PWWP module of human hepatoma-derived growth factor forms a domain-swapped dimer with much higher affinity for heparin

Hepatoma-derived growth factor (hHDGF)-related proteins (HRPs) comprise a new growth factor family sharing a highly conserved and ordered N-terminal PWWP module (residues 1-100, previously referred to as a HATH domain) and a variable disordered C-terminal domain. We have shown that the PWWP module is responsible for heparin binding and have solved its structure in solution. Here, we show that under physiological conditions, both the PWWP module and hHDGF can form dimers. Surface plasmon resonance (SPR) studies revealed that the PWWP dimer binds to heparin with affinity that is two orders of magnitude higher (K(d)=13 nM) than that of the monomeric PWWP module (K(d)=1.2 microM). The monomer-dimer equilibrium properties and NMR structural data together suggest that the PWWP dimer is formed through a domain-swapping mechanism. The domain-swapped PWWP dimer structures were calculated on the basis of the NMR data. The results show that the two PWWP protomers exchange their N-terminal hairpin to form a domain-swapped dimer. The two monomers in a dimer are linked by the long flexible L2 loops, a feature supported by NMR relaxation data for the monomer and dimer. The enhanced heparin-binding affinity of the dimer can be rationalized in the framework of the dimer structure.     

Solution structure of human acidic fibroblast growth factor and interaction with heparin-derived hexasaccharide

Fibroblast growth factors (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparansulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110–131 and 17–21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin- oligosaccharides in a similar manner.     

Solution structure of the PWWP domain of the hepatoma-derived growth factor family

Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded beta-barrel with a PWWP-specific long loop connecting beta2 and beta3 (PR-loop), followed by a helical region including two alpha-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the beta1/beta2 loop (beta-beta arch) and the beta3/beta4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.     

Downregulation of hepatoma-derived growth factor activates the Bad-mediated apoptotic pathway in human cancer cells

Hepatoma-derived growth factor (HDGF) is highly expressed in human cancer and its expression is correlated with poor prognosis of cancer. The growth factor is known to stimulate cell growth while the underlying mechanism is however not clear. Transfection with HDGF cDNA stimulated while its specific antisense oligonucleotides repressed the growth of human hepatocellular carcinoma HepG2 cells. Furthermore, knock-down of HDGF by antisense oligos also induced apoptosis in HepG2 cells and in other human cancer cells, e.g. human squamous carcinoma A431 cells. HDGF knock-down was found to induce the expression of the pro-apoptotic protein Bad and also inactivate ERK and Akt, which in turn led to dephosphorylation of Bad at Ser-112, Ser-136, and activation of the intrinsic apoptotic pathway, i.e. depolarization of the mitochondrial membrane, release of mitochondrial cytochrome c, increase in the processing of caspase 9 and 3. As HDGF knock-down not only suppresses the growth but also induces apoptosis in human cancer cells, HDGF may therefore serve as a survival factor for human cancer cells and a potential target for cancer therapy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative studies of the interaction of human and bovine platelet factor 4 with heparin using histidine NMR resonances as spectroscopic probes

ThepK _a values of His-38 and His-50 of the heparin-binding protein, bovine platelet factor 4, are 5.6 and 6.5, respectively, as determined by¹H NMR spectroscopy. The¹H NMR resonance of His-38 of bovine platelet factor 4 which exhibits the lowerpK _a value is perturbed upon heparin binding to a greater degree than the resonance of His-50. Human platelet factor 4 contains the homologous residues His-23 and His-35. ThepK _a values of the two histidine residues of human platelet factor 4 are 5.3 and 6.4. The¹H NMR resonance of the histidine of human platelet factor 4 exhibiting the lowerpK _a value also is perturbed upon heparin binding to a greater degree than the histidine resonance exhibiting the higherpK _a , thereby suggesting comparable heparin-protein interactions in bovine and human platelet factor 4.     

Solution structure of a phage-derived peptide antagonist in complex with vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mediator of angiogenesis and vasculogenesis. VEGF is involved pathologically in cancer, proliferative retinopathy and rheumatoid arthritis, and as such represents an important therapeutic target. Three classes of disulfide-constrained peptides that antagonize binding of the VEGF dimer to its receptors, KDR and Flt-1, were identified previously using phage display methods. NMR studies of a representative peptide from the most potent class of these peptide antagonists, v107 (GGNECDAIRMWEWECFERL), were undertaken to characterize its interactions with VEGF. v107 has no defined structure free in solution, but binding to VEGF induces folding of the peptide. The solution structure of the VEGF receptor-binding domain-v107 complex was determined using 3940 (1970 per VEGF monomer) internuclear distance and 476 (238 per VEGF monomer) dihedral angle restraints derived from NMR data obtained using samples containing either (13)C/(15)N-labeled protein plus excess unlabeled peptide or (13)C/(15)N-labeled peptide plus excess unlabeled protein. Residual dipolar coupling restraints supplemented the structure determination of the complex and were found to increase significantly both the global precision of VEGF in the complex and the agreement with available crystal structures of VEGF. The calculated ensemble of structures is of high precision and is in excellent agreement with the experimental restraints. v107 has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two v107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. The v107-binding site on VEGF overlaps partially with the binding site of KDR and is similar to that for domain 2 of Flt-1. The structure of the VEGF-v107 complex provides new insight into how binding to VEGF can be achieved that may be useful for the design of small molecule antagonists.     

HDGF的PWWP结构域改变对肿瘤细胞体外及体内增殖的影响

目的:研究肝癌衍生生长因子(Hepatoma-derived growth factor,HDGF)PWWP结构域(PWWP domain)改变对肿瘤细胞体外及体内增殖的影响。方法:构建HDGF的PWWP结构域突变体P24A,利用慢病毒感染细胞筛选稳定细胞系。采用CCK-8法和软琼脂克隆形成实验检测细胞的增殖情况。通过裸鼠皮下成瘤实验检测移植瘤的形成情况。结果:在肝癌Hep G2和结直肠癌DLD1稳定细胞株中,CCK-8法检测结果显示突变体P24A对细胞生长的抑制作用呈时间依赖性,其OD值在24、48、72和96 h均明显低于HDGF稳定细胞株(均P0.001)。克隆形成实验结果显示P24A组克隆数目明显小于HDGF组(P0.01)。异种移植瘤动物模型则证明P24A细胞株的瘤块生长速度(P0.001,P0.01,P0.01),瘤块大小及体积(P0.01)均明显低于HDGF细胞株。结论:PWWP结构域改变可能抑制HDGF发挥促进细胞增殖的作用。     

Micropattern-immobilization of heparin to regulate cell growth with fibroblast growth factor

Heparin was immobilized on a polystyrene plate in a specificpattern by photolithography. Heparin was coupled with azidoaniline. Thederivatized heparin was cast on the polystyrene plate from aqueoussolution. After drying, the plate was photo-irradiated in the presence of aphotomask. The micropatterning was confirmed by staining with a dye,ethydium bromide. Since heparin has negative charges, the cationic dyewas adsorbed on the regions where heparin was immobilized. In thepresence fibroblast growth factor (FGF), the growth of mouse fibroblastSTO cells was enhanced only on the heparin-immobilized regions. Thisresult indicated that micropattern-immobilized heparin activated FGF forcell growth activity.     

Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro. I. Evidence for reversible binding and inactivation of the platelet-derived growth factor by heparin

Summary We have investigated the effects of interactions between growth factors and heparin-like glycosaminoglycans on untransformed human arterial smooth muscle cells (hASMC) in vitro. The results indicate that heparin in the presence of serum mitogens prevents the cells from entering the S phase of the cell cycle by binding and inactivating reversibly some serum mitogen(s). Our results suggest that platelet-derived growth factor (PDGF) is one of them and that it is the most potent stimulator of hASMC growth in vitro. Thymidine incorporation as well as increase in DNA content was inhibited not only by the presence of heparin in serum-containing medium but also when serum was chromatographed on Heparin-Sepharose at physiologic salt concentrations before exposure to the cells. The mitogenic activity of the unretained serum fraction was restored by the addition of PDGF AA, AB, or BB dimers or of a fraction (RF I) that dissociated from Heparin-Sepharose at 0.2 to 0.6M NaCl. Radiolabeled recombinant PDGF (c-sis) dissociated from Heparin-Sepharose within a concentration range of NaCl similar to that of RF I. Neither the unretained material nor the RF I or PDGF dimers were effective alone. The effect of RF I was significantly decreased by the addition of an anti-PDGF IgG that is known to neutralize the PDGF mitogenic activity partially. Addition of heparin abolished DNA-synthesis when the PDGF dimers or RF I were combined with the unretained fraction. A second fraction (RF II) bound strongly to Heparin-Sepharose and eluted between 1.1 and 1.6M NaCl. The RF II also induced DNA synthesis but was neither as efficient as RF I nor depending on other serum fractions for growth promotion and it was not inhibited by anti-PDGF IgG. A similar strong affinity for Heparin-Sepharose was found for labeled basic fibroblast growth factor and we cannot exclude the possibility that RF II represent fibroblast growth factor. Under these culture conditions, inhibition of hASMC proliferation was directly correlated with the expression of smooth muscle specific alpha actin isoforms in stress fibers and the suppression of a proliferating cell-specific nuclear antigen. Conversely, stimulation of hASMC proliferation was associated with the opposite phenomenon. We conclude that heparin-like glycosaminoglycans influence growth and phenotype of hASMCs in vitro by binding and inactivating PDGF. Inasmuch as heparin-like substances constitute a significant proportion of the proteoglycan-associated glycosaminoglycans of the arterial wall, such mechanisms might be important for the development of atherosclerotic lesions.     

High resolution structure of the HDGF PWWP domain: a potential DNA binding domain

Hepatoma Derived Growth Factor (HDGF) is an endogenous nuclear-targeted mitogen that is linked with human disease. HDGF is a member of the weakly conserved PWWP domain family. This 70-amino acid motif, originally identified from the WHSC1 gene, has been found in more than 60 eukaryotic proteins. In addition to the PWWP domain, many proteins in this class contain known chromatin remodeling domains, suggesting a role for HDGF in chromatin remodeling. We have determined the NMR structure of the HDGF PWWP domain to high resolution using a combination of NOEs, J-couplings, and dipolar couplings. Comparison of this structure to a previously determined structure of the HDGF PWWP domain shows a significant difference in the C-terminal region. Comparison to structures of other PWWP domains shows a high degree of similarity to the PWWP domain structures from Dnmt3b and mHRP. The results of selected and amplified binding assay and NMR titrations with DNA suggest that the HDGF PWWP domain may function as a nonspecific DNA-binding domain. Based on the NMR titrations, we propose a model of the interaction of the PWWP domain with DNA.     

Solution structure of the ubiquitin-like domain of human DC-UbP from dendritic cells

The previously identified dendritic cell-derived ubiquitin-like protein (DC-UbP) was implicated in cellular differentiation and apoptosis. Sequence alignment suggested that it contains a ubiquitin-like (UbL) domain in the C terminus. Here, we present the solution NMR structure and backbone dynamics of the UbL domain of DC-UbP. The overall structure of the domain is very similar to that of Ub despite low similarity (<30%) in amino-acid sequence. One distinct feature of the domain structure is its highly positively charged surface that is different from the corresponding surfaces of the well-known UbL modifiers, Ub, NEDD8, and SUMO-1. The key amino-acid residues responsible for guiding polyubiquitinated proteins to proteasome degradation in Ub are not conserved in the UbL domain. This implies that the UbL domain of DC-UbP may have its own specific interaction partners with other yet unknown cellular functions related to the Ub pathway.     

NMR restraint analysis of transforming growth factor alpha: a key component for NMR structure refinement.

Structures of the protein, transforming growth factor alpha (TGF-alpha), have been derived from NMR data using distance geometry and subsequent energy refinement. Analysis of the sequential NOE distance bounds using a template algorithm provides a check for consistency in the calculation of bounds, stereospecific assignment of prochiral centers, and secondary structure assignment. Application of the template algorithm to the long range NOEs found within the NMR data sets collected at pH 6.3 and pH 3.4 is used to assess the confidence levels for the accuracy of the structures obtained from modeling. The method also provides critical insight in differentiating regions of the structure that are well defined from those that are not. Use of the restraint analysis protocol is shown to be a powerful adjunct to currently used methods for the assignment of protein structures from NMR data.     

Bovine retina contains three growth factor activities with different affinity to heparin: eye derived growth factor I, II, III

Several ocular tissues have been shown to contain growth factor activity designated under a generic name as Eye Derived Growth Factor. Purification from bovine retina was undertaken and a fraction which could induce target cells to proliferate at doses of 5 ng per ml of culture medium was obtained. Using heparin sepharose chromatography we now show that this mitogenic activity can be fractionated into three different activities. Crude extract of bovine retina used as starting material was separated into two major fractions, one with no affinity for heparin and which was named Eye Derived Growth Factor III, and one with a strong affinity for heparin and eluted from the column with 1.4 M NaCl named Eye Derived Growth Factor I. This fraction EDGF I induces cell proliferation at doses of 100 pg/ml of culture medium. A 10(5) fold purification was achieved by this single chromatography step. Cibacron Blue purified EDGF was also further fractionated by heparin sepharose. All biological activity was found to bind to heparin. One fraction eluted at 1 M NaCl named Eye Derived Growth Factor II had a biological activity at doses of 1 ng while the other growth factor was the EDGF I with biological activity at 25 pg. At this step of purification EDGF I runs as a single band on SDS polyacrylamide gel at a molecular weight of 17 000 d. These data strongly suggest that Eye Derived Growth Factors I and II are respectively similar to Brain Fibroblast Growth Factor and to Endothelial Cell Growth Factor from hypothalamus.     

Solution structure of the antifreeze-like domain of human sialic acid synthase

The structure of the C-terminal antifreeze-like (AFL) domain of human sialic acid synthase was determined by NMR spectroscopy. The structure comprises one alpha- and two single-turn 3(10)-helices and two beta-strands, and is similar to those of the type III antifreeze proteins. Evolutionary trace analyses of the type III antifreeze protein family suggested that the class-specific residues in the human and bacterial AFL domains are important for their substrate binding, while the class-specific residues of the fish antifreeze proteins are gathered on the ice-binding surface.     

Interactions of putative heparin-binding domains of basic fibroblast growth factor and its receptor, FGFR-1, with heparin using synthetic peptides

We have examined structure-function relationships that have been proposed to account for the heparin-binding properties of basic fibroblast growth factor and its receptor, FGFR-1, using synthetic peptides, DNA synthesis assays and binding assays in a resonant mirror biosensor. The results suggest that the interaction of FGFR-1 with heparin may not be physiologically relevant and that the site of interaction of the polysaccharide on bFGF is more complex than has been anticipated. © 1998 Rapid Science Ltd     

The structure of human acidic fibroblast growth factor and its interaction with heparin.

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.     

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.