Role of the aggregation factor in the regulation of phosphoinositide metabolism in sponges. Possible consequences on calcium efflux and on mitogenesis

The aggregation factor (AF) of the marine sponge Geodia cydonium recognizes the aggregation receptor (AR) which is inserted in the plasma membrane, under formation of species-specific aggregates. The specific cell-binding fragment of the AF was used to investigate for the first time the phosphoinositide metabolism in a lower avertebrate system. We found that after binding of the cell-binding fragment to the aggregation receptor a strong and rapid stimulation of the phosphate incorporation into phosphatidylinositol occurs followed by an increased turnover of phosphoinositides in the Geodia cells. The consequences of an increased degradation of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol-1,4,5-trisphosphate and diacylglycerol are 2-fold. First, after the addition of the extracellular stimulus the cytosolic Ca2+ concentration rises, resulting in a rapid increased Ca2+ efflux rate. The functional consequence of the increase of the extracellular Ca2+ level is an initiation of the aggregate formation that is mediated by the collagen assembly factor (= primary aggregation factor). Second, some experimental evidences are presented, showing that the other second messenger formed, diacylglycerol, causes a translocation of protein kinase C within the cell. Incubation of Geodia cells with the cell-binding fragment of the AF, or with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, resulted within 5 min after treatment in a 70% decrease in protein kinase C activity in the cytosolic fraction and in a 700% increase in enzyme activity in the membrane fraction. It is proposed that by membrane association protein kinase C becomes activated. As a result of this event a series of cellular proteins are phosphorylated, a process which ultimately leads to an unusually strong induction of DNA polymerase alpha activity. From these data we conclude that inositol trisphosphate and protein kinase C also play a fundamental role in cellular signal transduction in lower eukaryotes.     

Specific phosphorylation of proteins in pore complex-laminae from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester. Role of protein kinase C in the phosphorylation of DNA topoisomerase II.

We have recently shown that the aggregation factor (AF) from the sponge Geodia cydonium stimulates DNA synthesis in quiescent, dissociated cells from the same organism; this event was correlated with the release of the two second messengers: inositol trisphosphate and diacylglycerol. Here we describe that after binding of the AF to the plasma membrane-bound aggregation receptor, a rapid and drastic increase in the incorporation of 32Pi into a series of proteins in the pore complex-lamina fraction occurs. Addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, to quiescent cells resulted in a similar stimulation of phosphorylation of nuclear proteins. Among them we have selected one protein with a polypeptide Mr of 170,000 (pp170) for detailed studies. By immunoblotting pp170 was identified as DNA topoisomerase II. In vitro studies with nuclei and purified, homogeneous protein kinase C together with the required activators of this enzyme also showed a phosphorylation of pp170. After phosphorylation, DNA topoisomerase II activity was found to be 2.5-fold that of the non-phosphorylated enzyme. From these data we conclude that protein kinase C is involved in AF induced transmembrane signalling, ultimately leading to an initiation of DNA synthesis.     

Protein kinase C phosphorylates the sponge aggregation receptor after its binding to the homologous aggregation factor

The aggregation factor from the sponge Geodia cydonium functions also as a growth factor after binding to the aggregation receptor (= growth factor receptor) on the plasma membrane of homologous cells. We have recently shown that protein kinase C is involved in the pathway transducing the growth factor signal. Here we report that the aggregation receptor (a polypeptide with an Mr of 43,500) is phosphorylated by protein kinase C. Using a plasma membrane fraction only this phosphoprotein (pp) 43.5 became phosphorylated by kinase C. The phosphorylation of pp43.5 in intact cells in response to the binding of the aggregation factor to this polypeptide was a late event and occurred 10 to 15 h after addition of the aggregation factor. Based on studies with phorbol esters it appears to be very likely that protein kinase C also phosphorylates pp43.5 in vitro. The degree of phosphorylation of pp43.5 paralleled with both the extent of DNA synthesis and ras oncogene expression. The latter process resulted in a switch of the responsiveness of the cells to growth factors signals: 10 to 15 h after addition of the aggregation factor to dissociated cells, this factor lost its growth factor function while the homologous lectin gained the ability to stimulate cell proliferation (to be published). These results support the idea that phosphorylation of pp43.5 (= aggregation receptor) results in an inhibition of its function, i.e., the transduction of the growth factor (= aggregation factor) signal.     

Sponge aggregation factor: in situ localization by fluorescent monoclonal antibody techniques

The aggregation factor (AF) from sponges mediates a heterophilic interaction of homologous cells. Applying electron microscopical means, we succeeded only very rarely in identifying the 90 S AF particle in tissue sections from Geodia cydonium. By means of a fluorescent antibody technique, we have now localized the cell binding domain of the AF in situ. Previous studies in this laboratory have led to the identification of the 47-kDa cell binding protein of the AF, using the monoclonal antibody (mab) 5D2-D11 [Gramzow M, Bachmann M, Zahn RK, Uhlenbruck G, Dorn A, Müller WEG, J Cell Biol, 102: 1344-1349, 1986]. This mab and mab 7D5, directed against a 92-kDa protein in the AF complex, were chosen for the fluorescent studies. By using mab 5D2-D11, the plasma membranes of cells from different regions in the sponge could be brightly stained. However, mab 7D5 reacted only very weakly with the sponge surfaces. By applying the immuno-blotting technique it was furthermore demonstrated that the cell binding protein is present both in the associated form with AF complex and in a free state. Moreover, it was established that the 47-kDa binding protein is not present in homologous glycoconjugates, lectin, or collagen; these components are known to be involved in cell-matrix interaction.     

Identification and further characterization of the specific cell binding fragment from sponge aggregation factor

Monoclonal antibodies (McAbs) were raised against the aggregation factor (AF) from the marine sponge Geodia cydonium. Two clones were identified that secrete McAbs against the cell binding protein of the AF complex. Fab fragments of McAbs: 5D2-D11 completely abolished the activity of the AF to form secondary aggregates from single cells. The McAbs were determined to react with the AF in vitro; this interaction was prevented by addition of the aggregation receptor, isolated and purified from the same species. After dissociation of the AF by sodium dodecyl sulfate and 2-mercaptoethanol, followed by electrophoretical fractionation, a 47-kD protein was identified by immunoblotting which interacted with the McAbs: 5D2-D11. During this dissociation procedure, the sunburst structure of the AF was destroyed. In a second approach, the 47-kD protein was isolated by immunoprecipitation; 12 molecules of this protein species were calculated to be associated with the intact AF particle. The 47-kD AF fragment bound to dissociated Geodia cells with a high affinity (Ka of 7 X 10(8) M-1) even in the absence of Ca++ ions; the number of binding sites was approximately 4 X 10(6)/cell. This interaction was prevented by addition of the aggregation receptor to the 47-kD protein in the homologous cell system. Moreover, it was established that this binding occurs species-specifically. The 47-kD fragment of the AF was localized only extracellularly by indirect immunofluorescence staining in cryostat slices. These data suggest that the 47-kD protein is the cell binding molecule of the AF from Geodia.     

Species-specific association of the cell-aggregation molecule mediates recognition in marine sponges

Reaggregation of dissociated cells of marine sponges, resulting in reformation of functional sponges, is a calcium-dependent process mediated by large, proteoglycan-like molecules termed aggregation factors (AF). During aggregation, species-specific sorting of cells is often observed. We purified and characterized AFs from three different sponge species and investigated their role in species-specific aggregation using novel approaches. The calcium-dependent association between purified AFs is species-specific in most combinations, as was shown in overlay assays and bead-sorting assays with AFs immobilized onto colored beads. Species-specific interactions of living cells and AF-beads resulted in incorporation of only homospecific AF-beads into reforming cell aggregates. Sequences from peptides obtained from the AF core proteins could all be aligned to the sequence of one species, the Microciona prolifera AFp3 core protein. In contrast to this similarity, major species-specific differences were seen in carbohydrate composition and in the response of AFs to specific carbohydrate-recognizing antibodies. In summary, our data point to a prominent role for the calcium-dependent association of AFs in recognition processes during aggregation. As this association of AFs occurs via carbohydrate-carbohydrate interactions, we speculate that the specificity of those interactions may be fundamental to recognition mechanisms required for regeneration of individuals from dissociated cells and for rejection of foreign material by sponge individuals.     

Species-Specific Association of the Cell-Aggregation Molecule Mediates Recognition in Marine Sponges

Reaggrcgation of dissociated cells of marine sponges, resulting in reformation of functional sponges, is a calcium-dependent process mediated by large, proteoglycan-like molecules termed aggregation factors (AF). During aggregation, species-specific sorting of cells is often observed. We purified and characterized AFs from three different sponge species and investigated their role in species-specific aggregation using novel approaches. The calcium-dependent association between purified AFs is species-specific in most combinations, as was shown in overlay assays and bead-sorting assays with AFs immobilized onto colored beads. Species-specific interactions of living cells and AF-beads resulted in incorporation of only homospecific AF-beads into reforming cell aggregates. Sequences from peptides obtained from the AF core proteins could all be aligned to the sequence of one species, the Microciona prolifera AFp3 core protein. In contrast to this similarity, major species-specific differences were seen in carbohydrate composition and in the response of AFs to specific carbohydrate-recognizing antibodies. In summary, our data point to a prominent role for the calcium-dependent association of AFs in recognition processes during aggregation. As this association of AFs occurs via carbohydrate -carbohydrate interactions, we speculate that the specificity of those interactions may be fundamental to recognition mechanisms required for regeneration of individuals from dissociated cells and for rejection of foreign material by sponge individuals.     

Species-Specific Aggregation Factor in Sponges

An aggregation factor (AF) from the siliceous sponge Suberites domuncula has been isolated and purified by the following steps: Sepharose 2 B gel chromatography, sucrose gradient, Nonidet treatment, Sephadex G-100 gel chromatography and DEAE-Sephadex ion-exchange chromatography. By this procedure the AF was purified 1340-fold with a 63% yield nearly to homogeneity. The AF is originally associated with large particles, characterized by a sedimentation of 2200 S. These particles have been visualized electron microscopically; they are characterized by a filament-like shape of a length of 3400 Å and a cross-sectional diameter of 230 Å.
The purified, low-molecular weight AF has a buoyant density of 1.38 g/cm³ and an absorbance maximum at 282 nm. The isoelectric pH is approximately 5.75. The molecular weight of the AF has been determined to be 55,000. Chemical analysis revealed that AF consists mainly of protein.
The reaggregation process of Suberites cells to large aggregates (>1000 μm), mediated by Suberites AF, is strongly dependent on pH, ionic strength and the presence of calcium ions, and independent of incubation temperature between 0°C and 20°C. Aggregation can be inhibited by trypsin, D-glucosamine and dodecyl sulphate.
The Suberites AF is species-specific if tested in a system containing cells from the siliceous sponge Geodia cydonium .     

Sponge cell aggregation

Summary Dissociated sponge cell system has proved to be a useful model to study the process of cell aggregation both on cellular and subcellular level. The purpose of this review is to discuss recent results obtained from experiments with the marine sponge Geodia cydonium.Dissociated cells form functional aggregates during a process which can be sub-divided into three phases: first, formation of small primary aggregates in the presence of Ca²⁺; second, formation of secondary aggregates in the presence of an aggregation factor and third, reconstitution of a functional system of watercontaining channels by rearrangement in the secondary aggregates.On subcellular level a series of macromolecules are known which are involved in the control of aggregation and separation of sponge cells: Aggregation factor, aggregation receptor, anti-aggregation receptor, glucuronidase, ß-glucuronosyltransferase, ß- ß-galactosidase and a lectin. These components might be linked in the following sequence: (a) Activation of the aggregation receptor by its enzymic glucuronylation; (b) Adhesive recognition of the cells, mediated by the aggregation factor and the glucuronylated aggregation receptor; (c) Inactivation of the aggregation receptor by its deglucuronylation with the membrane-associated ß-glucuronidase; (d) Cell separation due to either the loss of the recognition site (glucuronic acid) of the aggregation receptor for the aggregation factor or to an inactivation of the aggregation factor by the anti-aggregation receptor. The activity of the anti-aggregation receptor is most likely controlled by the Geodia lectin.The events leading to cell-cell recognition cause a change in the following metabolic events: Increase of oxygen uptake, decrease of cyclic AMP level, increase of cyclic GMP level and stimulation of programmed syntheses.Abbreviations AF aggregation factor - CPP circular proteid particle - AR aggregation receptor - aAR anti-aggregation receptor - CMF calcium- and magnesium-free artificial sea water - ASW calcium- and magnesium-containing artificial sea water This paper is dedicated to PROF. DR. R. K. ZAHN on the occasion of his 60^th birthday.     

Origin of the integrin-mediated signal transduction. Functional studies with cell cultures from the sponge Suberites domuncula.

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals. Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated. In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside-in signaling molecules. The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established. Here we report that aggregation factor (AF)-mediated cell-cell adhesion is blocked by the RGDS peptide which is known to interact with beta integrin. Both RGDS and AF were found to stimulate DNA synthesis within 24 h. The beta subunit of the integrin receptor was cloned from S. domuncula; the estimated 91-kDa molecule comprises the characteristic signatures. Evolutionary conservation of the beta integrin was assessed by comparison with corresponding beta integrin subunits from evolutionary higher metazoan taxa. Addition of RGDS or of AF to isolated cells of S. domuncula causes a rapid (within 1-2 min) increase in the intracellular Ca2+ concentration which is further augmented in the presence of Ca2+. Furthermore, incubation of the cells with RGDS or AF causes an activation of the GTP-binding protein Ras. In addition it is shown that after a prolonged incubation of the cells with RGDS and AF the expression of the genes coding for Ras and for calmodulin is upregulated. These results suggest that the integrin receptor functions in the sponge system not only as adhesion molecule but also as a molecule involved in outside-in signaling.     

Species-specific aggregation factor in sponges. V. Influence on programmed syntheses.

Isolated cells from the siliceous sponge Geodia cydonium as well as small primary aggregates (diameter: 70 mum) consisting of them show no increase in rates of programmed syntheses and mitotic activity with time. After addition of a highly purified aggregation factor to a culture with primary aggregates which subsequently form secondary aggregates (diameter: larger than 1000 mum), a dramatic increase of DNA, RNA and protein synthesis occurs. Together with this increase, the cells show a high mitotic activity. The values for the mitotic coefficient reach a first maximum 8 h after the beginning of the secondary aggregation process. The stimulation of the mitotic activity of cells during the aggregation factor induced secondary aggregation process can be suppressed by inhibitors of RNA and protein synthesis as well as by a blocker of DNA synthesis. This finding may indicate that cells from the G0-population enter the proliferating cell pool via the G1-phase.     

Sponge aggregation factor: identification of the specific collagen-binding site by means of a monoclonal antibody

The aggregation factor (AF) from the sponge Geodia cydonium is known to be a complex proteinaceous particle, composed of a series of different (glyco)proteins (Mr lower than 150,000) around a 90S sunburst-like core structure. One of the low-Mr proteins is the 47-KD cell binding fragment. We describe a new monoclonal antibody (mAb), III1E6, raised against purified AF particles, which recognizes in tissue slices structures present both on the plasma membrane and in a network-like manner in the extracellular space. By applying immunoelectron microscopical, immunoblotting, and immunoaffinity chromatographical techniques, the mAb III1E6 was shown to recognize the core structure of the AF particle. Cell adhesion studies revealed that the mAb does not inhibit AF mediated cell-cell adhesion but abolishes AF-caused attachment of cells to collagen. Electron microscopic data show that III1E6 prevents association of AF particles with collagen fibrils. By applying the techniques of immunoblotting and of protein-protein recognition on the solid phase in vitro, we could formulate the following series of events: the AF particle recognizes, with its 47-KD cell binding fragment, the aggregation receptor protein in the plasma membrane and with its core structure the collagen fibrils. These fibrils interact optionally, either via the same route or via the collagen assembly factor, with an adjacent cell surface. These findings demonstrate that the AF particle is not only the key molecule for cell-cell adhesion but also a component of cell-matrix interactions.     

Ultrastructural study of differentiation processes during aggregation of purified sponge archaeocytes

Archaeocytes from the spongeEphydatia fluviatilis were dissociated and then isolated on Ficoll density gradients. Their aggregation and reconstitution processes were studied by transmission electron microscopy to determine their capabilities for differentiation.Archaeocyte aggregates follow a well defined sequence of differentiation to generate the characteristic structures of a sponge. Pinacoderm is the first structure to be regenerated and appears progressively at the surface of the 12 h aggregates. Pinacocytes which have differentiated in archaeocyte aggregates are identical to native ones except that the nucleolus remains in most cells. The choanocytes appear only after 24 h by a two step process. First, small cells (choanoblasts) are formed from archaeocytes by mitosis. These cells then transform into fully differentiated choanocytes possessing collars and flagella. The early choanocyte chambers are small, irregular and randomly dispersed in the aggregates. Finally, collencytes and sclerocytes begin to appear just before the aggregates spread on the substrate.The differentiation of a suspension of pure archaeocytes is a unique model system to study sponge cell differentiation and has allowed us to demonstrate that archaeocytes isolated from developed sponges maintain the capacity to differentiate even though this capacity is not usually expressed.     

Enzymatic removal of oxidized protein aggregates from erythrocyte membranes

Erythrocytes oxidized or aged in the circulation undergo membrane protein aggregation and anti-band 3 autoantibody binding to the cell surface. When human erythrocytes were mildly oxidized in vitro with 0.1 mM Fe(III) at 37 degrees C for 3 h, the aggregation of nonionic detergent C(12)E(8)-insoluble membrane protein and the binding of anti-band 3 IgG to the cell surface were increased. Incubation of membranes isolated from the oxidized cells increased the amount of protein aggregates by 5-fold after 6 h, while incubation for a further 12 h sharply decreased the amount of aggregates. In the presence of diisopropyl fluorophosphate (DFP), however, the increased amount of aggregates was maintained in the subsequent incubation. Western blot analysis of the aggregates using rabbit anti-band 3 showed that band 3 protein aggregates increased in the initial stage of incubation and decreased upon subsequent incubation, whereas the increased band 3 protein aggregates did not subsequently decrease when membranes were incubated in the presence of DFP. Incubation of the oxidized cells at 37 degrees C for 18 h caused reduction of the membrane protein aggregates and the (125)I-anti-band 3 IgG binding to the cell surface, while incubation in the presence of DFP did not cause these reductions. The results suggest that the oxidation-induced cell membrane protein aggregates were probably removed by 80-kDa serine protease, namely, oxidized protein hydrolase (OPH), in the oxidized cell membranes [Fujino et al. (1998) Biochim. Biophys. Acta 1374, 47-54; (1998) J. Biochem. 124, 1077-1085; (2000) Biochim. Biophys. Acta 1478, 102-112], and as a result the increased anti-band 3 binding to the cell surface was reduced.     

Congenital cataract causing mutants of αA-crystallin/sHSP form aggregates and aggresomes degraded through ubiquitin-proteasome pathway

Background

Mutations of human αA-crystallin cause congenital cataract by protein aggregation. How mutations of αA-crystallin cause disease pathogenesis through protein aggregation is not well understood. To better understand the cellular events leading to protein aggregation, we transfected cataract causing mutants, R12C, R21L, R21W, R49C, R54C, R116C and R116H, of human αA-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy.

Methodology/Principal Findings

YFP-tagged human αA-wild-type (αA-wt) was sub-cloned and the mutants were generated by site-directed mutagenesis. The αA-wt and the mutants were individually transfected or co-transfected with CFP-tagged αA-wt or αB-wild-type (αB-wt) in HeLa cells. Overexpression of these mutants forms multiple small dispersed cytoplasmic aggregates as well as aggresomes. Co-expression of αB-wt with these mutants significantly inhibited protein aggregates where as co-expression with αA-wt enhanced protein aggregates which seems to be due to co-aggregation of the mutants with αA-wt. Aggresomes were validated by double immunofluorescence by co-localization of γ-tubulin, a centrosome marker protein with αA-crystallin. Furthermore, increased ubiquitination was detected in R21W, R116C and R116H as assessed by western blot analyses. Immunostaining with an ubiquitin antibody revealed that ubiquitin inclusions in the perinuclear regions were evident only in R116C transfected cells. Pulse chase assay, after cycloheximide treatment, suggested that R116C degraded faster than the wild-type control.

Conclusions/Significance

Mutants of αA-crystallin form aggregates and aggresomes. Co-expression of αA-wt with the mutants increased aggregates and co-expression of αB-wt with the mutants significantly decreased the aggregates. The mutant, R116C protein degraded faster than wild-type control and increased ubiquitination was evident in R116C expressing cells.     

Distribution of cAMP-dependent protein kinase during development in Dictyostelium discoideum

During the developmental cycle of Dictyostelium discoideum cyclic AMP functions as both a chemotactic signal for aggregation and a regulatory molecule during later events of differentiation. Morphological and biochemical data suggest that cAMP may direct cells during morphogenesis and differentiation. We utilized microtechniques to determine the stage- and cell-specific levels of the cAMP-dependent protein kinase, the probable intracellular cAMP receptor. Kinase activity was low and non-cAMP-dependent in amoebae and early aggregates but increased and became cAMP-dependent in aggregates after the formation of tight cell contacts. Maximum kinase activity and cAMP dependency occurred during the slug and culmination stages. The only differential distribution of the kinase within a single stage occurred during culmination when the activity in the stalks was approximately one-fourth of that in the prespore mass. Preliminary evidence indicates that this difference is not due to an inhibitor. In all other stages tested cAMP-dependent protein kinase activity was equal in prespore and prestalk cells.     

Two cell surface proteins bind the sponge Microciona prolifera aggregation factor

Two extracellular matrix cell surface proteins which bind the proteoglycan-like aggregation factor from the marine sponge Microciona prolifera (MAF) and which may function as physiological receptors for MAF were identified and characterized for the first time. By probing nitrocellulose blots of nonreducing sodium dodecyl sulfate gels containing whole sponge cell protein with iodinated MAF, a 210- and a 68-kDa protein, which have native molecular masses of approximately 200-400 and 70 kDa, were identified. MAF binding to blots is species-specific. It is also sensitive to reduction and is completely abolished by pretreatment of live cells with proteases, as was cellular aggregation, indicating that the 210- and 68-kDa proteins may be located on the cell surface. The additional observations that the 68 kDa is an endoglycosidase F-sensitive glycoprotein and that antisera against whole sponge cells or membranes can immunoprecipitate the 210 kDa when prebound to intact cells are consistent with a cell surface location. Both proteins can be isolated from sponge cell membranes and from the sponge skeleton (insoluble extracellular matrix), but the 210-kDa MAF-binding protein can also be found in the soluble extracellular matrix (buffer washes of cells and skeleton) as well. A third MAF-binding protein of molecular mass 95 kDa was also found in the sponge extracellular matrix but rarely on cells. Both of the cell-associated 210- and 68-kDa proteins are nonintegral membrane proteins, based on Triton X-114 phase separation, flotation of liposomes containing sponge membrane lysates, and their extraction from membranes by buffer washes. Both proteins bind MAF affinity resins, indicating that they each exhibit a moderate affinity for MAF under native conditions. They can also be separated from each other and from the bulk of the protein in an octylpolyoxyethylene extract of membranes by fast protein liquid chromatography Mono Q anion exchange chromatography, as assessed by native dot blot and denaturing Western blot assays. Although neither protein bound to heparin, gelatin, hexosamine, or uronic acid-Sepharose resins, their affinity for an invertebrate proteoglycan, their roles in sponge cell adhesion, and their peripheral membrane protein natures suggest that they may represent early invertebrate analogs of cell-associated vertebrate extracellular matrix adhesion proteins, such as fibronectin or vitronectin, or else an entirely novel set of cell adhesion molecules.     

Only the reduced conformer of alpha-lactalbumin is inducible to aggregation by protein aggregates

Reduced apo-alpha-lactalbumin (r-LA) in the pre-molten globule state is soluble in neutral and reduced buffer at 25 degrees C but becomes aggregated when aggregates of various proteins are added. However, protein aggregates do not induce the aggregation of apo-alpha-lactalbumin in the molten globule state. The presence of the molecular chaperone protein disulfide isomerase or the "chemical chaperone" polyethyleneglycol inhibits the induced aggregation. Native proteins, aggregation-free folding intermediates, and soluble aggregates do not induce the aggregation. The interaction between r-LA and protein aggregates is hydrophobic in nature. These findings suggest that pre-molten globule state of LA is the target not only for chaperones but also for protein aggregates.     

Auranofin increases the affinity of phorbol dibutyrate receptors in chronic lymphocytic leukemia cells (B cells)

Previous studies have shown that auranofin (AF), a lipophilic gold I complex, modulates metabolic events in leukocytes stimulated by phorbol esters, whose major cellular binding site is now known to be the Ca++/phospholipid-dependent protein kinase (protein kinase C). In these experiments we have investigated the effect of AF on the binding of phorbol dibutyrate (PDBu) to human chronic lymphocytic leukemia (CLL) B cells. AF enhanced binding of PDBu to its receptor in CLL cells by a) causing an increase in the affinity of PDBu receptors from Kd 20.3 nM to 7.3 nM, and b) enhancing translocation of PDBu receptors to the cell membrane. The increase in PDBu binding induced by AF in whole cells was only partially reversible by EGTA or the intracellular Ca++ antagonist TMB-8. Studies performed with quin-2-labeled cells showed that 100 microM AF caused a mean (+/- SD) rise in cytosolic Ca++ levels from 0.41 (0.12) to 0.85 (0.33) (n = 5). Thus the mechanism by which AF increases binding of PDBu to its receptor appears to be partially dependent on Ca++. These effects of AF occurred at cellular levels achieved in mononuclear cells during chrysotherapy of patients with rheumatoid arthritis.