| 1. |
- Belting, Mattias, et al.
(författare)
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Glypican-1 is a vehicle for polyamine uptake in mammalian cells. A pivotal role for nitrosothiol-derived nitric oxide.
- 2003
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 278:47, s. 47181-47189
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Tidskriftsartikel (refereegranskat)abstract
- Polyamines (putrescine, spermidine, and spermine) are essential for growth and survival of all cells. When polyamine biosynthesis is inhibited, there is up-regulation of import. The mammalian polyamine transport system is unknown. We have previously shown that the heparan sulfate (HS) side chains of recycling glypican-1 (Gpc-1) can sequester spermine, that intracellular polyamine depletion increases the number of NO-sensitive N-unsubstituted glucosamines in HS, and that NO-dependent cleavage of HS at these sites is required for spermine uptake. The NO is derived from S-nitroso groups in the Gpc-1 protein. Using RNA interference technology as well as biochemical and microscopic techniques applied to both normal and uptake-deficient cells, we demonstrate that inhibition of Gpc-1 expression abrogates spermine uptake and intracellular delivery. In unperturbed cells, spermine and recycling Gpc-1 carrying HS chains rich in N-unsubstituted glucosamines were co-localized. By exposing cells to ascorbate, we induced release of NO from the S-nitroso groups, resulting in HS degradation and unloading of the sequestered polyamines as well as nuclear targeting of the deglycanated Gpc-1 protein. Polyamine uptake-deficient cells appear to have a defect in the NO release mechanism. We have managed to restore spermine uptake partially in these cells by providing spermine NONOate and ascorbate. The former bound to the HS chains of recycling Gpc-1 and S-nitrosylated the core protein. Ascorbate released NO, which degraded HS and liberated the bound spermine. Recycling HS proteoglycans of the glypican-type may be plasma membrane carriers for cargo taken up by caveolar endocytosis.
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| 2. |
- Belting, Mattias, et al.
(författare)
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Nuclear delivery of macromolecules: barriers and carriers.
- 2005
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Ingår i: Advanced Drug Delivery Reviews. - : Elsevier BV. - 0169-409X. ; 57:4, s. 505-527
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Forskningsöversikt (refereegranskat)abstract
- Recent evidence for efficient delivery of macromolecules, such as peptides and nucleic acids, from the cell exterior to the nucleus offers the interesting possibility of developing novel treatments directed at intranuclear targets. The findings should also stimulate the search for physiological ligands that utilize similar transport mechanisms to regulate pathobiological processes. Cytokines, growth factors and their receptors, as well as morphogens have all been shown to enter the nucleus to evoke biological responses in target cells. The rational design of intracellular drug delivery vehicles requires an increased understanding of the elaborate systems that mediate cellular communication and coordination with the extracellular environment without inflicting on the integrity of the cell. This review discusses some aspects of the carriers and barriers in macromolecular transport.
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| 3. |
- Belting, Mattias, et al.
(författare)
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Proteoglycans as endocytosis receptors for CPPs
- 2007
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Ingår i: Handbook of Cell-Penetrating Peptides, Second Edition. - 9780849350900 - 0849350905 ; , s. 219-219
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 4. |
- Belting, Mattias, et al.
(författare)
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Regulation of angiogenesis by tissue factor cytoplasmic domain signaling
- 2004
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Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1546-170X .- 1078-8956. ; 10:5, s. 502-509
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Tidskriftsartikel (refereegranskat)abstract
- Hemostasis initiates angiogenesis-dependent wound healing, and thrombosis is frequently associated with advanced cancer. Although activation of coagulation generates potent regulators of angiogenesis, little is known about how this pathway supports angiogenesis in vivo. Here we show that the tissue factor (TF)-VIIa protease complex, independent of triggering coagulation, can promote tumor and developmental angiogenesis through protease-activated receptor-2 (PAR-2) signaling. In this context, the TF cytoplasmic domain negatively regulates PAR-2 signaling. Mice from which the TF cytoplasmic domain has been deleted (TFDeltaCT mice) show enhanced PAR-2-dependent angiogenesis, in synergy with platelet-derived growth factor BB (PDGF-BB). Ocular tissue from diabetic patients shows PAR-2 colocalization with phosphorylated TF specifically on neovasculature, suggesting that phosphorylation of the TF cytoplasmic domain releases its negative regulatory control of PAR-2 signaling in angiogenesis. Targeting the TF-VIIa signaling pathway may thus enhance the efficacy of angiostatic treatments for cancer and neovascular eye diseases.
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| 5. |
- Ding, Kan, et al.
(författare)
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Modulations of glypican-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sites
- 2001
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:50, s. 46779-46791
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Tidskriftsartikel (refereegranskat)abstract
- Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Persson, S., and Fransson, L.-A. (1999) Biochem. J. 338, 317-323; Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2001) Proc. Natl. Acad. Sci. U. S. A., in press). Here, we have analyzed the effect of polyamine deprivation on the structure and polyamine affinity of the heparan sulfate chains in various glypican-1 glycoforms synthesized by a transformed cell line (ECV 304). Heparan sulfate chains of glypican-1 were either cleaved with heparanase at sites embracing the highly modified regions or with nitrite at N-unsubstituted glucosamine residues. The products were separated and further degraded by heparin lyase to identify sulfated iduronic acid. Polyamine affinity was assessed by chromatography on agarose substituted with the polyamine spermine. In heparan sulfate made by cells with undisturbed endogenous polyamine synthesis, free amino groups were restricted to the unmodified, unsulfated segments, especially near the core protein. Spermine high affinity binding sites were located to the modified and highly sulfated segments that were released by heparanase. In cells with up-regulated polyamine uptake, heparan sulfate contained an increased number of clustered N-unsubstituted glucosamines and sulfated iduronic acid residues. This resulted in a greater number of NO/nitrite-sensitive cleavage sites near the potential spermine-binding sites. Endogenous degradation by heparanase and NO-derived nitrite in polyamine-deprived cells generated a separate pool of heparan sulfate oligosaccharides with an exceptionally high affinity for spermine. Spermine uptake in polyamine-deprived cells was reduced when NO/nitrite-generated degradation of heparan sulfate was inhibited. The results suggest a functional interplay between glypican recycling, NO/nitrite-generated heparan sulfate degradation, and polyamine uptake.
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| 6. |
- Ding, Kan, et al.
(författare)
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N-unsubstituted glucosamine in heparan sulfate of recycling glypican-1 from suramin-treated and nitrite-deprived endothelial cells. mapping of nitric oxide/nitrite-susceptible glucosamine residues to clustered sites near the core protein
- 2001
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:6, s. 3885-3894
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Tidskriftsartikel (refereegranskat)abstract
- We have analyzed the content of N-unsubstituted glucosamine in heparan sulfate from glypican-1 synthesized by endothelial cells during inhibition of (a) intracellular progression by brefeldin A, (b) heparan sulfate degradation by suramin, and/or (c) endogenous nitrite formation. Glypican-1 from brefeldin A-treated cells carried heparan sulfate chains that were extensively degraded by nitrous acid at pH 3.9, indicating the presence of glucosamines with free amino groups. Chains with such residues were rare in glypican-1 isolated from unperturbed cells and from cells treated with suramin and, surprisingly, when nitrite-deprived. However, when nitrite-deprived cells were simultaneously treated with suramin, such glucosamine residues were more prevalent. To locate these residues, chains were first cleaved at linkages to sulfated l-iduronic acid by heparin lyase and released fragments were separated from core protein carrying heparan sulfate stubs. These stubs were then cleaved off at sites linking N-substituted glucosamines to d-glucuronic acid. These fragments were extensively degraded by nitrous acid at pH 3.9. When purified proteoglycan isolated from brefeldin A-treated cells was incubated with intact cells, endoheparanase-catalyzed degradation generated a core protein with heparan sulfate stubs that were similarly sensitive to nitrous acid. We conclude that there is a concentration of N-unsubstituted glucosamines to the reducing side of the endoheparanase cleavage site in the transition region between unmodified and modified chain segments near the linkage region to the protein. Both sites as well as the heparin lyase-sensitive sites seem to be in close proximity to one another.
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| 7. |
- Fransson, Lars-Åke, et al.
(författare)
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Novel aspects of glypican glycobiology.
- 2004
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Ingår i: Cellular and Molecular Life Sciences. - : Springer Science and Business Media LLC. - 1420-9071 .- 1420-682X. ; 61:9, s. 1016-1024
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Forskningsöversikt (refereegranskat)abstract
- Mutations in glypican genes cause dysmorphic and overgrowth syndromes in men and mice, abnormal development in flies and worms, and defective gastrulation in zebrafish and ascidians. All glypican core proteins share a characteristic pattern of 14 conserved cysteine residues. Upstream from the C-terminal membrane anchorage are 3–4 heparan sulfate attachment sites. Cysteines in glypican-1 can become nitrosylated by nitric oxide in a copper-dependent reaction. When glypican-1 is exposed to ascorbate, nitric oxide is released and participates in deaminative cleavage of heparan sulfate at sites where the glucosamines have a free amino group. This process takes place while glypican-1 recycles via a nonclassical, caveolin-1-associated route. Glypicans are involved in growth factor signalling and transport, e.g. of polyamines. Cargo can be unloaded from heparan sulfate by nitric oxide-dependent degradation. How glypican and its degradation products and the cargo exit from the recycling route is an enigma.
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| 8. |
- Magzoub, Mazin, et al.
(författare)
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N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis
- 2006
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 348:2, s. 379-385
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Tidskriftsartikel (refereegranskat)abstract
- A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1–24) and a basic domain (KKRPKP, residues 25–30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp–DNA–gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide’s ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases.
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| 9. |
- Mani, Katrin, et al.
(författare)
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HIV-Tat protein transduction domain specifically attenuates growth of polyamine deprived tumor cells.
- 2007
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Ingår i: Molecular Cancer Therapeutics. - 1538-8514. ; 6:2, s. 782-788
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Tidskriftsartikel (refereegranskat)abstract
- Polyamines are essential for tumor cell growth, and the polyamine pathway represents an attractive target for cancer treatment. Several polyamine transport proteins have been cloned and characterized in bacteria and yeast cells; however, the mechanism of polyamine entry into mammalian cells remains poorly defined, although a role for proteoglycans has been suggested. Here, we show that the HIV-Tat transduction peptide, which is known to enter cells via a proteoglycan-dependent pathway, efficiently inhibits polyamine uptake. Polyamine uptake–deficient mutant cells with intact proteoglycan biosynthesis (CHO MGBG) displayed unperturbed HIV-Tat uptake activity compared with wild-type cells, supporting the notion that HIV-Tat peptide interferes with polyamine uptake via competition for proteoglycan binding sites rather than a putative downstream transporter. HIV-Tat specifically inhibited growth of human carcinoma cells made dependent on extracellular polyamines by treatment with the polyamine biosynthesis inhibitor {alpha}-difluoromethylornithine; accordingly, the Tat peptide prevented intracellular accumulation of exogenous polyamines. Moreover, combined treatment with {alpha}-difluoromethylornithine and HIV-Tat efficiently blocked tumor growth in an experimental mouse model. We conclude that HIV-Tat transduction domain and polyamines enter cells through a common pathway, which can be used to target polyamine-dependent tumor growth in the treatment of cancer.
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| 10. |
- Mani, Katrin, et al.
(författare)
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Tumor attenuation by 2(6-hydroxynaphthyl)-{beta}-D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products.
- 2004
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 14:5, s. 387-397
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Tidskriftsartikel (refereegranskat)abstract
- We have previously reported that the heparan sulfate-priming glycoside 2-(6-hydroxynaphthyl)-ß-D-xylopyranoside selectively inhibits growth of transformed or tumor-derived cells. To investigate the specificity of this xyloside various analogs were synthesized and tested in vitro. Selective growth inhibition was dependent on the presence of a free 6-hydroxyl in the aglycon. Because cells deficient in heparan sulfate synthesis were insensitive to the xyloside, we conclude that priming of heparan sulfate synthesis was required for growth inhibition. In growth-inhibited cells, heparan sulfate chains primed by the active xyloside were degraded to products that contained anhydromannose and appeared in the nuclei. Hence the degradation products were generated by nitric oxide–dependent cleavage. Accordingly, nitric oxide depletion reduced nuclear localization of the degradation products and counteracted the growth-inhibitory effect of the xyloside. We propose that 2-(6-hydroxynaphthyl)-ß-D-xylopyranoside entered cells and primed synthesis of heparan sulfate chains that were subsequently degraded by nitric oxide into products that accumulated in the nucleus. In vivo experiments demonstrated that the xyloside administered subcutaneously, perorally, or intraperitoneally was adsorbed and made available to tumor cells located subcutaneously. Treatment with the xyloside reduced the average tumor load by 70–97% in SCID mice. The present xyloside may serve as a lead compound for the development of novel antitumor strategies.
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