Selectivity in glycosaminoglycan binding dictates the distribution and diffusion of fibroblast growth factors in the pericellular matrix

The range of biological outcomes generated by many signalling proteins in development and homeostasis is increased by their interactions with glycosaminoglycans, particularly heparan sulfate (HS). This interaction controls the localization and movement of these signalling proteins, but whether such control depends on the specificity of the interactions is not known. We used five fibroblast growth factors with an N-terminal HaloTag (Halo-FGFs) for fluorescent labelling, with well-characterized and distinct HS-binding properties, and measured their binding and diffusion in pericellular matrix of fixed rat mammary 27 fibroblasts. Halo-FGF1, Halo-FGF2 and Halo-FGF6 bound to HS, whereas Halo-FGF10 also interacted with chondroitin sulfate/dermatan sulfate, and FGF20 did not bind detectably. The distribution of bound FGFs in the pericellular matrix was not homogeneous, and for FGF10 exhibited striking clusters. Fluorescence recovery after photobleaching showed that FGF2 and FGF6 diffused faster, whereas FGF1 diffused more slowly, and FGF10 was immobile. The results demonstrate that the specificity of the interactions of proteins with glycosaminoglycans controls their binding and diffusion. Moreover, cells regulate the spatial distribution of different protein-binding sites in glycosaminoglycans independently of each other, implying that the extracellular matrix has long-range structure.     

A basic peptide derived from the HARP C-terminus inhibits anchorage-independent growth of DU145 prostate cancer cells

Heparin affin regulatory peptide (HARP) is an 18 kDa heparin-binding protein that plays a key role in tumor growth. We showed previously that the synthetic peptide P(111-136) composed of the last 26 HARP amino acids inhibited HARP-induced mitogenesis. Here, to identify the exact molecular domain involved in HARP inhibition, we investigated the effect of the shorter basic peptide P(122-131) on DU145 cells, which express HARP and its receptor protein tyrosine phosphatase beta/zeta (RPTPbeta/zeta). P(122-131) was not cytotoxic; it dose-dependently inhibited anchorage-independent growth of DU145 cells. Binding studies using biotinylated P(122-131) indicated that this peptide interfered with HARP binding to DU145 cells. Investigation of the mechanisms involved suggested interference, under anchorage-independent conditions, of P(122-131) with a HARP autocrine loop in an RPTPbeta/zeta-dependent fashion. Thus, P(122-131) may hold potential for the treatment of disorders involving RPTPbeta/zeta.     

Conserved intron positions in ancient protein modules

Background  

The timing of the origin of introns is of crucial importance for an understanding of early genome architecture. The Exon theory of genes proposed a role for introns in the formation of multi-exon proteins by exon shuffling and predicts the presence of conserved splice sites in ancient genes. In this study, large-scale analysis of potential conserved splice sites was performed using an intron-exon database (ExInt) derived from GenBank.     

Binding to intracellular targets of the metastasis-inducing protein, S100A4 (p9Ka)

Experimentally elevated levels of S100A4 induce a metastatic phenotype in benign mammary tumour cells in vivo. In humans, the presence of S100A4 in breast cancer cells correlates strongly with reduced patient survival. Potential interacting binding partners for S100A4 have now been examined using an optical biosensor. There was significant interaction of S100A4 with non-muscle myosin and p53, but not with actin, tropomyosin or tubulin. The results suggest that myosin and p53 are likely to be intracellular targets of S100A4. S100A4 had a greater affinity for wild-type or mutant arg-175-his p53 than for non-muscle myosin. The results suggest that S100A4 might induce metastasis by influencing the function of p53 as well as through its interaction with myosin and that any mechanism is independent of the mutational status of p53.     

Glycosaminoglycans differentially bind HARP and modulate its biological activity

Heparin affin regulatory peptide (HARP) is a polypeptide belonging to a family of heparin binding growth/differentiation factors. The high affinity of HARP for heparin suggests that this secreted polypeptide should also bind to heparan sulfate proteoglycans derived from cell surface and extracellular matrix defined as extracellular compartments. Using Western blot analysis, we detected HARP bound to heparan sulfate proteoglycans in the extracellular compartments of MDA-MB 231 and MC 3T3-E1 as well as NIH3T3 cells overexpressing HARP protein. Heparitinase treatment of BEL cells inhibited HARP-induced cell proliferation, and the biological activity of HARP in this system was restored by the addition of heparin. We report that heparan sulfate, dermatan sulfate, and to a lesser extent, chondroitin sulfate A, displaced HARP bound to the extracellular compartment. Binding analyses with a biosensor showed that HARP bound heparin with fast association and dissociation kinetics (kass = 1.6 x 10(6) M-1 s-1; kdiss = 0.02 s-1), yielding a Kd value of 13 nM; the interaction between HARP and dermatan sulfate was characterized by slower association kinetics (kass = 0.68 x 10(6) M-1 s-1) and a lower affinity (Kd = 51 nM). Exogenous heparin, heparan sulfate, and dermatan sulfate potentiated the growth-stimulatory activity of HARP, suggesting that corresponding proteoglycans could be involved in the regulation of the mitogenic activity of HARP.     

Hepatocyte growth factor/scatter factor and its interaction with heparan sulphate and dermatan sulphate

Hepatocyte growth factor (HGF)/scatter factor (SF) is a unique growth factor, in that it binds both heparan sulphate (HS) and dermatan sulphate (DS). The sequences in HS and DS that specifically interact with and modulate HGF/SF activity have not yet been fully identified. Ascidian DS, which uniquely possesses O-sulphation at C-6 (and not C-4) of its N -acetylgalactosamine unit, was analysed for HGF/SF-binding activity in the biosensor. The kinetic analysis revealed a strong, biologically relevant interaction with an equilibrium dissociation constant ( K (d)) of approx. 1 nM. An Erk activation assay also demonstrated stimulation of the MAP kinase pathway downstream of the Met receptor following addition of both HGF/SF and ascidian DS to the glycosaminoglycan-deficient CHO-745 mutant cell line. Furthermore, the activation of Met and the MAP kinase pathway by HGF/SF and ascidian DS leads to a cellular response in the form of migration.     

The peptide route to multifunctional gold nanoparticles

Extremely stable, peptide-capped gold nanoparticles with two different biomolecular recognition motifs expressed on their surface have been prepared, and their specific and selective binding to artificial, DNA-modified target particles and to DNA and protein microarrays has been demonstrated. Stabilization and biofunctionalization has been achieved in a single preparative step starting with citrate-stabilized gold hydrosols and a derivatization cocktail of peptide-capping ligands, which carry the functionalities of choice.     

Sepsis progression and outcome: a dynamical model

Background  

Sepsis (bloodstream infection) is the leading cause of death in non-surgical intensive care units. It is diagnosed in 750,000 US patients per annum, and has high mortality. Current understanding of sepsis is predominately observational and correlational, with only a partial and incomplete understanding of the physiological dynamics underlying the syndrome. There exists a need for dynamical models of sepsis progression, based upon basic physiologic principles, which could eventually guide hourly treatment decisions.     

Diversification of the Structural Determinants of Fibroblast Growth Factor-Heparin Interactions: IMPLICATIONS FOR BINDING SPECIFICITY*

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K_D values varying between 38 nm (FGF-18) and 620 nm (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 m⁻¹ s⁻¹ and FGF-9, 130,000 m⁻¹ s⁻¹). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.     

S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration

S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K(d) = 0.5 μM; IIB, K(d) = 8 μM; IIC, K(d) = 1.0 μM). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.