富自体浓缩生长因子生物学作用的研究进展

富自体浓缩生长因子(concentrated growth factor,CGF)为第三代血小板浓缩产物,是再生医疗领域中诱导组织再生分化的一种新型生物材料。CGF通过差速离心可释放出多种生长因子,如血小板衍生生长因子(plateletderived growth factor,PDGF)、转化生长因子-β(transforming growth factor-β,TGF-β)、类胰岛素生长因子(insulin-like growth factor,IGF)、血管内皮生长因子(vascular endothelial growth factor,VEGF)、表皮生长因子(epidermal growth fcator,EGF)及成纤维细胞生长因子(fibroblast growth factor,FGF)等,这些生长因子相互作用,可发挥促进组织再生的作用。本文就CGF在促进细胞增殖及分化、组织再生及修复、软组织愈合等生物学作用中的研究进展作一综述。     

Comparative modeling and molecular dynamics studies of the delta, kappa and mu opioid receptors

Molecular models of the trans-membrane domains of delta, kappa and mu opioid receptors, members of the G-protein coupled receptor (GPCR) superfamily, were developed using techniques of homology modeling and molecular dynamics simulations. Structural elements were predicted from sequence alignments of opioid and related receptors based on (i) the consensus, periodicities and biophysical interpretations of alignment- derived properties, and (ii) tertiary structure homology to rhodopsin. Initial model structures of the three receptors were refined computationally with energy minimization and the result of the first 210 ps of a 2 ns molecular dynamics trajectory at 300K. Average structures from the trajectory obtained for each receptor subtype after release of the initial backbone constraints show small backbone deviations, indicating stability. During the molecular dynamics phase, subtype-differentiated residues of the receptors developed divergent structures within the models, including changes in regions common to the three subtypes and presumed to belong to ligand binding regions. The divergent features developed by the model structures appear to be consistent with the observed ligand binding selectivities of the opioid receptors. The results thus implicate identifiable receptor microenvironments as primary determinants of some of the observed subtype specificities in opiate ligand binding and in functional effects of mutagenesis. Networks of interacting residues observed in the models are common to the opiate receptors and other GPCRs, indicating core interfaces that are potentially responsible for structural integrity and signal transduction. Analysis of extended molecular dynamics trajectories reveals concerted motions of distant parts of ligand-binding regions, suggesting motion-sensitive components of ligand binding. The comparative modeling results from this study help clarify experimental observations of subtype differences and suggest both structural and dynamic rationales for differences in receptor properties.      

Iterative Nonproteinogenic Residue Incorporation Yields α/β‐Peptides with a Helix–Loop–Helix Tertiary Structure and High Affinity for VEGF

Inhibition of specific protein–protein interactions is attractive for a range of therapeutic applications, but the large and irregularly shaped contact surfaces involved in many such interactions make it challenging to design synthetic antagonists. Here, we describe the development of backbone‐modified peptides containing both α‐ and β‐amino acid residues (α/β‐peptides) that target the receptor‐binding surface of vascular endothelial growth factor (VEGF). Our approach is based on the Z‐domain, which adopts a three‐helix bundle tertiary structure. We show how a two‐helix “mini‐Z‐domain” can be modified to contain β and other nonproteinogenic residues while retaining the target‐binding epitope by using iterative unnatural residue incorporation. The resulting α/β‐peptides are less susceptible to proteolysis than is their parent α‐peptide, and some of these α/β‐peptides match the full‐length Z‐domain in terms of affinity for receptor‐recognition surfaces on the VEGF homodimer.     

Adnectins: engineered target-binding protein therapeutics

Adnectins? are a new family of therapeutic proteins based on the 10th fibronectin type III domain, and designed to bind with high affinity and specificity to therapeutically relevant targets. Adnectins share with antibody variable domains a beta-sheet sandwich fold with diversified loops, but differ from antibodies in primary sequence and have a simpler, single-domain structure without disulfide bonds. As a consequence, Adnectins bind targets with affinity and specificity as high as those of antibodies, but are easier to manipulate genetically and compatible with bacterial expression systems. Adnectins that bind macromolecular targets with nanomolar and picomolar affinity have been selected using in vitro evolution methods, including mRNA display, phage display and yeast display. CT-322, a PEGylated, anti-angiogenic Adnectin that binds vascular endothelial growth factor (VEGF) receptor 2 and blocks its interaction with VEGF A, C and D, is being evaluated in Phase II clinical trials for efficacy in several oncology indications.     

Osteoblast Dysfunction in Non-Hereditary Sclerosing Bone Diseases

A review of the available literature was performed in order to summarize the existing evidence between osteoblast dysfunction and clinical features in non-hereditary sclerosing bone diseases. It has been known that proliferation and migration of osteoblasts are concerted by soluble factors such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), bone morphogenetic protein (BMP) but also by signal transduction cascades such as Wnt signaling pathway. Protein kinases play also a leading role in triggering the activation of osteoblasts in this group of diseases. Post-zygotic changes in mitogen-activated protein kinase (MAPK) have been shown to be associated with sporadic cases of Melorheostosis. Serum levels of FGF and PDGF have been shown to be increased in myelofibrosis, although studies focusing on Sphingosine-1-phosphate receptor was shown to be strongly expressed in Paget disease of the bone, which may partially explain the osteoblastic hyperactivity during this condition. Pathophysiological mechanisms of osteoblasts in osteoblastic metastases have been studied much more thoroughly than in rare sclerosing syndromes: striking cellular mechanisms such as osteomimicry or complex intercellular signaling alterations have been described. Further research is needed to describe pathological mechanisms by which rare sclerosing non hereditary diseases lead to osteoblast dysfunction.     

Structure-function analysis of a conserved aromatic cluster in the N- terminal domain of human epidermal growth factor

The importance of a cluster of conserved aromatic residues of human epidermal growth factor (hEGF) to the receptor binding epitope is suggested by the interaction of His10 and Tyr13 of the A-loop with Tyr22 and Tyr29 of the N-terminal beta-sheet to form a hydrophobic surface on the hEGF protein. Indeed, Tyr13 has previously been shown to contribute a hydrophobic determinant to receptor binding. The roles of His10, Tyr22 and Tyr29 were investigated by structure-function analysis of hEGF mutant analogues containing individual replacements of each residue. Substitutions with aromatic residues or a leucine at position 10 retained receptor affinities and agonist activities similar to wild- type indicating that an aromatic residue is not essential. Variants with polar, charged or aliphatic substitutions altered in size and/or hydrophobicity exhibited reduced binding and agonist activities. 1- Dimensional 1H NMR spectra of high, moderate and low-affinity analogues at position 10 suggested only minor alterations in hEGF native structure. In contrast, a variety of replacements were tolerated at position 22 or 29 indicating that neither aromaticity nor hydrophobicity of Tyr22 and Tyr29 is required for receptor binding. CD spectra of mutant analogues at position 22 or 29 indicated a correlation between loss of receptor affinity and alterations in hEGF structure. The results indicate that similar to Tyr13, His10 of hEGF contributes hydrophobicity to the receptor binding epitope, whereas Tyr22 and Tyr29 do not appear to be directly involved in receptor interactions. The latter conclusion, together with previous studies, suggests that hydrophobic residues on only one face of the N-terminal beta-sheet of hEGF are important in receptor recognition.      

Molecular dynamics of the 5-HT1a receptor and ligands

A 3-D model of the human 5-HT_1a receptor was constructed fromits amino acid sequence by computer graphics techniques, molecularmechanics calculations and molecular dynamics simulations. Themodel has seven -helical membrane spanning segments, which forma central core containing a putative ligand binding site. Electrostaticpotentials 1.4 Å outside the water accessible surfacewere mainly negative on the synaptic side of the receptor modeland at the postulated ligand binding site, and positive in thecytoplasmic domains. The negative electrostatic potentials aroundthe synaptic domains indicate that positively charged ligandsare attracted to the receptor by electrostatic forces. Moleculardynamics simulations of the receptor model with serotonin, ipsapirone,R(–)-methiothepin or S(+)- methiothepin in the centralcore suggested that up to 22 different amino acid residues mayform a ligand binding pocket, and contribute to the specificityof ligand recognition and binding.     

Protein Contacts and Ligand Binding in the Inward‐Facing Model of Human P‐Glycoprotein

The primary aim of this work was to analyze the contacts between residues in the nucleotide binding domains (NBDs) and at the interface between the transmembrane domains (TMDs) and the NBDs in the inward‐open homology model of human P‐glycoprotein (P‐gp). The analysis revealed communication nets through hydrogen bonding in the NBD and at the NBD–TMD interface of each half involving residues from the adenosine triphosphate (ATP) motifs and the coupling helices of the intracellular loops. Similar networks have been identified in P‐gp conformations generated by molecular dynamics simulation. Differences have been recorded in the networking between both halves of P‐gp. Many of the residue contacts have also been observed in the X‐ray crystal structures of other ATP binding cassette (ABC) transporters, which confirms their validity. Next, possible binding pockets involving residues of importance for the TMD–NBD communication were identified. By studying these pockets, binding sites were suggested for rhodamine 123 (R‐site) and prazosin (regulatory site) at the NBD–TMD interface that agreed with the experimental data on their location. Additionally, one more R‐site in the protein cavity was proposed, in accordance with the available biochemical data. Together with the previously suggested Hoechst 33342 site (H‐site), all sites were interpreted with respect to their effects on the protein ATPase activity, in correspondence with the experimental observations. Several residues involved in key contacts in the P‐gp NBDs were proposed for further targeted mutagenesis experiments.     

GRAFTER: a computational aid for the design of novel proteins

The GRAFTER suite of programs provides geometric search andevaluation functions that simplify and automate the processof identifying the best scaffolds for a particular structuralmotif. Three applications of the GRAFTER suite are presented.Potential grafts between repressor and 434 repressor were identifiedthat should change the DNA binding specificity of these repressors.These results are compared with site-directed mutagenesis experimentsthat have been shown to alter repressor-DNA binding specificity.Next, 26 loops from antibody structures were grouped into familiesof similar structure. Grafts of antibody loops onto a pre-existingscaffold are an essential component of antibody humanization.Finally, interleukin (lL)-4 was searched as a scaffold thatmight accept the graft of a five residue epitope from humangrowth hormone (hGH). The existence of a crystal structure ofthe hGH-hGH receptor complex, extensive mutagenesis studiesof the hGH residues that contribute to the energetics of ligand-receptorinteractions and the gross structural homology between hGH andIL-4 make this an appealing computational target. The approachpresented here could aid the development of novel enzymes andbinding proteins     

Synthetic DNA aptamers to detect protein molecular variants in a high-throughput fluorescence quenching assay

Real-time protein detection in homogeneous solutions is necessary in many biotechnology and biomedical studies. The recent development of molecular aptamers, combined with fluorescence techniques, may provide an easy and efficient approach to protein elucidation. This report describes the development of a fluorescence-based assay with synthetic DNA aptamers that can detect and distinguish molecular variants of proteins in biological samples in a high-throughput process. We used an aptamer with high affinity for the B chain of platelet-derived growth factor (PDGF), labeled it with a fluorophore and a quencher at the two termini, and measured fluorescence quenching by PDGF. The specific quenching can be used to detect PDGF at picomolar concentrations even in the presence of serum and other cell-derived proteins in cell culture media. This is the first successful application of a synthetic aptamer for the detection of tumor-related proteins directly from the tumor cells. We also show that three highly related molecular variants of PDGF (AA, AB, and BB dimers) can be distinguished from one another in this single-step assay, which can be readily adapted to a microtiter plate assay for high-throughput analysis. The use of fluorescence quenching as a measure of binding between the DNA probe and the target protein eliminates potential false signals that may arise in traditional fluorescence enhancement assays as a result of degradation of the DNA aptamer by contaminating nucleases in biological specimens. This assay is applicable to proteins that are not naturally DNA binding. The excellent specificity, ultrahigh sensitivity, and simplicity of this one-step assay addresses a growing need for high-throughput methods that detect changes in the expression of gene products and their variants in cell cultures and biological specimens.     

Anti‐HIV‐1 Peptide Derivatives Based on the HIV‐1 Co‐receptor CXCR4

The human immunodeficiency virus type 1 (HIV‐1) uses CD4 and the co‐receptor CCR5 or CXCR4 in the process of cell entry. The negatively charged extracellular domains of CXCR4 (CXCR4‐ED) interact with positive charges on the V3 loop of gp120, facilitating binding via electrostatic interactions. The presence of highly conserved positively charged residues in the V3 loop suggests that CXCR4‐ED‐derived inhibitors might be broadly effective inhibitors. Synthetic peptide derivatives were evaluated for anti‐HIV‐1 activity. The 39‐mer extracellular N‐terminal region (NT) was divided into three fragments with 10‐mer overlapping sites ( N1 – N3 ), and these linear peptides were synthesized. Peptide N1 contains Met 1–Asp 20 and shows significant anti‐HIV‐1 activity. Extracellular loops 1 and 2 (ECL1 and 2) were mimicked by cyclic peptides C1 and C2 , which were synthesized by chemoselective cyclization. Cyclic peptides C1 and C2 show higher anti‐HIV‐1 activity than their linear peptide counterparts, L1 and L2 . The cytotoxicities of C1 and C2 are lower than those of L1 and L2 . These results indicate that Met 1–Asp 20 segments of the NT and cyclic peptides of ECL1 and ECL2 are potent anti‐HIV‐1 drug candidates.     

Probing Structural Features and Binding Mode of 3-Arylpyrimidin-2,4-diones within Housefly γ-Aminobutyric Acid (GABA) Receptor

In order to obtain structural features of 3-arylpyrimidin-2,4-diones emerged as promising inhibitors of insect γ-aminobutyric acid (GABA) receptor, a set of ligand-/receptor-based 3D-QSAR models for 60 derivatives are generated using Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Index Analysis (CoMSIA). The statistically optimal CoMSIA model is produced with highest q(2) of 0.62, r(2) (ncv) of 0.97, and r(2) (pred) of 0.95. A minor/bulky electronegative hydrophilic polar substituent at the 1-/6-postion of the uracil ring, and bulky substituents at the 3'-, 4'- and 5'-positions of the benzene ring are beneficial for the enhanced potency of the inhibitors as revealed by the obtained 3D-contour maps. Furthermore, homology modeling, molecular dynamics (MD) simulation and molecular docking are also carried out to gain a better understanding of the probable binding modes of these inhibitors, and the results show that residues Ala-183(C), Thr-187(B), Thr-187(D) and Thr-187(E) in the second transmembrane domains of GABA receptor are responsible for the H-bonding interactions with the inhibitor. The good correlation between docking observations and 3D-QSAR analyses further proves the model reasonability in probing the structural features and the binding mode of 3-arylpyrimidin-2,4-dione derivatives within the housefly GABA receptor.     

Pericytes of Stria Vascularis Are Targets of Cisplatin-Induced Ototoxicity: New Insights into the Molecular Mechanisms Involved in Blood-Labyrinth Barrier Breakdown

The stria vascularis (SV) contributes to cochlear homeostasis and consists of three layers, one of which contains the blood-labyrinthic barrier (BLB), with a large number of bovine cochlear pericytes (BCPs). Cisplatin is a chemotherapeutic drug that can damage the SV and cause hearing loss. In this study, cell viability, proliferation rate, cytotoxicity and reactive oxygen species production were evaluated. The protein content of phospho-extracellular signal-regulated kinases (ERK) 1/2, total ERK 1/2, phospho-cytosolic phospholipase A₂ (cPLA₂), total cPLA₂ and cyclooxygenase 2 (COX-2) and the release of prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) from BCPs were analyzed. Finally, the protective effect of platelet-derived growth factor (PDGF-BB) on BCPs treated with cisplatin was investigated. Cisplatin reduced viability and proliferation, activated ERK 1/2, cPLA₂ and COX-2 expression and increased PGE2 and VEGF release; these effects were reversed by Dexamethasone. The presence of PDGF-BB during the treatment with cisplatin significantly increased the proliferation rate. No studies on cell regeneration in ear tissue evaluated the effect of the PDGF/Dex combination. The aim of this study was to investigate the effects of cisplatin on cochlear pericytes and propose new otoprotective agents aimed at preventing the reduction of their vitality and thus maintaining the BLB structure.     

碱性成纤维生长因子对皮肤损伤修复作用的研究进展

皮肤损伤是临床常见疾病之一,大多由撕裂、割伤或挫伤等急性创伤导致。在皮肤损伤修复过程中,多种细胞通过分泌细胞因子参与创面愈合,如碱性成纤维生长因子(fibroblast growth factor 2,FGF2或bFGF)、血小板源性生长因子(platelet-derived growth factor,PDGF)和转化生长因子β(transforming growth factor-β,TGF-β)等,其中FGF2可通过促进血管生成、肉芽形成,减少瘢痕形成,加速上皮化促进伤口愈合。FGF2半衰期短,在体内易受酶和皮肤微环境的影响,在伤口处保留时间短暂,降低了其促进伤口愈合的能力,因此,FGF2的稳定性对其临床用于皮肤损伤修复尤为重要。本文就FGF2的分子结构及功能、FGF2受体激活的信号转导途径、FGF2在伤口愈合方面的作用机制及其在整形方面的应用作一综述。     

Antitumor activity of SRI 62-834, a cyclic ether analog of ET-18-OCH3

SRI 62-834, an analog of the antitumor agent ET-18-OCH₃ in which the oxygen atom at carbon atom 2 has been incorporated into a five-membered heterocycle, has been prepared and evaluated as an antitumor agent. The compound exhibited good cytotoxicity in vitro against a variety of tumor cell lines and was as effective as ET-18-OCH₃ given orally in the mouse Meth A sarcoma model. SRI 62-834 was shown to be an inhibitor of platelet-derived growth factor (PDGF), possibly at the receptor level, and platelet-activating factor (PAF) at the receptor level.     

Analysis of high-affinity binding determinants in the receptor binding epitope of basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitope required for receptor binding of the growth factor. In previous studies, we identified Glu96 as an essential residue in this epitope using site-directed mutagenesis. Here we examined the role of solvent accessible neighboring residues of Glu96 of bFGF on receptor binding affinity. Wild-type bFGF and its muteins were cloned and expressed in Escherichia coli and evaluated for FGF receptor binding affinity. Replacement of Asn104 of bFGF by alanine reduced receptor binding affinity over 400-fold compared with wild-type bFGF. We next explored the effect of neighboring residues of Asn104 on receptor binding affinity-Muteins in which Arg97, Leu98, Glu99, Asn101, Asn102, Thr105 and Pro141 were individually replaced by alanine exhibited receptor binding similar to wild-type bFGF. By contrast, substitution of Tyr103 or Leu140 by alanine reduced receptor binding affinity about 400- and 150-fold, respectively, in accord with a previous report. We conclude that at least six solvent-accessible residues in bFGF are crucial for high-affinity receptor binding, as evidenced by at least a 10-fold diminution in the affinity of the corresponding alanine muteins. The polar residues Glu96 and Asn104 appear to form an area important for facilitating the initial contact between ligand and receptor, whereas Tyr24, Tyr103, Leu140 and Met142 form a hydrophobic patch that may stabilize the complex. The detailed structure of this functional epitope can be employed in the discovery and design of bFGF antagonists using computational methods.      

Molecular modelling of the ORL1 receptor and its complex with nociceptin

The opioid receptor like (ORL1) receptor is a G-protein coupled receptor superfamily, and regulates a plethora of neurophysiological functions. The structural requirements for receptor activation by its endogenous agonist, nociceptin (FGGFTGARKSARKLANQ), differ markedly from those of the kappa-opioid receptor and its putative peptide agonist, dynorphin A (YGGFLRRIRPKLKWDNQ). In order to probe the functional architecture of the ORL1 receptor, a molecular model of the receptor has been built, including the TM domain and the extra- and intracellular loops. An extended binding site able to accommodate nociceptin-(1-13), the shortest fully active analogue of nociceptin, has been characterized. The N-terminal FGGF tetrapeptide is proposed to bind in a highly conserved region, comprising two distinct hydrophobic pockets in a cavity formed by TM helices 3, 5, 6 and 7, capped by the acidic second extracellular (EL2) loop controlling access to the TM elements of the peptide binding site. The nociceptin conformation provides for the selective preference of the ORL1 receptor for nociceptin over dynorphin A, conferred by residue positions 5 and 6 (TG versus LR), and the favourable interaction of its highly positively charged core (residues 8-13) with the EL2 loop, thought to mediate receptor activation. The functional roles of the EL2 loop and the conserved N-terminal tetrapeptide opioid 'message' binding site are discussed in the context of the different structural requirements of the ORL1 and kappa-opioid receptors for activation.      

Identification of two new hydrophobic residues on basic fibroblast growth factor important for fibroblast growth factor receptor binding

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several types of vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitopes required for receptor binding of the growth factor. Using structure-based site-directed mutagenesis, two critical areas on the bFGF surface for the high affinity receptor binding have already been identified [Springer, B.A., Pantoliano, M.W., Barberal, F.A., Gunyuzlu, P.L., Thompson, L.D., Herblin, W.F., Rosenfeld, S.A. and Book, G.W. (1994) J. Biol. Chem., 269, 26879-26884; Zhu, H.Y., Ramnarayan, K., Anchin, J., Miao, Y., Sereno, A., Millman, L., Zheng, J., Balaji, V.N. and Wolff, M.E. (1995) J. Biol. Chem., 270, 21869-21874; Zhu, H.Y., Anchin, J., Ramnarayan, K., Zheng, J., Kawai, T., Mong, S. and Wolff, M.E. (1997) Protein Engng, 10, 417-421]. According to these studies, one receptor binding site includes two polar residues Glu96 and Asn104 on bFGF whereas the other includes four hydrophobic residues Tyr24, Tyr103, Leu140 and Met142. Using a protein modelling technique, we report here the identification of a new hydrophobic patch on bFGF which includes residues Tyr73, Val88 and Phe93. The role of this area on receptor binding affinity was evaluated by mutating each of these residues individually and determining the mutated protein's (mutein's) receptor binding affinity. In addition, we examined the role of two other hydrophobic residues, Phe30 and Leu138, on bFGF for high-affinity receptor binding. These two residues are the neighbors of the hydrophobic residues Tyr24 and Tyr103, respectively. Replacement of Val88 and Phe93 with alanine reduced the receptor binding affinity about 10- and 80-fold, respectively, compared with wild-type bFGF. In contrast, substitution of Phe30 and Leu138 with alanine has no effect on the receptor binding affinities. We conclude that the newly identified hydrophobic residues, Val88 and Phe93, are crucial for the receptor binding. The present data, together with the previous identification of four hydrophobic residues (Tyr24, Tyr103, Leu140 and Met142), suggests that there are two hydrophobic receptor binding sites on the bFGF surface. Our findings can be employed in the discovery and design of potent bFGF antagonists using computational methods.