Structural Analysis of the Human Fibroblast Growth Factor Receptor 4 Kinase

The family of fibroblast growth factor receptors (FGFRs) plays an important and well-characterized role in a variety of pathological disorders. FGFR4 is involved in myogenesis and muscle regeneration. Mutations affecting the kinase domain of FGFR4 may cause cancer, for example, breast cancer or rhabdomyosarcoma. Whereas FGFR1–FGFR3 have been structurally characterized, the structure of the FGFR4 kinase domain has not yet been reported. In this study, we present four structures of the kinase domain of FGFR4, in its apo-form and in complex with different types of small-molecule inhibitors. The two apo-FGFR4 kinase domain structures show an activation segment similar in conformation to an autoinhibitory segment observed in the hepatocyte growth factor receptor kinase but different from the known structures of other FGFR kinases. The structures of FGFR4 in complex with the type I inhibitor Dovitinib and the type II inhibitor Ponatinib reveal the molecular interactions with different types of kinase inhibitors and may assist in the design and development of FGFR4 inhibitors.     

A Structural Portrait of the PDZ Domain Family

PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family.     

成纤维细胞生长因子与其受体相互作用及其抑制剂的研究进展

成纤维细胞生长因子（FGF）有许多重要的生理功能，并与肿瘤的形成有关.为了弄清FGF与成纤维细胞生长因子受体(FGFR)相互作用的机制，人们对FGF和FGFR的各个结合结构域进行了深入、细致的研究，定位了aFGF、bFGF的肝素结合区、bFGF的受体结合区、FGF受体的肝素结合区、配体结合区和FGF受体相互结合区，提出了两个FGF与FGFR相互作用的模型，在此基础上设计了FGF的核酸类、糖类和多肽类抑制剂，为寻找新一代抗癌药物打下了理论基础.     

Travel depth, a new shape descriptor for macromolecules: application to ligand binding

Depth is a term frequently applied to the shape and surface of macromolecules, describing for example the grooves in DNA, the shape of an enzyme active site, or the binding site for a small molecule in a protein. Yet depth is a difficult property to define rigorously in a macromolecule, and few computational tools exist to quantify this notion, to visualize it, or analyze the results. We present our notion of travel depth, simply put the physical distance a solvent molecule would have to travel from a surface point to a suitably defined reference surface. To define the reference surface, we use the limiting form of the molecular surface with increasing probe size: the convex hull. We then present a fast, robust approximation algorithm to compute travel depth to every surface point. The travel depth is useful because it works for pockets of any size and complexity. It also works for two interesting special cases. First, it works on the grooves in DNA, which are unbounded in one direction. Second, it works on the case of tunnels, that is pockets that have no "bottom", but go through the entire macromolecule. Our algorithm makes it straightforward to quantify discussions of depth when analyzing structures. High-throughput analysis of macromolecule depth is also enabled by our algorithm. This is demonstrated by analyzing a database of protein-small molecule binding pockets, and the distribution of bound magnesium ions in RNA structures. These analyses show significant, but subtle effects of depth on ligand binding localization and strength.     

成纤维细胞生长因子9在抑郁症及其运动干预调节中的作用

成纤维细胞生长因子9（fibroblast growth factor 9, FGF9）是成纤维细胞生长因子（fibroblast growth factor,FGF）家族成员之一,属于一种自分泌或旁分泌生长因子。在脑组织中,FGF9主要表达于海马和皮质区,具有促进细胞增殖和维持细胞存活的功能。研究发现,FGF家族在抑郁症患者的多个脑区出现表达紊乱,FGF9在抑郁行为中扮演着负调控角色,但其介导抑郁行为的分子机制尚不清楚。本文综述了FGF9及其家族成员在抑郁中的作用; 围绕其受体（FGFR）信号在中枢神经系统中的功能特点,深入分析FGF9调节抑郁行为中的作用机制;结合运动抗抑郁的神经营养假说,提出经由FGFR/GSK3β/β-catenin通路的FGF信号,可能介导抑郁症的运动干预机制的假设。这些将为FGF9介导抑郁行为和运动抗抑郁的有关研究提供理论的基础和探索的思路。     

Structural and Thermodynamic Characterization of Pre- and Postpolymerization States in the F4 Fimbrial Subunit FaeG

Enterotoxigenic Escherichia coli expressing F4 fimbriae are the major cause of porcine colibacillosis and are responsible for significant death and morbidity in neonatal and postweaned piglets. Via the chaperone-usher pathway, F4 fimbriae are assembled into thin, flexible polymers mainly composed of the single-domain adhesin FaeG. The F4 fimbrial system has been labeled eccentric because the F4 pilins show some features distinct from the features of pilins of other chaperone-usher-assembled structures. In particular, FaeG is much larger than other pilins (27  versus ∼ 17 kDa), grafting an additional carbohydrate binding domain on the common immunoglobulin-like core. Structural data of FaeG during different stages of the F4 fimbrial biogenesis process, combined with differential scanning calorimetry measurements, confirm the general principles of the donor strand complementation/exchange mechanisms taking place during pilus biogenesis via the chaperone-usher pathway.     

Structural Analysis of Peroxide-Soaked MnSOD Crystals Reveals Side-On Binding of Peroxide to Active-Site Manganese

The superoxide dismutase (SOD) enzymes are important antioxidant agents that protect cells from reactive oxygen species. The SOD family is responsible for catalyzing the disproportionation of superoxide radical to oxygen and hydrogen peroxide. Manganese- and iron-containing SOD exhibit product inhibition whereas Cu/ZnSOD does not. Here, we report the crystal structure of Escherichia coli MnSOD with hydrogen peroxide cryotrapped in the active site. Crystallographic refinement to 1.55 Å and close inspection revealed electron density for hydrogen peroxide in three of the four active sites in the asymmetric unit. The hydrogen peroxide molecules are in the position opposite His26 that is normally assumed by water in the trigonal bipyramidal resting state of the enzyme. Hydrogen peroxide is present in active sites B, C, and D and is side-on coordinated to the active-site manganese. In chains B and D, the peroxide is oriented in the plane formed by manganese and ligands Asp167 and His26. In chain C, the peroxide is bound, making a 70° angle to the plane. Comparison of the peroxide-bound active site with the hydroxide-bound octahedral form shows a shifting of residue Tyr34 towards the active site when peroxide is bound. Comparison with peroxide-soaked Cu/ZnSOD indicates end-on binding of peroxide when the SOD does not exhibit inhibition by peroxide and side-on binding of peroxide in the product-inhibited state of MnSOD.     

两种类型人碱性成纤维生长因子的体外稳定性研究

目的:通过野生型bFGF和改构型bFGF蛋白溶液中的聚集过程的比较,初步探讨bFGF在水溶液中发生聚集的机理。方法:在选择合适溶液体系后,采用蛋白溶解度和促有丝分裂活性能力为指标,表征野生型bFGF和突变型bFGF聚合程度,分析共价聚合和非共价聚合所起的作用。结果:在相同的溶液体系中,野生型bFGF的聚集程度高于突变型bFGF。bFGF聚合程度与浓度有依赖性。沉淀结果分析非共价聚合占主要作用。结论:野生型bFGF在溶液中共价聚和和非共价聚合同时发生,两种bFGF聚合过程中非共价聚合占较大比例。突变半胱氨酸可以减少聚合发生。     

成纤维细胞生长因子的特性与分子功能

成纤维细胞生长因子(FGFs)是一类蛋白质活性肽,在有机体内外显示广谱的生物学活性和功能,在生物发育学、生理学与临床药理学方面具有重要的作用和意义．对 FGFs 家族成员肽的特征特性,FGFs 的染色体定位、基因结构与功能,FGFs 的信号分子作用与促分裂素作用,FGFs 的作用受体、主动和被动调节与拮抗机理进行了综述．     

Identification of a New Motif in Family B DNA Polymerases by Mutational Analyses of the Bacteriophage T4 DNA Polymerase

Structure-based protein sequence alignments of family B DNA polymerases revealed a conserved motif that is formed from interacting residues between loops from the N-terminal and palm domains and between the N-terminal loop and a conserved proline residue. The importance of the motif for function of the bacteriophage T4 DNA polymerase was revealed by suppressor analysis. T4 DNA polymerases that form weak replicating complexes cannot replicate DNA when the dGTP pool is reduced. The conditional lethality provides the means to identify amino acid substitutions that restore replication activity under low-dGTP conditions either by correcting the defect produced by the first amino acid substitution or by generally increasing the stability of polymerase complexes; the second type are global suppressors that can effectively counter the reduced stability caused by a variety of amino acid substitutions. Some amino acid substitutions that increase the stability of polymerase complexes produce a new phenotype—sensitivity to the antiviral drug phosphonoacetic acid. Amino acid substitutions that confer decreased ability to replicate DNA under low-dGTP conditions or drug sensitivity were identified in the new motif, which suggests that the motif functions in regulating the stability of polymerase complexes. Additional suppressor analyses revealed an apparent network of interactions that link the new motif to the fingers domain and to two patches of conserved residues that bind DNA. The collection of mutant T4 DNA polymerases provides a foundation for future biochemical studies to determine how DNA polymerases remain stably associated with DNA while waiting for the next available dNTP, how DNA polymerases translocate, and the biochemical basis for sensitivity to antiviral drugs.     

人碱性成纤维细胞生长因子突变体的高效表达

用PCR法将人碱性成纤维细胞生长因子(hbFGF)基因中编码第25、69和92位的半胱氨酸(Cys)密码子突变为丝氨酸(Ser)密码子,将突变的hbFGFcDNA片断与表达质粒pET3c连接,构建重组质粒pET3chbFGFSer25,69,92。hbFGFSer25,69,92在大肠杆菌BL21(DE3)中的表达量大于30%。通过阳离子交换和肝素亲和层析两步纯化,得到纯度大于95%的hbFGFSer25,69,92。MTT法测定纯化的产物活性表明,hbFGFSer25,69,92突变体促Balb/c细胞增殖的活性与野生型hbFGF相当,为下一步对hbFGFSer25,69,92突变体进行定点化学修饰打下了基础。     

Using Soft X-Rays for a Detailed Picture of Divalent Metal Binding in the Nucleosome

Divalent metals associate with DNA in a site-selective manner, which can influence nucleosome positioning, mobility, compaction, and recognition by nuclear factors. We previously characterized divalent metal binding in the nucleosome core using hard (short-wavelength) X-rays allowing high-resolution crystallographic determination of the strongest affinity sites, which revealed that Mn²⁺ associates with the DNA major groove in a sequence- and conformation-dependent manner. In this study, we obtained diffraction data with soft X-rays at the Mn²⁺ absorption edge for a core particle crystal in the presence of 10 mM MnSO₄, mimicking prevailing Mg²⁺ concentration in the nucleus. This provides an exceptional view of counterion binding in the nucleosome through identification of 45 divalent metal binding sites.In addition to that at the well-characterized major interparticle interface, only one other histone-divalent metal binding site is found, which corresponds to a symmetry-related counterpart on the ‘free’ H2B α1 helix C-terminus. This emphasizes the importance of the α-helix dipole in ion binding and suggests that the H2B motif may serve as a nucleation site in nucleosome compaction. The 43 sites associated with the DNA are characterized by (1) high-affinity direct coordination at the most electrostatically favorable major groove locations, (2) metal hydrate binding to the major groove, (3) direct coordination to phosphate groups at sites of high charge density, (4) metal hydrate binding in the minor groove, or (5) metal hydrate-divalent anion pairing. Metal hydrates are found within the minor groove only at locations displaying a narrow range of high-intermediate width and to which histone N-terminal tails are not associated or proximal. This indicates that divalent metals and histone tails can both collaborate and compete in minor groove association, which sheds light on nucleosome solubility and chromatin compaction behavior.     

Type 2 Fibroblast Growth Factor Receptor Signaling Preserves Stemness and Prevents Differentiation of Prostate Stem Cells from the Basal Compartment

Prostate stem cells (P-SCs) are capable of giving rise to all three lineages of prostate epithelial cells, which include basal, luminal, and neuroendocrine cells. Two types of P-SCs have been identified in both human and mouse adult prostates based on prostasphere or organoid cultures, cell lineage tracing, renal capsule implantation, and expression of luminal- and basal-specific proteins. The sphere-forming P-SCs are from the basal cell compartment that express P63, and are therefore designated as basal P-SCs (P-bSCs). Luminal P-SCs (P-lSCs) express luminal cytokeratins and Nkx3.1. Herein, we report that the type 2 FGF receptor (FGFR2) signaling axis is crucial for preserving stemness and preventing differentiation of P-bSCs. FGFR2 signaling mediated by FGFR substrate 2α (FRS2α) is indispensable for formation and maintenance of prostaspheres derived from P63⁺ P-bSCs. Ablation of Fgfr2 in P63⁺ cells in vitro causes the disintegration of prostaspheres. Ablation of Fgfr2 in vivo reduces the number of P63-expressing basal cells and enriches luminal cells. This suggests a basal stem cell-to-luminal cell differentiation. In addition, ablation of Fgfr2 in P63⁺ cells causes defective postnatal development of the prostate. Therefore, the data indicate that FGFR2 signaling is critical for preserving stemness and preventing differentiation of P-bSCs.     

牛脑成纤维细胞生长因子的分离纯化与鉴定

新鲜牛脑组织匀浆液经两步硫酸铵沉淀、CM-Sephadex C50 离子交换层析以及肝素-Sepharose 亲和层析,可得到纯化的酸性和碱性成纤维细胞生长因子(aFGF 和bFGF),分子量分别为13.2kD 和15.2—15.8kD.两种因子均可有效促进3T3细胞的 DNA 合成,ED₅₀分别为15.8ng/ml 和 0.32ng/ml.进一步对 aFGF 的等电点及氨基酸组成做了分析.     

Localization of Basic Fibroblast Growth Factor in Bovine Adrenal Chromaffin Cells

Abstract: We have investigated basic fibroblast growth factor (FGF-2) localization in and release from isolated bovine adrenal chromaffin cells. In contrast to previous reports, we found no evidence of fibroblast growth factor (FGF) storage in catecholamine-containing chromaffin granules. Subcellular fractionation studies did not show enrichment of FGF-2 immunoreactivity in granules, and cholinergic stimulation failed to release FGF-2 into the medium. Our results suggest that adrenal chromaffin cells resemble other FGF-2-synthesizing cell types with respect to FGF storage and secretion.     

Structural Basis of the Action of Glucosyltransferase Lgt1 from Legionella pneumophila

The glucosyltransferase Lgt1 is one of three glucosylating toxins of Legionella pneumophila, the causative agent of Legionnaires disease. It acts through specific glucosylation of a serine residue (S53) in the eukaryotic elongation factor 1A and belongs to type A glycosyltransferases. High-resolution crystal structures of Lgt1 show an elongated shape of the protein, with the binding site for uridine disphosphate glucose at the bottom of a deep cleft. Lgt1 shows only a low sequence identity with other type A glycosyltransferases, and structural conservation is limited to a central folding core that is usually observed within this family of proteins. Domains and protrusions added to the core motif represent determinants for the specific recognition and binding of the target. Manual docking experiments based on the crystal structures of toxin and target protein suggest an obvious mode of binding to the target that allows for efficient transfer of a glucose moiety.     

Deletion of the PH-domain and Leucine-rich Repeat Protein Phosphatase 1 (Phlpp1) Increases Fibroblast Growth Factor (Fgf) 18 Expression and Promotes Chondrocyte Proliferation

Endochondral ossification orchestrates formation of the vertebrate skeleton and is often induced during disease and repair processes of the musculoskeletal system. Here we show that the protein phosphatase Phlpp1 regulates endochondral ossification. Phlpp1 null mice exhibit decreased bone mass and notable changes in the growth plate, including increased BrdU incorporation and matrix production. Phosphorylation of known Phlpp1 substrates, Akt2, PKC, and p70 S6 kinase, were enhanced in ex vivo cultured Phlpp1^−/− chondrocytes. Furthermore, Phlpp1 deficiency diminished FoxO1 levels leading to increased expression of Fgf18, Mek/Erk activity, and chondrocyte metabolic activity. Phlpp inhibitors also increased matrix content, Fgf18 production and Erk1/2 phosphorylation. Chemical inhibition of Fgfr-signaling abrogated elevated Erk1/2 phosphorylation and metabolic activity in Phlpp1-null cultures. These results demonstrate that Phlpp1 controls chondrogenesis via multiple mechanisms and that Phlpp1 inhibition could be a strategy to promote cartilage regeneration and repair.