may be an important intracellular (possibly, autocrine) initiatory or facilitatory factor in compensatory hyperplastic lung growth after pneumonectomy.

Calmodulin and Calmodulin-Binding Proteins during Cell Fusion in Dictyostelium Discoideum: Developmental Regulation by Calcium Ions

The calcium-dependent regulatory protein calmodulin (CaM) mediates diverse cellular functions via a large number of calmodulin-binding and -dependent proteins (CaMBPs). The use of [35S]calmodulin, labeled during its expression (VU-1-CaM) in Escherichia coli, visualized over 25 CaMBPs in Dictyostelium discoideum. Seven, with Mrs of 155,000, 91,000, 85,000, 48,000, 46,000, 38,000, and 28,000, were present only during sexual development. In addition, intracellular calmodulin levels were low during gamete formation but rose during cell fusion in response to the presence of extracellular calcium. Thus, calmodulin appears to mediate gamete formation and fusion through two distinct mechanisms: first, via unique developmentally regulated CaMBPs, and, second, via the regulation of intracellular calmodulin levels. The identification of the CaMBP spectrin in sexually developing Dictyostelium cells suggests that this cytoskeleton/plasma membrane, cross-linking protein may function during biomembrane fusion in D. discoideum as it does in other organisms.

Selective Ca2+-dependent interaction of calmodulin with the head domain of synapsin 1

The calcium-dependent regulatory protein calmodulin is a critical element in the machinery regulating exocytosis at nerve terminals. Okabe & Sobue [(1987) FEBS Lett. 213, 184-188] showed that calmodulin interacts with one of the proteins intimately connected with the neuronal exocytotic process, i.e. synapsin 1. We have investigated the site at which calmodulin interacts with synapsin 1. We find that it is possible to generate chemically cross-linked Ca2(+)-dependent complexes between synapsin 1 and calmodulin in vitro, and have used covalent cross-linking in conjunction with calmodulin affinity chromatography to identify fragments of synapsin 1 that interact with calmodulin. Ca2(+)-dependent calmodulin binding is restricted to the 'head' domain (residues 1-453 in bovine synapsin 1). Within this domain the binding site is located in a unique 11 kDa Staphylococcus aureus V8 proteinase generated fragment. This fragment does not contain the site for cyclic-AMP-dependent phosphorylation and therefore does not represent the N-terminus of the protein.

ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast.

As a first step in understanding the cellular basis of maternal-fetal calcium transfer, we examined the characteristics of calcium uptake by a highly purified preparation of the syncytiotrophoblast basal (fetal facing) plasma membrane. In the presence of nanomolar concentrations of free calcium, basal membranes demonstrated substantial ATP-dependent calcium uptake [K0.5 = 119 nM, maximum velocity (Vmax) = 2 nM X min-1 X mg-1]. This uptake required magnesium, was not significantly affected by Na+ or K+ (50 mM), or sodium azide (10 mM). Intravesicular calcium was rapidly and completely released by the calcium ionophore A23187. Calcium transport was significantly stimulated by the calcium-dependent regulatory protein calmodulin. Placental membrane fractions enriched in endoplasmic reticulum (ER) and mitochondria also demonstrated ATP-dependent calcium uptake. In contrast to basal membrane, mitochondrial calcium uptake was completely inhibited by azide. The rate of calcium uptake by the ER was only 20% of that of basal membranes. We conclude that the placental basal plasma membrane possesses a high-affinity calcium transport system similar to that found in plasma membranes of a variety of cell types. This transporter is situated to permit it to function in vivo in maternal-fetal calcium transfer.

The effects of psychotropic drugs on synthesis of DNA and the infectivity of herpes simplex virus

Several drugs used in the treatment of psychiatric illness have been reported to have an antiviral effect. Perhaps the best known of these is lithium chloride, which is widely used in the treatment of depression, but is also an effective inhibitor of herpes simplex virus I (HSV I) replication (Skinner et al. 1980). However, other psychoactive drugs have also been shown to have antiviral properties, including chlorpromazine (Bohn et al. 1983), atropine (Alarcon et al. 1984), and haloperidol (Wunderlich et al. 1980). These and other studies prompted us to search for a relationship between the therapeutic properties of these drugs and their antiviral potency. This is of interest particularly in view of the viral hypotheses of mental illness (Crow 1978) and the serological evidence that has been put forward to support these hypotheses (Cleobury et al. 1971; Torrey et al. 1982). Several mechanisms for the antiviral activity of phenothiazines have been proposed. Phenothiazines and related drugs are potent inhibitors of calmodulin (Prozialeck and Weiss 1982), the calcium-dependent regulator protein that participates in many cellular calcium-dependent functions, including the activation of cyclic adenosine monophospha~ (c AMP) phosphodiesterase . Krizanova et al. (1982) have postulated that interference with these cellular processes prevents viral replication. They have shown that the inhibition of influenza virus replication in culture with chlo~rom~ine was reversed by the addition of calmodulin. Furthermore, Bohn et al. (1983) have demonstrated a calcium-dependent inhibition of measles virus budding by phenothiazines. Shaskan et al. (1985) have suggested that the dopamine receptor may be a cell surface receptor for herpes simplex virus entry into cells, so that antidopaminergic drugs could compete with virus binding sites. A further possible mechanism for inhibition of virus replication is suggested by the ability of phenothiazines to intercalate with nucleic acids (Allison and Hahn 1977); indeed, chlorpromazine is able to cause the loss of bacterial episomes and inhibition of the DNA polymerase of hepatitis B virus (Allison and Hahn 1977). Finally, the lipophilic nature of phenothiazines could affect adsorption or release of virus by inte~e~ng with the cellular or viral membrane.

Calmodulin and pancreatic B-cell function.

In September-October 1979, two independent reports indicated the presence of the calcium-dependent regulatory protein, calmodulin, in rat pancreatic islet extracts 34'39. The reported values for the islet content in calmodulin averaged 0.11 and 0.13 pmoles per islet. In one of these studies, it was demonstrated that calmodulin binds to a membrane-rich particulate fraction prepared from a rat islet homogenate and causes activation of adenylate cyclase in the same subcellular materiaP 9. Both the specific binding of ~25I-labelled calmodulin and the activation of adenylate cyclase were Ca2+-dependent processes 39. In the other study, the allegedly specific inhibitor of calmodulin, trifluoperazine, was found to inhibit glucose-stimulated insulin release, whilst failing to affect the oxidation of glucose by rat islets 34. These pilot observations thus documented the presence of calmodulin in islet cells and the capacity of calmodulin to affect the activity of a target enzymatic system in the islets, raising the view that calmodulin may be involved in stimulus-secretion coupling in the B-cell. Thereafter, from 1980 onwards, about 30 further publications and three reviews 28'3t'36 were devoted to the same topic. The knowledge gained from these further studies indicates that calmodulin may affect the activity of several enzymes in the islets. However the precise role played by calmodulin in the secretory process remains to be fully elucidated.

Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isoproterenol and cholera toxin.

The role of calcium in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats. Incubation of granulosa cells with various calcium channel blockers (verapamil, cobalt or manganese) and a calcium chelator (EGTA) resulted in marked decreases in progesterone production in response to follicle-stimulating hormone (FSH), cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cyclic AMP (Bt2cAMP). Cyclic AMP production, however, was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogenesis (0.1-1.0 mM and 125 microM, respectively). Two inhibitors of the calcium-dependent regulatory protein, calmodulin [trifluoperazine, 40 microM and 1[bis-(p-chlorophenyl)methyl] 3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy )-phenethyl]imidazolium chloride, ( R24571 ) 20 microM] significantly inhibited both cyclic AMP and progesterone production elicited by these stimulatory agents. Over the concentration range of 62.5 ng/ml-1.0 micrograms/ml, the calcium ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at approximately 0.14 microgram/ml. Maximal steroidogenic response to the calcium ionophore (1 microgram/ml) however, was only 50% of that evoked by FSH (0.33 microgram/ml). A23187 (0.5 microgram/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 microgram/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 microgram/ml). These findings support our earlier suggestion that the calcium-calmodulin system plays a central role in the gonadotropic regulation of ovarian steroidogenesis and suggest that a transmembrane flux of extracellular calcium may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.

Calmodulin localization in bone and cartilage

The localization of calmodulin (CaM), a calcium-dependent regulatory protein, was demonstrated in the following rat skeletal tissues by the indirect immunofluorescence method: a) growth plate cartilage of fetal and juvenile long bones, b) fetal epiphyseal cartilage, c) juvenile hyaline costal cartilage, d) neonatal mandibular condylar cartilage, e) neonatal diaphyseal lamellar bone. CaM was not detected in perichondrial and periosteal cells. Mature and mineralizing chondrocytes demonstrated the highest labelling intensities.

A high-affinity, calmodulin-sensitive (Ca 2+ + Mg 2+)-ATPase and associated calcium-transport pump in the Ehrlich ascites tumor cell plasma membrane

A unique cytoplast preparation from Ehrlich ascites tumor cells ( G. V. Henius, P. C. Laris, and J. D. Woodburn (1979) Exp. Cell. Res. 121, 337–345 ), highly enriched in plasma membranes, was employed to characterize the high-affinity plasma membrane calcium-extrusion pump and its associated adenosine triphosphatase (ATPase). An ATP-dependent calcium-transport system which had a high affinity for free calcium ( K 0.5 = 0.040 ± 0.005 μM) was identified. Two different calcium-stimulated ATPase activities were detected. One had a low ( K 0.5 = 136 ± 10 μM) and the other a high ( K 0.5 = 0.103 ± 0.077 μM) affinity for free calcium. The high-affinity enzyme appeared to represent the ubiquitous high-affinity plasma membrane (Ca 2+ + Mg 2+)-ATPase (calcium-stimulated, magnesium-dependent ATPase) seen in normal cells. Both calcium transport and the (Ca 2+ + Mg 2+)-ATPase were significantly stimulated by the calcium-dependent regulatory protein calmodulin, especially when endogenous activator was removed by treatment with the calcium chelator ethylene glycol bis(β-aminoethyl ether) N, N′-tetraacetic acid. Other similarities between calcium transport and the (Ca 2+ + Mg 2+)-ATPase included an insensitivity to ouabain (0.5 m m), lack of activation by potassium (20 m m) and a requirement for magnesium. These similar properties suggested that the (Ca 2+ + Mg 2+)-ATPase represents the enzymatic basis of the high-affinity calcium pump. The calcium pump/enzyme system was inhibited by orthovanadate at comparatively high concentrations (calcium transport: K 0.5 − 100 μM; (Ca 2+ + Mg 2+)-ATPase: K 0.5 > 100 μM). Upon Hill analysis, the tumor cell (Ca 2+ + Mg 2+)-ATPase failed to exhibit cooperative activation by calcium which is characteristic of the analogous enzyme in the plasma membrane of normal cells.

Calcium-dependent exocytosis in an in vitro secretory granule plasma membrane preparation from sea urchin eggs and the effects of some inhibitors of cytoskeletal function.

Egg cortical granules remain attached to the egg plasma membrane when the egg is ruptured. We present evidence that demonstrates that, when the cytoplasmic face of the egg plasma membrane is exposed to micromolar calcium concentrations, an exocytosis of the cortical granules occurs which corresponds to the cortical granule exocytosis seen when the egg is fertilized. The calcium sensitivity of the preparation is decreased by an increase in magnesium concentration and increased by a decrease in magnesium concentration. Exocytosis is inhibited by trifluoperazine (half inhibition at 6 microM), a drug that inhibits the action of the calcium-dependent regulatory protein calmodulin. Colchicine, vinblastine, nocodazole, cytochalasin B, phalloidin, N-ethylmaleimide-modified myosin subfragment 1, and antibody to actin are without effect on this in vitro exocytosis at concentrations that far exceed those required to disrupt microtubules and microfilaments. Conditions are such that penetration to the exocytotic site is optimal. It is unlikely, therefore, that either actin or tubulin participate intimately in exocytosis. Our data also exclude on quantitative grounds several other mechanisms postulated to account for the fusion of the secretory granule with the plasma membrane.

Inhibition of Cytokinin-Regulated Responses by Calmodulin-Binding Compounds

Two classes of compounds which bind to calmodulin in a calcium-dependent manner (neuroleptic drugs and local anaesthetics) were used to investigate the possible involvement of a calcium-dependent regulator protein in the action of the plant hormones cytokinins.The cytokinin-induced synthesis of betacyanin in Amaranthus tricolor seedlings was used as one test system. The calmodulin antagonists inhibited betacyanin synthesis with the following order of potency: fluphenazine > trifluoperazine = pimozide > chlorpromazine > dibucaine > penfluridol > haloperidol > tetracaine, over a concentration range (IC(50)) of 0.1 to 0.6 millimolar. Red light and fusicoccin increase betacyanin accumulation and are synergistic with cytokinins. These red light- and fusicoccin-dependent inductions were inhibited by lower concentrations of the drugs than cytokinin-dependent induction, and the order of potency of the drugs was not precisely the same. The results are compatible with the hypothesis that cytokinins may act, at least in part, by changing ion fluxes, with the additional involvement of a calmodulin.The second cytokinin-regulated response studied was growth in soybean callus culture. In this system, inhibition was observed with lower concentrations of drugs than in cytokinin-dependent betacyanin synthesis with an order of inhibitory potency of pimozide = trifluoperazine > penfluridol > chlorpromazine > haloperidol > tetracaine. The effective concentration range (IC(50)) was from 0.07 to 0.5 millimolar.Inhibition of betacyanin synthesis by 0.15 millimolar trifluoperazine and of soybean callus growth by 2 millimolar tetracaine were both reversible.

Drugs that block calmoduLin activity inhibit cell-to-cell coupling in the epidermis of Tenebrio molitor.

In many cell systems, the permeability of membrane junctions is modulated by the cytoplasmic level of free Ca++. To examine whether the calcium-dependent regulatory protein calmodulin is involved in this process, the ability of anticalmodulin drugs to influence the cell-to-cell passage of injected current and an organic tracer was tested using standard intracellular glass microelectrode techniques. Several antipsychotics and local anesthetics were found to block junctional communication in the epidermis of the beetle Tenebrio molitor. Treatment of the epidermis with chlorpromazine (0.25 mM) raised intercellular resistance two- to threefold within 20 to 25 min; cell-to-cell passage of electrical current was abolished within 41 +/- 5 min. Loss of electrotonic coupling was accompanied by a block in the cell-to-cell movement of the organic tracer carboxyfluorescein. The reaction is fully reversible, with normal electrotonic coupling being restored within 2 to 4 hr. Other antipsychotics and local anesthetics had similar effects on cell coupling. The order of potency found was: trifluoperazine greater than thioridazine greater than D-butaclamol greater than chlorprothixine = chlorpromazine greater than L-butaclamol greater than dibucaine greater than tetracaine. The relative uncoupling potencies of these drugs correlate well with their known ability to inhibit calmodulin-dependent phosphodiesterase activity. Other anesthetic compounds, procaine and pentobarbital, did not block cell-to-cell communication. Altering the extracellular Ca++ concentration did not affect the rate of uncoupling by antipsychotics, while chelation of extracellular Ca++ with EGTA raised electrotonic coupling. The effect of three metabolic inhibitors on coupling was also examined. Iodoacetate uncoupled the epidermal cells while DNP and cyanide did not. These results are discussed in terms of possible mechanisms by which calmodulin may control junctional communication in this tissue.

Assay of calmodulin with Bordetella pertussis adenylate cyclase

Low levels of the calcium-dependent regulator protein, calmodulin, may be measured utilizing membranes prepared from Bordetella pertussis which contain and adenylate cyclase which is activated by this protein. The activation is dose dependent and tissue levels of calmodulin can be determined over a range from 2 pg to 100 ng with good reliability. We demonstrate how this bioassay may be employed to measure the levels of calmodulin in a variety of protein and cellular preparations.