RESUMO
A transfection vector based on a peptide dendrimer (1) has been developed and its abilities for DNA binding and transport have been investigated. By attaching a fluorophore to the vector system (1*), several steps in the transfection process could be monitored directly. As DLS and AFM studies showed, the labeled vector 1* condensed DNA into tightly packed aggregates able to enter eukaryotic cells. Co-localization experiments revealed that the ligand/plasmid complex is taken up by the endosomal pathway followed by an endosomal escape or lysosomal degradation. Afterwards, the plasmid DNA seems to enter the nucleus due to a breakdown of the nuclear envelope during mitosis, as only cells that have recently undergone mitosis showed H2B-GFP expression.
Assuntos
Dendrímeros , Lisina/genética , Transfecção , Plasmídeos/genética , DNA/genética , ÂnionsRESUMO
Invited for the cover of this issue are Christoph Hirschhäuser and his colleagues from the University of Duisburg-Essen. The image depicts a biotin-labelled transfection vector selectively channelling DNA into a cancer cell. The QR code on the label will lead you to a video abstract (https://youtu.be/OgXfBPZTKGA). Read the full text of the article at 10.1002/chem.202104618.
Assuntos
DNA , DNA/química , TransfecçãoRESUMO
A transfection vector that can home in on tumors is reported. Whereas previous vectors that allow moderately cell selective gene transfection used larger systems, this small-molecule approach paved the way for precise structure-activity relationship optimization. For this, biotin, which mediates cell selectivity, was combined with the potent DNA-binding motif tetralysine-guanidinocarbonypyrrol via a hydrophilic linker, thus enabling SAR-based optimization. The new vector mediated biotin receptor (BR)-selective transfection of cell lines with different BR expression levels. Computer-based analyses of microscopy images revealed a preference of one order of magnitude for the BR-positive cell lines over the BR-negative controls.
Assuntos
Vetores Genéticos , Neoplasias , Biotina/metabolismo , Linhagem Celular , Humanos , TransfecçãoRESUMO
Targeting specific protein binding sites to interfere with protein-protein interactions (PPIs) is crucial for the rational modulation of biologically relevant processes. Survivin, which is highly overexpressed in most cancer cells and considered to be a key player of carcinogenesis, features two functionally relevant binding sites. Here, we demonstrate selective disruption of the Survivin/Histone H3 or the Survivin/Crm1 interaction using a supramolecular approach. By rational design we identified two structurally related ligands (LNES and LHIS ), capable of selectively inhibiting these PPIs, leading to a reduction in cancer cell proliferation.
Assuntos
Proteínas Inibidoras de Apoptose , Sítios de Ligação , Proliferação de Células , Proteínas Inibidoras de Apoptose/metabolismo , Ligação Proteica , Survivina/química , Survivina/metabolismoRESUMO
Survivin's dual function as apoptosis inhibitor and regulator of cell proliferation is mediated via its interaction with the export receptor CRM1. This protein-protein interaction represents an attractive target in cancer research and therapy. Here, we report a sophisticated strategy addressing Survivin's nuclear export signal (NES), the binding site of CRM1, with advanced supramolecular tweezers for lysine and arginine. These were covalently connected to small peptides resembling the natural, self-complementary dimer interface which largely overlaps with the NES. Several biochemical methods demonstrated sequence-selective NES recognition and interference with the critical receptor interaction. These data were strongly supported by molecular dynamics simulations and multiscale computational studies. Rational design of lysine tweezers equipped with a peptidic recognition element thus allowed to address a previously unapproachable protein surface area. As an experimental proof-of-principle for specific transport signal interference, this concept should be transferable to any protein epitope with a flanking well-accessible lysine.
Assuntos
Carioferinas/química , Carioferinas/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Survivina/química , Survivina/metabolismo , Sítios de Ligação , Proliferação de Células , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Modelos Moleculares , Sinais de Exportação Nuclear , Ligação Proteica , Conformação Proteica , Proteína Exportina 1RESUMO
We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation-induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission "on" signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ-potential and dynamic light-scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold-standard transfection reagent Lipofectamine® 2000.
Assuntos
Aminoglicosídeos/química , Transfecção/métodos , Aminoglicosídeos/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Lipídeos/química , Microscopia Confocal , Plasmídeos/química , Plasmídeos/metabolismo , Eletricidade Estática , Tobramicina/química , Tobramicina/farmacologiaRESUMO
The synthesis and self-assembly of a polymer featuring a self-complementary supramolecular binding motif guanidiniocarbonyl pyrrole carboxylate zwitterion (GCP-zwitterion) bearing lactose moieties are reported. The GCP-zwitterion acts as a cross-linker to facilitate self-assembly of the polymeric chain into nanoparticles (NPs) at neutral pH in an aqueous medium. The formation of polymeric NPs can be controlled by addition of external stimuli (acid or base), which disfavors self-assembly of the GCP-zwitterion because of protonation or deprotonation of the GCP units in the polymer chain. The small-sized (<40 nm) NPs have a hydrophobic cavity and accessible lactose units on the outer shell for multivalent lectin binding. The multivalent interaction between NPs and the lectin peanut agglutinin was confirmed by agglutination experiments. In addition, the stimuli-responsive property of NPs was exploited for the uptake and release of a hydrophobic guest Nile red. Furthermore, the selectivity toward different cell lines (HEK 296T, HeLa, and Hep2G) was tested, and a cellular uptake of cargo-loaded NPs was found for Hep2G cells bearing the lactose-specific asialogylcoprotein receptor, whereas all other cells showed no NP interaction.
Assuntos
Lectinas , Nanopartículas , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , PolímerosRESUMO
Correction for 'Nanoparticle binding attenuates the pathobiology of gastric cancer-associated Helicobacter pylori' by Dana Westmeier et al., Nanoscale, 2018, 10, 1453-1463.
RESUMO
The protein Survivin is highly upregulated in most cancers and considered to be a key player in carcinogenesis. We explored a supramolecular approach to address Survivin as a drug target by inhibiting the protein-protein interaction of Survivin and its functionally relevant binding partner Histone H3. Ligand L1 is based on the guanidiniocarbonyl pyrrole cation and serves as a highly specific anion binder in order to target the interaction between Survivin and Histone H3. NMR titration confirmed binding of L1 to Survivin's Histone H3 binding site. The inhibition of the Survivin-Histone H3 interaction and consequently a reduction of cancer cell proliferation were demonstrated by microscopic and cellular assays.
Assuntos
Histonas/metabolismo , Pirróis/química , Survivina/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Histonas/química , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Ligação Proteica , Pirróis/metabolismo , Pirróis/farmacologia , Survivina/químicaRESUMO
In this communication we present the use of a novel class of luminophores with aggregation-induced emission (AIE) properties based on the steroid estrone. These molecules were equipped with cationic residues yielding amphiphiles suitable for lipofection. To this end, self-assembled luminescent structures were formed in aqueous media and mixed with a red-fluorescent protein expressing plasmid, yielding lipoplexes with increased emission intensity. These luminescent lipoplexes were able to efficiently transfect HeLa and HEK 293T cells and were able to be tracked due to the aggregation induced-emission properties.
RESUMO
Nanomaterials are promising novel antibiotics, but often ineffective. We found that nanomaterial-bacteria complex formation occurred with various nanomaterials. The bactericidal activity of NMs strongly depends on their physical binding to (multidrug-resistant) bacteria. Nanomaterials' binding and antibiotic effect was reduced by various pathophysiological biomolecule coronas strongly inhibiting their antibiotic effects. We show from analytical to in vitro to in vivo that nanomaterial-based killing could be restored by acidic pH treatments. Here, complex formation of negatively-charged, plasma corona-covered, nanomaterials with bacteria was electrostatically enhanced by reducing bacteria's negative surface charge. Employing in vivo skin infection models, acidic pH-induced complex formation was critical to counteract Staphylococcus aureus infections by silver nanomaterials. We explain why nano-antibiotics show reduced activity and provide a clinically practical solution.
RESUMO
Fungal infections are a growing global health and agricultural threat, and current chemical antifungals may induce various side-effects. Thus, nanoparticles are investigated as potential novel antifungals. We report that nanoparticles' antifungal activity strongly depends on their binding to fungal spores, focusing on the clinically important fungal pathogen Aspergillus fumigatus as well as common plant pathogens, such as Botrytis cinerea. We show that nanoparticle-spore complex formation was enhanced by the small nanoparticle size rather than the material, shape or charge, and could not be prevented by steric surface modifications. Fungal resistance to metal-based nanoparticles, such as ZnO-, Ag-, or CuO-nanoparticles as well as dissolution-resistant quantum dots, was mediated by biomolecule coronas acquired in pathophysiological and ecological environments, including the lung surfactant, plasma or complex organic matters. Mechanistically, dose-dependent corona-mediated resistance occurred via reducing physical adsorption of nanoparticles to fungal spores. The inhibitory effect of biomolecules on the antifungal activity of Ag-nanoparticles was further verified in vivo, using the invertebrate Galleria mellonella as an A. fumigatus infection model. Our results explain why current nanoantifungals often show low activity in realistic application environments, and will guide nanomaterial designs that maximize functionality and safe translatability as potent antifungals for human health, biotechnology, and agriculture.
Assuntos
Antifúngicos , Aspergillus fumigatus/crescimento & desenvolvimento , Farmacorresistência Fúngica/efeitos dos fármacos , Nanopartículas Metálicas , Coroa de Proteína/química , Animais , Antifúngicos/química , Antifúngicos/farmacologia , Botrytis , Modelos Animais de Doenças , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Camundongos , Mariposas , Doenças das Plantas , Aspergilose Pulmonar/tratamento farmacológico , Aspergilose Pulmonar/metabolismo , Aspergilose Pulmonar/patologiaRESUMO
Airborne fungal pathogens, predominantly Aspergillus fumigatus, can cause severe respiratory tract diseases. Here we show that in environments, fungal spores can already be decorated with nanoparticles. Using representative controlled nanoparticle models, we demonstrate that various nanoparticles, but not microparticles, rapidly and stably associate with spores, without specific functionalization. Nanoparticle-spore complex formation was enhanced by small nanoparticle size rather than by material, charge, or "stealth" modifications and was concentration-dependently reduced by the formation of environmental or physiological biomolecule coronas. Assembly of nanoparticle-spore surface hybrid structures affected their pathobiology, including reduced sensitivity against defensins, uptake into phagocytes, lung cell toxicity, and TLR/cytokine-mediated inflammatory responses. Following infection of mice, nanoparticle-spore complexes were detectable in the lung and less efficiently eliminated by the pulmonary immune defense, thereby enhancing A. fumigatus infections in immunocompromised animals. Collectively, self-assembly of nanoparticle-fungal complexes affects their (patho)biological identity, which may impact human health and ecology.
Assuntos
Aspergillus fumigatus/imunologia , Citocinas/imunologia , Pulmão/imunologia , Nanopartículas , Aspergilose Pulmonar/imunologia , Esporos Fúngicos/imunologia , Células A549 , Animais , Humanos , Pulmão/patologia , Camundongos , Coroa de Proteína/imunologia , Aspergilose Pulmonar/patologia , Células THP-1RESUMO
The high-energy sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), generated by human PAPS synthase isoforms PAPSS1 and PAPSS2, is required for all human sulfation pathways. Sulfotransferase SULT2A1 uses PAPS for sulfation of the androgen precursor dehydroepiandrosterone (DHEA), thereby reducing downstream activation of DHEA to active androgens. Human PAPSS2 mutations manifest with undetectable DHEA sulfate, androgen excess, and metabolic disease, suggesting that ubiquitous PAPSS1 cannot compensate for deficient PAPSS2 in supporting DHEA sulfation. In knockdown studies in human adrenocortical NCI-H295R1 cells, we found that PAPSS2, but not PAPSS1, is required for efficient DHEA sulfation. Specific APS kinase activity, the rate-limiting step in PAPS biosynthesis, did not differ between PAPSS1 and PAPSS2. Co-expression of cytoplasmic SULT2A1 with a cytoplasmic PAPSS2 variant supported DHEA sulfation more efficiently than co-expression with nuclear PAPSS2 or nuclear/cytosolic PAPSS1. Proximity ligation assays revealed protein-protein interactions between SULT2A1 and PAPSS2 and, to a lesser extent, PAPSS1. Molecular docking studies showed a putative binding site for SULT2A1 within the PAPSS2 APS kinase domain. Energy-dependent scoring of docking solutions identified the interaction as specific for the PAPSS2 and SULT2A1 isoforms. These findings elucidate the mechanistic basis for the selective requirement for PAPSS2 in human DHEA sulfation.
Assuntos
Carcinoma Adrenocortical/metabolismo , Sulfato de Desidroepiandrosterona/metabolismo , Complexos Multienzimáticos/metabolismo , Sulfato Adenililtransferase/metabolismo , Sulfotransferases/metabolismo , Sítios de Ligação , Núcleo Celular/metabolismo , Cristalografia por Raios X , Citosol/metabolismo , Sulfato de Desidroepiandrosterona/química , Humanos , Simulação de Acoplamento Molecular , Complexos Multienzimáticos/química , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Sulfato Adenililtransferase/química , Sulfotransferases/química , Células Tumorais CultivadasRESUMO
Enteric bacteria may cause severe diseases, including gastric cancer-associated Helicobacter pylori. Their infection paths overlap with the oro-gastrointestinal uptake route for nanoparticles, increasingly occurring during environmental or consumer/medical exposure. By comprehensive independent analytical methods, such as live cell fluorescence, electron as well as atomic force microscopy and elemental analysis, we show that a wide array of nanoparticles (NPs) but not microparticles form complexes with H. pylori and enteric pathogens without the need for specific functionalization. The NP-assembly that occurred rapidly was not influenced by variations in physiological temperature, though affected by the NPs' physico-chemical characteristics. Improved binding was observed for small NPs with a negative surface charge, whereas binding could be reduced by surface 'stealth' modifications. Employing human gastric epithelial cells and 3D-organoid models of the stomach, we show that NP-coating did not inhibit H. pylori's cellular attachment. However, even the assembly of non-bactericidal silica NPs attenuated H. pylori infection by reducing CagA phosphorylation, cytoskeletal rearrangement, and IL-8 secretion. Here we demonstrate that NP binding to enteric bacteria may impact their pathobiology which could be further exploited to rationally modulate the (patho)biology of microbes by nanomaterials.
Assuntos
Helicobacter pylori/metabolismo , Helicobacter pylori/patogenicidade , Nanopartículas/metabolismo , Neoplasias Gástricas/microbiologia , Aderência Bacteriana , Células Epiteliais/microbiologia , Mucosa Gástrica/citologia , Humanos , Organoides/microbiologia , Dióxido de SilícioRESUMO
Nanotechnology provides the food industry with new ways to modulate various aspects of food. Hence, engineered nanoparticles (NPs) are increasingly added to food and beverage products as functional ingredients. However, the impact of engineered as well as naturally occurring NPs on both commensal and pathogenic microorganisms within the gastrointestinal tract (GI) is not fully understood. Here, well-defined synthetic NPs and bacterial models were used to probe nanoparticle-bacteria interactions, from analytical to in situ to in vitro. NP-bacteria complexation occurred most efficiently for small NPs, independent of their core material or surface charge, but could be reduced by NPs' steric surface modifications. Adsorption to bacteria could also be demonstrated for naturally occurring carbon NPs isolated from beer. Complex formation affected the (patho)biological behavior of both the NPs and bacteria, including their cellular uptake into epithelial cells and phagocytes, pathogenic signaling pathways, and NP-induced cell toxicity. NP-bacteria complex formation was concentration-dependently reduced when the NPs became coated with biomolecule coronas with sequential simulation of first oral uptake and then the GI. However, efficient NP adsorption was restored when the pH was sufficiently low, such as in simulating the conditions of the stomach. Collectively, NP binding to enteric bacteria may impact their (patho)biology, particularly in the stomach. Nanosized-food additives as well as naturally occurring NPs may be exploited to (rationally) shape the microbiome. The information contained in this article should facilitate a "safe by design" strategy for the development and application of engineered NPs as functional foods ingredients.
RESUMO
Transcription factor TFIIA is controlled by complex regulatory networks including proteolysis by the protease Taspase 1, though the full impact of cleavage remains elusive. Here, we demonstrate that in contrast to the general assumption, de novo produced TFIIA is rapidly confined to the cytoplasm via an evolutionary conserved nuclear export signal (NES, amino acids 21VINDVRDIFL30), interacting with the nuclear export receptor Exportin-1/chromosomal region maintenance 1 (Crm1). Chemical export inhibition or genetic inactivation of the NES not only promotes TFIIA's nuclear localization but also affects its transcriptional activity. Notably, Taspase 1 processing promotes TFIIA's nuclear accumulation by NES masking, and modulates its transcriptional activity. Moreover, TFIIA complex formation with the TATA box binding protein (TBP) is cooperatively enhanced by inhibition of proteolysis and nuclear export, leading to an increase of the cell cycle inhibitor p16INK, which is counteracted by prevention of TBP binding. We here identified a novel mechanism how proteolysis and nuclear transport cooperatively fine-tune transcriptional programs.
Assuntos
Núcleo Celular/metabolismo , Endopeptidases/metabolismo , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fator de Transcrição TFIIA/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular , Células HeLa , Humanos , Modelos Moleculares , Sinais de Exportação Nuclear , Conformação Proteica , Fator de Transcrição TFIIA/análise , Fator de Transcrição TFIIA/genética , Ativação Transcricional , Proteína Exportina 1RESUMO
Here we report on a novel system based on aromatic thioethers with unique luminescence properties. Fifteen different compounds were investigated in detail on their luminescence properties using UV/Vis absorption and steady-state and time-resolved luminescence spectroscopy. Excited state lifetimes as well as quantum yields were determined, and the toxicity towards HeLa cells was investigated. Besides X-ray analyses also quantum chemical calculations were performed to gain deeper insights in the unique behavior of this facile system. The studied compounds reveal remarkable fluorescence emission ranging from 437 to 588â nm as well as phosphorescence (up to 5â µs).
RESUMO
BACKGROUND: It is well established that effector T cell responses are crucial for the control of most virus infections, but they are often tightly controlled by regulatory T cells (Treg) to minimize immunopathology. NK cells also contribute to virus control but it is not known if their antiviral effect is influenced by virus-induced Tregs as well. We therefore analyzed whether antiretroviral NK cell functions are inhibited by Tregs during an acute Friend retrovirus infection of mice. RESULTS: Selective depletion of Tregs by using the transgenic DEREG mouse model resulted in improved NK cell proliferation, maturation and effector cell differentiation. Suppression of NK cell functions depended on IL-2 consumption by Tregs, which could be overcome by specific NK cell stimulation with an IL-2/anti-IL-2 mAb complex. CONCLUSIONS: The current study demonstrates that virus-induced Tregs indeed inhibit antiviral NK cell responses and describes a targeted immunotherapy that can abrogate the suppression of NK cells by Tregs.
Assuntos
Interleucina-2/imunologia , Células Matadoras Naturais/imunologia , Infecções por Retroviridae/imunologia , Linfócitos T Reguladores/imunologia , Animais , Animais Geneticamente Modificados , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Retroviridae/imunologia , Infecções por Retroviridae/virologiaRESUMO
INTRODUCTION: Atrial fibrillation (AF) is the most frequent cardiac arrhythmia. The potassium current IKs is essential for cardiac repolarization. Gain-of-function mutation in KCNQ1, the gene encoding the pore-forming α-subunit of the IKs channel (KV 7.1), was the first ion channel dysfunction to be associated with familial AF. We hypothesized that early-onset lone AF is associated with a high prevalence of mutations in KCNQ1. METHODS AND RESULTS: We bidirectionally sequenced the entire coding sequence of KCNQ1 in 209 unrelated patients with early-onset lone AF (<40 years) and investigated the identified mutations functionally in a heterologous expression system. We found 4 nonsynonymous KCNQ1 mutations (A46T, R195W, A302V, and R670K) in 4 unrelated patients (38, 31, 39, and 36 years, respectively). None of the mutations were present in the control group (n = 416 alleles). No other mutations were found in genes previously associated with AF. The mutations A46T, R195W, and A302V have previously been associated with long-QT syndrome. In line with previous reports, we found A302V to display a pronounced loss-of-function of the IKs current, while the other mutants exhibited a gain-of-function phenotype. CONCLUSIONS: Mutations in the IKs channel leading to gain-of-function have previously been described in familial AF, yet this is the first time a loss-of-function mutation in KCNQ1 is associated with early-onset lone AF. These findings suggest that both gain-of-function and loss-of-function of cardiac potassium currents enhance the susceptibility to AF.