IGD motifs, which are required for migration stimulatory activity of fibronectin type I modules, do not mediate binding in matrix assembly

Picomolar concentrations of proteins comprising only the N-terminal 70-kDa region (70K) of fibronectin (FN) stimulate cell migration into collagen gels. The Ile-Gly-Asp (IGD) motifs in four of the nine FN type 1 (FNI) modules in 70K are important for such migratory stimulating activity. The 70K region mediates binding of nanomolar concentrations of intact FN to cell-surface sites where FN is assembled. Using baculovirus, we expressed wildtype 70K and 70K with Ile-to-Ala mutations in (3)FNI and (5)FNI; (7)FNI and (9)FNI; or (3)FNI, (5)FNI, (7)FNI, and (9)FNI. Wildtype 70K and 70K with Ile-to-Ala mutations were equally active in binding to assembly sites of FN-null fibroblasts. This finding indicates that IGD motifs do not mediate the interaction between 70K and the cell-surface that is important for FN assembly. Further, FN fragment N-(3)FNIII, which does not stimulate migration, binds to assembly sites on FN-null fibroblast. The Ile-to-Ala mutations had effects on the structure of FNI modules as evidenced by decreases in abilities of 70K with Ile-to-Ala mutations to bind to monoclonal antibody 5C3, which recognizes an epitope in (9)FNI, or to bind to FUD, a polypeptide based on the F1 adhesin of Streptococcus pyogenes that interacts with 70K by the β-zipper mechanism. These results suggest that the picomolar interactions of 70K with cells that stimulate cell migration require different conformations of FNI modules than the nanomolar interactions required for assembly.     

Multi-factorial modulation of IGD motogenic potential in MSF (Migration Stimulating Factor)

Migration Stimulating Factor (MSF) is a genetically truncated isoform of fibronectin (Fn). MSF is a potent stimulator of fibroblast migration, whereas full length Fn is devoid of motogenic activity. MSF and Fn contain four IGD motifs, located in the 3rd, 5th, 7th and 9th type I modules; these modules are referred to as ³FnI, ⁵FnI, ⁷FnI and ⁹FnI, respectively. We have previously reported that mutation of IGD motifs in modules ⁷FnI and ⁹FnI of MSF is sufficient to completely abolish the motogenic response of target adult skin fibroblasts. We now report that the IGD sequences in ³FnI and ⁵FnI are also capable of exhibiting motogenic activity when present within fragments of MSF. When present within ^1-5FnI, these sequences require the presence of serum or vitronectin for their motogenic activity to be manifest, whereas the IGD sequences in ⁷FnI and ⁹FnI are bioactive in the absence of serum factors. All MSF and IGD-containing peptides stimulated the phosphorylation of the integrin binding protein focal adhesion kinase (FAK) but did not necessarily affect migration. These results suggest that steric hindrance determines the motogenic activity of MSF and Fn, and that both molecules contain cryptic bioactive fragments.     

Synthesis of an IGD peptidomimetic with motogenic activity

Rational design and synthesis of an IGD peptidomimetic substrate with significant motogenic activity.     

Hepatocyte growth Factor/Scatter factor (HGF/SF) is a regulator of fibronectin splicing in MDCK cells: comparison between the effects of HGF/SF and TGF-beta1 on fibronectin splicing at the EDA region.

EDA-containing fibronectin (EDA + FN) is selectively produced under several physiological and pathological conditions requiring tissue remodeling, where cells actively proliferate and migrate. Only a few growth factors, such as transforming growth factor (TGF)-beta1, have been reported to regulate FN splicing at the EDA region. In the present study, we showed for the first time that hepatocyte growth factor/scatter factor (HGF/SF), which is mainly produced by mesenchymal cells and functions as a motogenic and mitogenic factor for epithelial cells, modulates FN splicing at the EDA region in MDCK epithelial cells. HGF/SF treatment increased the ratio of EDA + FN mRNA to mRNA of FN that lacks EDA (EDA - FN) (EDA+/EDA- ratio) more than TGF-beta1 treatment did: at a range from 0.02 to 20 ng/ml, HGF/SF increased the ratio in a dose-dependent manner by up to 2. 1-fold compared with nontreated control, while TGF-beta1 stimulated the EDA+/EDA- ratio by 1.5-fold at the optimum dose of 10 ng/ml. However, TGF-beta1 increased total FN mRNA levels by 3-fold at 10 ng/ml, but HGF/SF did not. We previously demonstrated that fibroblasts cultured at low cell density expressed more EDA + FN than those at high cell density. The same effect of cell density was also observed in MDCK cells. Furthermore, at low cell density, HGF/SF stimulated EDA inclusion into FN mRNA more effectively than did TGF-beta1, whereas at high cell density, TGF-beta1 was more potent than HGF/SF. Simultaneous treatment of cells with HGF/SF and TGF-beta1 synergistically stimulated EDA inclusion into FN mRNA. This stimulation of EDA inclusion into FN mRNA by HGF/SF led to increased EDA + FN protein production and secretion by cells, which was demonstrated by immunoblotting. Thus, our studies have shown that HGF/SF is an enhancer of EDA inclusion into FN mRNA as is TGF-beta1. However, these two factors were different in their effects at low and high cell densities and also in their effects on total FN mRNA levels.     

Effects of covalently attached chondroitin sulfate on aggrecan cleavage by ADAMTS-4 and MMP-13

Aggrecan is degraded by several aggrecanase-1 (ADAMTS-4) isoforms differing in the number of sulfated glycosaminoglycan (sGAG)-binding motifs. ADAMTS-4 and MMPs cleave aggrecan more efficiently within the chondroitin sulfate (CS)-rich region than the interglobular domain (IGD). We investigated the influence of CS on aggrecan core protein cleavage by ADAMTS-4 (p68) and (p40) as well as MMP-13, which has no recognizable GAG-binding sites. Chondroitinase ABC-treated cartilage aggrecan was cleaved with ADAMTS-4 (p68) less efficiently than CS-substituted aggrecan within the CS-2 domain. Keratanase-treated aggrecan exhibited reduced IGD cleavage, but when both CS and KS were removed, the IGD cleavage was restored. This result suggests that KS in the IGD may compete with CS for ADAMTS-4 (p68) binding. In the absence of KS, however, p68 binding was shifted to the CS-2 domain. CS-deficient full-length recombinant aggrecan (rbAgg) was produced by chondroitinase ABC treatment, or by expression in the xylosyltransferase-deficient CHO-pgsA745 cell line. When digested with the ADAMTS-4 (p68), each of these preparations exhibited reduced CS-2 domain cleavage compared to CS-substituted CHO-K1 cell-derived aggrecan. Additionally, CS-deficient rbAgg showed increased IGD scission prior to cleavage within the CS-2 domain. ADAMTS-4 (p40) readily cleaved both rbAggs within the IGD, but cleaved poorly within the CS-2 domain, indicating little CS dependence. MMP-13, in contrast, cleaved the CS region and the IGD of both CS-substituted and CS-deficient rbAgg equally well. These data indicate that covalently bound CS enhances ADAMTS-4-mediated cleavage within the CS-rich region. MMP-13 also cleaves preferentially within the CS-region, but by an apparently CS-independent mechanism.     

Interaction with heparin and matrix metalloproteinase 2 cleavage expose a cryptic anti-adhesive site of fibronectin

We recently found that fibronectin (FN) had a functional site [YTIYVIAL sequence in the heparin-binding domain 2 (Hep 2)] that was capable of suppressing the integrin-mediated cell adhesion to extracellular matrix. However, our results also indicated that this anti-adhesive site seemed to be usually buried within the Hep 2 domain structure because of its hydrophobic nature, raising a question as to the physiological significance of the cryptic anti-adhesive activity of FN. The present study demonstrates that the cryptic anti-adhesive activity can be exposed through the physiological processes. A 30-kDa chymotryptic FN fragment derived from Hep 2 domain (Hep 2 fragment), which had no effect on adhesion of MSV-transformed nonproducer 3T3 cell line (KN(7)8) to FN, expressed the anti-adhesive activity after treatment with 6 M urea. Light scattering and circular dichroism measurements showed that the urea treatment induced the conformational change of the Hep 2 fragment from a more compact form to an unfolded one. Incubation of the Hep 2 fragment with heparin also induced similar conformational changes and expression of anti-adhesive activity. Additionally, both the urea and heparin treatments made the Hep 2 fragment and intact FN much more accessible to the polyclonal antibody (alphaIII14A), with a recognition site near the anti-adhesive site of FN. Specific cleavage of either the Hep 2 fragment or intact FN by matrix metalloproteinase 2 (MMP-2) released a 10-kDa fragment with the anti-adhesive activity, which was shown to have the exposed anti-adhesive site on the amino-terminal region. Thus, the cryptic anti-adhesive activity of FN can be expressed upon conformational change and proteolytic cleavage of Hep 2 domain.     

Motogenic Sites in Human Fibronectin Are Masked by Long Range Interactions

Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains ^4–5FnI and ³FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes ³FnIII and disrupts the observed ^4–5FnI-³FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites.Fibronectin (FN),4 a large multidomain glycoprotein found in all vertebrates, plays a vital role in cell adhesion, tissue development, and wound healing (1). It exists in soluble form in plasma and tissue fluids but is also present in fibrillar networks as part of the extracellular matrix. The structures of many FN domains of all three types, FnI, FnII, and FnIII, are known, for example (2–4). Although interactions between domains that are close in primary sequence have been demonstrated (3, 5), studies of multidomain fragments generally assume a beads-on-string model (2). There is, however, much evidence for the presence of long range order in soluble FN as a number of functional sites, termed cryptic, are not active in the native molecule, until exposed through conformational change. These include self-association sites (5–8), sites of protein interactions (9), and sites that control cellular activity (10, 11). Low resolution studies of the FN dimer suggest a compact conformation under physiological conditions (12–14); however, attempts to define large scale structure in FN by small angle scattering or electric birefringence (15–17) have yielded contradictory results. Interpretation of domain stability changes in terms of interaction sites (18) has also not been straightforward (2), possibly because of domain stabilization through nearest-neighbor effects (19, 20).A FN splicing variant produced in fetal and cancer patient fibroblasts, termed migration stimulation factor (MSF), stimulates migration of adult skin fibroblasts into type I collagen gels (10, 21) and breast carcinoma cells using the Boyden chamber (22). MSF comprises FN domains ¹FnI to ⁹FnI, a truncated ¹FnIII, and a small C-terminal extension; a recombinant FN fragment corresponding to ¹FnI-⁹FnI (Fn70kDa) displays the same activity (10). An overview of FN domain structure and nomenclature is presented in Fig. 1a. Further experiments sub-localized full motogenic activity to the gelatin binding domain of FN (GBD, domains ⁶FnI-⁹FnI) (23) and partial activity to a shorter fragment spanning domains ^7–9FnI (24). Two IGD tripeptides of domains ⁷FnI and ⁹FnI were shown to be essential through residue substitutions and reconstitution of partial motogenic activity in synthetic peptides (10, 24, 25); however, similar IGD tripeptides outside the GBD, on domains ³FnI and ⁵FnI, appear to have little effect (10, 23). Full-length adult FN does not affect cell migration in similar assays (10, 23); thus motogenic activity sites are presumed to be masked in the conformation adopted by soluble FN, although they could be exposed by molecular rearrangement.Open in a separate windowFIGURE 1.Motogenic activity of FN fragments. a, schematic representation of the FN domain structure (top) and enlargement of the FN N terminus (bottom). Type I domains are shown as pentagons; type II domains as hexagons; and type III domains as ovals. b, comparison of motogenic activity versus protein concentration of wild-type Fn70kDa and Fn100kDa fragments. Error bars are derived from duplicate experiments, and a gray band denotes migration activity of media without additives. c, similar comparisons for mutant Fn100kDa fragments. d, analytical size exclusion chromatography of large FN fragments. The trace of UV absorbance at 280 nm versus elution volume shown here indicates a larger hydrodynamic radius for Fn100kDa R222A compared with the wild type, consistent with our model (Fig. 6a).Here we show that a recombinant fragment, closely matching a truncated form of FN identified in zebrafish (26), as well as amphibians, birds, and mammals (27), does not stimulate cell migration. This fragment is similar to MSF but includes the first three FnIII domains (^1–3FnIII), suggesting that these domains are responsible for a conformational transition that masks the activity sites in this construct and probably in full-length FN. To identify the mechanism behind this transition, we performed structural studies by solution NMR spectroscopy and identified a specific long range interaction between domains ^4–5FnI and ³FnIII as essential for this masking effect. Interestingly, this interaction does not involve direct contacts with the GBD but possibly represses motogenic activity through chain compaction, evident in analytical size exclusion assays. Intramolecular interactions thus provide a mechanism by which conformational rearrangement induced, for example, by tension or splicing variation can result in cellular activity differences.     

MMPs are less efficient than ADAMTS5 in cleaving aggrecan core protein

Aggrecan degradation in articular cartilage occurs predominantly through proteolysis and has been attributed to the action of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families. Both families of enzymes cleave aggrecan at specific sites within the aggrecan core protein. One cleavage site within the interglobular domain (IGD), between Glu^373-374Ala and five additional sites in the chondroitin sulfate-2 (CS-2) region of aggrecan were characterized as “aggrecanase” (ADAMTS) cleavage sites, while cleavage between Ser^341-342Phe within the IGD of bovine aggrecan is attributed to MMP action. The objective of this study was to assess the cleavage efficiency of MMPs relative to ADAMTS and their contribution to aggrecan proteolysis in vitro. The analysis of aggrecan IGD degradation in bovine articular cartilage explants treated with catabolic cytokines over a 19-day period showed that MMP-mediated degradation of aggrecan within the IGD can only be observed following day 12 of culture. This delay is associated with the lack of activation of proMMPs during the first 12 days of culture. Analysis of MMP1, 2, 3, 7, 8, 9, 12, 13 and ADAMTS5 efficiencies at cleaving within the aggrecan IGD and CS-2 region in vitro was carried out by the digestion of bovine aggrecan with the various enzymes and Western blot analysis using aggrecan anti-G1 and anti-G3 antibodies. Of these MMPs, MMP12 was the most efficient at cleaving within the aggrecan IGD. In addition to cleavage in the IGD, MMP, 3, 7, 8 and 12 were also able to degrade the aggrecan CS-2 region. MMP3 and MMP12 were able to degrade aggrecan at the very C-terminus of the CS-2 region, cleaving the Glu^2047-2048Ala bond which was previously shown to be cleaved by ADAMTS5. However, in comparison to ADAMTS5, MMP3 was about 100 times and 10 times less efficient at cleaving within the aggrecan IGD and CS-2 regions, respectively. Collectively, our results showed that the delayed activation of proMMPs and the relatively low cleavage efficiency of MMPs can explain the minor contribution of these enzymes to aggrecan catabolism in vivo. This study also uncovered a potential role for MMPs in the C-terminal truncation of aggrecan.     

Characterization of a 59 kDa gelatin-binding fragment of buffalo plasma fibronectin

Limited proteolysis of buffalo plasma fibronectin (FN) by thermolysin yielded four gelatin-binding fragments of which, the major 59 kDa fragment, GBF1, was isolated by gelatin-Sepharose and heparin-Sepharose affinity columns. GBF1 appeared during early phase of thermolysin digestion and remained intact even after 4 hr of digestion. GBF1 may be similar to 56 kDa gelatin-binding fragment of FNs from human and hamster plasma. But, it is more resistant to thermolysin cleavage. The fragment binds to heparin with low affinity. On the basis of the structure of human plasma FN, the modular structure of GBF1 may be given as: 6Fn1 1Fn2 2Fn2 7Fn1 8Fn1 9Fn1 1Fn3. Biophysical properties of GBF1 suggest an expanded native conformation. The interaction of the fragment with gelatin is pH-dependent and independent of NaCl concentration.     

Rat plasma fibronectin contains two distinct chemotactic domains for fibroblastic cells

Mechanism of fibronectin (FN)-induced chemotaxis of fibroblastic cells has not been fully understood. The present study was performed to establish a molecular nature of the chemotactic region of rat plasma FN. The chemotactic dose-response pattern of intact FN for mouse embryo fibroblastic cells, NIH-L13 cells, which was represented as a "bell-shape" curve with a maximum activity at around 50 nM, changed to a "biphasic" mode through a proteolysis with thermolysin. Two distinct chemotactic components were isolated from the thermolytic fragments. One component, a fragment with a molecular mass of 110-150 kDa, was estimated to contain the central cell-binding domain and the carboxyl-terminal heparin-binding domain of the intact FN molecule. Cell migration stimulated by the 110-150-kDa fragment increased successively in a dose-dependent manner, and the capability to promote the migration was much higher than that of the intact FN (over 2-fold). The second chemotactic component, a fragment with a molecular mass of 21 kDa, was shown to reside in the carboxyl-terminal fibrin-binding domain. The 21-kDa fragment produced a bell-shape dose-response pattern, being consistent with the intact FN, whereas a maximum response occurred at a 100-fold lower concentration (0.5 nM) than that of the intact FN molecule. At higher concentrations, this fragment revealed an inhibitory activity for the cell migration in response to the 110-150-kDa fragment. No significant molecular interaction between these two active components was observed by polyacrylamide gel electrophoresis under nondenaturing conditions, suggesting that the 21-kDa fragment may act directly on the cell to inhibit the cell migration. These results suggest that rat plasma FN contains at least two chemotactically active components that regulate cooperatively chemotactic migration of fibroblastic cells.     

Importance of the Heparin-binding Domain of Fibronectin for Enhancing Cell Adhesion Activity of the Recombinant Fibronectin

We have previously shown that the recombinant human fibronectin (FN) fragment composed of central cell binding domains (CCBD) spanning the ninth and tenth type III domains promotes cell adhesion and proliferation of osteoblasts. In the present study, we investigated the biological potency of heparin-binding domain (HBD) of FN spanning the twelfth and fourteenth type III domains. The HBD of FN significantly enhances the RGD-containing CCBD-mediated cell adhesion and proliferation in HOS cells (P < 0.05).     

Stromelysin generates a fibronectin fragment that inhibits Schwann cell proliferation

Our previous report (Muir, D., S. Varon, and M. Manthorpe. 1990. J. Cell Biol. 109:2663-2672) described the isolation and partial characterization of a 55-kD antiproliferative protein found in Schwann cell (SC) and schwannoma cell line-conditioned media and we concluded that SC proliferation is under negative autocrine control. In the present study the 55-kD protein was found to possess metalloprotease activity and stromelysin immunoreactivity. The SC-derived metalloprotease shares many properties with stromelysin isolated from other sources including the ability to cleave fibronectin (FN). Furthermore, limited proteolysis of FN by the SC-derived protease generated a FN fragment which itself expresses a potent antiproliferative activity for SCs. The active FN fragment corresponds to the 29-kD amino-terminal region of the FN molecule which was also identified as an active component in SC CM. Additional evidence that a proteolytic fragment of FN can possess antiproliferative activity for SCs was provided by the finding that plasmin can generate an amino-terminal FN fragment which mimicked the activity of the SC metalloprotease-generated antiproliferative FN fragment. Both the 55-kD SC metalloprotease and the 29-kD FN fragment could completely and reversibly inhibit proliferation of SCs treated with various mitogens and both were largely ineffective at inhibiting proliferation by immortalized or transformed SC lines. Normal and transformed SC types do secrete the proform of stromelysin, however, transformed cultures do not produce activated stromelysin and thus cannot generate the antiproliferative fragment of FN. These results suggest that, once activated, a SC-derived protease similar to stromelysin cleaves FN and generates an antiproliferative activity which can maintain normal SC quiescence in vitro.     

Decision-Making for Risky Gains and Losses among College Students with Internet Gaming Disorder

Individuals with Internet gaming disorder (IGD) tend to exhibit disadvantageous risky decision-making not only in their real life but also in laboratory tasks. Decision-making is a complex multifaceted function and different cognitive processes are involved in decision-making for gains and losses. However, the relationship between impaired decision-making and gain versus loss processing in the context of IGD is poorly understood. The main aim of the present study was to separately evaluate decision-making for risky gains and losses among college students with IGD using the Cups task. Additionally, we further examined the effects of outcome magnitude and probability level on decision-making related to risky gains and losses respectively. Sixty college students with IGD and 42 matched healthy controls (HCs) participated. Results indicated that IGD subjects exhibited generally greater risk taking tendencies than HCs. In comparison to HCs, IGD subjects made more disadvantageous risky choices in the loss domain (but not in the gain domain). Follow-up analyses indicated that the impairment was associated to insensitivity to changes in outcome magnitude and probability level for risky losses among IGD subjects. In addition, higher Internet addiction severity scores were associated with percentage of disadvantageous risky options in the loss domain. These findings emphasize the effect of insensitivity to losses on disadvantageous decisions under risk in the context of IGD, which has implications for future intervention studies.     

Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor

Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN-induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell-binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESP, where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dose-dependent manner by a rabbit polyclonal antiserum to a 140-kD cell surface FN receptor. This antibody was more effective on normal than on transformed 3T3 cells. Neither the anti-FN receptor antiserum nor a monoclonal antibody to the cell-binding site of FN blocked migration induced by another potent chemoattractant, platelet-derived growth factor. These data indicate that FN-induced chemotaxis of 3T3 and SV3T3 cells is mediated via the RGDS cell-attachment site of FN and the 140-kD cell surface FN receptor. The interaction is specific and can be altered by transformation.     

Isolation and characterization of two monoclonal antibodies that recognize remote epitopes on the cell-binding domain of human fibronectin

Two monoclonal anti-fibronectin antibodies that inhibit fibronectin-mediated cell adhesion have been established and characterized. One antibody, FN12-8, inhibited attachment of rat kidney fibroblasts on the fibronectin-coated substrate in a concentration-dependent manner, attaining a maximal inhibition of greater than 85% at 850 micrograms/ml. Another antibody, FN30-8, caused about 70% inhibition at a concentration as low as 0.85 microgram/ml, although further increase of the antibody concentration did not significantly augment the inhibitory effect. Immunoblot analysis with defined proteolytic fragments revealed that both antibodies are directed to the cell-binding domain of fibronectin. The epitopes for these antibodies were further narrowed down using recombinant cell-binding fragments expressed in Escherichia coli. FN12-8 recognized the 11.5-kDa cell-binding fragment previously characterized by Pierschbacher et al. (1981, Cell 26, 259-267), suggesting that FN12-8 blocks the Arg-Gly-Asp (RGD) cell adhesion signal. FN30-8 could not bind this fragment but did recognize a longer cell-binding fragment containing additional greater than 111 amino acid residues upstream of the 11.5-kDa fragment. Since the RGD-dependent cell adhesion seems to require another signal located at a region 50-160 residues upstream of the 11.5-kDa fragment for full activity, FN30-8 may exert its inhibitory effect by blocking the latter signal.     

Crystal structure of a hemojuvelin-binding fragment of neogenin at 1.8Å

Neogenin is a type I transmembrane glycoprotein with a large ectodomain containing tandem immunoglobulin-like and fibronectin type III (FNIII) domains. Closely related to the tumor suppressor gene DCC, neogenin functions in critical biological processes through binding to various ligands, including netrin, repulsive guidance molecules, and the iron regulatory protein hemojuvelin. We previously reported that neogenin binds to hemojuvelin through its membrane-proximal fifth and sixth FNIII domains (FN5-6), with domain 6 (FN6) contributing the majority of critical binding interactions. Here we present the crystal structure of FN5-6, the hemojuvelin-binding fragment of human neogenin, at 1.8?. The two FNIII domains are orientated nearly linearly, a domain arrangement most similar to that of a tandem FNIII-containing fragment within the cytoplasmic tail of the β4 integrin. By mapping surface-exposed residues that differ between neogenin FN5-6 and the comparable domains from DCC, which does not bind hemojuvelin, we identified a potential hemojuvelin-binding site on neogenin FN6. Neogenin FN5, which does not bind hemojuvelin in isolation, exhibits a highly electropositive surface, which may be involved in interactions with negatively-charged polysaccharides or phospholipids in the membrane bilayer. The neogenin FN5-6 structure can be used to facilitate a molecular understanding of neogenin's interaction with hemojuvelin to regulate iron homeostasis and with hemojuvelin-related repulsive guidance molecules to mediate axon guidance.     

MTH1 Substrate Recognition—An Example of Specific Promiscuity

MTH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1.     

Design,synthesis and biological evaluation of novel human monoamine oxidase B inhibitors based on a fragment in an X-ray crystal structure

Herein we report our efforts of developing reversible selective hMAO-B inhibitors based on isatin, a fragment in an X-ray crystal structure. Five different scaffolds were designed and many compounds were synthesized. Among them, compound A3 demonstrated very high potency and isoform selectivity against hMAO-B, 11 and 13 times more potent (IC₅₀?=?3?nM) and 23.64 and 6.8 times more selective than the marked drugs, selegiline and safinamide. However, the endeavors to modify the polar 3-one group of isatin, that is in a hydrophobic environment in the binding site of hMAO-B, to small nonpolar hydrophobic groups did not bring about improved hMAO-B inhibitors, which may challenge our understanding of molecular interactions and molecular recognition in biological systems.     

Integrin-type fibronectin receptors of rat arterial smooth muscle cells: isolation, partial characterization and role in cytoskeletal organization and control of differentiated properties

The spreading of freshly isolated rat arterial smooth muscle cells (SMCs) on a substrate of fibronectin (FN) is associated with marked changes in fine structure and function of the cells, collectively referred to as a modulation from a contractile to a synthetic phenotype. Recent studies have indicated that this process is mediated via an interaction between the minimal cell-attachment sequence of FN (RGDS) and cell surface receptors. Here, we report the isolation of such receptors by sequential chromatography on affinity columns of wheat germ agglutinin (WGA) and a 105-kDa cell-binding fragment of FN (105-kDa fragment). The receptor was composed of two proteins with electrophoretic mobilities in SDS-polyacrylamide gels of 160 and 115 kDa under nonreducing conditions and 150 and 130 kDa under reducing conditions. Immunoprecipitation of surface-labeled cells with a rabbit antiserum against the beta chain of the rat hepatocyte FN receptor similarly yielded two proteins of 160 and 115 kDa. In metabolically labeled cells an additional component of 105 kDa was precipitated, presumably representing a precursor of the 115-kDa protein. Immunocytochemical studies demonstrated that SMCs grown on laminin formed FN fibrils and actin filament bundles in close alignment with cell surface receptors after a few days of culture. In cells seeded on the 105-kDa fragment, the receptors were already arranged in parallel with actin filaments on the first day of culture. Later on, the cells secreted FN and laid down FN fibrils along the receptors on the cell surface and the actin filament bundles in the cytoplasm. Taken together, the findings indicate that arterial SMCs are equipped with FN receptors that belong to the integrin family of proteins and consists of alpha (160-kDa) and beta (115-kDa) subunits. The receptor complexes apparently play an important role in determining the differentiated characteristics of the cells, possibly by mediating a linkage between the extracellular matrix and the cytoskeleton.     

网络游戏成瘾的奖赏加工缺陷及其神经机制

奖赏加工异常是网络游戏成瘾（internet gaming disorder，IGD）核心特征之一，近期研究结合认知神经科学技术对IGD的神经机制进行探讨发现，IGD与药物成瘾存在相似的神经基础，但仍然存在较多争议。本文从奖赏类型、奖赏加工阶段梳理了IGD人群奖赏加工的研究进展。IGD人群在游戏相关线索下，奖赏预期（reward anticipation）阶段的奖赏系统激活可能与注意偏向、情绪体验和渴求感增加有关。同时，IGD人群对自然奖赏表现出较为一致的低敏感性，该特征主要出现在结果评估（outcome evaluation）阶段。未来研究可以排除共病因素、结合生动的游戏奖赏刺激，进一步探究奖赏预期和结果评估异常如何推动IGD的发展。