Elucidation of the mechanism of the regulatory function of the Ig1 module of the fibroblast growth factor receptor 1

The extracellular part of the fibroblast growth factor (FGF) receptor (FGFR) consists of up to three Ig modules (Ig1-Ig3), in which the Ig2 and Ig3 modules determine affinity and specificity for FGF and heparin. The FGFR isoforms lacking the Ig1 module have higher affinity for FGF and heparin than the triple Ig-module isoforms, suggesting that the Ig1 module is involved in the regulation of the FGFR-ligand interaction. We show here by surface plasmon resonance and NMR analyses that the Ig1 module binds to the Ig2 module, and identify by NMR the binding sites involved in the Ig1-Ig2 interaction. The identified binding site in the Ig2 module was found to be in the area of the FGF-Ig2 and Ig2-heparin contact sites, thus providing direct structural evidence that the Ig1 module functions as a competitive autoinhibitor of the FGFR-ligand interaction. Furthermore, the Ig1 binding site of the Ig2 module overlaps the Ig2-Ig2 contact site. This suggests that the function of the Ig1 module is not only regulation of the FGFR-ligand binding affinity but also prevention of spontaneous FGFR dimerization (through a direct Ig2-Ig2 interaction) in the absence of FGF.     

FGFR1 regulates proliferation and metastasis by targeting CCND1 in FGFR1 amplified lung cancer

ABSTRACT

Aims: The analysis of the online databases revealed that CCND1 expression is correlated with poor prognosis in LSCC. We aimed to explore the function of CCND1 in tumor progression in LSCC.

Main methods: The expression of mRNA was measured using qRT-PCR. Protein expression was assessed by Western blot. Cell migration and invasion were assessed by transwell assay.

Key findings: CCND1 was co-overexpressed with FGFR1 in lung cancer patients. Overexpression of CCND1 promoted LSCC cell proliferation and metastasis. FGFR1 promoted the processes of EMT through AKT/MAPK signaling by targeting CCND1 in FGFR1-amplification cell lines.

Significance: IIn conclusion, our study demonstrated the regulatory mechanism between CCND1 and FGFR1 in FGFR1 amplified LSCC. Co-targeting CCND1 and FGFR1 could provide greater clinical benefits.     

胶质瘤中FGFR1OP和PTEN的表达及临床意义

探讨FCFR1OP和PTEN在胶质瘤中的表达情况和临床意义。选取54例胶质瘤手术切除标本,采用SP法进行免疫组织化学染色。FGFR1OP和PTEN的阳性表达率分别为85．2％和48．1％,且随着胶质瘤的恶性程度不同表现出明显的统计学差异（P〈0．05）。FGFR1OP的高表达与PTEN的低表达可能参与胶质瘤的生长分化和进展,并提示预后不良。     

MiR‐3116 sensitizes glioma cells to temozolomide by targeting FGFR1 and regulating the FGFR1/PI3K/AKT pathway

Glioma is a brain tumour that is often diagnosed, and temozolomide (TMZ) is a common chemotherapeutic drug used in glioma. Yet, resistance to TMZ is a chief hurdle towards curing the malignancy. The current work explores the pathways and involvement of miR‐3116 in the TMZ resistance. miR‐3116 and FGFR1 mRNA were quantified by real‐time PCR in malignant samples and cell lines. Appropriate assays were designed for apoptosis, viability, the ability to form colonies and reporter assays to study the effects of the miR‐3116 or FGFR1. The involvement of PI3K/AKT signalling was assessed using Western blotting. Tumorigenesis was evaluated in an appropriate xenograft mouse model in vivo. This work revealed that the levels of miR‐3116 dipped in samples resistant to TMZ, while increased miR‐3116 caused an inhibition of the tumour features mentioned above to hence augment TMZ sensitivity. miR‐3116 was found to target FGFR1. When FGFR1 was overexpressed, resistance to TMZ was augmented and reversed the sensitivity caused by miR‐3116. Our findings further confirmed PI3K/AKT signalling pathway is involved in this action. In conclusion, miR‐3116 sensitizes glioma cells to TMZ through FGFR1 downregulation and the PI3K/AKT pathway inactivation. Our results provide a strategy to overcome TMZ resistance in glioma treatment.     

Three-dimensional solution structure of mouse [Cd7]-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopy

Sequential 1H-NMR assignments of mouse [Cd7]-metallothionein-1 (MT1) have been carried out by standard homonuclear NMR methods and the use of an accordion-heteronuclear multiple quantum correlation (HMQC) experiment for establishing the metal, 113Cd2+, to cysteine connectivities. The three-dimensional structure was then calculated using the distance constraints from two-dimensional nuclear Overhauser effect (NOE) spectroscopy spectra and the Cys-Cd connectivities as input for a distance geometry-dynamical simulated annealing protocol in X-PLOR 3.851. Similar to the mammalian MT2 isoforms, the homologous primary structure of MT1 suggested two separate domains, each containing one metal cluster. Because there were no interdomain constraints, the structure calculation for the N-terminal beta- and the C-terminal alpha-domain were carried out separately. The structures are based on 409 NMR constraints, consisting of 381 NOEs and 28 cysteine-metal connectivities. The only elements of regular secondary structure found were two short stretches of 3(10) helices along with some half-turns in the alpha-domain. Structural comparison with rat liver MT2 showed high similarity, with the beta-domain structure in mouse MT1 showing evidence of increased flexibility compared to the same domain in MT2. The latter was reflected by the presence of fewer interresidue NOEs, no slowly exchanging backbone amide protons, and enhanced cadmium-cadmium exchange rates found in the beta-domain of MT1.     

The NMR solution structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi.

The 3-dimensional structure of the pheromone Er-1 isolated from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution by 1H NMR spectroscopy. The structure of this 40-residue protein was calculated with the distance geometry program DIANA on the basis of 503 upper distance constraints derived from nuclear Overhauser effects and 77 dihedral angle constraints derived from spin-spin coupling constants, and refined by restrained energy minimization with the program OPAL. The Er-1 solution structure is represented by a group of 20 conformers with an average RMS deviation relative to the mean structure of 0.55 A for the backbone atoms N, C alpha, and C', and 0.93 A for all heavy atoms of the complete polypeptide chain, residues 1-40. The molecular architecture is dominated by an up-down-up bundle of 3 alpha-helices formed by residues 2-9, 12-19, and 24-33. Although this core part coincides closely with the previously determined structure of the homologous pheromone Er-10, the C-terminal peptide segment adopts a novel conformation. This is of interest in view of previous suggestions, based on sequence comparisons, that this molecular region may be important for the different specificity of receptor recognition by different pheromones.     

The three-dimensional structure of the first EGF-like module of human factor IX: comparison with EGF and TGF-alpha.

The three-dimensional structure of the first epidermal growth factor (EGF)-like module from human factor IX has been determined in solution using two-dimensional nuclear magnetic resonance (in the absence of calcium and at pH 4.5). The structure was found to resemble closely that of EGF and the homologous transforming growth factor-alpha (TGF-alpha). Residues 60-65 form an antiparallel beta-sheet with residues 68-73. In the C-terminal subdomain a type II beta-turn is found between residues 74 and 77 and a five-residue turn is found between residues 79 and 83. Glu 78 and Leu 84 pair in an antiparallel beta-sheet conformation. In the N-terminal region a loop is found between residues 50 and 55 such that the side chains of both are positioned above the face of the beta-sheet. Residues 56-60 form a turn that leads into the first strand of the beta-sheet. Whereas the global fold closely resembles that of EGF, the N-terminal residues of the module (46-49) do not form a beta-strand but are ill-defined in the structure, probably due to the local flexibility of this region. The structure is discussed with reference to recent site-directed mutagenesis data, which have identified certain conserved residues as ligands for calcium.     

Solution structure of a PAN module from the apicomplexan parasite Eimeria tenella

Micronemes, specialised organelles found in all apicomplexan parasites, secrete molecules that are essential for parasite attachment and invasion of host cells. EtMIC5 is one such microneme protein that contains eleven tandemly repeating modules. These modules have homology with the PAN module superfamily. Members of this family are found in blood clotting proteins, some growth factors and some nematode proteins. This paper presents the structure of the 9^th PAN module in EtMIC5, determined using high resolution NMR. The structure shows similarities to and some differences from the N-terminal module of hepatocyte growth factor (HGF), the only previous member of the PAN family with known structure. AbbreviationsNMR – nuclear magnetic resonance; NOE – nuclear Overhauser enhancement; NOESY – NOE spectroscopy; COSY – correlated spectroscopy; TOCSY – total correlated spectroscopy; HSQC – hetero nuclear single quantum coherence; HMQC-J – hetero nuclear multiple quantum coherence-J coupling; MICs – microneme proteins; EtMIC5 – a microneme protein from Eimeria tenella; Apple9 – the ninth Apple repeat of EtMIC5; FXI – blood coagulation factor XI; PK – plasma prekallikrein; HGF – hepatocyte growth factor.     

Design,synthesis and biological evaluation of novel 1H-1,2,4-triazole,benzothiazole and indazole-based derivatives as potent FGFR1 inhibitors viafragment-based virtual screening

Abstract

Fibroblast growth-factor receptor (FGFR) is a potential target for cancer therapy. We designed three novel series of FGFR1 inhibitors bearing indazole, benzothiazole, and 1H-1,2,4-triazole scaffold via fragment-based virtual screening. All the newly synthesised compounds were evaluated in vitro for their inhibitory activities against FGFR1. Compound 9d bearing an indazole scaffold was first identified as a hit compound, with excellent kinase inhibitory activity (IC₅₀ = 15.0?nM) and modest anti-proliferative activity (IC₅₀ = 785.8?nM). Through two rounds of optimisation, the indazole derivative 9?u stood out as the most potent FGFR1 inhibitors with the best enzyme inhibitory activity (IC₅₀ = 3.3?nM) and cellular activity (IC₅₀ = 468.2?nM). Moreover, 9?u also exhibited good kinase selectivity. In addition, molecular docking study was performed to investigate the binding mode between target compounds and FGFR1.     

NMR solution structure of the acylphosphatase from Escherichia coli

The solution structure of Escherichia coli acylphosphatase (E. coli AcP), a small enzyme catalyzing the hydrolysis of acylphosphates, was determined by (1)H and (15)N NMR and restrained modelling calculation. In analogy with the other members of AcP family, E. coli AcP shows an alpha/beta sandwich domain composed of four antiparallel and one parallel beta-strand, assembled in a five-stranded beta-sheet facing two antiparallel alpha-helices. The pairwise RMSD values calculated for the backbone atoms of E. coli and Sulfolobus solfataricus AcP, Bovine common type AcP and Horse muscle AcP are 2.18, 5.31 and 5.12 A, respectively. No significant differences are present in the active site region and the catalytic residue side chains are consistently positioned in the structures.     

NMR solution structure of the isolated Apo Pin1 WW domain: comparison to the x-ray crystal structures of Pin1.

The NMR solution structure of the isolated Apo Pin1 WW domain (6-39) reveals that it adopts a twisted three-stranded antiparallel beta-sheet conformation, very similar to the structure exhibited by the crystal of this domain in the context of the two domain Pin1 protein. While the B factors in the apo x-ray crystal structure indicate that loop 1 and loop 2 are conformationally well defined, the solution NMR data suggest that loop 1 is quite flexible, at least in the absence of the ligand. The NMR chemical shift and nuclear Overhauser effect pattern exhibited by the 6-39 Pin1 WW domain has proven to be diagnostic for demonstrating that single site variants of this domain adopt a normally folded structure. Knowledge of this type is critical before embarking on time-consuming kinetic and thermodynamic studies required for a detailed understanding of beta-sheet folding.     

Study of the interaction of the Ig2 module of the fibroblast growth factor receptor, FGFR Ig2, with the fibroblast growth factor 1, FGF1, by means of NMR spectroscopy

Fibroblast growth factor (FGF) receptor (FGFR) consists extracellularly of three immunoglobulin (Ig) modules (Ig1-3). Currently, there are two competing models (symmetric and asymmetric) of the FGF-FGFR-heparin complex based on crystal structures. Indirect evidence exists in support of both models. However, it is not clear which model is physiologically relevant. Our aim was to obtain direct, non-crystallographic evidence in support of them. We found by nuclear magnetic resonance that Ig2 could bind to FGF1 not only via the primary site (present in both models), but also via the secondary site (present only in the symmetric model). Thus, our data support the symmetric model.     

NMR structure of the WIF domain of the human Wnt-inhibitory factor-1

The human Wnt-binding protein Wnt-inhibitory factor-1 (WIF-1) comprises an N-terminal WIF module followed by five EGF-like repeats. Here we report the three-dimensional structure of the WIF domain of WIF-1 determined by NMR spectroscopy. The fold consists of an eight-stranded beta-sandwich reminiscent of the immunoglobulin fold. Residual detergent (Brij-35) used in the refolding protocol was found to bind tightly to the WIF domain. The binding site was identified by intermolecular nuclear Overhauser effects observed between the WIF domain and the alkyl chain of the detergent. The results point to a possible role of WIF domains as a recognition motif of Wnt and Drosophila Hedgehog proteins that are activated by palmitoylation.     

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.     

FGF9/FGFR1 promotes cell proliferation,epithelial-mesenchymal transition,M2 macrophage infiltration and liver metastasis of lung cancer

Fibroblast growth factors 9 (FGF9) modulates cell proliferation, differentiation and motility for development and repair in normal cells. Abnormal activation of FGF9 signaling is associated with tumor progression in many cancers. Also, FGF9 may be an unfavorable prognostic indicator for non-small cell lung cancer patients. However, the effects and mechanisms of FGF9 in lung cancer remain elusive. In this study, we investigated the FGF9-induced effects and signal activation profiles in mouse Lewis lung carcinoma (LLC) in vitro and in vivo. Our results demonstrated that FGF9 significantly induced cell proliferation and epithelial-to-mesenchymal transition (EMT) phenomena (migration and invasion) in LLC cells. Mechanism-wise, FGF9 interacted with FGFR1 and activated FAK, AKT, and ERK/MAPK signal pathways, induced the expression of EMT key proteins (N-cadherin, vimentin, snail, MMP2, MMP3 and MMP13), and reduced the expression of E-cadherin. Moreover, in the allograft mouse model, intratumor injection of FGF9 to LLC-tumor bearing C57BL/6 mice enhanced LLC tumor growth which were the results of increased Ki67 expression and decreased cleaved caspase-3 expression compared to control groups. Furthermore, we have a novel finding that FGF9 promoted liver metastasis of subcutaneous inoculated LLC tumor with angiogenesis, EMT and M2-macrophage infiltration in the tumor microenvironment. In conclusion, FGF9 activated FAK, AKT, and ERK signaling through FGFR1 with induction of EMT to stimulate LLC tumorigenesis and hepatic metastasis. This novel FGF9/LLC allograft animal model may therefore be useful to study the mechanism of liver metastasis which is the worst prognostic factor for lung cancer patients with distant organ metastasis.     

Solution NMR structure of cementum protein 1 derived peptide (CEMP1-p1) and its role in the mineralization process

We report the characterization of the three-dimensional structure of the CEMP1-p1 peptide [MGTSSTDSQQAQHRRCSTSN: corresponding to residues 1–20 of the N-terminus of cementum protein 1 (CEMP1)]. This peptide imitates the capacity of CEMP1 to stimulate hydroxyapatite (HA) crystal nucleation and growth, and promotes the differentiation of periodontal ligament cells into a cementoblastic phenotype. Additionally, in experimental models of critical-sized calvarial defects in Wistar rats, CEMP1-p1 has shown osteogenic properties that enhanced the physiological deposition and maturation of newly formed bone. In this work, studies of CEMP1-p1 by circular dichroism (CD) and nuclear magnetic resonance (NMR) were performed in trifluoroethanol D2 (TFED2) and aqueous solution to determine the 3D structure of the peptide. Using the 3D model, experimental data from HA crystals formation and calcium fluorescence emission, we explain the biological mechanisms involved in CEMP1-p1 activity to promote calcium recruitment and its affinity to HA crystals. This information is valuable because it proposes, for the first time, a plausible molecular mechanism during the mineralization process, from a specific cementum protein-derived peptide.