RESUMEN
The mechanisms behind target vs. host cell recognition of the human antimicrobial peptide LL-37 remain ill-defined. Here, we have investigated the membrane disruption capacity of LL-37 using large unilamellar vesicles (LUVs) composed of varying mixtures of POPC, POPG and cholesterol to mimic target and host membranes respectively. We show that LL-37 is unable to induce leakage of entrapped calcein from zwitterionic POPC LUVs, whereas leakage from LUVs partially composed of POPG is fast and efficient. In accordance with typical antimicrobial peptide behavior, cholesterol diminished LL-37 induced leakage. By using linear dichroism and flow oriented LUVs, we found that LL-37 orients with the axis of its induced α-helix parallel to the membrane surface in POPC:POPG (7:3) LUVs. In the same system, we also observed a time-dependent increase of the parallel α-helix LD signal on timescales corresponding to the leakage kinetics. The increased LD may be connected to a peptide translocation step, giving rise to mass balance across the membrane. This could end the leakage process before it is complete, similar to what we have observed. Confocal microscopy studies of eukaryotic cells show that LL-37 is able to mediate the cell delivery of non-covalently linked fluorescent oligonucleotides, in agreement with earlier studies on delivery of plasmid DNA (Sandgren et al., J. Biol. Chem. 279 (2004) 17951). These observations highlight the potential dual functions of LL-37 as an antimicrobial agent against bacterial target cells and a cell-penetrating peptide that can deliver nucleic acids into the host cells.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Sistemas de Liberación de Medicamentos/métodos , Liposomas Unilamelares/química , Animales , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Chlorocebus aethiops , Colesterol/química , Fluoresceínas/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Humanos , Oligonucleótidos/química , Oligonucleótidos/farmacología , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Estructura Secundaria de Proteína , CatelicidinasRESUMEN
Naked DNA plasmid represents the simplest vehicle for gene therapy and DNA-based vaccination purposes; however, the molecular mechanisms of DNA uptake in mammalian cells are poorly understood. Here, we show that naked DNA uptake occurs via proteoglycan-dependent macropinocytosis, thus challenging the concept of a specific DNA-internalizing receptor. Cells genetically deficient in proteoglycans, which constitute a major source of cell-surface polyanions, exhibited substantially decreased uptake of likewise polyanionic DNA. The apparent paradox was explained by the action of DNA-transporting proteins present in conditioned medium. Complexes between these proteins and DNA require proteoglycans for cellular entry. Mass spectrometry analysis of cell medium components identified several proteins previously shown to associate with DNA and to participate in membrane transport of macromolecular cargo. The major pathway for proteoglycan-dependent DNA uptake was macropinocytosis, whereas caveolae-dependent and clathrin-dependent pathways were not involved, as determined by using caveolin-1 knock-out cells, dominant-negative constructs for dynamin and Eps15, and macropinocytosis-disruptive drugs, as well as confocal fluorescence co-localization studies. Importantly, a significant fraction of internalized DNA was translocated to the nucleus for expression. Our results provide novel insights into the mechanism of DNA uptake by mammalian cells and extend the emerging role of proteoglycans in macromolecular transport.
Asunto(s)
ADN/química , Pinocitosis , Proteoglicanos/metabolismo , Animales , Transporte Biológico , Células CHO , Núcleo Celular/metabolismo , Cricetinae , Cricetulus , Medios de Cultivo/farmacología , Humanos , Espectrometría de Masas , Microscopía Confocal , Microscopía Fluorescente , Polielectrolitos , Polímeros/química , Proteoglicanos/químicaRESUMEN
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.
Asunto(s)
Productos del Gen tat/farmacología , Fragmentos de Péptidos/farmacología , Poliaminas/metabolismo , Neoplasias de la Vejiga Urinaria/prevención & control , Animales , Células CHO , Proliferación Celular , Cromatografía de Afinidad , Cricetinae , Cricetulus , Eflornitina/farmacología , Femenino , Heparina/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteoglicanos/aislamiento & purificación , Proteoglicanos/metabolismo , Espermidina/farmacología , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
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.
Asunto(s)
Fragmentos de Péptidos/metabolismo , Pinocitosis/fisiología , Priones/metabolismo , Androstadienos/farmacología , Animales , Células CHO , Bovinos , Cricetinae , Cricetulus , Citocalasina D/farmacología , Estructura Terciaria de Proteína , Transporte de Proteínas , WortmaninaRESUMEN
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.
Asunto(s)
Membrana Celular/efectos de los fármacos , Clatrina/fisiología , Endocitosis/fisiología , Sustancias Macromoleculares/metabolismo , Membrana Nuclear/fisiología , Matriz Nuclear/fisiología , Proteoglicanos/fisiología , Transporte Biológico/fisiología , Membrana Celular/fisiología , Permeabilidad de la Membrana Celular , Endocitosis/efectos de los fármacos , Membrana Nuclear/metabolismo , Fagocitosis/fisiología , Proteoglicanos/metabolismoRESUMEN
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 (TF Delta CT 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.
Asunto(s)
Neovascularización Patológica , Neovascularización Fisiológica , Tromboplastina/fisiología , Animales , Aorta/patología , Oftalmopatías/patología , Oftalmopatías/fisiopatología , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Estructura Terciaria de Proteína , Receptor PAR-2/fisiología , Transducción de Señal , Tromboplastina/química , Tromboplastina/genéticaRESUMEN
The monoclonal antibody 10E4, which recognizes an epitope supposed to contain N-unsubstituted glucosamine, is commonly used to trace heparan sulfate proteoglycans. It has not been fully clarified if the N-unsubstituted glucosamine is required for antibody recognition and if all heparan sulfates carry this epitope. Here we show that the epitope can contain N-unsubstituted glucosamine and that nitric oxide-generated deaminative cleavage at this residue in vivo can destroy the epitope. Studies using flow cytometry and confocal immunofluorescence microscopy of both normal and transformed cells indicated that the 10E4 epitope was partially inaccessible in the heparan sulfate chains attached to glypican-1. The 10E4 antibody recognized mainly heparan sulfate degradation products that colocalized with acidic endosomes. These sites were greatly depleted of 10E4-positive heparan sulfate on suramin inhibition of heparanase. Instead, there was increased colocalization between 10E4-positive heparan sulfate and glypican-1. When both S-nitrosylation of Gpc-1 and heparanase were inhibited, detectable 10E4 epitope colocalized entirely with glypican-1. In nitric oxide-depleted cells, there was both an increased signal from 10E4 and increased colocalization with glypican-1. In suramin-treated cells, the 10E4 epitope was destroyed by ascorbate-released nitric oxide with concomitant formation of anhydromannose-containing heparan sulfate oligosaccharides. Immunoisolation of radiolabeled 10E4-positive material from unperturbed cells yielded very little glypican-1 when compared with specifically immunoisolated glypican-1 and total proteoglycan and degradation products. The 10E4 immunoisolates were either other heparan sulfate proteoglycans or heparan sulfate degradation products.
Asunto(s)
Endosomas/metabolismo , Epítopos/metabolismo , Glucosamina/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Óxido Nítrico/metabolismo , Anticuerpos Monoclonales , Línea Celular Tumoral , Epítopos/inmunología , Glucosamina/inmunología , Proteoglicanos de Heparán Sulfato/genética , Proteoglicanos de Heparán Sulfato/inmunología , Humanos , Microscopía Confocal , Microscopía Fluorescente , Óxido Nítrico Sintasa/metabolismo , Oligosacáridos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Suramina/farmacologíaRESUMEN
Antimicrobial peptides, such as LL-37, are found both in nonvertebrates and vertebrates, where they represent important components of innate immunity. Bacterial infections at epithelial surfaces are associated with substantial induction of LL-37 expression, which allows efficient lysis of the invading microbes. Peptide-mediated lysis results in the release of bacterial nucleic acids with potential pathobiological activity in the host. Here, we demonstrate that LL-37 targets extracellular DNA plasmid to the nuclear compartment of mammalian cells, where it is expressed. DNA transfer occurred at physiological LL-37 concentrations that killed bacterial cells, whereas virtually no cytotoxic or growth-inhibitory effects were observed in mammalian cells. Furthermore, LL-37 protected DNA from serum nuclease degradation. LL-37.DNA complex uptake was a saturable time- and temperature-dependent process and was sensitive to cholesterol-depleting agents that are known to disrupt lipid rafts and caveolae, as shown by flow cytometry. Confocal fluorescence microscopy studies showed localization of internalized DNA to compartments stained by cholera toxin B, a marker of lipid rafts, but failed to demonstrate any co-localization of internalized DNA with caveolin-positive endocytotic vesicles. Moreover, LL-37-mediated plasmid uptake and reporter gene expression were strictly dependent on cell surface proteoglycans. We conclude that the human antimicrobial peptide LL-37 binds to, protects, and efficiently targets DNA plasmid to the nuclei of mammalian cells through caveolae-independent membrane raft endocytosis and cell surface proteoglycans.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Endocitosis , Técnicas de Transferencia de Gen , Proteoglicanos/metabolismo , Animales , Células CHO , Células COS , Catelicidinas , División Celular , Línea Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Cricetinae , ADN/química , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Luciferasas/metabolismo , Microdominios de Membrana/química , Microscopía Confocal , Microscopía Fluorescente , Modelos Biológicos , Péptidos/química , Plásmidos/metabolismo , Proteoglicanos/química , Factores de TiempoRESUMEN
We have previously reported that the heparan sulfate-priming glycoside 2-(6-hydroxynaphthyl)-beta-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)-beta-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.
Asunto(s)
División Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Glicósidos/farmacología , Heparitina Sulfato/metabolismo , Óxido Nítrico/metabolismo , Animales , Células CHO , Núcleo Celular/metabolismo , Cricetinae , Cricetulus , Humanos , Ratones , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Proteoglicanos de Heparán Sulfato/metabolismo , Óxido Nítrico/metabolismo , Poliaminas/farmacocinética , Ácido Ascórbico/farmacología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proteoglicanos de Heparán Sulfato/química , Proteoglicanos de Heparán Sulfato/genética , Heparitina Sulfato/química , Humanos , Microscopía Fluorescente , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Compuestos Nitrosos/metabolismo , Poliaminas/metabolismo , Espermina/farmacocinética , TransfecciónRESUMEN
Polyamines are necessary for tumour cell growth. Inhibition of endogenous polyamine biosynthesis results in compensatory up-regulation of polyamine uptake. Here, the combined effect of suramin and the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) on human carcinoma cell proliferation was studied. Suramin selectively inhibited the growth of tumour cells made dependent on extracellular polyamines by DFMO-treatment. In an animal tumour model, low non-toxic doses of suramin resulted in a 2-fold increase in DFMO tumour growth reduction. Moreover, suramin bound strongly to polyamine-agarose and significantly inhibited polyamine uptake in DFMO-treated cells. Our results indicate that non-toxic doses of suramin augment tumour growth inhibition by DFMO, and that a combination of these well-studied anticancer drugs may represent an additional strategy for cancer treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma/patología , Eflornitina/uso terapéutico , Inhibidores de Crecimiento/uso terapéutico , Poliaminas/farmacología , Suramina/uso terapéutico , Neoplasias de la Vejiga Urinaria/patología , Animales , Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Cromatografía de Afinidad , Sinergismo Farmacológico , Eflornitina/farmacología , Femenino , Inhibidores de Crecimiento/farmacología , Heparina/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Sefarosa , Organismos Libres de Patógenos Específicos , Espermina/metabolismo , Suramina/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
New therapies based on gene transfer and protein delivery require a better understanding of the basic mechanisms of macromolecular membrane transport. We have studied cellular uptake of macromolecular polyanions, i.e. DNA and glycosaminoglycans, and a polybasic HIV-Tat derived peptide (GRKKRRQRRRPPQC) using fluorescence assisted cell sorting and confocal fluorescence microscopy. The transactivator of HIV transcription (Tat) peptide stimulated cellular uptake of both DNA and heparan sulfate in a time-, concentration-, and temperature-dependent manner. Peptide-polyanion complexes accumulated in large, acidic, cytoplasmic vesicles formed de novo. This was followed by transfer of polyanion into the nuclear compartment and subsequent disappearance of the endolysosomal vesicles. In the absence of polyanion the Tat peptide displayed rapid accumulation in the nuclear compartment. However, in the presence of polyanion the peptide was almost exclusively retained in cytoplasmic vesicles. Cell-surface proteoglycans played a pivotal role in the uptake of complexes exhibiting a relatively high peptide to polyanion ratio, corresponding to a net positive charge of the complexes. Uptake of polyanions per se or complexes with a relatively low peptide to polyanion ratio was favored by proteoglycan deficiency in the recipient cells, indicating the existence of distinct transport mechanisms. Moreover, expression of full-length HIV-Tat as well as exogenous addition of HIV-Tat peptide resulted in cellular accumulation of endogenous proteoglycans. We conclude that an HIV-Tat derived peptide efficiently targets extraneous DNA and glycosaminoglycans to the nuclear compartment and that proteoglycans serve a regulatory role in these processes, which may have implications for directed gene and drug delivery in vivo.