Heparan Sulfate Proteoglycans Play a Dual Role in Regulating Fibroblast Growth Factor-2 Mitogenic Activity in Human Breast Cancer Cells

The human breast cancer cell lines MCF-7 and MDA-MB-231 differ in their responsiveness to fibroblast growth factor-2 (FGF-2). This growth factor stimulates proliferation in well-differentiated MCF-7 cells, whereas the less well-differentiated MDA-MB-231 cells are insensitive to this molecule. To investigate the potential regulation of FGF-2 mitogenic activity by heparan sulfate proteoglycans (HSPG), we have treated human breast cancer cells by glycosaminoglycan degrading enzymes or a metabolic inhibitor of proteoglycan sulfation: sodium chlorate. The interaction between FGF-2 and proteoglycans was assayed by examining the binding of¹²⁵I-FGF-2 to breast cancer cell cultures as well as to cationic membranes loaded with HSPG. Using MCF-7 cells, we showed that heparinase treatment inhibited FGF-2 binding to HSPG and completely abolished FGF-2 induced growth; chlorate treatment of MCF-7 cells decreased FGF-2 binding to HSPG and cell responsiveness in a dose-dependent manner. This demonstrates a requirement of adequately sulfated HSPG for FGF-2 growth-promoting activity on MCF-7 cells. In highly invasive MDA-MB-231 cells which produce twice as much HSPG as MCF-7 cells and which are not normally responsive to exogenously added FGF-2, chlorate treatment decreased FGF-2 binding to HSPG and induced FGF-2 mitogenic effect. This chlorate effect was dose dependent and observed at concentrations of 10–30 mM;higher chlorate concentrations completely abolished the FGF-2 effect. This shows that the HSPG level of sulfation can also negatively regulate the biological activity of FGF-2. Taken together, these results demonstrate a crucial role for HSPG in both positive and negative control of FGF-2 mitogenic activity in breast cancer cell proliferation.     

The proliferative and migratory activities of breast cancer cells can be differentially regulated by heparan sulfates

To explore how heparan sulfate (HS) controls the responsiveness of the breast cancer cell lines MCF-7 and MDA-MB-231 to fibroblast growth factors (FGFs), we have exposed them to HS preparations known to have specificity for FGF-1 (HS glycosaminoglycan (HSGAG A)) or FGF-2 (HSGAGB). Proliferation assays confirmed that MCF-7 cells were highly responsive to FGF-2 complexed with GAGB, whereas migration assays indicated that FGF-1/HSGAGA combinations were stimulatory for the highly invasive MDA-MB-231 cells. Quantitative polymerase chain reaction for the levels of FGF receptor (FGFR) isoforms revealed that MCF-7 cells have greater levels of FGFR1 and that MDA-MB-231 cells have greater relative levels of FGFR2. Cross-linking demonstrated that FGF-2/HSGAGB primarily activated FGFR1, which in turn up-regulated the activity of mitogen-activated protein kinase; in contrast, FGF-1/HSGAGA led to the phosphorylation of equal proportions of both FGFR1 and FGFR2, which in turn led to the up-regulation of Src and p125(FAK). MDA-MB-231 cells were particularly responsive to vitronectin substrates in the presence of FGF-1/HSGAGA, and blocking antibodies established that they used the alpha(v)beta(3) integrin to bind to it. These results suggest that the clustering of particular FGFR configurations on breast cancer cells induced by different HS chains leads to distinct phenotypic behaviors.     

Identification of common and specific growth factor binding sites in heparan sulfate proteoglycans

The structural complexity within heparan sulfate has suggested that it contains multiple protein-specific binding sites. To evaluate the selectivity of growth factor binding to heparan sulfate, we conducted a detailed study of the intercompetition of fibroblast growth factor-2 (FGF-2) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) binding to heparan sulfate (HS) on bovine aortic smooth muscle cells. Radioligand binding assays were conducted, and an analytical method was developed for determining the apparent binding constants and numbers of specific and shared binding sites within HS. These studies revealed the presence of two general classes of HS-binding sites for FGF-2 and HB-EGF. The major class (approximately 10(6) sites per cell) was able to bind to either growth factor with relatively low affinity (K(d) = 12 and 44 nM for FGF-2 and HB-EGF, respectively) and was termed "common" binding sites. However, both FGF-2 and HB-EGF also showed specific high affinity (0.6 and 6.1 nM for FGF-2 and HB-EGF, respectively) binding to a minor subset (118,000 and 28,000 sites per cell for FGF-2 and HB-EGF, respectively) of "unique" binding sites, which were unable to bind the other growth factor. These studies indicate that growth factor binding to HS involves multiple binding sites of variable affinity, density, and selectivity. The approach outlined in this study could be applied to aid in the evaluation of the relative biological roles of these selective and nonselective growth factor binding domains within HS.     

Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2.

Proteins that belong to the fibroblast growth factor (FGF) family regulate proliferation, migration, and differentiation of many cell types. Several FGFs, including the prototype factors FGF-1 and FGF-2, depend on interactions with heparan sulfate (HS) proteoglycans for activity. We have assessed tissue-derived HS fragments for binding to FGF-1 and FGF-2 to identify the authentic saccharide motifs required for interactions. Sequence information on a range of N-sulfated HS octasaccharides spanning from low to high affinity for FGF-1 was obtained. All octasaccharides with high affinity for FGF-1 (> or =0.5 m NaCl required for elution) contained an internal IdoUA(2-OSO(3))-GlcNSO(3)(6-OSO(3))-IdoUA(2-OSO(3))-trisaccharide motif. Octasaccharides with a higher overall degree of sulfation but lacking the specific trisaccharide motif showed lower affinity for FGF-1. FGF-2 was shown to bind to a mono-O-sulfated HS 6-mer carrying a single internal IdoUA(2-OSO(3))-unit. However, a di-O-sulfated -IdoUA(2-OSO(3))-GlcNSO(3)-IdoUA(2-OSO(3))-trisaccharide sequence within a HS 8-mer gave stronger binding. These findings show that not only the number but also the positions of individual sulfate groups determine affinity of HS for FGFs. Our findings support the notion that FGF-dependent processes can be modulated in vivo by regulated expression of distinct HS sequences.     

Lateral mobility and specific binding to GABA(A) receptors on hippocampal neurons monitored by fluorescence correlation spectroscopy

The binding behavior of a fluorescently labeled muscimol derivative to the GABA(A) receptor was analyzed at rat hippocampal neurons by fluorescence correlation spectroscopy. After muscimol had been labeled with the fluorophore Alexa Fluor 532, specific binding constants for binding of the dye-labeled ligand (Mu-Alexa) to the GABA(A) receptor were determined. We found a high specific binding affinity of Mu-Alexa with a K(D) value of 3.4 +/- 0.5 nM and a rate constant of ligand-receptor dissociation (k(diss)) of (5.37 +/- 0.95) x 10(-2) s(-1). A rate constant of ligand-receptor association (k(ass)) of (1.57 +/- 0.28) x 10(7) L mol(-1) s(-1) was calculated. The following diffusion coefficients were observed: D(free) = 233 +/- 20 microm(2)/s (n = 66) for free diffusing Mu-Alexa, D(bound1) = 2.8 +/- 0.9 microm(2)/s (n = 64) for the lateral mobility, and D(bound2) = 0.14 +/- 0.05 microm(2)/s (n = 56) for the hindered mobility of the GABA(A) receptor-ligand complex in the cell membrane. Saturation of Mu-Alexa binding was observed at a concentration of 50 nM. A maximum number of binding sites [B(max) = 18.4 +/- -0.4 nM (n = 5)] was found. Similar K(i) values of 4.5 +/- 1.0 nM for nonlabeled muscimol and 8.8 +/- 1.8 nM for Mu-Alexa were found by RRAs using [(3)H]muscimol as a radioligand. A concentration-dependent increase in the level of specific Mu-Alexa binding was demonstrated by the positive cooperative activity of co-incubated midazolam, which was selectively found in GABA(A) receptor-ligand complexes with hindered mobility.     

High affinity binding of Bcl-xL to cytochrome c: possible relevance for interception of translocated cytochrome c in apoptosis

The release of cytochrome c from mitochondria and apoptosis relies on several preferential and selective interactions involving the Bcl-2 family of proteins. There is, however, no direct evidence for the interaction of cytochrome c with these proteins at any stage of apoptosis. To investigate if any pro-survival protein from the Bcl-2 family could intercept cytochrome c after its translocation from mitochondria, the interaction of cytochrome c with bacterially expressed human Bcl-x(L) was studied at pH 7. In size-exclusion chromatography, purified full-length His(6)-tagged Bcl-x(L) migrated as both dimer and monomer, of which the monomeric fractions were used for experiments. Coimmunoprecipitation studies show that cytochrome c interacts with Bcl-x(L). The extent of caspase activity in cell lysate elicited by externally added cytochrome c is reduced when a preincubated mixture of Bcl-x(L) and cytochrome c is used instead. Equilibrium binding monitored by optical absorption of cytochrome c as a function of titrating concentrations of Bcl-x(L) yields the association constant, K(ass) = 8.4(+/- 4) x 10(6) M(-1) (binding affinity, K(diss) = 1/K(ass) approximately 120 nM) which decreases at high ionic strength. The rates for binding of Bcl-x(L) to cytochrome c, studied by stopped-flow kinetics at pH 7, show that the bimolecular rate constant for binding, k(bi) = 0.24 x 10(6) M(-1) s(-1). Values of the thermodynamic and kinetic parameters for Bcl-x(L)-cytochrome c interaction are very similar to those known for regulatory protein-protein interactions in apoptosis.     

Sulfated derivatives of Escherichia coli K5 polysaccharides as modulators of fibroblast growth factor signaling

Heparan sulfate (HS) proteoglycans are intimately involved in the regulation of fibroblast growth factor (FGF) signaling. HS and the related glycosaminoglycan heparin interact with FGFs and FGF receptors (FGFRs), and it is believed that both interactions are required for productive FGF signaling. Attempts to inhibit FGF activity have been made with modified heparin preparations, various heparin-like polysaccharide analogues and other polyanionic molecules, which may all act by interfering with the physiological HS-FGF-FGFR interactions on the cell surface. Here, we have studied the potential of sulfated derivatives of a bacterial polysaccharide (capsular polysaccharide from Escherichia coli K5 (K5PS)) in the modulation of FGF-heparin/HS interactions and FGF signaling. We demonstrate that O-sulfated and N,O-sulfated species of K5PS, with high degrees of sulfation, displaced FGF-1, FGF-2, and FGF-8b from heparin. However, only O-sulfated K5PS efficiently inhibited the FGF-induced proliferation of S115 mammary carcinoma cells and 3T3 fibroblasts, whereas N,O-sulfated K5PS had little or no inhibitory effect. Studies with CHO677 cells lacking endogenous HS, as well as with chlorate-treated S115 cells expressing undersulfated HS, indicated that whereas exogenously administered heparin and N,O-sulfated K5PS restored the cellular response toward FGF stimulation, O-sulfated K5PS was largely devoid of such stimulatory activity. Our data suggest that highly O-sulfated species of K5PS may be efficient inhibitors of FGF signaling.     

Kinetics of interaction of trypsin with an anionic inhibitor of trypsin BWI-1a from buckwheat seeds

The kinetics of binding of bovine trypsin to a proteinaceous inhibitor of trypsin from buckwheat seeds (BWI-1a) has been studied. The association rate constant (k(ass)) was 2.2 x 10(6) M-1 x sec-1 and the dissociation rate constant (k(off)) of the enzyme--inhibitor complex was 3.5 x 10(-3) sec-1; the inhibition constant Ki was 1.5 nM. The inhibitor BWI-1a is of the slow, tightly binding type. The mechanism of the inhibition of bovine trypsin by the trypsin inhibitor BWI-1a was studied. The mechanism of inhibition was found to involve two steps according to the kinetic data.     

Increased stability and lifetime of the complex formed between DNA and meta-phenyl-substituted Hoechst dyes as studied by fluorescence titrations and stopped-flow kinetics

The large increase in fluorescence upon binding of five para- and meta-phenyl substituted hydroxy and methoxy derivatives of the Hoechst dye with poly[d(A-T)], d(CGCGAATTCGCG)2, and its corresponding T4-looped 28-mer hairpin was used to monitor the binding by equilibrium titrations and by stopped-flow kinetics. The affinity increases in the same order for the three DNAs: p-OH相似文献   

Effect of chlorate on the sulfation of lipoprotein lipase and heparan sulfate proteoglycans. Sulfation of heparan sulfate proteoglycans affects lipoprotein lipase degradation

In avian-cultured adipocytes 76% of the newly synthesized lipoprotein lipase is degraded before release into the medium (Cupp, M., Bensadoun, A., and Melford, K. (1987) J. Biol. Chem. 262, 6383-6388). The same group (Cisar, L. A., Hoogewerf, A. J., Cupp, M., Rapport, C. A., and Bensadoun, A. (1989) J. Biol. Chem. 264, 1767-1774) has proposed that the interaction of lipoprotein lipase with a class of cell surface heparan sulfate proteoglycans is necessary for degradation to occur. To test further this hypothesis, the binding capacity of the plasma membrane for the lipase was decreased by inhibiting the sulfation of glycosaminoglycans with sodium chlorate, an inhibitor of sulfate adenyltransferase. Chlorate decreased sulfate incorporation into trypsin-releasable heparan sulfate proteoglycans to 20% of control levels. The amount of uronic acid in the trypsin-releasable heparan sulfate proteoglycans remained constant. Therefore, chlorate decreased sulfation density on heparan sulfate chains by approximately 5-fold. In the same fractions, chlorate increased the median heparan sulfate Mr measured on Sephacryl S-300. Chlorate decreased the maximum binding of 125I-lipoprotein lipase to adipocytes by 4-fold, but no significant effects on the affinity constants were observed. Chlorate increased lipoprotein lipase secretion in a dose-dependent relationship up to 30 mM. Utilizing a pulse-chase protocol, it was shown that lipase synthesis in control and chlorate-treated cells was not significantly different and that the increased secretion could be accounted for by a decreased lipoprotein lipase degradation rate. In control cells 77 +/- 11% of the synthesized enzyme was degraded whereas in chlorate-treated cells degradation was reduced to 42 +/- 9% of the synthesized amount. The present study shows that decreased sulfation of heparan sulfate proteoglycans decreases the maximum binding of the lipase for the adipocyte cell surface. Consistent with the model that binding of lipoprotein lipase to cell surface heparan sulfate is required for lipase degradation, degradation is reduced in chlorate-treated cultures. In this report it is also shown that chlorate inhibits lipoprotein lipase sulfation and that desulfation of the enzyme has no effect on its catalytic efficiency or on its binding to cultured adipocytes.     

Characterization of high-affinity [3H]TBZOH binding to the human platelet vesicular monoamine transporter.

The present study indicates that human platelets can be used as an accessible peripheral model not only for the plasma membrane serotonin transporter, but also for the vesicular monoamine transporter. The vesicular monoamine transporter (VMAT2) is responsible for the accumulation of monoamines in the synaptic vesicles. VMAT2 differs from the plasma membrane transporters in its capability to recognize serotonin, histamine, norepinephrine and dopamine with almost the same affinity. Dihydrotetrabenazine (TBZOH) is a very potent inhibitor of VMAT2 that binds with high affinity to this transporter. [3H]TBZOH has been used as a ligand to label VMAT2 in human, bovine and rodent brain. In this study we characterized the pharmacodynamic and pharmacokinetic parameters of [3H]TBZOH binding in human platelets as compared to rat brain. The density (Bmax) and affinity (Kd) of [3H]TBZOH specific binding was assessed by Scatchard analysis. Association and dissociation rate constants (k(on), K(off)) were assessed by kinetic binding studies. In this study high-affinity and saturable binding sites for [3H]TBZOH were demonstrated in human platelets. Both the affinity of [3H]TBZOH to its binding site in platelets (Kd = 3.2+/-0.5 nM) and the kinetic rate constants (K(on) = 2.8 x 10(7) M(-1) min(-1); K(off) = 0.099 min(-1)) were similar to that in rat brain (Kd(striatum) = 1.5 nM; Kd(cerebral cortex) = 1.35 nM; K(on) = 2 x 10(7) M(-1) min(-1); K(off) = 0.069 min(-1)). Only the VMAT2 blockers tetrabenazine and reserpine inhibited [3H]TBZOH specific binding.     

Concurrent reduction in the sulfation of heparan sulfate and basement membrane assembly in a cell model system

Basement membranes (BMs) are specialized extracellular matrices that have important roles in cell attachment, migration, growth and differentiation. The murine teratocarcinoma cell line, M1536-B3, has been shown to produce a model BM composed of laminin, entactin and heparan sulfate proteoglycans but lacking collagen. Therefore, M1536-B3 cells are an excellent model system in which to study the role of non-collagenous components in BM assembly. We have used these cells to test for a requirement of mature heparan sulfate (HS) chains in BM assembly. Growth of M1536-B3 cells in the presence of chlorate, an inhibitor of activated sulfate synthesis, resulted in a dose-dependent decrease in the sulfation of glycosaminoglycans and reduction in the charge density of the isolated HS. The undersulfated HS from chlorate-treated cells had a decreased binding capacity for laminin when compared with control HS. Concurrent with these changes in sulfation, chlorate treatment of M1536-B3 cells resulted in the failure of BM assembly, which was restored upon removal of the chlorate from the growth medium. These results were not due to major alterations in cell attachment, spreading, growth, protein synthesis, or to an inability of the cells to synthesize and secrete laminin. These data suggest that the sulfation of HS and its subsequent ability to interact with other BM components play major roles in the assembly and structure of BMs.     

Effect of depolarization on binding kinetics of scorpion alpha-toxin highlights conformational changes of rat brain sodium channels.

Binding of scorpion alpha-toxins to receptor site 3 on voltage-gated sodium channels inhibits sodium current inactivation and is voltage-dependent. To reveal the direct effect of depolarization, we analyzed binding kinetics of the alpha-toxin Lqh-II (from Leiurus quinquestriatus hebraeus) to rat brain synaptosomes and effects on rat brain II (rBII) channels expressed in mammalian cells. Our results indicated that the 33-fold decrease in toxin affinity for depolarized (0 mV, 90 mM [K(+)](out), K(d) = 5.85 +/- 0.5 nM) versus polarized (-55 mV, 5 mM [K(+)](out), K(d) = 0.18 +/- 0.04 nM) synaptosomes at steady state results from a 48-fold reduction in the association rate (k(on) at 5 mM [K(+)] = (12.0 +/- 4) x 10(6) M(-1) s(-1) and (0.25 +/- 0.03) x 10(6) M(-1) s(-1) at 90 mM [K(+)](out)) with nearly no change in the dissociation rate. Electrophysiological analyses of rBII channels expressed in mammalian cells revealed that approximately 75% and 40% of rBII occupied fast- and slow-inactivated states, respectively, at resting membrane potential of synaptosomes (-55 mV), and Lqh-II markedly increased the steady-state fast and slow inactivation. To mimic electrophysiological conditions we induced fast depolarization of toxin-bound synaptosomes, which generated a biphasic unbinding of Lqh-II from toxin-receptor complexes. The first fast off rate closely resembled values determined electrophysiologically for rBII in mammalian cells. The second off rate was similar to the voltage-independent steady-state value, attributed to binding to the slow-inactivated channel states. Thus, the Lqh-II voltage-dependent affinity highlights two independent mechanisms representing conformational changes of sodium channels associated with transitions among electrically visible and invisible inactivated states.     

Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library

Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, DeltaHexA-GlcNSO(3)-(HexA-GlcNSO(3))(3)) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin. 2-O- and 6-O-sulfated Octa-I were prepared by enzymatically transferring one to three 2-O-sulfate groups and one to three 6-O-sulfate groups per molecule, respectively, to Octa-I. Another octasaccharide containing 3 units of HexA(2SO(4))-GlcNSO(3)(6SO(4)) was prepared also from heparin. This octasaccharide library was subjected to affinity chromatography for interactions with fibroblast growth factor (FGF)-2, -4, -7, -8, -10, and -18, hepatocyte growth factor, bone morphogenetic protein 6, and vascular endothelial growth factor, respectively. Based upon differences in the affinity to those octasaccharides, the growth factors could be classified roughly into five groups: group 1 needed 2-O-sulfate but not 6-O-sulfate (FGF-2); group 2 needed 6-O-sulfate but not 2-O-sulfate (FGF-10); group 3 had the affinity to both 2-O-sulfate and 6-O-sulfate but preferred 2-O-sulfate (FGF-18, hepatocyte growth factor); group 4 required both 2-O-sulfate and 6-O-sulfate (FGF-4, FGF-7); and group 5 hardly bound to any octasaccharides (FGF-8, bone morphogenetic protein 6, and vascular endothelial growth factor). The approach using the oligosaccharide library may be useful to define specific structures required for binding to various heparin-binding proteins. Octasaccharides with the high affinity to FGF-2 and FGF-10 had the activity to release them, respectively, from their complexes with HS. Thus, the library may provide new reagents to specifically regulate bindings of the growth factors to HS.     

Potentiation and inhibition of bFGF binding by heparin: a model for regulation of cellular response

Basic fibroblast growth factor (bFGF) binds to cell surface tyrosine kinase receptor proteins and to heparan sulfate proteoglycans. The interaction of bFGF with heparan sulfate on the cell surface has been demonstrated to impact receptor binding and biological activity. bFGF receptor binding affinity is reduced on cells that do not express heparan sulfate. The addition of soluble heparin or heparan sulfate has been demonstrated to rescue the bFGF receptor binding affinity on heparan sulfate deficient cells yet has also been shown to inhibit binding under some conditions. While the chemical requirements of the heparin-bFGF-receptor interactions have been studied in detail, the possibility that heparin enhances bFGF binding in part by physically associating with the cell surface has not been fully evaluated. In the study presented here, we have investigated the possibility that heparin binding to the cell surface might play a role in modulating bFGF receptor binding and activity. Balb/c3T3 cells were treated with various concentrations of sodium chlorate, so as to express a range of endogenous heparan sulfate sites, and [(125)I]bFGF binding was assessed in the presence of a range of heparin concentrations. Low concentrations of heparin (0.1-30 nM) enhanced bFGF receptor binding to an extent that was inversely proportional to the amount of endogenous heparan sulfate sites present. At high concentrations (10 microM), heparin inhibited bFGF receptor binding in cells under all conditions. The ability of heparin to stimulate and inhibit bFGF-receptor binding correlated with altered bFGF-stimulated tyrosine kinase activity and cell proliferation. Under control and chlorate-treated conditions, [(125) I]heparin was observed to bind with a high affinity to a large number of binding sites on the cells (K(d) = 57 and 50 nM with 3.5 x 10(6) and 3.6 x 10(6) sites/cell for control and chlorate-treated cells, respectively). A mathematical model of this process revealed that the dual functions of heparin in bFGF binding were accurately represented by heparin cell binding-mediated stimulation and soluble heparin-mediated inhibition of bFGF receptor binding.     

Real time analysis of interaction between inositol 1,4, 5-trisphosphate receptor type I and its ligand.

Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger that releases intracellular Ca(2+) by binding to its specific receptor, inositol 1,4,5-trisphosphate receptor (IP(3)R), in a wide range of cellular processes. We report here large-scale expression and purification of N-terminal 604 amino acids of IP(3)R type 1 (T604) expressed in E. coli, which contains the ligand binding domain. Surface plasmon resonance biosensor studies showed that purified T604 could bind to its ligands with binding specificity identical to that of full-length native IP(3)R type 1. Kinetic parameters of T604 for IP(3) consisted of a fast association rate constant (K(ass) = 1.2 x 10(6) M(-1) s(-1)) and a rapid dissociation rate constant (k(diss) = 1 s(-1)), and the equilibrium dissociation constant was determined to be 336 nM, at 150 mM NaCl and pH 7.4. However, association and dissociation patterns depended on the pH level and ionic strength. These results pave the way toward detail analysis of structure-function analysis of the ligand binding domain of IP(3)R type 1 for its ligands.     

Full-length and N-TIMP-3 display equal inhibitory activities toward TNF-alpha convertase

We previously reported that tumor necrosis factor-alpha converting enzyme (TACE) was specifically inhibited by TIMP-3 but not TIMP-1, -2, and -4. Further mutagenesis studies showed that the N-terminal domain of TIMP-3 (N-TIMP-3) retained full inhibitory activity towards TACE. Full-length TIMP-3 and N-TIMP-3 exhibited indistinguishable values for the association rate constant and inhibitory affinity constant for the active catalytic domain of TACE (k(on) approximately 10(5) M(-1) s(-1) and K(app)(i) approximately 0.20 nM). Moreover, their k(on) (approximately 10(4) M(-1) s(-1)) and K(app)(i) (approximately 1.0 nM) values with a longer form of TACE (which encompasses the complete ectodomain including disintegrin, EGF and Crambin-like domains) were also shown to be similar. Detailed kinetic analyses indicated that TIMP-3 associated more quickly and with tighter final binding with TACE devoid of these C-terminal domains. We conclude that, unlike the interaction between many MMPs and TIMPs, the C-terminal domains of TIMP-3 and TACE are not essential in the formation of a tight binary complex.     

Poly(A)-binding protein increases the binding affinity and kinetic rates of interaction of viral protein linked to genome with translation initiation factors eIFiso4F and eIFiso4F·4B complex

VPg of turnip mosaic virus (TuMV) was previously shown to interact with translation initiation factor eIFiso4F and play an important role in mRNA translation [Khan, M. A., et al. (2008) J. Biol. Chem.283, 1340-1349]. VPg competed with cap analogue for eIFiso4F binding and competitively inhibited cap-dependent translation and enhanced cap-independent translation to give viral RNA a significant competitive advantage. To gain further insight into the cap-independent process of initiation of protein synthesis, we examined the effect of PABP and/or eIF4B on the equilibrium and kinetics of binding of VPg to eIFiso4F. Equilibrium data showed the addition of PABP and/or eIF4B to eIFiso4F increased the binding affinity for VPg (K(d) = 24.3 ± 1.6 nM) as compared to that with eIFiso4F alone (K(d) = 81.3 ± 0.2.4 nM). Thermodynamic parameters showed that binding of VPg to eIFiso4F was enthalpy-driven and entropy-favorable with the addition of PABP and/or eIF4B. PABP and eIF4B decreased the entropic contribution by 67% for binding of VPg to eIFiso4F. The decrease in entropy involved in the formation of the eIFiso4F·4B·PABP-VPg complex suggested weakened hydrophobic interactions for complex formation and an overall conformational change. The kinetic studies of eIFiso4F with VPg in the presence of PABP and eIF4B show 3-fold faster association (k(2) = 182 ± 9.0 s(-1)) compared to that with eIFiso4F alone (k(2) = 69.0 ± 1.5 s(-1)) . The dissociation rate was 3-fold slower (k(-2) = 6.5 ± 0.43 s(-1)) for eIFiso4F with VPg in the presence of PABP and eIF4B (k(-2) = 19.0 ± 0.9 s(-1)). The addition of PABP and eIF4B decreased the activation energy of eIFiso4F with VPg from 81.0 ± 3.0 to 44.0 ± 2.4 kJ/mol. This suggests that the presence of both proteins leads to a rapid, stable complex, which serves to sequester initiation factors.     

Kinetic analysis of the interactions between troponin C and the C-terminal troponin I regulatory region and validation of a new peptide delivery/capture system used for surface plasmon resonance

Using surface plasmon resonance (SPR)-based biosensor analysis and fluorescence spectroscopy, the apparent kinetic constants, k(on) and k(off), and equilibrium dissociation constant, K(d), have been determined for the binding interaction between rabbit skeletal troponin C (TnC) and rabbit skeletal troponin I (TnI) regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). To carry out SPR analysis, a new peptide delivery/capture system was utilized in which the TnI peptides were conjugated to the E-coil strand of a de novo designed heterodimeric coiled-coil domain. The TnI peptide conjugates were then captured via dimerization to the opposite strand (K-coil), which was immobilized on the biosensor surface. TnC was then injected over the biosensor surface for quantitative binding analysis. For fluorescence spectroscopy analysis, the environmentally sensitive fluoroprobe 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid (1,5-IAEDANS) was covalently linked to Cys98 of TnC and free TnI peptides were added. SPR analysis yielded equilibrium dissociation constants for TnC (plus Ca(2+)) binding to the C-terminal TnI regulatory peptides TnI(96-131) and TnI(96-139) of 89nM and 58nM, respectively. The apparent association and dissociation rate constants for each interaction were k(on)=2.3x10(5)M(-1)s(-1), 2.0x10(5)M(-1)s(-1) and k(off)=2.0x10(-2)s(-1), 1.2x10(-2)s(-1) for TnI(96-131) and TnI(96-139) peptides, respectively. These results were consistent with those obtained by fluorescence spectroscopy analysis: K(d) being equal to 130nM and 56nM for TnC-TnI(96-131) and TnC-TnI(96-139), respectively. Interestingly, although the inhibitory region peptide (TnI(96-115)) was observed to bind with an affinity similar to that of TnI(96-131) by fluorescence analysis (K(d)=380nM), its binding was not detected by SPR. Subsequent investigations examining salt effects suggested that the binding mechanism for the inhibitory region peptide is best characterized by an electrostatically driven fast on-rate ( approximately 1x10(8) to 1x10(9)M(-1)s(-1)) and a fast off-rate ( approximately 1x10(2)s(-1)). Taken together, the determination of these kinetic rate constants permits a clearer view of the interactions between the TnC and TnI proteins of the troponin complex.     

Kinetic analysis of the mass transport limited interaction between the tyrosine kinase lck SH2 domain and a phosphorylated peptide studied by a new cuvette-based surface plasmon resonance instrument

We explored the use of a newly developed cuvette-based surface plasmon resonance (SPR) instrument (IBIS) to study peptide-protein interactions. We studied the interaction between the SH2 domain of lck and a phosphotyrosine peptide EPQY*EEIPIYL which was immobilized on a sensor chip. No indications for mass transport limitation (MTL) were observed when standard kinetic approaches were used. However, addition of competing peptide during dissociation revealed a high extent of rebinding. A dissociation rate constant (k(d)) of 0.6+/-0.1 s(-1) was obtained in the presence of large amounts of peptide. A simple bimolecular binding model, applying second-order kinetics for the cuvette system, could not adequately describe the data. Fits were improved upon including a step in the model which describes diffusion of the SH2 domain from the bulk to the sensor, especially for a surface with high binding capacity. From experiments in glycerol-containing buffers, it appeared that the diffusion rate decreased with higher viscosity. It is demonstrated that MTL during association and dissociation can be described by the same diffusion rate. A binding constant (K(D)) of 5.9+/-0.8 nM was obtained from the SPR equilibrium signals by fitting to a Langmuir binding isotherm, with correction for loss of free analyte due to binding. An association rate constant k(a) of 1.1(+/-0.2)x10(8) M(-1) x s(-1) was obtained from k(d)/K(D). The values for k(a) and k(d) obtained in this way were 2-3 orders larger than that from standard kinetic analysis, ignoring MTL. We conclude that in a cuvette the extent of MTL is comparable to that in a flow system.