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1.
There is considerable current interest in the design of encodable molecules that regulate intracellular protein circuitry and/or activity, ideally with a high level of specificity. Src homology 3 (SH3) domains are ubiquitous components of multidomain signaling proteins, including many kinases, and are attractive drug targets because of the important role their interactions play in diseases as diverse as cancer, osteoporosis, and inflammation. Here we describe a set of miniature proteins that recognize distinct SH3 domains from Src family kinases with high affinity. Three of these molecules discriminate effectively between the SH3 domains of Src and Fyn, which are expressed ubiquitously, and two of these three activate Hck kinase with potencies that rival HIV Nef, one of the most potent kinase activators known. These results suggest that miniature proteins represent a viable, encodable strategy for selective activation of Src family kinases in a variety of cell types.  相似文献   

2.
BACKGROUND: The ability to control specific protein-protein interactions conditionally in vivo would be extremely helpful for analyzing protein-protein interaction networks. SH3 (Src homology 3) modular protein binding domains are found in many signaling proteins and they play a crucial role in signal transduction by binding to proline-rich sequences. RESULTS: Random in vitro mutagenesis coupled with yeast two-hybrid screening was used to identify mutations in the second SH3 domain of Nck that render interaction with its ligand temperature sensitive. Four of the mutants were functionally temperature sensitive in mammalian cells, where temperature sensitivity was correlated with a pronounced instability of the mutant domains at the nonpermissive temperature. Two of the mutations affect conserved residues in the hydrophobic core (Val133 and Val160), suggesting a general strategy for engineering temperature-sensitive SH3-containing proteins. Indeed mutagenesis of the corresponding positions in another SH3 domain, that of Crk-1, rendered the full-length Crk-1 protein temperature sensitive for function and stability in mammalian cells. CONCLUSIONS: Construction of temperature-sensitive SH3 domains is a novel approach to regulating the function of SH3 domains in vivo. Such mutants will be valuable in dissecting SH3-mediated signaling pathways. Furthermore, the methodology described here to isolate temperature-sensitive domains should be widely applicable to any domain involved in protein-protein interactions.  相似文献   

3.
Current sustained delivery strategies of protein therapeutics are limited by the fragility of the protein, resulting in minimal quantities of bioactive protein delivered. In order to achieve prolonged release of bioactive protein, an affinity-based approach was designed which exploits the specific binding of the Src homology 3 (SH3) domain with short proline-rich peptides. Specifically, methyl cellulose was modified with SH3-binding peptides (MC-peptide) with either a weak affinity or strong affinity for SH3. The release profile of SH3-rhFGF2 fusion protein from hyaluronan MC-SH3 peptide (HAMC-peptide) hydrogels was investigated and compared to unmodified controls. SH3-rhFGF2 release from HAMC-peptide was extended to 10 days using peptides with different binding affinities compared to the 48 h release from unmodified HAMC. This system is capable of delivering additional proteins with tunable rates of release, while maintaining bioactivity, and thus is broadly applicable.  相似文献   

4.
A human cDNA phage display library screen, using a phosphopeptide designed to mimic the activation loop phosphotyrosine of the Src tyrosine kinase, has identified the N-terminal SH2 domain of the p85 regulatory subunit of phosphatidyl inositol-3 kinase (PI3K) as an interacting recognition domain. Activation loop phosphorylation is known to play a conformational role in kinase activation, but is largely not thought to play a role in protein/protein recognition. Affinity chromatography and biochemical evaluation in mouse fibroblast cells has confirmed the dependence of this interaction on both the Src activation loop phosphotyrosine and the N-terminal SH2 domain of PI3K.  相似文献   

5.
The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) binds two separate ligands: a phosphotyrosine-containing peptide and the Itk Src homology 3 (SH3) domain. Binding specificity for these ligands is regulated via cis/trans isomerization of the Asn 286-Pro 287 imide bond in the Itk SH2 domain. In this study, we develop a novel method of analyzing chemical shift perturbation and cross-peak volumes to measure the affinities of both ligands for each SH2 conformer. We find that the cis imide bond containing SH2 conformer exhibits a 3.5-fold higher affinity for the Itk SH3 domain compared with binding of the trans conformer to the same ligand, while the trans conformer binds phosphopeptide with a 4-fold greater affinity than the cis-containing SH2 conformer. In addition to furthering the understanding of this system, the method presented here will be of general application in quantitatively determining the specificities of conformationally heterogeneous systems that use a molecular switch to regulate binding between multiple distinct ligands.  相似文献   

6.
The noncovalent binding of various peptide ligands to pp60src (Src) SH2 (Src homology 2) domain protein (12.9 ku) has been used as a model system for development of electrospray ionization mass spectrometry (ESI-MS) as a tool to study noncovalently bound complexes. SH2 motifs in proteins are critical in the signal transduction pathways of the tyrosine kinase growth factor receptors and recognize phosphotyrosine-containing proteins and peptides. ESI-MS with a magnetic sector instrument and array detection has been used to detect the protein-peptide complex with low-picomole sensitivity. The relative abundances of the multiply charged ions for the complex formed between Src SH2 protein and several nonphosphorylated and phosphorylated peptides have been compared. The mass spectrometry data correlate well to the measured binding constants derived from solution-based methods, indicating that the mass spectrometry-based method can be used to assess the affinity of such interactions. Solution-phase equilibrium constants may be determined by measuring the amount of bound and unbound species as a function of concentration for construction of a Scatchard graph. ESI-MS of a solution containing Src SH2 with a mixture of phosphopeptides showed the expected protein-phosphopeptide complex as the dominant species in the mass spectrum, demonstrating the method’s potential for screening mixtures from peptide libraries.  相似文献   

7.
In the year 1994, the protein MIA (melanoma inhibitory activity) was found to be strongly expressed and secreted by malignant melanomas and subsequent studies revealed that MIA has an important function in melanoma development and invasion. Multidimensional NMR-spectroscopy and x-ray crystallography revealed that recombinant human MIA adopts a Src homology 3 (SH3) domain-like fold in solution, a structure with two perpendicular antiparallel three- and five-stranded beta-sheets. SH3 domains are protein modules that are found in many intracellular signalling proteins and mediate protein-protein interactions by binding to proline-rich peptide sequences. Unlike previously described protein structures with SH3 domain folds, MIA is a secreted single-domain protein of 12 kDa that contains an additional antiparallel beta-sheet and two disulfide bonds. Furthermore, the charge surrounding the canonical binding site differs from that of classical SH3 domains. The two disulfide bonds are crucial for correct folding and function as revealed by mutation analysis. Therefore, MIA appears to be the first extracellular protein adopting an SH3 domain-like fold. MIA was shown to interact with fibronectin, and MIA-interacting peptide ligands identified by phage display screening are similar to the consensus sequence of type III human fibronectin repeats, especially FN14. Interestingly, recent data revealed that MIA can also directly bind to integrin alpha 4 beta 1 and alpha 5 beta1 and that it modulates integrin activity negatively. These findings suggest an interesting role of the SH3-domain proteins in the extracellular compartment. Recently, MIA homologous proteins with a sequence identity of 44% and a sequence homology of approximately 80% were determined (TANGO, MIA-2, OTOR). This clearly suggests that this structural device is used more frequently, in processes ranging from developmental changes to the interference of cell attachment in the extracellular matrix. Detailed studies are necessary to determine the exact function of the MIA homologous proteins. It will be interesting to know whether additional protein families can be identified which are secreted and carry SH3 domain-like modules, in addition to elucidate what the specific physiological targets of this protein family are.  相似文献   

8.
Combinatorial chemistry is a laboratory emulation of natural recombination and selection processes. Strategies in this developing discipline involve the generation of diverse, molecular libraries through combinatorial synthesis and the selection of compounds that possess a desired property. Such approaches can facilitate the identification of ligands that bind to biological receptors, promoting our chemical understanding of cellular processes. This article illustrates that the coupling of combinatorial synthesis, multidimensional NMR spectroscopy, and biochemical methods has enhanced our understanding of a protein receptor used commonly in signal transduction, the Src Homology 3 (SH3) domain. This novel approach to studying molecular recognition has revealed a set of rules that govern SH3–ligand interactions, allowing models of receptor–ligand complexes to be constructed with only a knowledge of the polypeptide sequences. Combining combinatorial synthesis with structural methods provides a powerful new approach to understanding how proteins bind their ligands in general.  相似文献   

9.
The sensitization of Eu(III) and Tb(III) by ethylenediaminetetraaceticacid (EDTA)-derivatized tryptophan (Trp), 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW) has been examined. These Trp analogs were utilized in the present study because they can be incorporated into proteins in place of native Trp residues and because they absorb strongly beyond 305 nm (where Trp absorbance goes to zero), allowing selective excitation of such species in the presence of other Trp-containing proteins. All three indole derivatives were able to sensitize Tb(III) luminescence, with the relative sensitization being in the order Trp > 5HW > 7AW. On the other hand, only the 7AW-EDTA complex was able to sensitize Eu(III) luminescence, likely owing to a better spectral overlap between 7AW emission and Eu(III) absorbance. The sensitized emission of Tb(III) and Eu(II) displayed the expected long emission lifetimes at 545 nm [for Tb(III)] and 617 nm [for Eu(III)], indicating that long-lifetime lanthanide emission could be produced using nonnatural amino-acid donors. Thus, 7AW- and 5HW-sensitized lanthanide emissions should prove to be useful in biophysical studies, such as the use of fluorescence energy transfer to probe biomolecular interactions in vivo.  相似文献   

10.
A "global" strategy for the acquisition of selective high affinity inhibitors for the Src kinase subfamily of tyrosine kinases is described. Members of the Src family exhibit a strong amino acid sequence homology. However, recent studies have revealed differences in the relative spatial relationships of the three distinct protein-binding domains present in these enzymes. We have constructed an inhibitor, using an amalgamation of combinatorial methods and directed design, which simultaneously associates with the active site and an ancillary protein-binding region (SH2 domain). The inhibitor exhibits high inhibitory potency and selectivity for the Group A versus Group B subset of Src kinases.  相似文献   

11.
The Src homology 3 (SH3) domains are small protein-protein interaction domains that mediate a range of important biological processes and are considered valuable targets for the development of therapeutic agents. We have been developing 2-aminoquinolines as ligands for SH3 domains--so far the only reported examples of entirely small-molecule ligands for the SH3 domains. The highest affinity 2-aminoquinolines so far identified are 6-substituted compounds. In this article, the synthesis of several new 2-aminoquinolines, including 5-, 6- and 7-substituted compounds, for Tec SH3 domain ligand binding studies is presented. As a part of the synthetic investigation, the utility of different methods for the synthesis of 2-aminoquinolines was explored and potentially powerful methods were identified for the synthesis of 2-aminoquinolines with diverse functionality. Of the compounds prepared, the 5-substituted-2-aminoquinolines generally bound with similar affinities to unsubstituted 2-aminoquinoline, whilst the 7-substituted compounds generally bound with similar or lower affinity than unsubstituted 2-aminoquinoline. However, the 6-substituted-2-aminoquinolines generally bound with significantly higher affinity than unsubstituted 2-aminoquinoline. In addition, one 6-substituted-N-benzylated-2-aminoquinoline was also tested for SH3 binding and some evidence for the formation of additional contacts at other regions of the SH3 domain was found. These results provide new and useful SAR information that should greatly assist with the challenge of developing high affinity small-molecule ligands for the SH3 domains.  相似文献   

12.
A stepwise library-based strategy has been employed to acquire a potent ligand for the SH3 domain of Fyn, a Src kinase family member that plays a key role in T cell activation. The easily automated methodology is designed to identify potential interaction sites that circumscribe the protein/peptide binding region on the SH3 domain. The library protocol creates peptide/nonpeptide chimeras that are able to bind to these interaction sites that are otherwise inaccessible to natural amino acid residues. The peptide-derived lead and the Fyn-SH3 domain form a complex that exhibits a K(D) of 25 +/- 5 nM, approximately 1000-fold more potent than that displayed by the corresponding conventional peptide ligand. Furthermore, the lead ligand exhibits selectivity against SH3 domains derived from other Src kinases, in spite of a sequence identity of approximately 80%.  相似文献   

13.
Phosphonomethylphenylalanine (Pmp, 2) has shown wide utility as a hydrolytically stable phosphtyrosyl (pTyr, 1) mimetic, particularly in Src homology 2 (SH2) domain-binding peptides. (2S,3R)-3-(4-(phosphonomethyl)phenyl)pipecolic acid (3) represents a variant of Pmp having φ and χ1 torsion angles constrained through incorporation into the piperidinyl ring structure contained within pipecolic acid. Reported herein is the enantioselective preparation of 3, in an orthogonally protected form (4) suitable for use in peptide synthesis. Stereochemistries at both the 2- and 3-positions are derived inductively from a single chiral center provided by the commercially available Evans chiral auxiliary, (4S)-4-benzyl-1,3-oxazolidin-2-one. Incorporation of 4 into a Grb2 SH2 domain-directed tripeptide (18) showed that Grb2 SH2 domain-binding affinity was reduced relative to the parent Pmp-containing tripeptide (19). Although conformational constraint did not enhance affinity in this case, novel amino acid analogue 4 may serve as a useful tool for the induction of defined phosphotyrosyl geometry in peptides directed at other signal transduction targets.  相似文献   

14.
《Chemistry & biology》1998,5(10):529-538
Background: Many intracellular signal-transduction pathways are regulated by specific protein-protein interactions. These interactions are mediated by structural domains within signaling proteins that modulate a protein's cellular location, stability or activity. For example, Src-homology 2 (SH2) domains mediate protein-protein interactions through short contiguous amino acid motifs containing phosphotyrosine. As SH2 domains have been recognized as key regulatory molecules in a variety of cellular processes, they have become attractive drug targets.Results: We have developed a novel mechanism-based cellular assay to monitor specific SH2-domain-dependent protein-protein interactions. The assay is based on a two-hybrid system adapted to function in mammalian cells where the SH2 domain ligand is phosphorylated, and binding to a specific SH2 domain can be induced and easily monitored. As examples, we have generated a series of mammalian cell lines that can be used to monitor SH2-domain-dependent activity of the signaling proteins ZAP-70 and Src. We are utilizing these cell lines to screen for immunosuppressive and anti-osteoclastic compounds, respectively, and demonstrate here the utility of this system for the identification of small-molecule, cell-permeant SH2 domain inhibitors.Conclusions: A mechanism-based mammalian cell assay has been developed to identify inhibitors of SH2-domain-dependent protein-protein interactions. Mechanism-based assays similar to that described here might have general use as screens for cell-permeant, nontoxic inhibitors of protein-protein interactions.  相似文献   

15.
BACKGROUND: Src homology 3 (SH3) domains bind sequences bearing the consensus motif PxxP (where P is proline and x is any amino acid), wherein domain specificity is mediated largely by sequences flanking the PxxP core. This specificity is limited, however, as most SH3 domains show high ligand cross-reactivity. We have recently shown that diverse N-substituted residues (peptoids) can replace the prolines in the PxxP motif, yielding a new source of ligand specificity. RESULTS: We have tested the effects of combining multiple peptoid substitutions with specific flanking sequences on ligand affinity and specificity. We show that by varying these different elements, a ligand can be selectively tuned to target a single SH3 domain in a test set. In addition, we show that by making multiple peptoid substitutions, high-affinity ligands can be generated that completely lack the canonical PxxP motif. The resulting ligands can potently disrupt natural SH3-mediated interactions. CONCLUSIONS: Peptide-peptoid hybrid scaffolds yield SH3 ligands with markedly improved domain selectivity, overcoming one of the principal challenges in designing inhibitors against these domains. These compounds represent important leads in the search for orthogonal inhibitors of SH3 domains, and can serve as tools for the dissection of complex signaling pathways.  相似文献   

16.
We have constructed a phage-displayed library based on the human fibronectin tenth type III domain (FN3) scaffold by randomizing residues in its FG and BC loops. Screening against the SH3 domain of human c-Src yielded six different clones. Five of these contained proline-rich sequences in their FG loop that resembled class I (i.e., +xxPxxP) peptide ligands for the Src SH3 domain. The sixth clone lacked the proline-rich sequence and showed particularly high binding specificity to the Src SH3 domain among various SH3 domains tested. Competitive binding, loop replacement, and NMR perturbation experiments were conducted to analyze the recognition properties of selected binders. The strongest binder was able to pull down full-length c-Src from murine fibroblast cell extracts, further demonstrating the potential of this scaffold for use as an antibody mimetic.  相似文献   

17.
Reversed-phase HPLC was coupled on-line to a rapid, competitive affinity probe capillary electrophoresis (APCE) assay to screen mixtures for compounds that inhibit protein-ligand interactions. The Fyn Src homology 2 (SH2) domain and its phosphopeptide binding partner were used as a model interaction for demonstration of this technique. In the method, mixtures containing possible inhibitors of binding were separated by HPLC at a flow rate of 0.3 mL/min. A small portion of effluent was directed to a fluidic tee where it was mixed on-line with Fyn SH2 domain and a fluorescent phosphopeptide ("affinity probe") known to bind selectively to Fyn SH2 domain. Electropherograms of the reaction mixture were collected on-line at approximately 6s intervals using a flow-gated interface to control injections onto the capillary electrophoresis with laser-induced fluorescence system. The resulting electropherograms contained two peaks, one corresponding to the free affinity probe and the other a complex of the affinity probe and Fyn SH2 domain. Compounds that bound the protein were detected as a decrease in the peak height of the complex and an increase in the peak height of affinity probe with relative standard deviations of <5%. The assay was shown to resolve multiple peptidergic inhibitors and selectively detect them within a complex mixture of peptides. Signals were dependent upon both concentration of active peptide and its potency in binding inhibition. Detection limits were in the range of 2-11 microM depending upon the peptide. Common organic solvents used in HPLC were shown to have minimal effect in the on-line measurement up to approximately 60% in the mobile phase.  相似文献   

18.
C-Terminal peptide thioesters are key intermediates in the synthesis/semisynthesis of proteins and of cyclic peptides by native chemical ligation. They are prepared by solid-phase peptide synthesis (SPPS) or biosynthetically by protein splicing techniques. Until recently, the chemical synthesis of C-terminal alpha-thioester peptides by SPPS was largely restricted to the use of Boc/Benzyl chemistry due to the poor stability of the thioester bond to the basic conditions required for the deprotection of the N(alpha)-Fmoc group. In the present work, we describe a new method for the SPPS of C-terminal thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. This step converts the acyl hydrazine group into a highly reactive acyl diazene intermediate which reacts with an alpha-amino acid alkyl thioester (H-AA-SR) to yield the corresponding peptide alpha-thioester in good yield. This method has been successfully used to prepare a variety of peptide thioesters, cyclic peptides, and a fully functional Src homology 3 (SH3) protein domain.  相似文献   

19.
Catalysts hold promise as tools for chemical protein modification. However, the application of catalysts or catalyst-mediated reactions to proteins has only recently begun to be addressed, mainly in in vitro systems. By radically improving the affinity-guided DMAP (4-dimethylaminopyridine) (AGD) catalysts that we previously reported (Koshi, Y.; Nakata, E.; Miyagawa, M.; Tsukiji, S.; Ogawa, T.; Hamachi, I. J. Am. Chem. Soc. 2008, 130, 245.), here we have developed a new organocatalyst-based approach that allows specific chemical acylation of a receptor protein on the surface of live cells. The catalysts consist of a set of 'multivalent' DMAP groups (the acyl transfer catalyst) fused to a ligand specific to the target protein. It was clearly demonstrated by in vitro experiments that the catalyst multivalency enables remarkable enhancement of protein acylation efficiency in the labeling of three different proteins: congerin II, a Src homology 2 (SH2) domain, and FKBP12. Using a multivalent AGD catalyst and optimized acyl donors containing a chosen probe, we successfully achieved selective chemical labeling of bradykinin B(2) receptor (B(2)R), a G-protein coupled receptor, on the live cell-surface. Furthermore, the present tool allowed us to construct a membrane protein (B(2)R)-based fluorescent biosensor, the fluorescence of which is enhanced (tuned on) in response to the antagonist ligand binding. The biosensor should be applicable to rapid and quantitative screening and assay of potent drug candidates in the cellular context. The design concept of the affinity-guided, multivalent catalysts should facilitate further development of diverse catalyst-based protein modification tools, providing new opportunities for organic chemistry in biological research.  相似文献   

20.
BACKGROUND: The observations that Src(-/-) mice develop osteopetrosis and Src family tyrosine kinase inhibitors decrease osteoclast-mediated resorption of bone have implicated Src in the regulation of osteoclast-resorptive activity. We have designed and synthesized a compound, AP22161, that binds selectively to the Src SH2 domain and demonstrated that it inhibits Src-dependent cellular activity and inhibits osteoclast-mediated resorption. RESULTS: AP22161 was designed to bind selectively to the Src SH2 domain by targeting a cysteine residue within the highly conserved phosphotyrosine-binding pocket. AP22161 was tested in vitro for binding to SH2 domains and was found to bind selectively and with high affinity to the Src SH2 domain. AP22161 was further tested in mechanism-based cellular assays and found to block Src SH2 binding to peptide ligands, inhibit Src-dependent cellular activity and diminish osteoclast resorptive activity. CONCLUSIONS: These results indicate that a compound that selectively inhibits Src SH2 binding can be used to inhibit osteoclast resorption. Furthermore, AP22161 has the potential to be further developed for treating osteoporosis.  相似文献   

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