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1.
Cell-penetrating peptides can deliver macromolecular cargo into cells and show promise as vectors for intracellular drug delivery. Internalization occurs predominantly via endocytosis, but the exact uptake mechanisms are not fully understood. We show quantitatively how penetratin, a 16-residue cationic peptide, stimulates fluid-phase endocytosis and triggers its own uptake into Chinese hamster ovarian cells, using a 70 kDa dextran to indicate macropinocytosis. The total cellular endocytotic rate is significantly less affected and we therefore propose up-regulation of macropinocytosis to occur at the expense of other types of endocytosis. By comparing penetratin to its analogs PenArg and PenLys, enriched in arginines and lysines, respectively, we show how these side-chains contribute to uptake efficiency. The degree of peptide and dextran uptake follows similar patterns regarding peptide concentration and arginine/lysine content (PenArg > penetratin > PenLys), indicating that a high content of arginines is beneficial but not necessary for stimulating endocytosis.  相似文献   

2.
Arginine-rich peptides, including octaarginine (R8), HIV-1 Tat, and branched-chain arginine-rich peptides, belong to one of the major classes of cell-permeable peptides which deliver various proteins and macromolecules to cells. The importance of the endocytic pathways has recently been demonstrated in the cellular uptake of these peptides. We have previously shown that macropinocytosis is one of the major pathways for cellular uptake and that organization of the F-actin accompanies this process. In this study, using proteoglycan-deficient CHO cells, we have demonstrated that the membrane-associated proteoglycans are indispensable for the induction of the actin organization and the macropinocytic uptake of the arginine-rich peptides. We have also demonstrated that the cellular uptake of the Tat peptide is highly dependent on heparan sulfate proteoglycan (HSPG), whereas the R8 peptide uptake is less dependent on HSPG. This suggests that the structure of the peptides may determine the specificity for HSPG, and that HSPG is not the sole receptor for macropinocytosis. Comparison of the HSPG specificity of the branched-chain arginine-rich peptides in cellular uptake has suggested that the charge density of the peptides may determine the specificity. The activation of the Rac protein and organization of the actin were observed within a few minutes after the peptide treatment. These data strongly suggest the possibility that the interaction of the arginine-rich peptides with the membrane-associated proteoglycans quickly activates the intracellular signals and induces actin organization and macropinocytotis.  相似文献   

3.
Non-invasive detection of prostate cancer or metastases still remains a challenge in the field of molecular imaging. In our recent work of screening arginine- or lysine-rich peptides for intracellular delivery of a therapeutic agent into prostate cancer cells, an arginine-rich cell permeable peptide (NH2GR11) was found with an unexpectedly preferential uptake in prostate cancer cell lines. The goal of this work was to develop this peptide as a positron emission tomography (PET) imaging probe for specific detection of distant prostate cancer metastases. The optimal length of arginine-rich peptides was evaluated by the cell uptake efficiency of three fluorescein isothiocyanate (FITC)-tagged oligoarginines (NHGR9, NHGR11, and NHGR13) in four human prostate cell lines (LNCaP, PZ-HPV-7, DU145, and PC3). Of the three oligoarginines, NH2GR11 showed the highest cell uptake and internalization efficiency with its subcellular localization in cytosol. The biodistribution of FITC-NHGR9, FITC-NHGR11, and FITC-NHGR13 performed in control nude mice displayed the unique preferential accumulation of FITC-NHGR11 in the prostate tissue. Further in vivo evaluation of FITC-NHGR11 in PC3 tumor-bearing nude mice revealed elevated uptake of this peptide in tumors as compared to other organs. In vivo pharmacokinetics evaluated with 64Cu-labeled NH2GR11 showed that the peptide was rapidly cleared from the blood (t 1/2 = 10.7 min) and its elimination half-life was 17.2 h. The PET imaging specificity of 64Cu-labled NH2GR11 was demonstrated for the detection of prostate cancer in a comparative imaging experiment using two different human cancer xenograft models.  相似文献   

4.
A laminin-binding peptide (peptide G), predicted from the cDNA sequence for a 33-kDa protein related to the 67-kDa laminin receptor, specifically inhibits binding of laminin to heparin and sulfatide. Since the peptide binds directly to heparin and inhibits interaction of another heparin-binding protein with the same sulfated ligands, this inhibition is due to direct competition for binding to sulfated glycoconjugates rather than an indirect effect of interaction with the binding site on laminin for the 67-kDa receptor. Direct binding of laminin to the peptide is also inhibited by heparin. This interaction may result from contamination of the laminin with heparan sulfate, as binding is enhanced by the addition of substoichiometric amounts of heparin but inhibited by excess heparin and two heparin-binding proteins. Furthermore, laminin binds more avidly to a heparin-binding peptide derived from thrombospondin than to the putative receptor peptide. Adhesion of A2058 melanoma cells on immobilized peptide G is also heparin-dependent, whereas adhesion of the cells on laminin is not. Antibodies to the beta 1-integrin chain or laminin block adhesion of the melanoma cells to laminin but not to peptide G. Thus, the reported inhibition of melanoma cell adhesion to endothelial cells by peptide G may result from inhibition of binding of laminin or other proteins to sulfated glycoconjugate receptors rather than from specific inhibition of laminin binding to the 67-kDa receptor.  相似文献   

5.
Mouse blastocysts in serum-free culture for 24-48 h become attachment-competent, adhere to fibronectin- or laminin-coated surfaces, and subsequently form trophoblast outgrowths. The blastocyst laminin receptor was characterized in outgrowth studies using modified laminin. Trophoblast cells interacted with the peptide portion of laminin, but not the oligosaccharide moiety since its adhesive activity was reduced by boiling or trypsin treatment, but not by treatments that removed or modified its carbohydrate. Laminin outgrowth-promoting activity was further localized within its structural domains by use of the well-characterized proteolytic fragments of laminin, E1-4, and E8, and a synthetic peptide, CDPGYIGSR. The E1-4 fragment of laminin did not promote embryo outgrowth. However, the E8 fragment, which contains a heparin-binding domain as well as sites recognized during cell adhesion and neurite outgrowth, vigorously promoted outgrowth in both the presence and absence of heparin, heparan sulfate, or heparinase. Consistent with these results, outgrowth on intact laminin was not inhibited by CDPGYIGSR, a sequence within the E1-4 fragment that is known to mediate the adhesion of some cell types. It is concluded from these results that early trophoblast cells adhere to peptide in the E8 domain of laminin using a mechanism that is independent of the one used for adhesion to fibronectin.  相似文献   

6.
The mechanism of the arginine-rich peptide-mediated cellular uptake is currently a controversial issue. Several factors, including the type of peptide, the nature of the cargo, and the linker between them, appear to affect uptake. One of the less studied factors, which may affect the uptake mechanism, is the effect of peptide density on the surface of the cargo. Here, we examined the mechanism of cellular uptake and intracellular trafficking of liposomes modified with different densities of the octaarginine (R8) peptide. Liposomes modified with a low R8 density were taken up mainly through clathrin-mediated endocytosis, leading to extensive lysosomal degradation, whereas those modified with a high R8 density were taken up mainly through macropinocytosis and were less subject to lysosomal degradation. Furthermore, the high density R8-liposomes were able to stimulate the macropinocytosis-mediated uptake of other particles. When plasmid DNA was condensed and encapsulated in R8-liposomes, the levels of gene expression were three orders of magnitude higher for the high density liposomes. The enhanced gene expression by the high density R8-liposomes was highly impaired by blocking uptake through macropinocytosis. The different extents of gene expression from different densities of the R8 peptide on the liposomes could be explained principally by the existence of an intracellular trafficking route, but not by the uptake amount, of internalized liposomes. These results show that the density of the R8 peptide on liposomes determines the uptake mechanism and that this is directly linked to intracellular trafficking, resulting in different levels of gene expression.  相似文献   

7.
PURPOSE: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. RESULTS: [(14)C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145 binding but unexpectedly not to its uptake. CONCLUSIONS: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.  相似文献   

8.
To study the contribution of T‐cell receptors (TCR) to resulting T‐cell responses, we studied three different human αβ TCRs, reactive to the same gp100‐derived peptide presented in the context of HLA‐A*0201. When expressed in primary CD8 T cells, all receptors elicited classic antigen‐induced IFN‐γ responses, which correlated with TCR affinity for peptide–MHC in the order T4H2 > R6C12 > SILv44. However, SILv44 elicited superior IL‐17A release. Importantly, in vivo, SILv44‐transgenic T cells mediated superior antitumor responses to 888‐A2 + human melanoma tumor cells upon adoptive transfer into tumor‐challenged mice while maintaining IL‐17 expression. Modeling of the TCR ternary complexes suggested architectural differences between SILv44 and the other complexes, providing a potential structural basis for the observed differences. Overall, the data reveal a more prominent role for the T‐cell receptor in defining host T‐cell physiology than traditionally assumed, while parameters beyond IFN‐γ secretion and TCR affinity ultimately determine the reactivity of tumor‐reactive T cells.  相似文献   

9.
Peptide 11, CDPGYIGSR-NH2, is a segment of laminin which blocks tumor cell invasion. A high affinity laminin receptor in tumor cells is thought to be blocked by the carboxyl-terminal YIGSR, and conformational energy calculations suggest that the glycine in YIGSR allows an important conformational bend. We replaced the YIGSR glycine residue in peptide 11 with either D-alanine or L-alanine to allow or disfavor the proposed glycine bend. We found the Gly7-->D-Ala7 analog to be equal to peptide 11 in inhibiting tumor cell invasion of basement membrane matrix. The Gly7-->L-Ala7 analog was much less capable of invasion inhibition. Two-dimensional 1H-1H NMR was used to study the solution conformations of the peptide 11 analogs. NOESY experiments revealed close NH-NH contacts in peptide 11 and the D-Ala7 analog, but not in the L-Ala7 analog. Molecular dynamics generated low energy structures with excellent NOE agreement for peptide 11 and its analogs. Both peptide 11 and the D-Ala7 analog, but not the less active L-Ala7 analog, were predicted to have similar bends around Gly7 or D-Ala7. These results suggest that a bend in the YIGSR region of peptide 11 may be important for the binding of laminin to its metastasis-associated receptor.  相似文献   

10.
Therapeutic vaccination with dendritic cells (DC) is an emerging investigational therapy for eradication of minimal residual disease in acute myeloid leukemia. Various strategies are being explored in manufacturing DC vaccines ex vivo, e.g., monocyte-derived DC (MoDC) loaded with leukemia-associated antigens (LAA). However, the optimal source of LAA and the choice of DC-activating stimuli are still not well defined. Here, loading with leukemic cell preparations (harboring both unknown and known LAA) was explored in combination with a DC maturation-inducing cytokine cocktail (CC; IL-1β, IL-6, TNF-α, and PGE2) and Toll-like receptor ligands (TLR-L) to optimize uptake. Since heat shock induced apoptotic blasts were more efficiently taken up than lysates, we focused on uptake of apoptotic leukemic cells. Uptake of apoptotic blast was further enhanced by the TLR7/8-L R848 (20–30%); in contrast, CC-induced maturation inhibited uptake. CC, and to a lesser extent R848, enhanced the ability of MoDC to migrate and stimulate T cells. Furthermore, class II-associated invariant chain peptide expression was down-modulated after R848- or CC-induced maturation, indicating enhanced processing and presentation of antigenic peptides. To improve both uptake and maturation, leukemic cells and MoDC were co-incubated with R848 for 24 h followed by addition of CC. However, this approach interfered with CC-mediated MoDC maturation as indicated by diminished migratory and T cell stimulatory capacity, and the absence of IL-12 production. Taken together, our data demonstrate that even though R848 improved uptake of apoptotic leukemic cells, the sequential use of R848 and CC is counter-indicated due to its adverse effects on MoDC maturation.  相似文献   

11.
Endocytosis mediates the uptake of extracellular proteins, micronutrients and transmembrane cell surface proteins. Importantly, many viruses, toxins and bacteria hijack endocytosis to infect cells. The canonical pathway is clathrin-mediated endocytosis (CME) and is active in all eukaryotic cells to support critical house-keeping functions. Unconventional mechanisms of endocytosis exit in parallel of CME, to internalize specific cargoes and support various cellular functions. These clathrin-independent endocytic (CIE) routes use three distinct mechanisms: acute signaling-induced membrane remodeling drives macropinocytosis, activity-dependent bulk endocytosis (ADBE), massive endocytosis (MEND) and EGFR non-clathrin endocytosis (EGFR-NCE). Cargo capture and local membrane deformation by cytosolic proteins is used by fast endophilin-mediated endocytosis (FEME), IL-2Rβ endocytosis and ultrafast endocytosis at synapses. Finally, the formation of endocytic pits by clustering of extracellular lipids or cargoes according to the Glycolipid-Lectin (GL-Lect) hypothesis mediates the uptake of SV40 virus, Shiga and cholera toxins, and galectin-clustered receptors by the CLIC/GEEC and the endophilin-A3-mediated CIE.  相似文献   

12.
Several purified glycoproteins including laminin, fetuin, and human chorionic gonadotropin promote dose-dependent and saturable adhesion of Mycoplasma pneumoniae when adsorbed on plastic. Adhesion to the proteins is energy dependent as no attachment occurs in media without glucose. Adhesion to all of the proteins requires sialic acid, and only those proteins with alpha 2-3-linked sialic acid are active. The alpha-subunit of human chorionic gonadotropin also promotes attachment, suggesting that a simple biantennary asparagine-linked oligosaccharide is sufficient for binding. Soluble laminin, asparagine-linked sialyloligosaccharides from fetuin, and 3'-sialyllactose but not 6'-sialyllactose inhibit attachment of M. pneumoniae to laminin. M. pneumoniae also bind to sulfatide adsorbed on plastic. Dextran sulfate, which inhibits M. pneumoniae binding to sulfatide, does not inhibit attachment on laminin, and 3'-sialyllactose does not inhibit binding to sulfatide, suggesting that two distinct receptor specificities mediate binding to these two carbohydrate receptors. Both 3'-sialyllactose and dextran sulfate partially inhibit M. pneumoniae adhesion to a human colon adenocarcinoma cell line (WiDr) at concentrations that completely inhibit binding to laminin or sulfatide, respectively, and in combination they inhibit binding of M. pneumoniae to these cells by 90%. Thus, both receptor specificities contribute to M. pneumoniae adhesion to cultured human cells.  相似文献   

13.
Endothelin (ET)-1 is an important peptide in cancer progression stimulating cellular proliferation, tumor angiogenesis and metastasis. ET-1 binds with high affinity to the ETA receptor (R) and ETBR on cancer cells. High levels of tumor ET-1 and ETAR are associated with poor survival of lung cancer patients. Here the effects of ET-1 on epidermal growth factor (EGF)R and HER2 transactivation were investigated using non-small cell lung cancer (NSCLC) cells. ETAR mRNA was present in all 10 NSCLC cell lines examined. Addition of ET-1 to NCI-H838 or H1975 cells increased EGFR, HER2 and ERK tyrosine phosphorylation within 2 min. The increase in EGFR and HER2 transactivation caused by ET-1 addition to NSCLC cells was inhibited by lapatinib (EGFR and HER2 tyrosine kinase inhibitor (TKI)), gefitinib (EGFR TKI), ZD4054 or BQ-123 (ETAR antagonist), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or Tiron (superoxide scavenger). ET-1 addition to NSCLC cells increased cytosolic Ca2+ and reactive oxygen species. ET-1 increased NSCLC clonal growth, whereas BQ123, ZD4054, lapatinib or gefitinib inhibited proliferation. The results indicate that ET-1 may regulate NSCLC cellular proliferation in an EGFR- and HER2-dependent manner.  相似文献   

14.
A critical unmet need exists for methods to quantitatively measure endogenous pancreatic β-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity to islet β-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5), neuropeptide Y-2 receptor (NPY-2R), and glucagon-like peptide 1 receptor (GLP-1R). In vitro specificity ratios gave the following receptor rank order: PK-1R > GLP-1R > NPY-2R > mGluR5. The biodistribution rank order of selectivity to the pancreas was found to be PK-1R > VMAT2 ∼ GLP-1R > mGluR5. Favorable islet selectivity and biodistribution characteristics suggest several GPCRs as potential targets for PET imaging of pancreatic BCM.  相似文献   

15.
16.
Erythrocytes l-arginine uptake is conveyed by y+ and y+L membrane transport systems. Pre-incubation with N-ethylmaleimide for 10 min at 37°C inhibits the y+ system. The aim of this study was to determine the ideal pre-incubation temperature in evaluating y+ and y+L systems. Cells were pre-incubated with or without N-ethylmaleimide for 10 min at 4°C and 37°C. l-Arginine uptake was quantified by radioisotope and standard erythrocytes membrane flux methodology. Results demonstrate that erythrocytes l-arginine content is depleted by pre-incubation at 37°C for 10 min, thus changing the V max measurement. The inhibitory effect of N-ethylmaleimide pre-incubation was temperature independent and already complete after 1 min of incubation. No significant difference in kinetic parameters was detected between cells pre-incubated at 37°C or 4°C, under zero-trans conditions. In conclusion, we suggest that measurement of erythrocytes l-arginine uptake by y+ and y+L systems could be carried out without N-ethylmaleimide pre-incubation at 37°C.  相似文献   

17.
Functional domains of the 67-kDa laminin receptor precursor   总被引:9,自引:0,他引:9  
We report the characterization of two functional domains of the metastasis-associated 67-kDa laminin receptor (67-LR). Using synthetic peptides deduced from the cDNA sequence of the 37-kDa precursor of the laminin receptor (37-LRP) as well as their corresponding affinity-purified polyclonal antibodies, we identified a unique laminin binding site as well as a membrane-associated domain of the receptor. In laminin dot blot and solid phase radioligand assays, a 20 amino acid synthetic peptide (IPCNNKGAHSVGLMWWMLAR, amino acid residues 161-180, designated peptide G) specifically bound to laminin with high affinity (Kd = 5 x 10(-8) M). Peptide G also specifically eluted the 67-LR from a laminin affinity column. Peptide G and laminin reacted with a 1:1 stoichiometry, suggesting that there is one recognition site on laminin for the peptide G domain. Immunofluorescence studies, performed on permeabilized and nonpermeabilized human A2058 melanoma cells using 10 different affinity-purified antibodies to distinct regions of the 37-LRP, identified an unusually short membrane-associated domain that was consistent with a computer predicted transmembrane domain (residues 86-101). Our data demonstrate for the first time that the 37-LRP has two functional domains consistent with the characteristics of the mature 67-LR. Furthermore, we propose peptide G as a potential inhibitor of tumor cell interactions with laminin.  相似文献   

18.
19.
Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. We describe AGE uptake in LLC-PK1 and HK2 proximal tubule cell lines by macropinocytosis, a non-specific, endocytic mechanism. AGE–BSA induced dorsal circular actin ruffles and amiloride-sensitive dextran–TRITC uptake, significantly increased AGE–BSA–FITC uptake (167 ± 20% vs BSA control, p < 0.01) and was ezrin-dependent. AGE–BSA–FITC uptake was significantly inhibited by amiloride and inhibitors of Arf6, Rac1, racGEF Tiam1, PAK1 and actin polymerisation. AGE–BSA–FITC, Arf6 and PIP2 co-localised within dorsal circular actin ruffles. AGE–BSA increased PAK1 kinase activity (212 ± 41% vs control, p < 0.05) and protein levels of Tiam1, a Rac1 activator. AGE–BSA significantly increased TGF-β1 protein levels (160 ± 6%, p < 0.001 vs BSA), which were significantly inhibited by inhibitors of Arf6 (82 ± 19%, p < 0.001 vs AGE) and actin polymerisation (107 ± 11%, p < 0.001 vs AGE), suggesting AGEs partially exert their profibrotic effects via macropinocytosis. PAK1 and PIP5Kγ siRNA significantly decreased AGE–BSA–FITC uptake (81 ± 6% and 64 ± 7%, respectively, p < 0.05 vs control for both), and AGE-stimulated TGF-β1 protein release (99 ± 15% and 49 ± 8% of control, p < 0.05 and p < 0.001, respectively). Inhibition of AGE uptake by macropinocytosis inhibitors and a neutralising TGF-β antibody, reversed the AGE-induced decrease in surface Na+K+ATPase, suggesting AGE uptake by macropinocytosis may contribute to diabetic kidney fibrosis and/or EMT by modulating this pump. Understanding methods of cellular uptake and signalling by AGEs may lead to novel therapies for diabetic nephropathy.  相似文献   

20.
Aptamers represent an emerging strategy to deliver cargo molecules, including dyes, drugs, proteins or even genes, into specific target cells. Upon binding to specific cell surface receptors aptamers can be internalized, for example by macropinocytosis or receptor mediated endocytosis. Here we report the in vitro selection and characterization of RNA aptamers with high affinity (Kd = 20 nM) and specificity for the human IL-6 receptor (IL-6R). Importantly, these aptamers trigger uptake without compromising the interaction of IL-6R with its natural ligands the cytokine IL-6 and glycoprotein 130 (gp130). We further optimized the aptamers to obtain a shortened, only 19-nt RNA oligonucleotide retaining all necessary characteristics for high affinity and selective recognition of IL-6R on cell surfaces. Upon incubation with IL-6R presenting cells this aptamer was rapidly internalized. Importantly, we could use our aptamer, to deliver bulky cargos, exemplified by fluorescently labeled streptavidin, into IL-6R presenting cells, thereby setting the stage for an aptamer-mediated escort of drug molecules to diseased cell populations or tissues.  相似文献   

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