Ankylosing spondylitis, late osteoarthritis, vascular calcification, chondrocalcinosis and pseudo gout: toward a possible drug therapy.

In this review we consider diseases associated with pathological mineralization/ossification, namely, ankylosing spondylitis (AS), osteoarthritis (OA), generalized artery calcification of infancy (GACI), vascular calcification as well as chondrocalcinosis (CC) and pseudo gout. Deciphering the key enzymes implicated in the calcification process is an objective of prime importance and the ultimate goal is to synthesize inhibitors of these enzymes in order to provide efficient alternate therapeutic strategies that will slow down the pathologic mineralization and complement the arsenal of anti-inflammatory drugs. One of the difficulties in the definition of diseases associated with pathologic mineralization/ossification lies in the controversial relationship between the type of calcification and the nature of the disease. Here, we propose to clarify this relationship by making a distinction between diseases associated with hydroxyapatite (HA) and calcium pyrophosphate dihydrate (CPPD) deposits. AS, OA, GACI and vascular calcification are usually characterized by mineralization/ossification associated with HA deposits, while CC and pseudo gout are mostly characterized by CPPD deposits. Although both HA and CPPD deposits may occur concomitantly, as in chronic pyrophosphate arthritis or in OA with CPPD, they are formed as a result of two antagonistic processes indicating that treatment of distinct diseases can be only achieved by disease-specific drug therapies. The hydrolysis of PPi, an inhibitor of HA formation, is mostly controlled by tissue non-specific alkaline phosphatase TNAP, while PPi production in the extracellular medium is controlled by ANK, a PPi transporter, and/or NPP1 which generates PPi from nucleotide triphosphates. Low PPi concentration may lead to a preferential deposition of HA while high PPi concentration will favor the formation of CPPD deposits. Thus, HA and CCPD deposition cannot occur concomitantly because they are determined by the Pi/PPi ratio which, in turn, depends on the relative activities of antagonistic enzymes, TNAP hydrolyzing PPi or ANK and NPP1 producing PPi. TNAP inhibitors could prevent HA formation in AS, in late OA, in GACI, as well as in vascular calcifications, while ANK or NPP1 inhibitors could slow down CCPD deposition in CC and pseudo gout.

N- and C-terminal halves of human annexin VI differ in ability to form low pH-induced ion channels.

Human recombinant annexin VI (AnxVI) or its N- (AnxVIA) and C-terminal (AnxVIB) fragments were expressed in E. coli. Their ability to form voltage-dependent ion channels in membranes, induced by low pH, was measured to verify the hypothesis that, upon acidification, the hydrophobicity of AnxVI at a specific domain significantly increases allowing the AnxVI interaction with lipids in a Ca(2+)-independent manner. By theoretically analyzing changes in protein hydrophobicity, we found that hydrophobicity of AnxVIA significantly differed from that of AnxVIB at low pH. These predictions were confirmed experimentally by using planar lipid bilayers and liposome pull-down assay. We found striking difference between AnxVIA and AnxVIB in the ion channel activity, as well as in the membrane binding, suggesting that the halves of AnxVI maybe functionally different. Moreover, we calculated and predicted that the ion channel activity at low pH should appear in other human annexins, as AnxII, AnxV (as known), AnxVIII, and AnxXIII. The possibility that AnxVI acts as cytosolic component of a transmembrane pH-sensing mechanism is proposed.

Conformational states of annexin VI in solution induced by acidic pH.

Acidic pH-induced folding of annexin (Anx)VI in solution was investigated in order to study the mechanism of formation of ion channels by the protein in membranes. Using 2-(p-toluidino)naphthalene-6-sulfonic acid as a hydrophobic probe, it was demonstrated that AnxVI exerts a large change in hydrophobicity at acidic pH. Moreover, circular dichroism spectra indicated that the native state of AnxVI changes at acidic pH towards a state characterized by a significant loss of alpha-helix content and appearance of new beta-structures. These changes are reversible upon an increase of pH. It is postulated that the structural folding of AnxVI could explain how a soluble protein may undergo transition into a molecule able to penetrate the membrane hydrophobic region. The physiological significance of these observations is discussed.

UDP hydrolase activity associated with the porcine liver annexin fraction.

In the crude fraction of porcine liver annexins, we identified annexin IV (AnxIV), AnxII and AnxVI of MW (molecular weight) of 32, 36 and 68 kDa, respectively, an albumin of MW of 61.5 kDa and an UDP hydrolase (UDPase) of MW of 62 kDa, related to the human UDPase from Golgi membranes. The latter enzyme exhibits its highest specificity towards UDP and GDP but not ADP and CDP, and it is stimulated by Mg(2+) and Ca(2+). AnxVI itself, although it binds purine nucleotides, does not exhibit hydrolytic activity towards nucleotides. Taken together, these results suggest that AnxVI may interact in vivo with a nucleotide-utilizing enzyme, UDPase. This is in line with observations made by other investigators that various annexins are able to interact with nucleotide-utilizing proteins, such as protein kinases, GTPases, cytoskeletal proteins and p120(GAP). Such interactions could be of particular importance in modulating the biological activities of these proteins in vivo.

Annexins as nucleotide-binding proteins: facts and speculations.

Annexins are ubiquitous multifunctional Ca2+ and phospholipid-binding proteins whose mechanism of function remains largely unknown. The accumulated in vitro experimental evidence indicates that ATP and GTP are functional ligands for nucleotide-sensitive annexin isoforms. Such nucleotide binding could modulate Ca2+ homeostasis, vesicular transport and/or signal transduction pathways and link them to cellular energy metabolism. Alternatively, since annexins are able to interact with other nucleotide-utilizing proteins, such as various kinases, GTPases and structural proteins, these proteins could influence the guanine nucleotide exchange metabolism and/or control the activity of various G proteins. The nucleotide-binding properties of annexins may affect the development or maintenance of some pathologies and diseases in which changes in physiological concentrations of purine nucleotides or disruption of Ca2+ homeostasis are crucial targets.

GTP-binding properties of the membrane-bound form of porcine liver annexin VI.

Annexin VI (AnxVI) of molecular mass 68-70 kDa belongs to a multigenic family of ubiquitous Ca2+- and phospholipid-binding proteins. In this report, we describe the GTP-binding properties of porcine liver AnxVI, determined with a fluorescent GTP analogue, 2'-(or 3')-O-(2,4,6-trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP). The optimal binding of TNP-GTP to AnxVI was observed in the presence of Ca2+ and asolectin liposomes, as evidenced by a 5.5-fold increase of TNP-GTP fluorescence and a concomitant blue shift (by 17 nm) of its maximal emission wavelength. Titration of AnxVI with TNP-GTP resulted in the determination of the dissociation constant (Kd) and binding stoichiometry that amounted to 1.3 microM and 1:1 TNP-GTP/AnxVI, mole/mole, respectively. In addition, the intrinsic fluorescence of the membrane-bound form of AnxVI was quenched by TNP-GTP and this was accompanied by fluorescence resonance energy transfer (FRET) from AnxVI Trp residues to TNP-GTP. This indicates that the GTP-binding site within the AnxVI molecule is probably located in the vicinity of a Trp-containing domain of the protein. By controlled proteolysis of human recombinant AnxVI, followed by purification of the proteolytic fragments by affinity chromatography on GTP-agarose, we isolated a 35 kDa fragment corresponding to the N-terminal half of AnxVI containing Trp192. On the basis of these results, we suggest that AnxVI is a GTP-binding protein and the binding of the nucleotide may have a regulatory impact on the interaction of annexin with membranes, e.g. formation of ion channels by the protein.

Novel function of human RLIP76: ATP-dependent transport of glutathione conjugates and doxorubicin.

Active transport of conjugated and unconjugated electrophiles out of cells is essential for cellular homeostasis. We have previously identified in human tissues a transporter, DNP-SG [S-(2, 4-dinitrophenyl)glutathione] ATPase, capable of carrying out this function [Awasthi et al. (1998) Biochemistry 37, 5231-5238, 5239-5248]. We now report the cloning of DNP-SG ATPase. The sequence of the cDNA clone was identical to that of human RLIP76, a known Ral-binding protein. RLIP76 expressed in E. coli was purified by DNP-SG affinity chromatography. Purified recombinant RLIP76: (1) had ATPase activity stimulated by DNP-SG or doxorubicin (DOX), and the K(m) values of RLIP76 for ATP, DOX, and DNP-SG were similar to those reported for DNP-SG ATPase; (2) upon reconstitution with asolectin as well as with defined lipids, catalyzed ATP-dependent transport of DNP-SG and DOX with kinetic parameters similar to those of DNP-SG ATPase; (3) when transfected into K562 cells, resulted in increased resistance to DOX, and increased ATP-dependent transport of DNP-SG and DOX by inside-out membrane vesicles from transfected cells; (4) direct uptake of purified RLIP76 protein into mammalian cells from donor proteoliposomes confers DOX resistance. These results indicate that RLIP76, in addition to its role in signal transduction, can catalyze transport of glutathione conjugates and xenobiotics, and may contribute to the multidrug resistance phenomenon.

Lipid-binding proteins as stabilizers of membrane microdomains--possible physiological significance.

Below the melting point temperature of lipids, artificial lipid membranes usually exist in the ordered gel phase. Above these temperatures lipid acyl chains become fluid and disordered (liquid-crystalline phase). Depending on the chemical composition of artificial membranes, phase separation may occur, leading to the formation of transient or stable membrane domains. A similar phase separation of lipids into ordered and disordered domains has been observed in natural membranes at physiological temperature range. Moreover, it has been reported that certain proteins prefer certain organization of lipids, as for example glycosylphosphatidylinositol-anchored proteins or Src family of tyrosine kinases. The aim of present review is to discuss the possibility that some lipid microdomains are induced or stabilized by lipid-binding proteins that under certain conditions, for example due to a rise of cytosolic Ca2+ or pH changes, may attach to the membrane surface, inducing clustering of lipid molecules and creation of ordered lipid microdomains. These domains may than attract other cytosolic proteins, either enzymes or regulatory proteins. It is, therefore, postulated that lipid microdomains play important roles within a cell, in signal transduction and enzymatic catalysis, and also in various pathological states, as Alzheimer's disease, anti-phosphatidylserine syndrome, or development of multidrug resistance of cancer cells.

Affinity labeling of annexin VI with a triazine dye, Cibacron blue 3GA. Probable interaction of the dye with C-terminal nucleotide-binding site within the annexin molecule.

Annexin VI (AnxVI) from porcine liver, a member of the annexin family of Ca(2+)- and membrane-binding proteins, has been shown to bind ATP in vitro with a K(d) in the low micromolar concentration range. However, this protein does not contain within its primary structure any ATP-binding consensus motifs found in other nucleotide-binding proteins. In addition, binding of ATP to AnxVI resulted in modulation of AnxVI function, which was accompanied by changes in AnxVI affinity to Ca2+ in the presence of ATP. Using limited proteolytic digestion, purification of protein fragments by affinity chromatography on ATP-agarose, and direct sequencing, the ATP-binding site of AnxVI was located in a C-terminal half of the AnxVI molecule. To further study AnxVI-nucleotide interaction we have employed a functional nucleotide analog, Cibacron blue 3GA (CB3GA), a triazine dye which is commonly used to purify multiple ATP-binding proteins and has been described to modulate their activities. We have observed that AnxVI binds to CB3GA immobilized on agarose in a Ca(2+)-dependent manner. Binding is reversed by EGTA and by ATP and, to a lower extent, by other adenine nucleotides. CB3GA binds to AnxVI also in solution, evoking reversible aggregation of protein molecules, which resembles self-association of AnxVI molecules either in solution or on a membrane surface. Our observations support earlier findings that AnxVI is an ATP-binding protein.

ATP-Binding site of annexin VI characterized by photochemical release of nucleotide and infrared difference spectroscopy.

Structural changes induced by nucleotide binding to porcine liver annexin VI (AnxVI) were probed by reaction-induced difference spectroscopy (RIDS). Photorelease of the nucleotide from ATP[Et(PhNO2)] produced RIDS of AnxVI characterized by reproducible changes in the amide I region. The magnitude of the infrared change was comparable to RIDS of other ATP-binding proteins, such as Ca(2+)-ATPase and creatine and arginine kinases. Analysis of RIDS revealed the existence of ATP-binding site(s) (K(d) < 1 microM) within the AnxVI molecule, comprising five to six amino acid residues located in the C-terminal portion of the protein molecule. The binding stoichiometry of ATP:AnxVI was determined as 1:1 (mol/mol). ATP, in the presence of Ca2+, induced changes in protein secondary structure reflected by a 5% decrease in alpha-helix content of the protein in favor of unordered structure. Such changes may influence the affinity of AnxVI for Ca2+ and modulate its interaction with membranes.

Annexin VI interacts with adenine nucleotides and their analogs.

Annexin VI (AnxVI), a member of the annexin family of Ca2+- and membrane-binding proteins, has been shown to interact in vitro with adenine nucleotides. Furthermore, it has been proposed that within the AnxVI molecule a nucleotidde-binding domain exists, which is located in the C-terminal half of the protein, in the vicinity of Trp343. By comparison of exposure of tryptophan and multiple tyrosine residues upon nucleotide binding, as revealed by quenching of intrinsic fluorescence of AnxVI by ATP, ADP or cAMP, it can be concluded that the binding of nucleotides evokes changes in the protein tertiary structure. Moreover, in the course of present study we have found that AnxVI binds to a non-hydrolysable analog of ATP, the triazine dye Cibacron blue 3GA (CB3GA), immobilized on agarose. Binding reveals negative cooperativity with respect to protein concentration and is Ca2+-dependent. Binding is prevented by ATP. CB3GA binds to AnxVI also in solution, evoking the formation of annexin multimers. On the basis of this observation it can be suggested that interaction of CB3GA with AnxVI is useful to examine, with some limitations, the self-association of annexin molecules implying to play a role in interacting of AnxVI with biological membranes.

Annexin VI: an intracellular target for ATP.

Annexin VI (AnxVI), an Ca2+- and phospholipid-binding protein, interacts in vitro with ATP in a calcium-dependent manner. Experimental evidence indicates that its nucleotide-binding domain which is localized in the C-terminal half of the protein differs structurally from ATP/GTP-binding motifs found in other nucleotide-binding proteins. The amino-acid residues of AnxVI directly involved in ATP binding have not been yet defined. Binding of ATP to AnxVI induces changes in the secondary and tertiary structures of protein, affecting the affinity of AnxVI for Ca2+ and, in consequence, influencing the Ca2+-dependent activities of AnxVI: binding to F-actin and to membranous phospholipids, and self-association of the annexin molecules. These observations suggest that ATP is a functional ligand for AnxVI in vivo, and ATP-sensitive AnxVI may play the role of a factor coupling vesicular transport and calcium homeostasis to cellular metabolism.

Modulation of annexin VI--driven aggregation of phosphatidylserine liposomes by ATP.

Annexin (Anx) VI has been implicated in mediating the endosome aggregation and vesicle fusion in secreting epithelia during exocytosis. In addition, AnxVI of porcine liver is an ATP-binding protein, and ATP in vitro modulates its interaction with membranes and cytoskeletal elements (Bandorowicz-Pikula and Awasthi, FEBS Lett. 409 (1997) 300-306). In this study, we examined the effect of ATP on phosphatidylserine (PtdSer) aggregation in the presence of annexin and on calcium-dependent binding of protein to liposomes, and found that ATP stimulates the former process, although it increases the calcium concentration necessary for half-maximal binding of AnxVI to membranes. These results were corroborated by the experiments with fluorescent analog of ATP, in which binding of ATP to AnxVI was affected by binding of Ca2+ and/or phospholipids to the protein. Taken together they favour an idea of ATP being a functional ligand for AnxVI, which even in the relative absence of Ca2+ may modulate interaction of AnxVI with PtdSer-enriched membranes.

Apoptosis-like, reversible changes in plasma membrane asymmetry and permeability, and transient modifications in mitochondrial membrane potential induced by curcumin in rat thymocytes.

Curcumin (diferuoylmethane) is a natural compound with anticarcinogenic activities which is able to exert either proapoptotic or antiapoptotic effects in different cell types. This paper focuses on the sequence and extent of primary events induced by curcumin, in comparison with those occurring during dexamethasone-induced apoptosis in rat thymocytes. It also presents annexin VI-FITC as a new probe for studying membrane asymmetry. Curcumin readily penetrates into the cytoplasm, and is able to accumulate in membranous structures such as plasma membrane, endoplasmic reticulum and nuclear envelope. Curcumin-treated cells exhibit typical features of apoptotic cell death, including shrinkage, transient phosphatidylserine exposure, increased membrane permeability and decrease in mitochondrial membrane potential. However, nuclei morphology, DNA fragmentation, the extent and time-course of membrane changes are different from those observed during dexamethasone-induced apoptosis, suggesting that, despite many similarities, the mode of action and the events triggered by curcumin are different from those occurring during typical apoptosis.

ATP-Dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity.

Dinitrophenyl S-glutathione (DNP-SG) ATPase is a 38 kDa membrane protein expressed in erythrocytes and other tissues. Although stimulation of ATP hydrolysis catalyzed by DNP-SG ATPase has been demonstrated in the presence of several structurally unrelated amphiphilic ions, structural and functional properties of this protein have not been well-defined. In the present study, we have developed an improved protocol for the purification of DNP-SG ATPase and investigated its kinetic and substrate-binding properties. The purification procedure was based on highly specific elution of the 38 kDa protein from DNP-SG affinity resin in the presence of ATP. The protein could not be eluted using either ADP or adenosine-5'-[beta,gamma-methylene]triphosphate (methylene-ATP), a nonhydrolyzable analogue of ATP. Doxorubicin (DOX), a weakly basic anthracycline chemotherapy agent, was found to be the preferred activator for stimulation of ATP hydrolysis by the enzyme. ATP binding to the enzyme was demonstrated using 8-azido-ATP photoaffinity labeling and binding of trinitrophenyl (TNP)-ATP, a fluorescent analogue of ATP. The photoaffinity labeling of DNP-SG ATPase (38 kDa) was saturable with respect to 8-azido ATP (Kd = 2 microM), indicating that the enzyme was capable of specific and saturable binding to ATP. DNP-SG binding was evident from the purification procedure itself and was also demonstrable by quenching of tryptophan fluorescence. Results of quenching of tryptophan fluorescence as well as radioactive isotope-binding studies indicated that DOX was bound to the purified protein as well.

ATP-Dependent human erythrocyte glutathione-conjugate transporter. II. Functional reconstitution of transport activity.

Purified dinitrophenyl S-glutathione (DNP-SG) ATPase was reconstituted into artificial liposomes prepared from soybean asolectin. Electron micrography confirmed the formation of unilamellar vesicles with an average radius of 0.25 micron. Intravesicular volume estimated by incorporation of radiolabled inulin into the vesicles was found to be 19.7 +/- 1.3 microL/mL reconstitution solution. Accumulation of the glutathione-conjugate of CDNB, DNP-SG, and of doxorubicin (DOX) in the proteoliposomes was increased in the presence of ATP as compared to equimolar ADP or adenosine 5'-[beta,gamma-methylene]triphosphate tetralithium. ATP-dependent transmembrane movement of DOX and DNP-SG into DNP-SG ATPase-reconstituted vesicles was saturable with respect to time, sensitive to the osmolarity of the assay medium, and temperature dependent. The energy of activation was found to be 12 and 15 kcal/mol for DNP-SG and DOX, respectively. Optimal temperature for transport was 37 degrees C. Saturable transport was demonstrated for DNP-SG (Vmax of 433 +/- 20 nmol/min/mg of protein, KmATP = 2.4 +/- 0. 3 mM and KmDNP-SG = 36 +/- 5 microM) as well as DOX (Vmax = 194 +/- 19 nmol/min/mg of protein, KmATP = 2.5 +/- 0.6 mM and KmDOX = 2.4 +/- 0.7 microM). The kinetic data for both DNP-SG and DOX transport were consistent with a random bi-bi sequential reaction mechanism. DOX was found to be a competitive inhibitor of DNP-SG transport with Kis of 1.2 +/- 0.2 microM and DNP-SG was found to be a competitive inhibitor of DOX transport with Kis of 13.3 +/- 2.6 microM.