Conversion of a paracrine fibroblast growth factor into an endocrine fibroblast growth factor

FGFs 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. Here we show that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. In support of our model, we convert a paracrine FGF into an endocrine ligand by diminishing heparan sulfate-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor-binding site to home the ligand into the target tissue. In addition to serving as a proof of concept, the ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.     

The structural biology of growth factor receptor activation

Stimulation of cells by growth factors triggers cascades of signalling that result in cellular responses such as growth, differentiation, migration and survival. Many growth factors signal through receptor tyrosine kinases, leading to dimerization, trans-phosphorylation and activation of tyrosine kinases that phosphorylate components further downstream of the signal transduction cascade. Using insulin-like growth factor, nerve growth factor, hepatocyte growth factor and fibroblast growth factor as examples, we show that the globular architecture of the growth factors is essential for receptor binding. We describe how nerve growth factor (NGF) is a symmetrical dimer that binds four storage proteins (two -NGF and two γ-NGF) to give a symmetrical hetero-hexameric 7SNGF organised around the β-NGF dimer. It binds the extracellular domains of two receptor molecules in a similar way, so dimerising the receptor. Hepatocyte growth factor/scatter factor (HGF/SF) probably binds its receptor as a dimer stabilised by interactions with heparan sulfate, and fibroblast growth factor (FGF) binds its receptor as a dimer cross-linked by heparan sulfate. Surprisingly, insulin and insulin-like growth factor (IGF) bind in the monomeric form to receptors that are already covalent dimers. We propose that, in general, weak binary interactions between growth factor and individual domains of receptors are enhanced by cooperative interactions with further receptor domains, and sometimes other components like heparan, to give rise to specific multi-protein/domain complexes.     

Molecular cloning, overexpression and characterization of human interleukin 1alpha

Interleukin-1 alpha (IL-1alpha) regulates a wide range of important cellular processes. In this study for the first time, we report the cloning, expression, biophysical, and biological characterization of the human interleukin-1alpha. Human IL-1alpha has been expressed in Escherichia coli in high yields ( approximately 4mg per liter of the bacterial culture). The protein was purified to homogeneity ( approximately 98% purity) using affinity chromatography and size exclusion chromatography. Results of the steady-state fluorescence and 2D NMR experiments show that the recombinant IL-1alpha is in a folded conformation. Far-UV circular dichroism (CD) data suggest that IL-1alpha is an all beta-sheet protein with a beta-barrel architecture. Isothermal titration calorimetry (ITC) experiments show that the recombinant IL-1alpha binds strongly (K(d) approximately 5.6 x 10(-7) M) to S100A13, a calcium binding protein that chaperones the in vivo release of IL-1alpha into the extracellular compartment. Recombinant IL-1alpha was observed to exhibit strong cytostatic effect on human umbilical vascular endothelial cells. The findings of the present study not only pave way for an in-depth structural investigation of the molecular mechanism(s) underlying the non-classical release of IL-1alpha but also provide avenues for the rational design of potent inhibitors against IL-1alpha mediated pathogenesis.     

Cloning, overexpression, and characterization of cobrotoxin

Cobrotoxin (CBTX) is a highly toxic short neurotoxin, isolated from the Taiwan cobra (Naja naja atra) venom. In the present study for the first time we report the cloning and expression of CBTX in high yields (12mg/L) in Escherichia coli. CBTX fused to the IgG-binding domain of protein A (IgG-CBTX) was expressed in the soluble form. The misfolded CBTX portion (of the overexpressed fusion protein) was refolded under optimal redox conditions. The fusion protein (IgG-CBTX) was observed to undergo auto-catalytic cleavage to yield CBTX with additional 5 amino acids upstream of its N-terminal end. The far UV and near UV circular dichroism spectra of the recombinant CBTX were identical to those of the toxin isolated from the crude venom source. Recombinant CBTX was isotope labeled (15N and 13C) and all the resonances ('H, 13C, and 15N) in the protein have been unambiguously assigned. ' H '5N HSQC spectrum of recombinant CBTX revealed that the protein is in a biologically active conformation. 1H-15Nchemical shift perturbation data showed that recombinant CBTX binds to a peptide derived from the alpha7 subunit of the Torpedo acetylcholine receptor (AchR) with high affinity. The AchR peptide is found to bind to residues located at the tip of Loop-2 in CBTX. The results of the present study provide an avenue to understand the structural basis for the high toxicity exhibited by CBTX. In addition, complete resonance assignments in CBTX (reported in this study) are expected to trigger intensive research towards the design of new pharmacological agents against certain neural disorders.     

Expression of fibroblast growth factor receptor 3 by fibroblast growth factor 2 in cultured chick embryo chondrocytes

Although fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 3 (FGFR3) both inhibit longitudinal bone growth, little is known about the relationship between FGF2 and FGFR3. Accordingly, the current study examined the expression of FGFR3 mRNA after the administration of FGF2 using cultured chondrocytes from day 17 chick embryos to evaluate the relationship between FGF2 and FGFR3. The chondrocytes were isolated from the caudal one-third portion (LS) of sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and lower portion of the proximal tibial growth plate (Ti) of day 17 chick embryo. The expression of FGFR1, FGFR3, and type II and X collagen mRNA in the chondrocytes from the LS, USP, USC, and Ti was determined. FGFR1 was not expressed in the LS and USP chondrocytes, yet strongly expressed in the USC and Ti chondrocytes. With a treatment of FGF2, the expression of FGFR1 slightly increased in the USC chondrocytes and was not related with the concentration of FGF2 in the Ti chondrocytes. FGFR3 was expressed in all the chondrocyte types, yet strongly increased in the LS, USC, USP, and Ti in that order according to the concentration of FGF2. For the LS and USP chondrocytes, the expression of FGFR3 with FGF2 increased in a 4-day culture, yet decreased in a 6-day culture, whereas for the USC chondrocytes, the expression of FGFR3 mRNA with FGF2 increased in a 2-day culture, yet decreased in a 4-day culture, suggesting that the hypertrophic chondrocytes were more numerous and sensitive compared to the proliferative chondrocytes. For all the chondrocyte types, FGF2 appeared to be up-regulated to FGFR3, as the expression of FGFR3 mRNA increased with a higher concentration of FGF2 until a peak level. In conclusion, FGF2 was found to up-regulate to FGFR3 until the peak level of FGFR3 mRNA expression, while in hypertrophic chondrocytes, FGFR3 appeared to cause the differentiaton of chondrocytes, resulting in the inhibition of longitudinal bone growth after the peak level of FGFR3 mRNA expression.     

Bacterial expression and characterization of the ligand-binding domain of the vitamin D receptor

The ligand-binding domain of the rat vitamin D receptor (amino acids 115-423) was expressed as an amino-terminal His-tagged protein in a bacterial expression system and purified over Ni-nitrilotriacetic acid resin and a Mono S column. The purified protein bound its ligand, 1,25-dihydroxyvitamin D3, with high affinity, similar to that of the full-length protein. Saturation of the protein with ligand quenched 90% of the tryptophan fluorescence, consistent with the purified protein being uniformly able to bind ligand. Addition of ligand produced no change in the tryptophan fluorescence lifetime, suggesting static quenching as the mechanism of fluorescence decrease. The near-UV circular dichroism spectrum showed a large increase in signal following the addition of ligand, consistent with a change in the environment of aromatic amino acid side chains. The far-UV circular dichroism spectrum was consistent with a protein of high alpha-helical content. Sedimentation equilibrium experiments demonstrated that the protein formed higher-order complexes, and the distribution of the protein among these complexes was significantly shifted by addition of ligand.     

Mutational identification of fibroblast growth factor receptor 1 and fibroblast growth factor receptor 2 genes in craniosynostosis in Indian population

OBJECTIVE:

The Objective of this study was to identify the association of mutation of fibroblast growth factor receptor 1 (FGFR1), FGFR2 genes with syndromic as well as non-syndromic craniosynostosis in Indian population.

MATERIALS AND METHODS:

Retrospective analysis of our records from January 2008 to December 2012 was done. A total of 41 cases satisfying the inclusion criteria and 51 controls were taken for the study. A total volume of 3 ml blood from the patient as well as parents was taken. Deoxyribonucleic acid extracted using phenol chloroform extraction method followed by polymerase chain reaction-restriction fragment length polymorphism method.

RESULTS:

There were 33 (80.4%) non-syndromic cases of craniosynostosis while 8 (19.5%) were syndromic. Out of these 8 syndromic cases, 4 were Apert syndrome, 3 were Crouzon syndrome and 1 Pfeiffer syndrome. Phenotypically the most common non-syndromic craniosynostosis was scaphocephaly (19, 57.7%) followed by plagiocephaly in (14, 42.3%). FGFR1 mutation (Pro252Arg) was seen in 1 (2.4%) case of non-syndromic craniosynostosis while no association was noted either with FGFR1 or with FGFR2 mutation in syndromic cases. None of the control group showed any mutation.

CONCLUSION:

Our study proposed that FGFR1, FGFR2 mutation, which confers predisposition to craniosynostosis does not exist in Indian population when compared to the western world.     

Insulin-like growth factor binding protein-2: NMR analysis and structural characterization of the N-terminal domain

The insulin-like growth factor binding proteins are a family of six proteins (IGFBP-1 to -6) that bind insulin-like growth factors-I and -II (IGF-I/II) with high affinity. In addition to regulating IGF actions, IGFBPs have IGF-independent functions. IGFBP-2, the largest member of this family, is over-expressed in many cancers and has been proposed as a possible target for the development of novel anti-cancer therapeutics. The IGFBPs have a common architecture consisting of conserved N- and C-terminal domains joined by a variable linker domain. The solution structure and dynamics of the C-terminal domain of human IGFBP-2 have been reported (Kuang Z. et al. J. Mol. Biol. 364, 690-704, 2006) but neither the N-domain (N-BP-2) nor the linker domain have been characterised. Here we present NMR resonance assignments for human N-BP-2, achieved by recording spectra at low protein concentration using non-uniform sampling and maximum entropy reconstruction. Analysis of secondary chemical shifts shows that N-BP-2 possesses a secondary structure similar to that of other IGFBPs. Although aggregation hampered determination of the solution structure for N-BP-2, a homology model was generated based on the high degree of sequence and structure homology exhibited by the IGFBPs. This model was consistent with experimental NMR and SAXS data and displayed some unique features such as a Pro/Ala-rich non-polar insert, which formed a flexible solvent-exposed loop on the surface of the protein opposite to the IGF-binding interface. NMR data indicated that this loop could adopt either of two alternate conformations in solution - an entirely flexible conformation and one containing nascent helical structure. This loop and an adjacent poly-proline sequence may comprise a potential SH3 domain interaction site for binding to other proteins.     

Molecular modeling of the GABAC receptor ligand-binding domain

We have constructed a molecular model of the ligand-binding domain of the GABA(C) receptor, which is a member of the Cys-loop ligand-gated ion channel family. The extracellular domains of these receptors share similar sequence homology (20%) with Limnaea acetylcholine-binding protein for which an X-ray crystal structure is available. We used this structure as a template for homology modeling of the GABA(C) receptor extracellular domain using FUGUE and MODELLER software. FlexX was then used to dock GABA into the receptor ligand-binding site, resulting in three alternative energetically favorable orientations. Residues located no more than 5 A from the docked GABA were identified for each model; of these, three were found to be common to all models with 14 others present only in certain models. Using data from experimental studies, we propose that the most likely orientation of GABA is with its amine close to Y198, and its carboxylate close to R104. These studies have therefore provided a model of the ligand-binding domain, which will be useful for both GABA(C) and GABA(A) receptor studies, and have also yielded an experimentally testable hypothesis of the location of GABA in the binding pocket. [Figure: see text].     

Production and characterization of the extracellular domain of recombinant human fibroblast growth factor receptor 4

Among the members of the fibroblast growth factor receptor family the FGFR4 has demonstrated strong dependence on heparin-like material for its activation by fibroblast growth factors. We have produced and characterized a recombinant human FGFR4 extracellular domain (FGFR4ed), in order to study its biochemical properties in isolated conditions. The FGFR4ed was expressed in an insect cell system and purified from the culture medium by Ni(2+)-affinity and gel filtration chromatography. Pure FGFR4ed was tested for FGF- and heparin-binding by covalent crosslinking experiments and by biosensor analysis. In solution, FGFR4ed formed complexes with acidic FGF (FGF-1) and basic FGF (FGF-2), both in the presence and absence of heparin. Immobilized FGFR4 also bound FGF-8 besides FGF-1 and FGF-2. Furthermore, heparin alone induced receptor oligomerization on the surface of the receptor coupled chip. Thus, the recombinant FGFR4ed revealed properties described for the cellular form of this receptor and can be used for interaction studies.     

Immunohistochemical detection of insulin-like growth factor-I, transforming growth factor-beta2, basic fibroblast growth factor and epidermal growth factor-receptor expression in developing rat ovary

The aim of this study was to determine the immunohistochemical expression and localization of insulin-like growth factor-I (IGF-I), transforming growth factor-β2 (TGF-β2), basic fibroblast growth factor (bFGF) and epidermal growth factor-receptor (EGF-R) in developing rat ovaries.Eighteen female Wistar rats were enrolled in this study; newborn (n = 6), one-month-old (n = 6) and adult (n = 6) rats. Formalin-fixed and parafin-embedded ovarian tissues were stained with antibodies against IGF-I, TGF-β2, bFGF and EGF-R, immunohistochemically. The ovarian cells were evaluated by semi-quantitative scoring system under light microscope.The staining of IGF-I, TGF-β2, bFGF and EGF-R were most intense in the oocytes and were heavily at one-month-old rats. A moderate immunostaining in theca cells and corpus luteii reacted with IGF-I in adult rats. Furthermore the staining intensity for IGF-I was moderate in granulosa cells of newborn rat ovaries. We detected also a moderate staining for TGF-β2 in corpus luteii of adult rats. In addition, we found a bFGF immunostaining mainly in oocytes of follicles of young and adult rats. Immunostaining for EGF-R was moderate in granulosa cells of one-month-old rats.In conclusion, this study suggests that growth factors play a pivotal role in ovarian function, especially in follicular development. The role of growth factor in controlling degeneration or growth (or both) of ovary follicles remain as explained.     

Molecular cloning,expression, and characterization of bovine tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz-type serine proteinase inhibitor that is secreted by all cells of the vasculature, and presumably plays a role in the regulation of plasmin-mediated matrix remodeling. In this report, we describe the cloning and expression of a full-length cDNA for bovine TFPI-2 that exhibits 72% sequence identity with that of human TFPI-2. Following a 22 residue signal peptide, the mature protein contains 212 amino acids with 18 cysteines, three putative N-glycosylation sites, and one putative O-glycosylation site. The deduced sequence of mature bovine TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxy-terminal tail highly enriched in basic amino acids. Recombinant bovine TFPI-2 was expressed in HEK 293 cells and resolved into two isoforms, designated as alpha-TFPI-2 (M(r) 33 kDa) and beta-TFPI-2 (M(r) 31 kDa), which presumably represent differentially glycosylated forms of the inhibitor. Similar to human TFPI-2, both bovine TFPI-2 isoforms exhibited strong inhibitory activity towards trypsin and plasmin, and weak inhibitory activity towards the factor VIIa-tissue factor complex.     

The FGFR D3 domain determines receptor selectivity for fibroblast growth factor 21

Islet amyloid polypeptide (IAPP; also known as amylin) is responsible for islet amyloid formation in type 2 diabetes, and IAPP-induced toxicity is believed to contribute to the loss of β-cell mass associated with the late stages of type 2 diabetes. Islet amyloid formation may also play a role in graft failure after transplantation. IAPP is produced as a prohormone, pro-islet amyloid polypeptide (proIAPP), and processed in the secretory granules of the pancreatic β-cells. Partially processed forms of proIAPP are found in amyloid deposits; most notable is a 48-residue intermediate, proIAPP_1-48, which includes the N-terminal pro-extension, but which has been properly processed at the C-terminus. Incomplete processing may play a role in islet amyloid formation by promoting interactions with sulfated proteoglycans of the extracellular matrix, which, in turn, promote amyloid formation. We show that acid fuchsin (3-(1-(4-amino-3-methyl-5-sulphonatophenyl)-1-(4-amino-3-sulphonatophenyl)methylene)cyclohexa-1,4-dienesulphonic acid), a simple sulfonated triphenyl methyl derivative, is a potent inhibitor of amyloid formation by the proIAPP_1-48 intermediate. The more complicated triphenyl methane derivative fast green FCF {ethyl-[4-[[4-[ethyl-[(3-sulfophenyl)methyl]amino]phenyl]-(4-hydroxy-2-sulfophenyl)methylidene]-1-cyclohexa-2,5-dienylidene]-[(3-sulfophenyl)methyl]azanium} also inhibits amyloid formation by IAPP and the proIAPP processing intermediate. Both compounds inhibit amyloid formation by mixtures of the proIAPP intermediate and the model glycosaminoglycan heparan sulfate. Acid fuchsin also inhibits glycosaminoglycan-mediated amyloid formation by mature IAPP. The ability to inhibit amyloid formation is not simply due to the compounds being sulfonated, since the sulfonated inhibitor of amyloid-β, tramiprosate, is not an inhibitor of amyloid formation by proIAPP_1-48.     

1H, 13C and 15N assignment of D2 domain of human fibroblast growth factor receptor 4

Fibroblast growth factor receptor (FGFR) 4 has been associated with progression of melanoma, breast, head and neck and hepatocellular carcinoma and is therefore an interesting target for therapeutic intervention (Ho et al. in J Hepatol 50:118–127, 2009). The extracellular D2 domain of the FGFR4 receptor contains a heparin binding site and the main interaction site with the fibroblast growth factor. We report the sequential backbone and side chain resonance assignment of the D2 domain of human FGFR4.     

Hrs regulates the endocytic sorting of the fibroblast growth factor receptor 2b

The keratinocyte growth factor receptor or fibroblast growth factor receptor 2b (KGFR/FGFR2b) is activated by the specific interaction with the keratinocyte growth factor (KGF/FGF7), which targets the receptor to the degradative pathway, and the fibroblast growth factor 10 (FGF10/KGF2), which drives the receptor to the juxtanuclear recycling route. Hrs plays a key role in the regulation of the endocytic degradative transport of ubiquitinated receptor tyrosine kinases, but the direct involvement of this protein in the regulation of FGFR endocytosis has not been investigated yet. We investigated here the possible role of Hrs in the alternative endocytic pathways of KGFR. Quantitative immunofluorescence microscopy and biochemical analysis showed that both overexpression and siRNA interference of Hrs inhibit the KGF-triggered KGFR degradation, blocking receptor transport to lysosomes and causing its rapid reapparance at the plasma membrane. In contrast, the FGF10-induced KGFR targeting to the recycling compartment is not affected by Hrs overexpression or depletion. Coimmunoprecipitation approaches indicated that Hrs is recruited to KGFR only after KGF treatment, although it is not tyrosine phosphorylated by the ligand. In conclusion, Hrs regulates the KGFR degradative pathway, but not its juxtanuclear recycling transport. In addition, the results suggest that Hrs recruitment to the receptor, but not its ligand-induced phosphorylation, could be required for its function.     

Acidic fibroblast growth factor (FGF) potentiates glial-mediated neurotoxicity by activating FGFR2 IIIb protein

Previous studies indicate that astrocytes are the brain cells that express acidic fibroblast growth factor (aFGF) and that the expression is increased upon activation. However, there has been no study investigating the significance of this phenomenon. Here we report that aFGF treatment of IFNγ-stimulated human astrocytes, and LPS/IFNγ-stimulated human microglia, enhances their secretion of inflammatory cytokines and other materials toxic to human neuroblastoma SH-SY5Y cells. The mechanism of aFGF enhancement involves stimulation of the receptor FGFR2 IIIb. We show by RT-PCR that this receptor, but not other FGF receptors, is robustly expressed by astrocytes and microglia. We establish by Western blotting, and immunohistochemistry on postmortem human brain tissue that the FGFR2 IIIb protein is expressed by both of these glial cell types. We blocked the inflammatory stimulant action of aFGF by transfecting microglia and astrocytes with a small inhibitory RNA (siRNA) to FGFR2 IIIb as well as by removal of aFGF using an anti-aFGF antibody. Treatment with bFGF in combination with the stimulants was without effect, but together with aFGF, it partially counteracted the action of aFGF, indicating that it may be a weak antagonist of FGFR2 IIIb. The inflammatory effect was also attenuated by treatment with inhibitors of protein kinase C, Src tyrosine kinase, and MEK-1/2 indicating the involvement of these intracellular pathways. Our data suggest that inhibition of expression or release of aFGF could have therapeutic potential by inhibiting inflammation in neurodegenerative diseases such as Alzheimer disease where many neuroinflammatory molecules are prominently expressed.