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1.
CD47, a cell surface transmembrane Ig superfamily member, is an extracellular ligand for signal regulatory protein (SIRPalpha). Interactions between CD47 and SIRPalpha regulate many important immune cell functions including neutrophil (PMN) transmigration. Here we report identification of a novel function-blocking peptide, CERVIGTGWVRC, that structurally mimics an epitope on CD47 and binds to SIRPalpha. The CERVIGTGWVRC sequence was identified by panning phage display libraries on the inhibitory CD47 mAb, C5D5. In vitro PMN migration assays demonstrated that peptide CERVIGTGWVRC specifically inhibited PMN migration across intestinal epithelial monolayers and matrix in a dose-dependent fashion. Further studies using recombinant proteins indicated that the peptide specifically blocks CD47 and SIRPalpha binding in a dose-dependent fashion. Protein binding assays using SIRPalpha domain-specific recombinant proteins demonstrated that this peptide directly bound to the distal-most Ig loop of SIRPalpha, the same loop where CD47 binds. In summary, these findings support the relevance of CD47-SIRPalpha interactions in regulation of PMN transmigration and provide structural data predicting the key residues involved on the surface of CD47. Such peptide reagents may be useful for studies on experimental models of inflammation and provide a template for the design of anti-inflammatory agents.  相似文献   

2.
CD47 is a widely distributed cell-surface protein that acts a marker of self through interactions of myeloid and neural cells. We describe the high-resolution X-ray crystallographic structures of the immunoglobulin superfamily domain of CD47 alone and in complex with the N-terminal ligand-binding domain of signal regulatory protein alpha (SIRPalpha). The unusual and convoluted interacting face of CD47, comprising the N terminus and loops at the end of the domain, intercalates with the corresponding regions in SIRPalpha. We have also determined structures of the N-terminal domains of SIRPbeta, SIRPbeta(2), and SIRPgamma; proteins that are closely related to SIRPalpha but bind CD47 with negligible or reduced affinity. These results explain the specificity of CD47 for the SIRP family of paired receptors in atomic detail. Analysis of SIRPalpha polymorphisms suggests that these, as well as the activating SIRPs, may have evolved to counteract pathogen binding to the inhibitory SIRPalpha receptor.  相似文献   

3.
NIT2, a positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes that encode nitrogen catabolic enzymes. NIT2 binding sites in the promoter regions of nit3, alc and lao have at least two GATA sequence elements. We have examined the binding affinity of the NIT2 protein for the yeast DAL5 wild-type upstream activation sequence UASNTR, which contains two GATA elements, and for a series of mutated binding sites, each differing from the wild-type site by a single base. Substitution for individual nucleotides within 5′ or 3′ sequences that flank the GATA elements had only modest effects upon NIT2 binding. In contrast, nearly all substitutions within the GATA elements almost completely eliminated NIT2 binding, demonstrating the importance of the GATA sequence for NIT2 binding. Four high-affinity binding sites for the NIT2 protein were found within a central region of the nit-2 gene itself.  相似文献   

4.
NIT2, a positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes that encode nitrogen catabolic enzymes. NIT2 binding sites in the promoter regions of nit3, alc and lao have at least two GATA sequence elements. We have examined the binding affinity of the NIT2 protein for the yeast DAL5 wild-type upstream activation sequence UASNTR, which contains two GATA elements, and for a series of mutated binding sites, each differing from the wild-type site by a single base. Substitution for individual nucleotides within 5 or 3 sequences that flank the GATA elements had only modest effects upon NIT2 binding. In contrast, nearly all substitutions within the GATA elements almost completely eliminated NIT2 binding, demonstrating the importance of the GATA sequence for NIT2 binding. Four high-affinity binding sites for the NIT2 protein were found within a central region of the nit-2 gene itself.  相似文献   

5.
CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8α and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8α, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.  相似文献   

6.
Two closely related proteins, signal regulatory protein alpha (SIRPalpha; SHPS-1/CD172) and SIRPbeta, have been described in humans. The existence of a third SIRP protein has been suggested by cDNA sequence only. We show that this third SIRP is a separate gene that is expressed as a protein with unique characteristics from both alpha and beta genes and suggest that this gene should be termed SIRPgamma. We have expressed the extracellular region of SIRPgamma as a soluble protein and have shown that, like SIRPalpha, it binds CD47, but with a lower affinity (K(d), approximately 23 microM) compared with SIRPalpha (K(d), approximately 2 microM). mAbs specific to SIRPgamma show that it was not expressed on myeloid cells, in contrast to SIRPalpha and -beta, being expressed instead on the majority of T cells and a proportion of B cells. The short cytoplasmic tail of SIRPgamma does not contain any known signaling motifs, nor does it contain a characteristic lysine, as with SIRPbeta, that is required for DAP12 interaction. DAP12 coexpression is a requirement for SIRPbeta surface expression, whereas SIRPgamma is expressed in its absence. The SIRPgamma-CD47 interaction may therefore not be capable of bidirectional signaling as with the SIRPalpha-CD47, but, instead, use unidirectional signaling via CD47 only.  相似文献   

7.
In an effective immune response, CD8+ T cell recognition of virally derived Ag, bound to MHC class I, results in killing of infected cells. The CD8alphabeta heterodimer acts as a coreceptor with the TCR, to enhance sensitivity of the T cells to peptide/MHC class I, and is two orders of magnitude more efficient as a coreceptor than the CD8alphaalpha. To understand the important interaction between CD8alphabeta and MHC class I, we created a panel of CD8beta mutants and identified mutations in the CDR1, CDR2, and CDR3 loops that decreased binding to MHC class I tetramers as well as mutations that enhanced binding. We tested the coreceptor function of a subset of reducing and enhancing mutants using a T cell hybridoma and found similar reducing and enhancing effects. CD8beta-enhancing mutants could be useful for immunotherapy by transduction into T cells to enhance T cell responses against weak Ags such as those expressed by tumors. We also addressed the question of the orientation of CD8alphabeta with MHC class I using CD8alpha mutants expressed as a heterodimer with wild-type CD8alpha or CD8beta. The partial rescuing of binding with wild-type CD8beta compared with wild-type CD8alpha is consistent with models in which either the topology of CD8alphaalpha and CD8alphabeta binding to MHC class I is different or CD8alphabeta is capable of binding in both the T cell membrane proximal and distal positions.  相似文献   

8.
The herpes simplex virus (HSV) ICP47 protein inhibits the MHC class I antigen presentation pathway by inhibiting the transporter associated with antigen presentation (TAP) which translocates peptides across the endoplasmic reticulum membrane. At present, ICP47 is the only inhibitor of TAP. Here, we show that ICP47 produced in bacteria can block human, but not mouse, TAP, and that heat denaturation of ICP47 has no effect on its ability to block TAP. ICP47 inhibited peptide binding to TAP without affecting ATP binding, consistent with previous observations that the peptide binding and ATP binding sites of TAP are distinct. ICP47 bound to TAP with a higher affinity (KD approximately 5 x 10(-8) M) than did peptides, and ICP47 did not dissociate from TAP. ICP47 was not transported by TAP and remained sensitive to proteases added from the cytosolic surface of the membrane. Peptides acted as competitive inhibitors of ICP47 binding to TAP, and this inhibition required a 100- to 1000-fold molar excess of peptide. These results demonstrate that ICP47 binds to a site which includes the peptide binding domain of TAP and remains bound to this site in a stable fashion.  相似文献   

9.
The gene for the alpha i1 subunit of human guanine nucleotide binding (G) protein was mapped by in situ hybridization to chromosome 7 at band q21. The regional chromosomal location of the human alpha i1 gene was confirmed using human/mouse somatic-cell hybrid lines containing portions of human chromosome 7. Because the alpha i1 gene mapped near the cystic fibrosis locus and because an abnormal G protein might be expected to contribute to the pathophysiology of this disease, the alpha i1 gene was mapped with respect to the cystic fibrosis locus as defined by the Met oncogene and anonymous DNA marker pJ3.11. The location of the alpha i1 gene proved to be distinct from that of the cystic fibrosis locus.  相似文献   

10.
Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2-3 mM) in exchange and zero-trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero-trans influx assays (inhibition constant values of 5-12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.  相似文献   

11.
Signal regulatory protein (SIRP) alpha is a membrane receptor that sends inhibitory signals to myeloid cells by engagement of CD47. The high resolution x-ray structure of the N-terminal ligand binding domain shows it to have a distinctive immunoglobulin superfamily V-like fold. Site-directed mutagenesis suggests that CD47 is bound at a surface involving the BC, FG, and DE loops, which distinguishes it from other immunoglobulin superfamily surface proteins that use the faces of the fold, but resembles antigen receptors. The SIRP interaction is confined to a single domain, and its use of an extended DE loop strengthens the similarity with T cell receptor binding and the suggestion that they are closely related in evolution. The employment of loops to form the CD47-binding surface provides a mechanism for small sequence changes to modulate binding specificity, explaining the different binding properties of SIRP family members.  相似文献   

12.
C-reactive protein (CRP) is thought to play an important role in immunomodulation. The exact biologic function of this pentraxin protein is, however, still unclear. Here we report experiments designed to further characterize the binding properties of CRP. Using purified human CRP it could be shown that CRP immobilized onto polystyrene surfaces or onto latex beads binds distinct plasma glycoproteins including IgG, asialofetuin, asialo-beta 2-glycoprotein I and, likewise, synthetic glycoproteins as a lectin, exhibiting binding specificity for terminal galactosyl residues of the glycoprotein glycans. Binding of CRP to IgA, IgM, IgG, asialofetuin, asialo-beta 2-glycoprotein I and to synthetic glycoproteins requires immobilization onto surfaces of both CRP and the ligand. Fibronectin and fibrinogen are bound by surface-immobilized CRP also in soluble phase. Comparing various mono-, di-, and trisaccharides as competitive inhibitors of the lectin binding activity of CRP, only beta-D-Gal-(1-3)-D-GalNAc, beta-D-Gal-(1-4)-D-GalNAc, and beta-D-Gal-(1-4)-beta-D-Gal-(1-4)-D-GlcNAc had significant inhibitory power at a concentration of 8 mmol/liter. Binding activity of CRP was pH-dependent with an optimum at pH 5 to 6 and was reduced by 90% when pH was shifted from 6 to the physiologic pH value of 7.4. CRP exhibited lectin-like properties with binding specificity for galactosyl residues also when bound to K-562 erythroleukemia cells. It is therefore suggested that CRP immobilized onto surfaces exhibits lectin activity toward galactosyl groups preferentially in a mildly acidic environment as present at sites of inflammation.  相似文献   

13.
The nature of the steroid binding site in alpha1-acid glycoprotein (orosomucoid) was investigated by chemical modification of individual amino acids and subsequent examination of the binding affinity for progesterone. Equilibrium dialyses were performed under conditions that excluded contact with human skin. Reaction of the lysyl residues with trinitrobenzenesulfonic acid or arylisocyanates resulted in a reduction of active sites. In an alternate approach, one lysyl residue of alpha1-acid glycoprotein was protected from modification by trinitrobenzenesulfonic acid when progesterone was present to form the complex with alpha1-acid glycoprotein. We conclude that a lysyl residue is located in the binding site. Reaction of tetranitromethane with the tyrosine groups in alpha1-acid glycoprotein also reduced the number of active binding sites for progesterone. Again, a partial protection of this modification was seen in the presence of progesterone and other delta4-3-ketosteroids. The progesterone binding activity observed in the tyrosine-modified alpha1-acid glycoprotein by equilibrium dialysis and by fluorescence quenching titration can be interpreted best by the presence of one tyrosyl residue in the binding site, and involvement of a second tyrosine nearby. Modification of tryptophan in alpha1-acid glycoprotein by mild acid hydrolysis, N-bromosuccinimide, hydroxynitrobenzylbromide, and formic acid resulted in a decreased steroid binding; the formylation reaction was fully reversible. The approximate distance between progesterone and the tryptophan involved in the binding was calculated to be between 9.1 A and 14.1 A. When alpah1-acid glycoprotein was cleaved by the cyanogen bromide procedure according to Ikenaka et al. (1972, Biochemistry 11, 3817-3829), both the amino and the carboxyl fragment had a weak progesterone binding affinity which could be measured in 4 M NaCl. This result thus failed to specify the location of the steroid binding site in alpha1-acid glycoprotein. However, the closeness of tryptophan, lysine and tyrosine in the primary and presumably the tertiary structure of alpha1-acid glycoprotein is in agreement with the properties of the binding site suggested by our studies.  相似文献   

14.
15.
Recent studies have demonstrated that CD47 plays an important role in regulating human neutrophil (PMN) chemotaxis. Two ligands for CD47, thrombospondin and SIRPalpha, have been described. However, it is not known if SIRP-CD47 interactions play a role in regulating PMN migration. In this study, we show that SIRPalpha1 directly binds to the immunoglobulin variable domain loop of purified human CD47 and that such SIRP-CD47 interactions regulate PMN transmigration. Specifically, PMN migration across both human epithelial monolayers and collagen-coated filters was partially inhibited by anti-SIRP monoclonal antibodies. Similar kinetics of inhibition were observed for PMN transmigration in the presence of soluble, recombinant CD47 consisting of the SIRP-binding loop. In contrast, anti-CD47 monoclonal antibodies inhibited PMN transmigration by markedly different kinetics. Results of signal transduction experiments suggested differential regulation of PMN migration by SIRP versus CD47 by phosphatidylinositol 3-kinase and tyrosine kinases, respectively. Immunoprecipitation followed by Western blotting after SDS-PAGE under nonreducing conditions suggested that several SIRP protein species may be present in PMN. Stimulation of PMN with fMLP resulted in increased surface expression of these SIRP proteins, consistent with the existence of intracellular pools. Taken together, these results demonstrate that PMN migration is regulated by CD47 through SIRPalpha-dependent and SIRPalpha-independent mechanisms.  相似文献   

16.
a1 protein alters the DNA binding specificity of alpha 2 repressor   总被引:44,自引:0,他引:44  
C Goutte  A D Johnson 《Cell》1988,52(6):875-882
The alpha 2 protein of S. cerevisiae, the product of the MAT alpha 2 gene, represses a set of cell-type-specific genes (the a-specific genes) by binding to an operator sequence upstream of each gene. We demonstrate that a second yeast regulatory protein, a1, the product of the MATa1 gene, can alter the binding specificity of alpha 2 so that it no longer recognizes the a-specific gene operator, but instead acquires the ability to recognize a different operator sequence found upstream of haploid-specific genes. Thus, under the influence of a1, alpha 2 can repress haploid-specific genes. An alpha cell expresses alpha 2 but not a1, so that alpha 2 turns off only the a-specific genes. An a/alpha cell makes both a1 and alpha 2, in a ratio that ensures that alpha 2 is distributed between two distinct binding modes: the alpha 2 binding mode and the a1-alpha 2 binding mode. Thus in an a/alpha cell, alpha 2 represses two distinct classes of genes.  相似文献   

17.
The binding constants for interaction of various thryoxine analogues with the thyroxine binding site on human thyroxine-binding globulin have been determined. Equilibrium dialysis, at pH 7.4 and 37 degrees C, was used to measure the competitive effects of different iodothyronine compounds on the binding of 125I-labeled thyroxine to highly purified thyroxine-binding globulin. Relative to L-thyroxine, K = 6 . 10(9) M-1, the association constants of some important analogues were D-thyroxine, 1.04 . 10(9) M-1, 3,5-diiodo-3'-isopropyl-L-thyronine, 4.9 . 10(8) M-1; L-triiodothyronine, 3.3 . 10(8) M-1, 3,3',5'-DL-triiodothyronine (reverse triiodothyronine), 3.1. 10(8) M-1; tetraiodothyropropionic acid, 2.7 . 10(8) M-1; tetraiodothyroacetic acid, 2.6 . 10(8) M-1; 3', 5'- diiodo-DL-thyronine, 8.3 . 10(7) M-1; and 3,5-diiodo-DL-thyronine, 7.1 . 10(7) M-1. Calculation of the deltaG0 values for binding of the analogues indicates that a major contribution to the free energy favoring binding is made by the alanine side chain of thyroxine. A change in configuration of the alpha-amino group from the L to D form causes an unfavorable change of 1 kcal/mol in the free energy of binding. Removal of the alpha-amino group as in tetraiodothyropropionic acid causes an unfavorable change of 1.9 kcal/mol in the free energy of binding. With regard to ring substituents, the results indicate that the two inner 3,5-iodines make about the same contribution to binding as the two outer 3', 5'-iodines.  相似文献   

18.
Fused in sarcoma (FUS) is involved in many processes of RNA metabolism. FUS and another RNA binding protein, TDP-43, are implicated in amyotrophic lateral sclerosis (ALS). It is significant to characterize the RNA recognition motif (RRM) of FUS as its nucleic acid binding properties are unclear. More importantly, abolishing the RNA binding ability of the RRM domain of TDP43 was reported to suppress the neurotoxicity of TDP-43 in Drosophila. The sequence of FUS-RRM varies significantly from canonical RRMs, but the solution structure of FUS-RRM determined by NMR showed a similar overall folding as other RRMs. We found that FUS-RRM directly bound to RNA and DNA and the binding affinity was in the micromolar range as measured by surface plasmon resonance and NMR titration. The nucleic acid binding pocket in FUS-RRM is significantly distorted since several critical aromatic residues are missing. An exceptionally positively charged loop in FUS-RRM, which is not found in other RRMs, is directly involved in the RNA/DNA binding. Substituting the lysine residues in the unique KK loop impaired the nucleic acid binding and altered FUS subcellular localization. The results provide insights into the nucleic acid binding properties of FUS-RRM and its potential relevance to ALS.  相似文献   

19.
We have previously found that the protein which bound to the essential region of human autonomously replicating sequences (ARS) could be the c-myc proto-oncogene product. Here we examined the binding specificity of human ARS binding protein purified from Burkitt's lymphoma Raji cells. Among several oligodeoxynucleotides, this protein bound to the fragments containing the sequence of 5'-CAPyCTCTNA-3'. Competition analysis revealed that the ARS binding protein could recognize not only the nucleotide sequences but also the high ordered structure.  相似文献   

20.
Using ligation-mediated polymerase chain reaction and in vivo footprinting methods to study the status of DNA-protein interactions at hypersensitive site 3.5 (HS3.5) of the locus control region in K562 and HEL cells, we found that there was protein occupancy in vivo at HS3.5 in both cell lines and the status of DNA-protein interaction was different between K562 and HEL. These data provide direct evidence that specific nuclear factor-DNA complexes form in vivo at functionally important sequence motifs of the HS3.5 in erythroid cells. This indicates that HS3.5 may play an important role in the regulation of the beta-globin gene cluster. K562 is a human erythroleukemia cell line in which the embryonic epsilon-globin gene is predominantly expressed, while the HEL cell line expresses predominantly the fetal beta-globin genes. Thus, HS3.5 might also be involved in the regulation of developmental stage-specific expression of beta-globin genes. Our results are also consistent with the model that each hypersensitive site acts as a functional unit and HS3.5 may facilitate the formation of the HS3 functional unit.  相似文献   

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