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1.
Arap3 is a phosphatidylinositol 3 kinase effector protein that plays a role as GTPase activator (GAP) for Arf6 and RhoA. Arap3 contains a sterile alpha motif (Sam) domain that has high sequence homology with the Sam domain of the EphA2‐receptor (EphA2‐Sam). Both Arap3‐Sam and EphA2‐Sam are able to associate with the Sam domain of the lipid phosphatase Ship2 (Ship2‐Sam). Recently, we reported a novel interaction between the first Sam domain of Odin (Odin‐Sam1), a protein belonging to the ANKS (ANKyrin repeat and Sam domain containing) family, and EphA2‐Sam. In our latest work, we applied NMR spectroscopy, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize the association between Arap3‐Sam and Odin‐Sam1. We show that these two Sam domains interact with low micromolar affinity. Moreover, by means of molecular docking techniques, supported by NMR data, we demonstrate that Odin‐Sam1 and Arap3‐Sam might bind with a topology that is common to several Sam‐Sam complexes. The revealed structural details form the basis for the design of potential peptide antagonists that could be used as chemical tools to investigate functional aspects related to heterotypic Arap3‐Sam associations.  相似文献   

2.
The EphA2 receptor controls diverse physiological and pathological conditions and its levels are often upregulated in cancer. Targeting receptor overexpression, through modulation of endocytosis and consequent degradation, appears to be an appealing strategy for attacking tumor malignancy. In this scenario, the Sam domain of EphA2 plays a pivotal role because it is the site where protein regulators of endocytosis and stability are recruited by means of heterotypic Sam–Sam interactions. Because EphA2‐Sam heterotypic complexes are largely based on electrostatic contacts, we have investigated the possibility of attacking these interactions with helical peptides enriched in charged residues. Several peptide sequences with high predicted helical propensities were designed, and detailed conformational analyses were conducted by diverse techniques including NMR, CD, and molecular dynamics (MD) simulations. Interaction studies were also performed by NMR, surface plasmon resonance (SPR), and microscale thermophoresis (MST) and led to the identification of two peptides capable of binding to the first Sam domain of Odin. These molecules represent early candidates for the generation of efficient Sam domain binders and antagonists of Sam–Sam interactions involving EphA2.  相似文献   

3.
We investigated the derivation of non‐natural peptide triazole dual receptor site antagonists of HIV‐1 Env gp120 to establish a pathway for developing peptidomimetic antiviral agents. Previously we found that the peptide triazole HNG‐156 [R‐I‐N‐N‐I‐X‐W‐S‐E‐A‐M‐M‐CONH2, in which X=ferrocenyltriazole‐Pro (FtP)] has nanomolar binding affinity to gp120, inhibits gp120 binding to CD4 and the co‐receptor surrogate mAb 17b, and has potent antiviral activity in cell infection assays. Furthermore, truncated variants of HNG‐156, typified by UM‐24 (Cit‐N‐N‐I‐X‐W‐S‐CONH2) and containing the critical central stereospecific LX‐LW cluster, retain the functional characteristics of the parent peptide triazole. In the current work, we examined the possibility of replacing natural with unnatural residue components in UM‐24 to the greatest extent possible. The analogue with the critical “hot spot” residue Trp 6 replaced with L ‐3‐benzothienylalanine (Bta) (KR‐41), as well as a completely non‐natural analogue containing D ‐amino acid substitutions outside the central cluster (KR‐42, DCit‐DN‐DN‐DI‐X‐Bta‐DS‐CONH2), retained the dual receptor site antagonism/antiviral activity signature. The results define differential functional roles of subdomains within the peptide triazole and provide a structural basis for the design of metabolically stable peptidomimetic inhibitors of HIV‐1 Env gp120.  相似文献   

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5.
Affinity maturation of protein‐targeting peptides is generally accomplished by homo‐ or heterodimerization of known peptides. However, applying a heterodimerization approach is difficult because it is not clear a priori what length or type of linker is required for cooperative binding to a target. Thus, an efficient and simple affinity maturation method for converting low‐affinity peptides into high‐affinity peptides would clearly be advantageous for advancing peptide‐based therapeutics. Here, we describe the development of a novel affinity maturation method based on a robust β‐hairpin scaffold and combinatorial phage‐display technology. With this strategy, we were able to increase the affinity of existing peptides by more than four orders of magnitude. Taken together, our data demonstrate that this scaffold‐assisted approach is highly efficient and effective in generating high‐affinity peptides from their low‐affinity counterparts.  相似文献   

6.
Human liver‐expressed antimicrobial peptide 2 (LEAP‐2) is a cationic antimicrobial peptide (CAMP) believed to have a protective role against bacterial infection. Little is known about the structure–activity relationships of LEAP‐2 or its mechanism of action. In this study we describe the structure of LEAP‐2, analyze its interaction with model membranes, and relate them to the antimicrobial activity of the peptide. The structure of LEAP‐2, determined by NMR spectroscopy, reveals a compact central core with disorder at the N and C termini. The core comprises a β‐hairpin and a 310‐ helix that are braced by disulfide bonds between Cys17–28 and Cys23–33 and further stabilized by a network of hydrogen bonds. Membrane‐affinity studies show that LEAP‐2 membrane binding is governed by electrostatic attractions, which are sensitive to ionic strength. Truncation studies show that the C‐terminal region of LEAP‐2 is irrelevant for membrane binding, whereas the N‐terminal (hydrophobic domain) and core regions (cationic domain) are essential. Bacterial‐growth‐inhibition assays reveal that the antimicrobial activity of LEAP‐2 correlates with membrane affinity. Interestingly, the native and reduced forms of LEAP‐2 have similar membrane affinity and antimicrobial activities; this suggests that disulfide bonds are not essential for the bactericidal activity. This study reveals that LEAP‐2 has a novel fold for a CAMP and suggests that although LEAP‐2 exhibits antimicrobial activity under low‐salt conditions, there is likely to be another physiological role for the peptide.  相似文献   

7.
In order to fabricate a new polymer matrix for application in biochips and to understand the mechanism of adsorption of proteins on conducting polymers, we prepared polypyrrole (PPy) functionalized with ω‐(N‐pyrrolyl)‐octylthiol moieties. The chemical structure of the polymer could be controlled by varying the concentration of pyrrole added as the monomer. Initially, ω‐(N‐pyrrolyl)‐octylthiol was self‐assembled into a monolayer on a gold surface. Thereafter, a layer of uniform and smooth PPy was obtained by the chemical copolymerization of pyrrole and the ω‐(N‐pyrrolyl)‐octylthiol. Bovine serum albumin (BSA) adsorption on the polymer was investigated using surface plasmon resonance spectroscopy and cyclic voltammograms. The chemical structure and monomer components of the as‐prepared films were characterized using Fourier transform infrared spectroscopy and X‐ray photoelectron spectroscopy. Water contact angle measurements were used to assess the surface wettability of the films throughout the preparative procedure. The kinetics of BSA adsorption onto the polymer could be controlled by varying the copolymer thickness and the pH value of the buffer solutions used. Moreover, the electroactivity was changed upon BSA binding. The results suggest that the new conducting polymer may potentially be applied as a more sensitive and reliable matrix in protein sensors. Copyright © 2011 Society of Chemical Industry  相似文献   

8.
Galectin‐1 (Gal‐1), a ubiquitous β‐galactoside‐binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal‐1 depend on its affinity for β‐galactoside‐containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr‐Xxx‐Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal‐1–asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr‐Xxx‐Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr‐Xxx‐Tyr peptides studied do not bind to Gal‐1, whereas their binding to ASF is clearly detected. 15N,1H HSQC titrations with 15N‐labeled Gal‐1 confirm the absence of any peptide–Gal‐1 interaction. These data indicate that the Tyr‐Xxx‐Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.  相似文献   

9.
Prevention of amyloid β peptide (Aβ) deposition via facilitation of Aβ binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer’s disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, to improve the affinity of this protein to monomeric Aβ by a factor of 3 (BBRC, 510(2), 248–253). Using plasmon resonance spectroscopy, we show here that another HSA ligand related to AD pathogenesis, serotonin (SRO), increases the affinity of the Aβ monomer to HSA by a factor of 7/17 for Aβ40/Aβ42, respectively. Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA’s affinity to monomeric Aβ, despite slowdown of the association and dissociation processes. Crosslinking with glutaraldehyde and dynamic light scattering experiments reveal that, compared with the TRP-induced effects, SRO binding causes more marked changes in the quaternary structure of HSA. Furthermore, molecular docking reveals distinct structural differences between SRO/TRP complexes with HSA. The disintegration of the serotonergic system during AD pathogenesis may contribute to Aβ release from HSA in the central nervous system due to impairment of the SRO-mediated Aβ trapping by HSA.  相似文献   

10.
Amyloid‐β (Aβ) peptide is the major component found in senile plaques of Alzheimer's disease patients. The 42‐residue fragment Aβ(1–42) is proposed to be one of the most pathogenic species therein. Here, the soluble Aβ(1–42) species were analyzed by various liquid‐state NMR methods. Transient formation of a micelle species was observed at the onset of the aggregation kinetics. This micelle is dissolved after approximately one day. Subsequent loss of this species and the formation of protofibrils are proposed to be the route of fibril formation. Consequently, the observed micelle species is suggested to be on an off‐pathway mechanism. Furthermore, characterization of the NMR‐observable soluble species shows that it is a random‐coil‐like entity with low propensities for four β‐strands. These β‐strands correlate with the β‐strand segments observed in Aβ fibrils. This finding indicates that the 3D structure of the fibrils might already be predisposed in the soluble species.  相似文献   

11.
SAR by NMR : A series of indole compounds derived from 5‐bromo‐1H‐indole‐3‐acetohydroxamic acid were synthesized. Their inhibitory activities were evaluated against purified peptide deformylases (PDFs), and their antibacterial activities against B. subtilis, E. coli (wild type and tolC), and a variety of pathogens were also determined. The potency of the best inhibitors was related to the NMR footprints of the respective acids with 15N‐labeled E. coli Ni‐PDF.

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12.
The cover picture shows a “reverse” indole derivative in complex with Bacillus stearothermophilus peptide deformylase (PDF). This compound was selected from a structure–activity relationship study as a potent inhibitor of bacterial PDFs and shows antibacterial activity toward Bacillus subtilis as well as other pathogens such as Streptococcus pneumoniae and Staphylococcus aureus. For more details, see the Full Paper by I. Artaud et al. on p. 261 ff.

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13.
Copper‐induced structural rearrangements of Aβ40 structure and its redox properties are described in this study. Electrochemical and fluorescent methods are used to characterise the behaviour of Aβ–Cu species. The data suggest that time‐dependent folding of Aβ–Cu species may cause changes in the redox potentials.

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14.
15.
Look at what the cat(ionic motif) dragged in! We report a general strategy to increase the cell permeability of β3‐peptides. Introduction of a minimal cationic motif within the folded structure of a high‐affinity β3‐peptide ligand for hDM2 led to molecules with high 314‐helical structure, high hDM2 affinity and sufficient cell permeability to upregulate p53‐dependent genes in live mammalian cells. Minimally cationic β3‐peptides represent the critical first step towards a class of protease‐resistant peptidomimetics that might modulate intracellular biological pathways.

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16.
β‐Amyloid (Aβ) aggregation is causally linked to neuronal pathology in Alzheimer's disease; therefore, several small molecules, antibodies, and peptides have been tested as anti‐Aβ agents. We developed two compounds based on the Aβ‐binding domain of transthyretin (TTR): a cyclic peptide cG8 and an engineered protein mTTR, and compared them for therapeutically relevant properties. Both mTTR and cG8 inhibit fibrillogenesis of Aβ, with mTTR inhibiting at a lower concentration than cG8. Both inhibit aggregation of amylin but not of α‐synuclein. They both bind more Aβ aggregates than monomer, and neither disaggregates preformed fibrils. cG8 retained more of its activity in the presence of biological materials and was more resistant to proteolysis than mTTR. We examined the effect of mTTR or cG8 on Aβ binding to human neurons. When mTTR was co‐incubated with Aβ under oligomer‐forming conditions, Aβ morphology was drastically changed and Aβ‐cell deposition significantly decreased. In contrast, cG8 did not affect morphology but decreased the amount of Aβ deposited. These results provide guidance for further evolution of TTR‐mimetic anti‐amyloid agents.  相似文献   

17.
18.
G‐quadruplexes and i‐motifs are tetraplex structures present in telomeres and the promoter regions of oncogenes. The possibility of producing nanodevices with pH‐sensitive functions has triggered interest in modified oligonucleotides with improved structural properties. We synthesized C‐rich oligonucleotides carrying conformationally restricted (2′S)‐2′‐deoxy‐2′‐C‐methyl‐cytidine units. The effect of this modified nucleoside on the stability of intramolecular i‐motifs from the vertebrate telomere was investigated by UV, CD, and NMR spectroscopy. The replacement of selected positions of the C‐core with C‐modified residues induced the formation of stable intercalated tetraplexes at near‐neutral pH. This study demonstrates the possibility of enhancing the stability of the i‐motif by chemical modification.  相似文献   

19.
20.
Truncated and mutated amyloid‐β (Aβ) peptides are models for systematic study—in homogeneous preparations—of the molecular origins of metal ion effects on Aβ aggregation rates, types of aggregate structures formed, and cytotoxicity. The 3D geometry of bis‐histidine imidazole coordination of CuII in fibrils of the nonapetide acetyl‐Aβ(13–21)H14A has been determined by powder 14N electron spin echo envelope modulation (ESEEM) spectroscopy. The method of simulation of the anisotropic combination modulation is described and benchmarked for a CuII‐bis‐cis‐imidazole complex of known structure. The revealed bis‐cis coordination mode, and the mutual orientation of the imidazole rings, for CuII in Ac‐Aβ(13–21)H14A fibrils are consistent with the proposed β‐sheet structural model and pairwise peptide interaction with CuII, with an alternating [‐metal‐vacancy‐]n pattern, along the N‐terminal edge. Metal coordination does not significantly distort the intra‐β‐strand peptide interactions, which provides a possible explanation for the acceleration of Ac‐Aβ(13–21)H14A fibrillization by CuII, through stabilization of the associated state and low‐reorganization integration of β‐strand peptide pair precursors.  相似文献   

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