共查询到20条相似文献,搜索用时 31 毫秒
1.
Cornelia Tolg Sara R. Hamilton Lyndsey Morningstar Jing Zhang S. Zhang Kenneth V. Esguerra Patrick G. Telmer Len G. Luyt Rene Harrison James B. McCarthy Eva A. Turley 《The Journal of biological chemistry》2010,285(34):26461-26474
An oncogenic form of RHAMM (receptor for hyaluronan-mediated motility, mouse, amino acids 163–794 termed RHAMMΔ163) is a cell surface hyaluronan receptor and mitotic spindle protein that is highly expressed in aggressive human cancers. Its regulation of mitotic spindle integrity is thought to contribute to tumor progression, but the molecular mechanisms underlying this function have not previously been defined. Here, we report that intracellular RHAMMΔ163 modifies the stability of interphase and mitotic spindle microtubules through ERK1/2 activity. RHAMM−/− mouse embryonic fibroblasts exhibit strongly acetylated interphase microtubules, multi-pole mitotic spindles, aberrant chromosome segregation, and inappropriate cytokinesis during mitosis. These defects are rescued by either expression of RHAMM or mutant active MEK1. Mutational analyses show that RHAMMΔ163 binds to α- and β-tubulin protein via a carboxyl-terminal leucine zipper, but in vitro analyses indicate this interaction does not directly contribute to tubulin polymerization/stability. Co-immunoprecipitation and pulldown assays reveal complexes of RHAMMΔ163, ERK1/2-MEK1, and α- and β-tubulin and demonstrate direct binding of RHAMMΔ163 to ERK1 via a D-site motif. In vitro kinase analyses, expression of mutant RHAMMΔ163 defective in ERK1 binding in mouse embryonic fibroblasts, and blocking MEK1 activity collectively confirm that the effect of RHAMMΔ163 on interphase and mitotic spindle microtubules is mediated by ERK1/2 activity. Our results suggest a model wherein intracellular RHAMMΔ163 functions as an adaptor protein to control microtubule polymerization during interphase and mitosis as a result of localizing ERK1/2-MEK1 complexes to their tubulin-associated substrates. 相似文献
2.
N. P. Akentieva S. S. Shushanov 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2017,11(1):24-34
The ability of two synthetic peptides GEGEEGEE and DFGEEAEE to inhibit the invasiveness of tumor cells has been studied. Using confocal microscopy, it was demonstrated that these peptides specifically bind to receptor for hyaluronic acid mediated motility (RHAMM) on the prostate cancer cell surface. Effect of peptides on invadopodia formation in cancer cells was studied by fluorescent gelatin degradation. Using confocal microscopy, images of the cells were obtained and the degree of their invasiveness was analyzed by assessing the gelatin degradation area using ImageJ software. It was found that pre-incubation of tumor cells with the peptides at a concentration of 40 μg/mL (2 × 10–7 M) inhibited the invasiveness by more than 80%. Thus, it was shown that peptides GEGEEGEE and DFGEEAEE may be potential antitumor agents. 相似文献
3.
Alexandra Hauser-Kawaguchi Cornelia Tolg Teresa Peart Mark Milne Eva A. Turley Leonard G. Luyt 《Bioorganic & medicinal chemistry》2018,26(18):5194-5203
The receptor for hyaluronan mediated motility (RHAMM, gene name HMMR) belongs to a group of proteins that bind to hyaluronan (HA), a high-molecular weight anionic polysaccharide that has pro-angiogenic and inflammatory properties when fragmented. We propose to use a chemically synthesized, truncated version of the protein (706–767), 7?kDa RHAMM, as a target receptor in the screening of novel peptide-based therapeutic agents. Chemical synthesis by Fmoc-based solid-phase peptide synthesis, and optimization using pseudoprolines, results in RHAMM protein of higher purity and yield than synthesis by recombinant protein production. 7?kDa RHAMM was evaluated for its secondary structure, ability to bind the native ligand, HA, and its bioactivity. This 62-amino acid polypeptide replicates the HA binding properties of both native and recombinant RHAMM protein. Furthermore, tubulin-derived HA peptide analogues that bind to recombinant RHAMM and were previously reported to compete with HA for interactions with RHAMM, bind with a similar affinity and specificity to the 7?kDa RHAMM. Therefore, in terms of its key binding properties, the 7?kDa RHAMM mini-protein is a suitable replacement for the full-length recombinant protein. 相似文献
4.
Hyaluronan (HA) is an important regulator of cell locomotion. We show that ras -transformed cells, termed 245 cells, respond to HA with an increase in random locomotion. We show that two HA receptors, RHAMM (receptor for hyaluronan-mediated motility) and CD44, are present on these ras -transformed fibroblasts. RHAMM is expressed as a 58-kDa protein and is distributed primarily as patches over lamellae. CD44 occurs largely as an 85- to 90-kDa protein that is distributed more or less evenly over the cell surface with small amounts concentrated at the tips of lamellae. CD44 and RHAMM both bind biotinylated HA in a transblot assay, indicating that they are both potential fibroblast HA receptors. CD44 binds approximately five times more HA than RHAMM as determined by densitometric analysis of transblots, indicating that this protein is the major HA receptor on fibroblasts. We assessed the role of these receptors in mediating the stimulatory effects of HA on cell motility by using antibody neutralization. Several antibodies to CD44 were used that inhibit HA/CD44 interactions. None of these had an effect on locomotory responses to HA, indicating that CD44 is not directly involved in mediating locomotion in response to HA on ras-transformed cells. In contrast, antibodies specific to RHAMM completely inhibited locomotion, indicating that RHAMM is the primary mediator of HA-promoted locomotion of ras -transformed cells. 相似文献
5.
JINWEN JIANG SHUBH D. SHARMA VICTOR J. HRUBY DAVID L. BENTLEY JODY L. FINK MAC E. HADLEY 《Pigment cell & melanoma research》1996,9(5):240-247
The objectives of this research were to determine whether melanocortin receptors are characteristic (constant) membrane markers of human epidermal melanocytes. Methodologies were developed to visualize melanotropin receptors by scanning electron microscopy (SEM). Multiple copies (up to a hundred) of [Nle4,D-Phe7]α-MSH, a superpotent analog of α-melanocyte stimulating hormone (α-MSH), were conjugated to a macromo-lecular carrier (latex beads: microspheres). Incubation in the presence of the melanotropin-conjugated microspheres resulted in binding of human normal epidermal melanocytes to the beads. Almost every (possibly all) melanocyte possesses melanocortin receptors as visualized by SEM. Specificity of binding of the macromolecular conjugate was demonstrated by several studies: 1) Binding of melanocytes to the microspheres was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; 2) microspheres lacking bound ligand did not bind to the melanocytes; 3) micro-spheres that were first treated with reducing agents (e.g., dithiothreitol) did not subsequently bind to melanocytes; 4) another peptide hormone ligand (e.g., a substance-P analog) attached to the latex beads failed to bind to the cells; 5) B16/F10 mouse melanoma cells known to express melanocortin receptors bound to the microspheres; and 6) cells of nonmelanocyte origin (e.g., mammary cancer cells, small-cell lung cancer cells, fibroblasts) did not bind to the macromolecular conjugate. One exception was that human epidermal keratinocytes also expressed melanocortin receptors as determined by all the criteria established above for epidermal melanocytes. Thus, cell specific melanocortin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes. 相似文献
6.
Human fetal lung fibroblasts were tested for their ability to accumulate zinc and to bind bacteria after 20–24 generations in tissue culture and again after 56–57 generations when the fibroblasts are near the end of their capacity for cell division. Uptake of 65ZnCl2 (measured under unsaturated conditions as accumulation per cell and per mg cell protein during a 1-hour incubation) was stable between 20 and 24 generations, and over a 3-day period of confluency. However, there was a 40 percent reduction of zinc uptake per mg of fibroblast protein at 56–57 generations, when the cells were larger. In contrast, incubation of labeled bacteria for 1 hour with the fibroblasts resulted in a constant proportion binding per unit cell mass at 20–24 and 56–57 generations. It is concluded that bacterial binding sites on the cell surface do not diminish with aging of fibroblasts, whereas uptake of zinc per unit cytoplasm undergoes reduction as a consequence of the aging process. 相似文献
7.
The addition of the glucocorticoid analog dexamethasone (DX) to serum-free cultures of human fibroblasts caused a twofold enhancement of the mitogenic response to epidermal growth factor (EGF), although DX by itself was not mitogenic. A basis for this effect was suggested by studies showing that DX also increased the cellular binding of 125I-EGF. DX increased the ability of the cells to bind 125I-EGF only at low physiological concentrations of this polypeptide. Thus, data from 125I-EGF binding to cells incubated without DX produced a linear Scatchard plot, whereas the data from 125I-EGF binding to DX-treated cells led to an upwardly curvilinear Scatchard plot. Measurements of 125I-EGF association with the cell surface and cytoplasm indicated that this binding change involved an alteration of cell surface EGF receptors. The binding change appeared not to involve negatively cooperative interactions between EGF receptors, nor a change in the number of receptors. The binding alteration could be explained by a model in which DX converted 25–30% of the cell surface EGF receptors to a form having a fourfold increased affinity. 相似文献
8.
Mandal SM Migliolo L Das S Mandal M Franco OL Hazra TK 《Journal of cellular biochemistry》2012,113(1):184-193
Nowadays, novel pharmacies have been screened from plants. Among them are the peptides, which show multiple biotechnological activities. In this report, a small peptide (Ala–Trp–Lys–Leu–Phe–Asp–Asp–Gly–Val) with a molecular mass of 1,050 Da was purified from Cycas revoluta seeds by using reversed‐phase liquid chromatography. This peptide shows clear deleterious effects against human epidermoid cancer (Hep2) and colon carcinoma cells (HCT15). It caused inhibition of cancer cell proliferation and further disruption of nucleosome structures, inducing apoptosis by direct DNA binding. A remarkable antibacterial activity was also observed in this same peptide. Nevertheless, no significant lysis of normal RBC cells was observed in the presence of peptide. Additionally, an acetylation at the N‐termini portion is able to reduce both activities. Bioinformatics tools were also utilized for construction of a three‐dimensional model showing a single amphipathic helix. Since in vitro binding studies show that the target of this peptide seems to be DNA, theoretical docking studies were also performed to better understand the interaction between peptide and nucleic acids and also to shed some light on the acetyl group role. Firstly, binding studies showed that affinity contacts basically occur due to electrostatic attraction. The complex peptide‐ssDNA was clearly oriented by residues Ala1, Lys3, and Asp6, which form several hydrogen bonds that are able to stabilize the complex. When acetyl was added, hydrogen bonds are broken, reducing the peptide affinity. In summary, it seems that information here provided could be used to design a novel derivative of this peptide which a clear therapeutic potential. J. Cell. Biochem. 113: 184–193, 2012. © 2011 Wiley Periodicals, Inc. 相似文献
9.
E. V. Navolotskaya V. B. Sadovnikov D. V. Zinchenko V. I. Vladimirov Y. A. Zolotarev 《Russian Journal of Bioorganic Chemistry》2018,44(4):403-407
We prepared 125I-labeled cholera toxin B subunit (125I-labeled CT-B, specific activity 98 Ci/mmol) and found that its binding to rat IEC-6 intestinal epithelial cells was high-affinity (Kd 1.9 nM). The binding of labeled protein was completely inhibited by unlabeled thymosin-α1 (TM-α1), interferon-α2 (IFN-α2), and synthetic peptide LKEKK, which corresponds to residues 16–20 in TM-α1 and 131–135 in IFN-α2 (Ki 1.2, 0.9, and 1.6 nM, respectively), but was not inhibited by synthetic peptide KKEKL with inverted amino acid sequence (Ki > 10 μM). Thus, TM-α1, IFN-α2, and the LKEKK peptide bind with high affinity and specificity to CT-B receptor on rIEC-6 cells. It was found that CT-B and the LKEKK peptide at concentrations of 10–1000 nM increased nitric oxide production and soluble guanylate cyclase activity in the cells in a dose-dependent manner. 相似文献
10.
Background
Ovarian cancer is the most lethal gynecological malignancy, and the ovarian clear cell carcinoma subtype (OCCA) demonstrates a particularly poor response to standard treatment. Improvements in ovarian cancer outcomes, especially for OCCA, could be expected from a clearer understanding of the molecular pathology that might guide strategies for earlier diagnosis and more effective treatment.Methodology/Principal Findings
Cell-SELEX technology was employed to develop new molecular probes for ovarian cancer cell surface markers. A total of thirteen aptamers with Kd''s to ovarian cancer cells in the pico- to nanomolar range were obtained. Preliminary investigation of the targets of these aptamers and their binding characteristics was also performed.Conclusions/Significance
We have selected a series of aptamers that bind to different types of ovarian cancer, but not cervical cancer. Though binding to other cancer cell lines was observed, these aptamers could lead to identification of biomarkers that are related to cancer. 相似文献11.
Fukui M Ueno K Suehiro Y Hamanaka Y Imai K Hinoda Y 《Cancer immunology, immunotherapy : CII》2006,55(5):538-546
Receptor for hyaluronan-mediated motility (RHAMM) is overexpressed in various tumors with high frequency, and was recently identified as an immunogenic antigen by serologic screening of cDNA expression libraries. In this study, we explored whether RHAMM is a potential target for dendritic cell (DC) immunotherapy. We constructed a plasmid for transduction of in vitro-transcribed mRNAs into DCs to efficiently transport the intracellular protein RHAMM into MHC class II compartments by adding a late endosomal/lysosomal sorting signal to the RHAMM gene. Immunization of mice with modified RHAMM mRNA-transfected DCs (DC/RHAMM) induced killing activity against RHAMM-positive tumor cells in splenocytes. To examine whether CD4+ and/or CD8+ T cells were required for this antitumor immunity, an anti-CD4 or anti-CD8 antibody was administered to mice after immunization with DC/RHAMM. Depletion of CD4+ T cells significantly diminished the induction of tumor cell-killing activity in splenocytes, whereas CD8+ T cell depletion had no effect. We then investigated the therapeutic effect of DC/RHAMM in a 3-day tumor model of EL4. DC/RHAMM was administered to mice on days 3, 7 and 10 after EL4 tumor inoculation. The treatment markedly inhibited tumor growth compared to control DCs. Moreover, antibody-mediated depletion of CD4+ T cells completely abrogated the therapeutic effect of DC/RHAMM, whereas depletion of CD8+ T cells had no effect. The results of this preclinical study indicate that DCs transfected with a modified RHAMM mRNA targeted to MHC class II compartments can induce CD4+ T cell-mediated antitumor activity in vivo. 相似文献
12.
Immunogenicity of HLA-A1-restricted peptides derived from S100A4 (metastasin 1) in melanoma patients
Valeska Hofmeister-Mueller Claudia S. Vetter-Kauczok Ramona Ullrich Katharina Meder Eugene Lukanidin Eva-Bettina Broecker Per thor Straten Mads Hald Andersen David Schrama Juergen C. Becker 《Cancer immunology, immunotherapy : CII》2009,58(8):1265-1273
S100A4 (metastasin 1) belongs to the S100 family of Ca2+ binding proteins. While not present in most differentiated adult tissues, S100A4 is upregulated in the micromilieu of tumors.
It is primarily expressed by tumor-associated macrophages, fibroblasts, and tumor endothelial cells. Due to its strong induction
in tumors S100A4 is a promising target for cancer immunotherapy. By reverse immunology, using epitope prediction programs,
we identified 3 HLA-A1-restricted peptide epitopes (S100A4 A1-1, A1-2, and A1-3) which are subject to human T cell responses
as detected in peripheral blood of melanoma patients by means of IFN-γ ELISPOT and cytotoxicity assays. In addition, IFN-γ
responses to S100A4 A1-2 can not only be induced by stimulation of T cells with peptide-loaded DC but also by stimulation
with S100A4 protein-loaded DC, indicating that this epitope is indeed generated by processing of the endogenously expressed
protein. In addition, S100A4 A1-2 reactive T cells demonstrate lysis of HLA-A1+ fibroblasts in comparison to HLA-A1− fibroblasts. In summary, this HLA-A1-restricted peptide epitope is a candidate for immunotherapeutical approaches targeting
S100A4-expressing cells in the tumor stroma. 相似文献
13.
Yu M Henning R Walker A Kim G Perroy A Alessandro R Virador V Kohn EC 《Journal of cellular and molecular medicine》2012,16(10):2369-2378
Recent work identified L‐asparaginase (L‐ASP) as a putative therapeutic target for ovarian cancer. We suggest that L‐ASP, a dysregulator of glycosylation, would interrupt the local microenvironment, affecting the ovarian cancer cell—endothelial cell interaction and thus angiogenesis without cytotoxic effects. Ovarian cancer cell lines and human microvascular endothelial cells (HMVEC) were exposed to L‐ASP at physiologically attainable concentrations and subjected to analyses of endothelial tube formation, invasion, adhesion and the assessment of sialylated proteins involved in matrix‐associated and heterotypic cell adhesion. Marked reduction in HMVEC tube formation in vitro, HMVEC and ovarian cancer cell invasion, and heterotypic cell‐cell and cell‐matrix adhesion was observed (P < 0.05–0.0001). These effects were associated with reduced binding to ß1integrin, activation of FAK, and cell surface sialyl LewisX (sLex) expression. No reduction in HMVEC E‐selectin expression was seen consistent with the unidirectional inhibitory actions observed. L‐ASP concentrations were non‐toxic to either ovarian cancer or HMVEC lines in the time frame of the assays. However, early changes of autophagy were observed in both cell types with induction of ATG12, beclin‐1, and cleavage of LC‐3, indicating cell injury did occur. These data and the known mechanism of action of L‐ASP on glycosylation of nascent proteins suggest that L‐ASP reduces of ovarian cancer dissemination and progression through modification of its microenvironment. The reduction of ovarian cancer cell surface sLex inhibits interaction with HMVEC and thus HMVEC differentiation into tubes, inhibits interaction with the local matrix reducing invasive behaviour, and causes cell injury initiating autophagy in tumour and vascular cells. 相似文献
14.
《Journal of receptor and signal transduction research》2013,33(5):307-317
AbstractSeveral neuropeptides have been shown to regulate the function of cells involved in immune response and inflammation. Neurotensin is a 13 amino acid neuropeptide localized primarily to the nervous system and gut. Neurotensin also stimulates mast cell degranulation and enhances phagocytic and cytolytic capability of macrophages, suggesting that this peptide regulates inflammatory and immune responses. Fibroblasts play an important role in inflammation and tissue healing, and these processes may be regulated by several neuropeptides that have been shown to bind to fibroblasts. However neurotensin receptors have not been identified on fibroblasts. Human embryonic lung fibroblasts (HELF) were examined for binding and biological effects of neurotensin. 125I-neurotensin binding to adherent and confluent human embryonic lung fibroblasts (HELF), plated in 12mm diameter wells was specific and saturable. Computer-assisted resolution of the binding data demonstrated two classes of binding sites: a high affinity, low capacity site (Kd = 1.6×10?11 M, 19.5×107 sites/well), and a low- affinity, high-capacity site (Kd = 10?8 M, 4×109 sites/well). Neurotensin stimulated immediate, transient, dose-dependent increases of cytosolic calcium in HELF (threshold dose: 1011 M), suggesting release of calcium from intracellular stores. The novel finding of neurotensin receptors on fibroblasts provides further support for this neuropeptide's role as a regulator of inflammatory and immune responses. 相似文献
15.
M. ALI OUAISSI J. P. KUSNIERZ H. GRAS-MASSE H. DROBECQ P. VELGE J. CORNETTE A. CAPRON A. TARTAR 《The Journal of eukaryotic microbiology》1988,35(1):111-114
ABSTRACT. The binding of synthetic peptides modeled from the sequence of the cell attachment site of fibronectin to T. cruzi trypomastigote surface receptors was investigated by fluorescence-activated cell-sorting analysis using fluorescein-labeled peptides. Peptides with the sequence Arg-Gly-Asp-Ser bound to the parasite surface. A low percentage of fresh parasites recently liberated from infected fibroblasts had the capacity to bind the peptide. In contrast, these parasites showed a time-dependent several-fold increase in their ability to bind the Arg-Gly-Asp-Ser-containing peptides during extracellular incubation. From these observations, it appears that the expression of surface receptors on a particular, mature stage of the parasite parallels its ability to adhere to and infect host cells. 相似文献
16.
There are multiple stages in the life cycle of Plasmodium that invade host cells. Molecular machinery involved is such host–pathogen interactions constitute excellent drug targets and/or vaccine candidates. A screen using a phage display library has previously demonstrated presence of enolase on the surface of the Plasmodium ookinete. Phage-displayed peptides that bound to the ookinete contained a conserved motif (PWWP) in their sequence. Here, direct binding of these peptides with recombinant Plasmodium falciparum enolase (rPfeno) was investigated. These peptides showed specific binding to rPfeno, but failed to bind to other enolases. Plasmodium spp enolases are distinct in having an insert of five amino acids (104EWGWS108) that is not found in host enolases. The possibility of this insert being the recognition motif for the PWWP containing peptides was examined, (i) by comparing the binding of the peptides with rPfeno and a deletion variant Δ-rPfeno lacking 104EWGWS108, (ii) by measuring the changes in proton chemical shifts of PWWP peptides on binding to different enolases and (iii) by inter-molecular docking experiment to locate the peptide binding site. Results from these studies showed that the pentapeptide insert of Pfeno indeed constitutes the binding site for the PWWP domain containing peptide ligands. Search for sequences homologous to phage displayed peptides among peritrophic matrix proteins resulted in identification of perlecan, laminin, peritrophin and spacran. The possibility of these PWWP domain-containing proteins in the peritrophic matrix of insect gut to interact with ookinete cell surface enolase and facilitate the invasion of mosquito midgut epithelium is discussed. 相似文献
17.
Arvizo RR Giri K Moyano D Miranda OR Madden B McCormick DJ Bhattacharya R Rotello VM Kocher JP Mukherjee P 《PloS one》2012,7(3):e33650
Background
We introduce a promising methodology to identify new therapeutic targets in cancer. Proteins bind to nanoparticles to form a protein corona. We modulate this corona by using surface-engineered nanoparticles, and identify protein composition to provide insight into disease development.Methods/Principal Findings
Using a family of structurally homologous nanoparticles we have investigated the changes in the protein corona around surface-functionalized gold nanoparticles (AuNPs) from normal and malignant ovarian cell lysates. Proteomics analysis using mass spectrometry identified hepatoma-derived growth factor (HDGF) that is found exclusively on positively charged AuNPs (+AuNPs) after incubation with the lysates. We confirmed expression of HDGF in various ovarian cancer cells and validated binding selectivity to +AuNPs by Western blot analysis. Silencing of HDGF by siRNA resulted s inhibition in proliferation of ovarian cancer cells.Conclusion
We investigated the modulation of protein corona around surface-functionalized gold nanoparticles as a promising approach to identify new therapeutic targets. The potential of our method for identifying therapeutic targets was demonstrated through silencing of HDGF by siRNA, which inhibited proliferation of ovarian cancer cells. This integrated proteomics, bioinformatics, and nanotechnology strategy demonstrates that protein corona identification can be used to discover novel therapeutic targets in cancer. 相似文献18.
Chuying Ma Guangfu Yin Danhong Yan Xueling He Li Zhang Yan Wei Zhongbing Huang 《Journal of peptide science》2013,19(12):730-736
Discovery of peptide ligands that can target human ovarian cancer and deliver chemotherapeutics offers new opportunity for cancer therapy. The advent of phage‐displayed peptide library facilitated the screening of such peptides. In vivo screening that set in a microanatomic and functional context was applied in our study, and a novel peptide WSGPGVWGASVK targeting ovarian cancer was isolated. The phage clone PC3‐1 displaying peptide WSGPGVWGASVK can gain effective access to accumulate in the tumor sites after intravenous injection while reducing its accumulation in normal organs. Positive immunostaining of PC3‐1 was located in both sites of tumor cells and tumor blood vessels, which resulted in a diffuse binding pattern through the tumor. In vitro study results confirmed the capability of peptide WSGPGVWGASVK binding to and being internalized by both tumor cells and angiogenic endothelial cells. Flow cytometry analysis revealed that the peptide bound to SKOV3 cells with Kd value of 5.43 ± 0.4 μM. Taken together, it suggested that peptide WSGPGVWGASVK is a lead candidate for delivering therapeutics to penetrate into tumors. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
19.
S E Dana M S Brown J L Goldstein 《Biochemical and biophysical research communications》1977,74(4):1369-1376
125I-Low density lipoprotein (125I-LDL)1 binds tightly to glass beads at physiologic pH and ionic strength. This binding shows saturability, high affinity (half maximal binding achieved at 10–15 μg protein/ml), and specificity (unlabeled LDL but not HDL or albumin competes with 125I-LDL for binding to the glass beads). In contrast to the binding of 125I-LDL to the physiologic LDL receptor on the surface of human fibroblasts and lymphocytes, the binding of 125I-LDL to bind to inert substances such as glass must be considered in the interpretation of studies in which 125I-LDL binding to membrane receptors is measured. The data emphasize the importance of correlating observed 125I-LDL binding with a physiologic action of the lipoprotein. 相似文献
20.
Catarina E. Hioe Denise M. McKinney Jeffrey A. Frelinger Minnie McMillan 《Immunogenetics》1994,40(3):222-229
In order to investigate the role of residues inside and outside the peptide binding cleft of the L2 molecule in peptide presentation to cytotoxic T lymphocytes (CTL), we constructed a series of point mutations in the L
d
gene. We determined the effects of the mutations in the Ld molecule on the binding and recognition of an Ld-restricted CTL epitope derived from the nucleoprotein (NP) of the lymphocytic phoriomeningitis virus (LCMV). Each of the mutations within the Ld peptide binding cleft resulted in a complete loss of CTL recognition. Addition of the LCMV NP peptide to cells expressing these mutants did not increase surface Ld expression, suggesting that the mutations altered peptide binding. Mutations involving pockets D and E within the cleft affected LCMV peptide binding and recognition as drastically as those in pocket B, which was predicted to interact with a main anchor residue of the peptide. In striking contrast, the mutations located outside the cleft did not change either recognition or binding. These results demonstrate that the Ld residues in the peptide binding cleft are the main determinants dictating LCMV NP peptide binding, and that the residues in each of the pockets within the cleft play a role in this interaction. Surprisingly, one mutation outside the peptide binding cleft, T92S, abrogated CTL lysis of target cells treated with the LCMV NP peptide, but not virus-infected cells. These data show that this mutation selectively altered the presentation of the LCMV NP peptide introduced to the cell exogenously, but not endogenously. This implies that the pathway by which peptides associate with class I molecules within the cell differs from that of exogenous peptide binding. 相似文献