The Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) proteins, particularly STAT3, are among the most promising new targets for cancer therapy. In addition to interleukin-6 (IL-6) and its family members, multiple pathways, including G-protein-coupled receptors (GPCRs), Toll-like receptors (TLRs) and microRNAs were recently identified to regulate JAK-STAT signalling in cancer. Well known for its role in tumour cell proliferation, survival, invasion and immunosuppression, JAK-STAT3 signalling also promotes cancer through inflammation, obesity, stem cells and the pre-metastatic niche. In addition to its established role as a transcription factor in cancer, STAT3 regulates mitochondrion functions, as well as gene expression through epigenetic mechanisms. Newly identified regulators and functions of JAK-STAT3 in tumours are important targets for potential therapeutic strategies in the treatment of cancer.
DNA-incorporating hydrophobic moieties can be synthesized by either solid-phase or solution-phase coupling. On a solid support the DNA is protected, and hydrophobic units are usually attached employing phosphoramidite chemistry involving a DNA synthesizer. On the other hand, solution coupling in aqueous medium results in low yields due to the solvent incompatibility of DNA and hydrophobic compounds. Hence, the development of a general coupling method for producing amphiphilic DNA conjugates with high yield in solution remains a major challenge. Here, we report an organic-phase coupling strategy for nucleic acid modification and polymerization by introducing a hydrophobic DNA-surfactant complex as a reactive scaffold. A remarkable range of amphiphile-DNA structures (DNA-pyrene, DNA-triphenylphosphine, DNA-hydrocarbon, and DNA block copolymers) and a series of new brush-type DNA side-chain homopolymers with high DNA grafting density are produced efficiently. We believe that this method is an important breakthrough in developing a generalized approach to synthesizing functional DNA molecules for self-assembly and related technological applications.
The conformational change of the influenza virus hemagglutinin (HA) protein mediating the fusion between the virus envelope and the endosomal membrane was hypothesized to be induced by protonation of specific histidine residues since their pKas match the pHs of late endosomes (pKa of ?6.0). However, such critical key histidine residues remain to be identified. We investigated the highly conserved His184 at the HA1-HA1 interface and His110 at the HA1-HA2 interface of highly pathogenic H5N1 HA as potential pH sensors. By replacing both histidines with different amino acids and analyzing the effect of these mutations on conformational change and fusion, we found that His184, but not His110, plays an essential role in the pH dependence of the conformational change of HA. Computational modeling of the protonated His184 revealed that His184 is central in a conserved interaction network possibly regulating the pH dependence of conformational change via its pKa. As the propensity of histidine to get protonated largely depends on its local environment, mutation of residues in the vicinity of histidine may affect its pKa. The HA of highly pathogenic H5N1 viruses carries a Glu-to-Arg mutation at position 216 close to His184. By mutation of residue 216 in the highly pathogenic as well as the low pathogenic H5 HA, we observed a significant influence on the pH dependence of conformational change and fusion. These results are in support of a pKa-modulating effect of neighboring residues.
The surface functionalization of fatty acid vesicles will allow their use as nanoreactors for complex chemistry. In this report, the tethering of several DNA conjugates to decanoic acid vesicles for molecular recognition and synthetic purposes was explored. Due to the highly dynamic nature of these structures, only one novel bola-amphiphile DNA conjugate could interact efficiently with or spontaneously pierce into the vesicle bilayers without jeopardizing their self-assembly or stability. This molecule was synthesized via a Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (click reaction), and consists of a single hydrocarbon chain of 20 carbons having on one end a triazole group linked to the 5'-phosphate of the nucleic acid and on the other side a hydroxyl-group. Its insertion was so effective that a fluorescent label on the DNA complementary to the conjugate could be used to visualize fatty acid structures.
The HA (haemagglutinin) of influenza viruses must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. We previ-ously showed with Förster Resonance Energy Transfer (FRET) that deletion of the two rafttargeting features of HA, S-acylation at the cytoplasmic tail and the hydrophobic amino acids VIL in the outer part of the transmembrane region (TMR) lead to reduced raft association. In addition, exchange of VIL, but not of the S-acylation sites severely retards transport of HA through the Golgi. Here we have further characterized the ill-defined signal in the TMR. A sequence comparison suggests that leucine of VIL might be part of a cholesterol consensus motif (CCM) that is known to bind cholesterol to 7-TM-receptors. The signal also comprises a lysine and a tryptophan on one and a tyrosine on another TMR-helix and is conserved in group 2 HAs. Mutations in the CCM retard Golgi-localized processing of HA, such as acquisition of Endo-H resistant carbohydrates in the medial-Golgi and proteolytic cleavage in the TGN. The delay in transport of HA to and from the medial Golgi varied with the mutation suggesting that different transport steps are affected. All mutants analyzed by FRET also showed reduced association with rafts at the plasma membrane. Thus, the raft targeting signal of HA encompasses not only hydrophobic, but also aromatic and positively charged residues. We speculate that binding to cholesterol might facilitate intracellular transport of HA and association with rafts.
The interactions between cholesterol and other membrane molecules determine important membrane properties. It was shown that even small changes in the molecular structure of cholesterol have a crucial influence on these interactions. We recently reported that in addition to alterations in the tetracyclic ring structure, the iso-branched side chain of cholesterol also has a significant impact on membrane properties (Scheidt et al., 2013). Here we used synthetic cholesterol analogs to investigate the influence of an unbranched aliphatic side chain of different length. The (2)H NMR order parameter of the phospholipid chains and therefore the molecular packing of the phospholipid molecules shows a significant dependence on the sterol's alkyl side chain length, while, membrane permeation studied by a dithionite ion permeation assay and lateral diffusion measured by (1)H MAS pulsed field gradient NMR are less influenced. To achieve the same molecular packing effect similar to that of an iso-branched aliphatic side chain, a longer unbranched side chain (n-dodecyl instead of n-octyl) at C17 of cholesterol is required. Obviously, sterols having a branched iso-alkyl chain with two terminal methyl groups exhibit altered cholesterol-phospholipid interactions compared to analogous molecules with a straight unbranched chain.
In mammals, circadian rhythms are generated by delayed negative feedback, in which period (PER1-PER3) and cryptochrome (CRY1, CRY2) proteins gradually accumulate in the nucleus to suppress the transcription of their own genes. Although the importance of nuclear import and export signals for the subcellular localization of clock proteins is well established, little is known about the dynamics of these processes as well as their importance for the generation of circadian rhythms. We show by pharmacological perturbations of oscillating cells that nuclear import and export are of crucial importance for the circadian period. Live-cell fluorescence microscopy revealed that nuclear import of the key circadian protein PER2 is fast and further accelerated by CRY1. Moreover, PER2 nuclear import is crucially dependent on a specific nuclear-receptor-binding motif in PER2 that also mediates nuclear immobility. Nuclear export, however, is relatively slow, supporting a model of PER2 nuclear accumulation by rapid import, slow export and substantial nuclear degradation.
This paper describes the synthesis and characterization of polymer-peptide conjugates to be used as infection-resistant coating for biomaterial implants and devices. Antiadhesive polymer brushes composed of block copolymer Pluronic F-127 (PF127) were functionalized with antimicrobial peptides (AMP), able to kill bacteria on contact, and arginine-glycine-aspartate (RGD) peptides to promote the adhesion and spreading of host tissue cells. The antiadhesive and antibacterial properties of the coating were investigated with three bacterial strains: Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa. The ability of the coating to support mammalian cell growth was determined using human fibroblast cells. Coatings composed of the appropriate ratio of the functional components: PF127, PF127 modified with AMP, and PF127 modified with RGD showed good antiadhesive and bactericidal properties without hampering tissue compatibility.
Optically active bio-composite blends of conjugated polymers or oligomers are fabricated by complexing them with bovine submaxilliary mucin (BSM) protein. The BSM matrix is exploited to host hydrophobic extended conjugated ?-systems and to prevent undesirable aggregation and render such materials water soluble. This method allows tuning the emission color of solutions and films from the basic colors to the technologically challenging white emission. Furthermore, electrically driven light emitting biological devices are prepared and operated.
The matrix protein M1 plays a pivotal role in the budding of influenza virus from the plasma membrane (PM) of infected cells. This protein interacts with viral genetic material and envelope proteins while binding to the inner leaflet of the PM. Its oligomerization is therefore closely connected to the assembly of viral components and the formation of new virions. Of interest, the molecular details of M1 interaction with lipids and other viral proteins are far from being understood, and it remains to be determined whether the multimerization of M1 is affected by its binding to the PM and interaction with its components. To clarify the connection between M1 oligomerization and binding to lipid membranes, we applied a combination of several quantitative microscopy approaches. First, we used number and brightness (N&B) microscopy to characterize protein multimerization upon interaction with the PM of living cells. Second, we used controlled biophysical models of the PM (i.e., supported bilayers) to delve into the details of M1-lipid and M1-M1 interactions by employing a combination of raster image correlation spectroscopy (RICS), fluorescence correlation spectroscopy (FCS), and atomic force microscopy (AFM). Our results show that M1 oligomer formation is strongly enhanced by membrane binding and does not necessarily require the presence of other viral proteins. Furthermore, we propose a specific model to explain M1 binding to the lipid bilayer and the formation of multimers.
Intracellular therapeutic targets that define tumor immunosuppression in both tumor cells and T cells remain intractable. Here, we have shown that administration of a covalently linked siRNA to an aptamer (apt) that selectively binds cytotoxic T lymphocyte-associated antigen 4 (CTLA4(apt)) allows gene silencing in exhausted CD8? T cells and Tregs in tumors as well as CTLA4-expressing malignant T cells. CTLA4 expression was upregulated in CD8? T cells in the tumor milieu; therefore, CTLA4(apt) fused to a STAT3-targeting siRNA (CTLA4(apt)-STAT3 siRNA) resulted in internalization into tumor-associated CD8? T cells and silencing of STAT3, which activated tumor antigen-specific T cells in murine models. Both local and systemic administration of CTLA4(apt)-STAT3 siRNA dramatically reduced tumor-associated Tregs. Furthermore, CTLA4(apt)-STAT3 siRNA potently inhibited tumor growth and metastasis in various mouse tumor models. Importantly, CTLA4 expression is observed in T cells of patients with blood malignancies, and CTLA4(apt)-STAT3 siRNA treatment of immunodeficient mice bearing human T cell lymphomas promoted tumor cell apoptosis and tumor growth inhibition. These data demonstrate that a CTLA4(apt)-based siRNA delivery strategy allows gene silencing in both tumor-associated T cells and tumor cells and inhibits tumor growth and metastasis.
Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs.
Site-specific derivatization of chemically equivalent functional groups has recently been facilitated by the introduction of high-affinity aptamers as non-covalent protective groups. More specifically, a series of RNA aptamers have proven to be highly efficient in enhancing the regioselectivity of reactions with the aminoglycoside antibiotic neomycin?B, which carries several chemically indistinguishable amino and hydroxy groups. Since small-molecule targets tend to exhibit multiple modes of binding with a single aptamer, the impact of secondary binding sites on the regioselectivity should be considered. To address this issue, we investigated a series of well-characterized RNA aptamers that bind neomycin?B and propose a mechanism that accounts for the regioselective outcome of these transformations. We further demonstrate that the regioselectivity induced by non-covalent aptamer protective groups is determined by the number of binding sites, their affinity, and the mode of interaction with the guest molecule.
Cyclic peptides containing a disulfide bridge were identified as a simple and versatile coordination sphere for asymmetric catalysis. Upon complexation with Cu(2+) ions they catalyze Diels-Alder and Friedel-Crafts reactions with high enantioselectivities of up to 99%?ee and 86%?ee, respectively. Moreover, the peptides ligands were systematically optimized with the assistance of "Alanine Scanning". This biomolecular design could greatly expand the choice of peptide scaffolds for artificial metallopeptide catalysts.
Polymer wrapping is a highly effective method of selecting semiconducting carbon nanotubes and dispersing them in solution. Semi-aligned semiconducting carbon nanotube networks are obtained by blade coating, an effective and scalable process. The field-effect transistor (FET) performance can be tuned by the choice of wrapping polymer, and the polymer concentration modifies the FET transport characteristics, leading to a record on/off ratio of 10(8) .
The large multiprotein complex, photosystem I (PSI), which is at the heart of light-dependent reactions in photosynthesis, is integrated as the active component in a solid-state organic photovoltaic cell. These experiments demonstrate that photoactive megadalton protein complexes are compatible with solution processing of organic-semiconductor materials and operate in a dry non-natural environment that is very different from the biological membrane.
Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance--PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol--using 2H solid-state nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.
Enveloped viruses often use membrane lipid rafts to assemble and bud, augment infection and spread efficiently. However, the molecular bases and functional consequences of the partitioning of viral glycoproteins into microdomains remain intriguing questions in virus biology. Here, we measured Foerster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) to study the role of distinct membrane proximal regions of the human immunodeficiency virus glycoprotein gp41 for lipid raft partitioning in living Chinese hamster ovary cells (CHO-K1). Gp41 was labelled with a fluorescent protein at the exoplasmic face of the membrane, preventing any interference of the fluorophore with the proposed role of the transmembrane and cytoplasmic domains in lateral organization of gp41. Raft localization was deduced from interaction with an established raft marker, a fluorescently tagged glycophosphatidylinositol anchor and the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial lateral sorting determinant in CHO-K1 cells. Interestingly, the raft association of gp41 indicates a substantial cell-to-cell heterogeneity of the plasma membrane microdomains. In complementary fluorescence polarization microscopy, a distinct CRAC requirement was found for the oligomerization of the gp41 variants. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral sorting of viral glycoproteins for virus assembly at cellular membranes.
Upon endocytosis in its cellular host, influenza A virus transits via early to late endosomes. To efficiently release its genome, the composite viral shell must undergo significant structural rearrangement, but the exact sequence of events leading to viral uncoating remains largely speculative. In addition, no change in viral structure has ever been identified at the level of early endosomes, raising a question about their role. We performed AFM indentation on single viruses in conjunction with cellular assays under conditions that mimicked gradual acidification from early to late endosomes. We found that the release of the influenza genome requires sequential exposure to the pH of both early and late endosomes, with each step corresponding to changes in the virus mechanical response. Step 1 (pH 7.5-6) involves a modification of both hemagglutinin and the viral lumen and is reversible, whereas Step 2 (pH <6.0) involves M1 dissociation and major hemagglutinin conformational changes and is irreversible. Bypassing the early-endosomal pH step or blocking the envelope proton channel M2 precludes proper genome release and efficient infection, illustrating the importance of viral lumen acidification during the early endosomal residence for influenza virus infection.
Influenza virus assembles in the budozone, a cholesterol-/sphingolipid-enriched ("raft") domain at the apical plasma membrane, organized by hemagglutinin (HA). The viral protein M2 localizes to the budozone edge for virus particle scission. This was proposed to depend on acylation and cholesterol binding. We show that M2-GFP without these motifs is still transported apically in polarized cells. Employing FRET, we determined that clustering between HA and M2 is reduced upon disruption of HA's raft-association features (acylation, transmembranous VIL motif), but remains unchanged with M2 lacking acylation and/or cholesterol-binding sites. The motifs are thus irrelevant for M2 targeting in cells.
Perovskite-based organic-inorganic hybrids hold great potential as active layers in electronics or optoelectronics or as components of biosensors. However, many of these applications require thin films grown with good control over structure and thickness-a major challenge that needs to be addressed. The work presented here is an effort towards this goal and concerns the layer-by-layer deposition at ambient conditions of ferromagnetic organic-inorganic hybrids consisting of alternating CuCl4 -octahedra and organic layers. The Langmuir-Blodgett technique used to assemble these structures provides intrinsic control over the molecular organization and film thickness down to the molecular level. Magnetic characterization reveals that the coercive field for these thin films is larger than that for solution-grown layered bulk crystals. The strategy presented here suggests a promising cost effective route to facilitate the excellently controlled growth of sophisticated materials on a wide variety of substrates that have properties relevant for the high density storage media and spintronic devices.
Developing molecular systems with functions analogous to those of macroscopic machine components, such as rotors, gyroscopes and valves, is a long-standing goal of nanotechnology. However, macroscopic analogies go only so far in predicting function in nanoscale environments, where friction dominates over inertia. In some instances, ratchet mechanisms have been used to bias the ever-present random, thermally driven (Brownian) motion and drive molecular diffusion in desired directions. Here, we visualize the motions of surface-bound molecular rotors using defocused fluorescence imaging, and observe the transition from hindered to free Brownian rotation by tuning medium viscosity. We show that the otherwise random rotations can be biased by the polarization of the excitation light field, even though the associated optical torque is insufficient to overcome thermal fluctuations. The biased rotation is attributed instead to a fluctuating-friction mechanism in which photoexcitation of the rotor strongly inhibits its diffusion rate.
A series of thioether profragrances was prepared by reaction of different sulfanylalkanoates with ?-damascone and tested for their release efficiencies in a fabric-softener and an all-purpose cleaner application. Dynamic headspace analysis on dry cotton and on a ceramic plate revealed that the performance of the different precursors depended on the structure, but also on the particular conditions encountered in different applications. Moreover, profragrances derived from other ?,?-unsaturated fragrance aldehydes and ketones were synthesized analogously and evaluated using the same test protocol. Thioethers were found to be suitable precursors to release the corresponding fragrances, but neither the quantity of profragrance deposited from an aqueous environment onto the target surface, nor the amount of fragrance released after deposition could be linearly correlated to the hydrophilicity or hydrophobicity of the compounds. Different sets of compounds were found to be the best performers for different types of applications. Only one of the compounds evaluated in the present work, namely the thiolactic acid derivative of ?-damascone, efficiently released the corresponding fragrance in both of the tested applications. Profragrance development for functional perfumery thus remains a partially empirical endeavour. More knowledge (and control) of the various application conditions are required for an efficient profragrance design.
The development of antiviral agents is one of the major challenges in medical science. So far, small monovalent molecular drugs that inhibit the late steps in the viral replication cycle, i.e., virus budding, have not worked well which emphasizes the need for alternative approaches. Polyvalently presented viral receptors, however, show potential as good inhibitors of virus-cell binding, which is the first step in the viral infection cycle. By gradually increasing the size of ligand functionalized gold nanoparticles, up to virus-like dimensions, we are now able to quantify the polyvalent enhancement of virus-cell binding inhibition and to identify varying mechanisms of virus inhibition with different efficacies: by employing a new binding assay we found that surface area-normalized polysulfated gold nanoparticles of diameters equal to and larger than the virus diameter (>50 nm) more efficiently inhibit the binding of vesicular stomatitis virus (VSV) to cells than smaller particles. On a per particle basis, larger sized gold nanoparticles were surprisingly shown to inhibit the viral infection up to two orders of magnitude more efficiently than smaller particles, which suggests different mechanisms of virus inhibition. Based on complementary electron microscopic data, we noticed that larger gold nanoparticles act as efficient cross-linkers between virions, whereas smaller gold nanoparticles decorate the surface of individual virus particles. Our systematic study accentuates the need for the design of biodegradable, virus-sized inhibitors capitalizing on polyvalent binding.
Increasing evidence suggests that premetastatic niches, consisting mainly of myeloid cells, provide microenvironment critical for cancer cell recruitment and survival to facilitate metastasis. While CD8(+) T cells exert immunosurveillance in primary human tumors, whether they can exert similar effects on myeloid cells in the premetastatic environment is unknown. Here, we show that CD8(+) T cells are capable of constraining premetastatic myeloid cell accumulation by inducing myeloid cell apoptosis in C57BL/6 mice. Ag-specific CD8(+) T-cell cytotoxicity against myeloid cells in premetastatic lymph nodes is compromised by Stat3. We demonstrate here that Stat3 ablation in myeloid cells leads to CD8(+) T-cell activation and increased levels of IFN-? and granzyme B in the premetastatic environment. Furthermore, Stat3 negatively regulates soluble Ag cross-presentation by myeloid cells to CD8(+) T cells in the premetastatic niche. Importantly, in tumor-free lymph nodes of melanoma patients, infiltration of activated CD8(+) T cells inversely correlates with STAT3 activity, which is associated with a decrease in number of myeloid cells. Our study suggested a novel role for CD8(+) T cells in constraining myeloid cell activity through direct killing in the premetastatic environment, and the therapeutic potential by targeting Stat3 in myeloid cells to improve CD8(+) T-cell immunosurveillance against metastasis.
Macrophages represent an important cellular target of HIV-1. Interestingly, they are also believed to play a potential role counteracting its infection. However, HIV-1 is known to impair macrophage immune functions such as antibody-mediated phagocytosis. Here, we present immunoliposomes that can bind HIV-1 virus-like particles (HIV-VLPs) while being specifically phagocytosed by macrophages, thus allowing the co-internalization of HIV-VLPs. These liposomes are decorated with anti-Env antibodies and contain phosphatidylserine (PS). PS mediates liposome internalization by macrophages via a mechanism not affected by HIV-1. Hence, PS-liposomes mimic apoptotic cells and are internalized into the macrophages due to specific recognition, carrying the previously bound HIV-VLPs. With a combination of flow cytometry, confocal live-cell imaging and electron microscopy we demonstrate that the PS-immunoliposomes presented here are able to elicit efficient HIV-VLPs phagocytosis by macrophages and might represent a new nanotechnological approach to enhance HIV-1 antigen presentation and reduce the ongoing inflammation processes.
Lipophilic nucleic acids have become a versatile tool for structuring and functionalization of lipid bilayers and biological membranes as well as cargo vehicles to transport and deliver bioactive compounds, like interference RNA, into cells by taking advantage of reversible hybridization with complementary strands. This contribution reviews the different types of conjugates of lipophilic nucleic acids, and their physicochemical and self-assembly properties. Strategies for choosing a nucleic acid, lipophilic modification, and linker are discussed. Interaction with lipid membranes and its stability, dynamic structure and assembly of lipophilic nucleic acids upon embedding into biological membranes are specific points of the review. A large diversity of conjugates including lipophilic peptide nucleic acid and siRNA provides tailored solutions for specific applications in bio- and nanotechnology as well as in cell biology and medicine, as illustrated through some selected examples.
We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1) infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC). We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG) enrichment tests to uncover co-regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.
Androgen receptor (AR) signaling is important for prostate cancer progression. However, androgen-deprivation and/or AR targeting-based therapies often lead to resistance. Here we demonstrate that loss of AR expression results in STAT3 activation in prostate cancer cells. AR downregulation further leads to development of prostate cancer stem-like cells (CSC), which requires STAT3. In human prostate tumor tissues, elevated cancer stem-like cell markers coincide with those cells exhibiting high STAT3 activity and low AR expression. AR downregulation-induced STAT3 activation is mediated through increased IL-6 expression. Treating mice with soluble IL-6 receptor fusion protein or silencing STAT3 in tumor cells significantly reduced prostate tumor growth and CSCs. Together, these findings indicate an opposing role of AR and STAT3 in prostate CSC development.
Copper catalyzed azide-alkyne cycloaddition (CuAAC) was employed to synthesize DNA block copolymers (DBCs) with a range of polymer blocks including temperature-responsive poly(N-isoproylacrylamide) (poly(NIPAM)) and highly hydrophobic poly(styrene). Exceptionally high yields were achieved at low DNA concentrations, in organic solvents, and in the absence of any solid support. The DNA segment of the DBC remained capable of sequence-specific hybridization: it was used to assemble a precisely defined nanostructure, a DNA tetrahedron, with pendant poly(NIPAM) segments. In the presence of an excess of poly(NIPAM) homopolymer, the tetrahedron-poly(NIPAM) conjugate nucleated the formation of large, well-defined nanoparticles at 40 °C, a temperature at which the homopolymer precipitated from solution. These composite nanoparticles were observed by dynamic light scattering and cryoTEM, and their hybrid nature was confirmed by AFM imaging. As a result of the large effective surface area of the tetrahedron, only very low concentrations of the conjugate were required in order for this surfactant-like behavior to be observed.
Significant efforts have been devoted to the development of techniques allowing the investigation of viral mRNA progression during the replication cycle. We herein describe the use of sequence-specific FIT-PNA (Forced Intercalation Peptide Nucleic Acids) probes which contain a single intercalator serving as an artificial fluorescent nucleobase. FIT-PNA probes are not degraded by enzymes, neither by nucleases nor by proteases, and provide for both high sensitivity and high target specificity under physiological conditions inside the infected living host cell.
IZI-06.1 is a humanized anti-TNFR1 single-chain fragment variable (scFv) that selectively inhibits binding of tumor necrosis factor (TNF) and lymphotoxin alpha to tumor necrosis factor receptor 1 (TNFR1) but not TNFR2. Recently, IZI-06.1 was converted into a fully human IgG1 antibody (ATROSAB) for the treatment of inflammatory diseases. Here, we compare the bivalent ATROSAB with a monovalent scFv-human serum albumin (HSA) fusion protein lacking any antibody-associated effector functions and possessing approximately only half the molecular mass of an IgG, which should facilitate accumulation in inflamed tissues. Furthermore, the half-life of the scFv should be strongly extended while maintaining monovalent binding, avoiding a possible signal transduction by receptor cross-linking in the absence of TNF. The scFv-HSA fusion protein was produced by stably transfected Chinese hamster ovary cells and purified by affinity chromatography. The fusion protein bound specifically to TNFR1 in enzyme-linked immunosorbent assay and TNFR1-transfected mouse embryonic fibroblasts. Affinity determined by quartz crystal microbalance was reduced compared with ATROSAB, which resulted also in a reduced inhibitory activity. Compared with the scFv fragment, the half-life of the fusion protein was significantly increased, although not reaching the long half-life of ATROSAB. In summary, the scFv-HSA may provide an alternative to the full-length IgG1 with the ability to selectively inhibit TNFR1 and exploiting the pharmacokinetic properties of albumin.
The influence of cholesterols alkyl side chain on membrane properties was studied using a series of synthetic cholesterol derivatives without a side chain or with a branched side chain consisting of 5 to 14 carbon atoms. Cholesterols side chain is crucial for all membrane properties investigated and therefore essential for the membrane properties of eukaryotic cells.
Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type I and type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (LAO-HisQMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg(2+) ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ. In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems.
Lipid analogues carrying three nitrilotriacetic acid (tris-NTA) head groups were developed for the selective targeting of His-tagged proteins into liquid ordered (lo ) or liquid disordered (ld ) lipid phases. Strong partitioning into the lo phase of His-tagged proteins bound to tris-NTA conjugated to saturated alkyl chains (tris-NTA DODA) was achieved, while tris-NTA conjugated to an unsaturated alkyl chain (tris-NTA SOA) predominantly resided in the ld phase. Interestingly, His-tag-mediated lipid crosslinking turned out to be required for efficient targeting into the lo phase by tris-NTA DODA. Robust partitioning into lo phases was confirmed by using viral lipid mixtures and giant plasma membrane vesicles. Moreover, efficient protein targeting into lo and ld domains within the plasma membrane of living cells was demonstrated by single-molecule tracking, thus establishing a highly generic approach for exploring lipid microdomains in situ.
Influenza A virus strains adopt different host specificities mainly depending on their hemagglutinin (HA) protein. Via HA, the virus binds sialic acid receptors of the host cell and, upon endocytic uptake, HA triggers fusion between the viral envelope bilayer and the endosomal membrane by a low pH-induced conformational change leading to the release of the viral genome into the host cell cytoplasm. Both functions are crucial for viral infection enabling the genesis of new progeny virus. Adaptation to different hosts in vitro was shown to require mutations within HA altering the receptor binding and/or fusion behavior of the respective virus strain. Human adapted influenza virus strains (H1N1, H3N2, H2N2) as well as recent avian influenza virus strains (H5, H7 and H9 subtypes) which gained the ability to infect humans mostly contained mutations in the receptor binding site (RBS) of HA enabling increased binding affinity of these viruses to human type (?-2,6 linked sialic acid) receptors. Thus, the receptor binding specificity seems to be the major requirement for successful adaptation to the human host; however, the RBS is not the only determinant of host specificity. Increased binding to a certain cell type does not always correlate with infection efficiency. Furthermore, viruses carrying mutations in the RBS often resulted in reduced viral fitness and were still unable to transmit between mammals. Recently, the pH stability of HA was reported to affect the transmissibility of influenza viruses. This review summarizes recent findings on the adaptation of influenza A viruses to the human host and related amino acid substitutions resulting in altered receptor binding specificity and/or modulated fusion pH of HA. Furthermore, the role of these properties (receptor specificity and pH stability of HA) for adaptation to and transmissibility in the human host is discussed.
Previously, [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD)-based fluorophores used as highly sensitive fluorescence lifetime probes reporting on their microenvironmental polarity have been described. Now, a new generation of DBD dyes has been developed. Although they are still sensitive to polarity, in contrast to the former DBD dyes, they have extraordinary spectroscopic properties even in aqueous surroundings. They are characterized by long fluorescence lifetimes (10-20?ns), large Stokes shifts (?100?nm), high photostabilities, and high quantum yields (>0.56). Here, the spectroscopic properties and synthesis of functionalized derivatives for labeling biological targets are described. Furthermore, thio-reactive maleimido derivatives of both DBD generations show strong intramolecular fluorescence quenching. This mechanism has been investigated and is found to undergo a photoelectron transfer (PET) process. After reaction with a thiol group, this fluorescence quenching is prevented, indicating successful bonding. Being sensitive to their environmental polarity, these compounds have been used as powerful fluorescence lifetime probes for the investigation of conformational changes in the maltose ATP-binding cassette transporter through fluorescence lifetime spectroscopy. The differing tendencies of the fluorescence lifetime change for both DBD dye generations promote their combination as a powerful toolkit for studying microenvironments in proteins.
The field of DNA nanotechnology has progressed rapidly in recent years and hence a large variety of 1D-, 2D- and 3D DNA nanostructures with various sizes, geometries and shapes is readily accessible. DNA-based nanoobjects are fabricated by straight forward design and self-assembly processes allowing the exact positioning of functional moieties and the integration of other materials. At the same time some of these nanosystems are characterized by a low toxicity profile. As a consequence, the use of these architectures in a biomedical context has been explored. In this review the progress and possibilities of pristine nucleic acid nanostructures and DNA hybrid materials for drug delivery will be discussed. For the latter class of structures, a distinction is made between carriers with an inorganic core composed of gold or silica and amphiphilic DNA block copolymers that exhibit a soft hydrophobic interior.
To characterize the structure and dynamics of cholesterol in membranes, fluorescent analogs of the native molecule have widely been employed. The cholesterol content in membranes is in general manipulated by using water-soluble cyclodextrins. Since the interactions between cyclodextrins and fluorescent-labeled cholesterol have not been investigated in detail so far, we have compared the cyclodextrin-mediated membrane extraction of three different fluorescent cholesterol analogs (one bearing a NBD and two bearing BODIPY moieties). Extraction of these analogs was followed by measuring the Förster resonance energy transfer between a rhodamine moiety linked to phosphatidylethanolamine and the labeled cholesterol. The extraction kinetics revealed that the analogs are differently extracted from membranes. We examined the orientation of the analogs within the membrane and their influence on lipid condensation using NMR and EPR spectroscopies. Our data indicate that the extraction of fluorescent sterols from membranes is determined by several parameters, including their impact on lipid order, their hydrophobicity, their intermolecular interactions with surrounding lipids, their orientation within the bilayer, and their affinity with the exogenous acceptor.
One of the barriers to the development of protein therapeutics is effective delivery to mammalian cells. The proteins must maintain a careful balance of polar moieties to enable administration and distribution and hydrophobic character to minimize cell toxicity. Numerous strategies have been applied to this end, from appending additional cationic peptides to supercharging the protein itself, sometimes with limited success. Here we present a strategy that combines these methods, by equipping a protein with supercharged elastin-like polypeptide (ELP) tags. We monitored cellular uptake and cell viability for GFP reporter proteins outfitted with a range of ELP tags and demonstrated enhanced uptake that correlates with the number of positive charges, while maintaining remarkably low cytotoxicity and resistance to degradation in the cell. GFP uptake proceeded mainly through caveolae-mediated endocytosis and we observed GFP emission inside the cells over extended time (up to 48 h). Low toxicity combined with high molecular weights of the tag opens the way to simultaneously optimize cell uptake and pharmacokinetic parameters. Thus, cationic supercharged ELP tags show great potential to improve the therapeutic profile of protein drugs leading to more efficient and safer biotherapeutics.
Cellular pathways involving ?-synuclein (?S) seem to be causative for development of Parkinsons disease. Interactions between ?S and lipid membranes appear to be important for the physiological function of the protein and influence the pathological aggregation of ?S leading to the formation of amyloid plaques. Upon membrane binding the unstructured ?S folds into amphipathic helices. In our work we characterized the penetration depth and probed the local environment of Trp-residues introduced along the ?S sequence. We could show that while the entire helix is well embedded in the lipid bilayer, segments with a shallower penetration and supposable higher flexibility exist.
Efficient selection of semiconducting SWCNTs of large diameter range (0.8-1.6 nm) on demand is demonstrated. Different diameters of SWCNT are systematically selected by tuning the alkyl side-chain lengths of the wrapping polymers of similar backbone. The exceptional quality and high concentration of the SWCNTs is validated by the outstanding optical properties and the highly performing random network ambipolar field-effect transistors.
Recombinant supercharged polypeptides (SUPs) with low cytotoxicity are developed and applied to rejuvenate the lubrication of naturally occurring salivary conditioning films (SCFs). SUPs with 72 positive charges adsorbed and rigidified the SCFs and recruited mucins to form a hydrated layer. These SCFs with SUPs have higher mechanical strength and sustain lubricating effect for longer duration compared with only SCFs.
A new amphiphilic membrane marker based on a water-soluble dendritic polyglycerol perylene imido dialkylester has been designed, synthesized, and its optical properties characterized. In water it forms fluorescently quenched micellar self-aggregates, but when incorporated into a lipophilic environment, it monomerizes, and the highly fluorescent properties of the perylene core are recovered. These properties make it an ideal candidate for the imaging of artificial and cellular membranes as demonstrated by biophysical studies.
Molecular rods consisting of a hydrophobic backbone and terminally varying functional groups have been synthesized for applications for the functionalization of membranes. In the present study, we employ a spin-labeled analogue of a recently described new class of molecular rods to characterize their dynamic interactions with membranes. By using the different approaches of ESR and NMR spectroscopy, we show that the spin moiety of the membrane-embedded spin-labeled rod is localized in the upper chain/glycerol region of membranes of different compositions. The rod is embedded within the membrane in a tilted orientation to adjust for the varying hydrophobic thicknesses of these bilayers. This orientation does not perturb the membrane structure. The water solubility of the rod is increased significantly in the presence of certain cyclodextrins. These cyclodextrins also allow the rods to be extracted from the membrane and incorporated into preformed membranes. The latter will improve the future applications of these rods in cellular systems as stable membrane-associated anchors for the functionalization of membrane surfaces.
Selective inhibition of TNFR1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, while leaving TNFR2 untouched, thus allowing for cell survival and tissue homeostasis. ATROSAB is a humanized antagonistic anti-TNFR1 antibody developed for the treatment of inflammatory diseases.
Functional interfaces of biomolecules and inorganic substrates like semiconductor materials are of utmost importance for the development of highly sensitive biosensors and microarray technology. However, there is still a lot of room for improving the techniques for immobilization of biomolecules, in particular nucleic acids and proteins. Conventional anchoring strategies rely on attaching biomacromolecules via complementary functional groups, appropriate bifunctional linker molecules, or non-covalent immobilization via electrostatic interactions. In this work, we demonstrate a facile, new, and general method for the reversible non-covalent attachment of amphiphilic DNA probes containing hydrophobic units attached to the nucleobases (lipid-DNA) onto SAM-modified gold electrodes, silicon semiconductor surfaces, and glass substrates. We show the anchoring of well-defined amounts of lipid-DNA onto the surface by insertion of their lipid tails into the hydrophobic monolayer structure. The surface coverage of DNA molecules can be conveniently controlled by modulating the initial concentration and incubation time. Further control over the DNA layer is afforded by the additional external stimulus of temperature. Heating the DNA-modified surfaces at temperatures >80 °C leads to the release of the lipid-DNA structures from the surface without harming the integrity of the hydrophobic SAMs. These supramolecular DNA layers can be further tuned by anchoring onto a mixed SAM containing hydrophobic molecules of different lengths, rather than a homogeneous SAM. Immobilization of lipid-DNA on such SAMs has revealed that the surface density of DNA probes is highly dependent on the composition of the surface layer and the structure of the lipid-DNA. The formation of the lipid-DNA sensing layers was monitored and characterized by numerous techniques including X-ray photoelectron spectroscopy, quartz crystal microbalance, ellipsometry, contact angle measurements, atomic force microscopy, and confocal fluorescence imaging. Finally, this new DNA modification strategy was applied for the sensing of target DNAs using silicon-nanowire field-effect transistor device arrays, showing a high degree of specificity toward the complementary DNA target, as well as single-base mismatch selectivity.
Influenza virus assembly and budding occur in the budozone, a coalesced raft domain in the plasma membrane. The viral transmembrane protein M2 is implicated in virus particle scission, the ultimate step in virus budding, probably by wedge-like insertion of an amphiphilic helix into the membrane. In order to do this, M2 is hypothesized to be targeted to the edge of the budozone, mediated by acylation and cholesterol binding. It was recently shown that acylation and cholesterol binding affect the membrane association of the cytoplasmic tail of M2 and targeting of the protein to coalesced rafts. This study tested whether combined removal of the acylation site (C50) and the cholesterol recognition/interaction amino acid consensus motifs (key residues Y52 and Y57) in the amphiphilic helix of M2 influenced virus formation. Recombinant influenza viruses were generated in the influenza strain A/WSN/33 background with mutations in one or both of these features. In comparison with the wild-type, all mutant viruses showed very similar growth kinetics in various cell types. Wild-type and mutant viruses differed in their relative M2 content but not regarding the major structural proteins. The morphology of the viruses was not affected by mutating M2. Moreover, wild-type and mutant viruses showed comparable competitive fitness in infected cells. Lastly, a global comparison of M2 sequences revealed that there are natural virus strains with M2 devoid of both lipid-association motifs. Taken together, these results indicate that the acylation and cholesterol-binding motifs in M2 are not crucial for the replication of influenza virus in cell culture, indicating that other factors can target M2 to the budding site.
To develop their biological activity, bioactive volatile compounds, such as pheromones or fragrances, have to evaporate from surfaces. Because these surfaces are usually exposed to natural daylight, the preparation of non-volatile precursors using photoremovable protecting groups is an ideal tool to control the release of caged volatile molecules from various surfaces by light-induced covalent bond cleavage. Many photoreactions occur under mild environmental conditions and are highly selective. To break covalent bonds under typical application conditions, the photoreaction has to proceed at ambient daylight, to tolerate the presence of oxygen and to run in polar media (e.g. in water). The amount of volatiles generated from photochemical delivery systems depends on the light intensity to which the systems are exposed. Both photoisomerisations and photofragmentations have successfully been investigated for the slow release of caged pheromones and fragrances from their corresponding precursors.
Sic1, cyclin-dependent kinase inhibitor of budding yeast, is synthesized in anaphase and largely degraded at the S-phase onset to regulate timing of DNA synthesis. Sic1 interacts with phase-specific B-type cyclin (Clb)-kinase (Cdk1) complexes, central regulators in cell cycle control. Its appearance is timed to mediate reduction in kinase activities at appropriate stages. Clbs are unstable proteins with extremely short half-lives. Interactions of Sic1 with Clbs have been detected both in vitro and in vivo by high-throughput genome-wide screenings. Furthermore, we have recently shown that Sic1 regulates waves of Clbs, acting as a timer in their appearance, thus controlling Cdk1-Clbs activation. The molecular mechanism is not yet fully understood but is hypothesized to occur via stoichiometric binding of Sic1 to Cdk1-Clb complexes. Using Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy (FLIM), we showed association of Sic1 to Clb cyclins in living yeast cells. This finding is consistent with the notion that inhibition of kinase activity can occur over the whole cell cycle progression despite variable Sic1 levels. Specifically, Sic1/Clb3 interaction was observed for the first time, and Sic1/Clb2 and Sic1/Clb5 pairs were confirmed, but no Sic1/Clb4 interaction was found, which suggests that, despite high functional homology between Clbs, only some of them can target Sic1 function in vivo.
STAT3 has important functions in both tumor cells and the tumor microenvironment to facilitate cancer progression. The STAT regulatory kinase Janus-activated kinase (JAK) has been strongly implicated in promoting oncogenesis of various solid tumors, including the use of JAK kinase inhibitors such as AZD1480. However, direct evidence that JAK drives STAT3 function and cancer pathogenesis at the level of the tumor microenvironment is yet to be established clearly. In this study, we show that AZD1480 inhibits STAT3 in tumor-associated myeloid cells, reducing their number and inhibiting tumor metastasis. Myeloid cell-mediated angiogenesis was also diminished by AZD1480, with additional direct inhibition of endothelial cell function in vitro and in vivo. AZD1480 blocked lung infiltration of myeloid cells and formation of pulmonary metastases in both mouse syngeneic experimental and spontaneous metastatic models. Furthermore, AZD1480 reduced angiogenesis and metastasis in a human xenograft tumor model. Although the effects of AZD1480 on the tumor microenvironment were important for the observed antiangiogenic activity, constitutive activation of STAT3 in tumor cells themselves could block these antiangiogenic effects, showing the complexity of the JAK/STAT signaling network in tumor progression. Together, our results indicated that AZD1480 can effectively inhibit tumor angiogenesis and metastasis mediated by STAT3 in stromal cells as well as tumor cells.
This review provides an overview of a relatively new class of bio-conjugates, DNA amphiphiles, which consist of oligonucleotides covalently bonded to synthetic hydrophobic units. The reader will find the basic principles for the structural design and preparation methods of the materials. Moreover, the self-assembly into superstructures of higher order will be highlighted. Finally, some potential applications will be described.
The development of targeted and triggerable delivery systems is of high relevance for anticancer therapies. We report here on reduction-sensitive liposomes composed of a novel multifunctional lipidlike conjugate, containing a disulfide bond and a biotin moiety, and natural phospholipids. The incorporation of the disulfide conjugate into vesicles and the kinetics of their reduction were studied using dansyl-labeled conjugate 1 in using the dansyl fluorescence environmental sensitivity and the Fo?rster resonance energy transfer from dansyl to rhodamine-labeled phospholipids. Cleavage of the disulfide bridge (e.g., by tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), l-cysteine, or glutathione (GSH)) removed the hydrophilic headgroup of the conjugate and thus changed the membrane organization leading to the release of entrapped molecules. Upon nonspecific uptake of vesicles by macrophages, calcein release from reduction-sensitive liposomes consisting of the disulfide conjugate and phospholipids was more efficient than from reduction-insensitive liposomes composed only of phospholipids. The binding of streptavidin to the conjugates did not interfere with either the subsequent reduction of the disulfide bond of the conjugate or the release of entrapped molecules. Breast cancer cell line BT-474, overexpressing the HER2 receptor, showed a high uptake of the reduction-sensitive doxorubicin-loaded liposomes functionalized with the biotin-tagged anti-HER2 antibody. The release of the entrapped cargo inside the cells was observed, implying the potential of using our system for active targeting and delivery.
The budding yeast genome comprises roughly 6000 genes generating a number of about 10?000 mRNA copies, which gives a general estimation of 1-2 mRNA copies generated per gene. What does this observation implicate for cellular processes and their regulation? Whether the number of mRNA molecules produced is important for setting the amount of proteins implicated in a particular function is at present unknown. In this context, we studied cell cycle control as one of the highly fine tuned processes that guarantee the precise timing of events essential for cell growth. Here, we developed a stochastic model that addresses the effect of varying the mRNA amount of Sic1, inhibitor of the Cdk1-Clb5 kinase activity, and the resulting noise on Sic1/Clb5 balance at the G1/S transition. We considered a range of SIC1 transcripts number according to our experimental data derived from the MS2 mRNA tagging system. Computational simulation revealed that an increased amount of SIC1 mRNAs lead to an amplified dispersion of Sic1 protein levels, suggesting mRNA control being critical to set timing of Sic1 downregulation and, therefore, S phase onset. Moreover, Sic1/Clb5 balance is strongly influenced by Clb5 production in both daughter and mother cells in order to maintain the characteristic time of S phase entry overall the population. Furthermore, CLB5 mRNA molecules calculated to reproduce temporal dynamics of Sic1 and Clb5 for daughter and mother cells agree with recent data obtained from more complex networks. Thus, the results presented here provide novel insights into the influence that the mRNA amount and, indirectly, the transcription process exploit on cell cycle progression.
DNA-polymer conjugates have been recognized as versatile functional materials in many different fields ranging from nanotechnology to diagnostics and biomedicine. They combine the favorable properties of nucleic acids and synthetic polymers. Moreover, joining both structures with covalent bonds to form bioorganic hybrids allows for the tuning of specific properties or even the possibility of evolving completely new functions. One important class of this type of material is amphiphilic DNA block copolymers, which, due to microphase separation, can spontaneously adopt nanosized micelle morphologies with a hydrophobic core and a DNA corona. These DNA nano-objects have been explored as vehicles for targeted gene and drug delivery, and also as programmable nanoreactors for organic reactions. Key to the successful realization of these potential applications is that (1) DNA block copolymer conjugates can be fabricated in a fully automated fashion by employing a DNA synthesizer; (2) hydrophobic compounds can be loaded within their interior; and (3) they can be site-specifically functionalized by a convenient nucleic acid hybridization procedure. This chapter aims to broaden the range of biodiagnostic and biomedical applications of these materials by providing a comprehensive outline of the preparation and characterization of multifunctional DNA-polymer nanoparticles.
The genetic instability of cancer cells frequently causes drug resistance. We established mouse cancer models, which allowed targeting of an oncogene by drug-mediated inactivation or monospecific CD8(+) effector T (T(E)) cells. Drug treatment of genetically unstable large tumors was effective but selected resistant clones in the long term. In contrast, T(E) cells completely rejected large tumors (?500 mm(3)), if the target antigen was cancer-driving and expressed in sufficient amounts. Although drug-mediated oncogene inactivation selectively killed the cancer cells and left the tumor vasculature intact, which likely facilitated survival and growth of resistant clones, T(E) cell treatment led to blood vessel destruction and probably "bystander" elimination of escape variants, which did not require antigen cross-presentation by stromal cells.
The influenza virus transmembrane protein M2 is a proton channel, but also plays a role in the scission of nascent virus particles from the plasma membrane. An amphiphilic helix in the CT (cytoplasmic tail) of M2 is supposed to insert into the lipid bilayer, thereby inducing curvature. Palmitoylation of the helix and binding to cholesterol via putative CRAC (cholesterol recognition/interaction amino acid consensus) motifs are believed to target M2 to the edge of rafts, the viral-budding site. In the present study, we tested pre-conditions of this model, i.e. that the CT interacts with membranes, and that acylation and cholesterol binding affect targeting of M2. M2-CT, purified as a glutathione transferase fusion protein, associated with [3H]photocholesterol and with liposomes. Mutation of tyrosine residues in the CRAC motifs prevented [(3)H]photocholesterol labelling and reduced liposome binding. M2-CT fused to the yellow fluorescent protein localized to the Golgi in transfected cells; membrane targeting was dependent on CRAC and (to a lesser extent) on palmitoylation. Preparation of giant plasma membrane vesicles from cells expressing full-length M2-GFP (green fluorescent protein) showed that the protein is partly present in the raft domain. Raft targeting required palmitoylation, but not the CRAC motifs. Thus palmitoylation and cholesterol binding differentially affect the intrinsic membrane binding of the amphiphilic helix.
The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter.
Molecular rods are synthetical molecules consisting of a hydrophobic backbone which are functionalized with varying terminal groups. Here, we report on the interaction of a recently described new class of molecular rods with lipid and biological membranes. In order to characterize this interaction, different fluorescently labeled rods were synthesized allowing for the application of fluorescence spectroscopy and microscopy based approaches. Our data show that the rods are incorporated into membranes with a perpendicular orientation to the membrane surface and enrich preferentially in liquid-disordered lipid domains. These characteristics underline that rods can be applied as stable membrane-associated anchors for functionalizing membrane surfaces.
This paper describes the preparation and characterization of polymer-protein conjugates composed of a synthetic triblock copolymer with a central polypropylene oxide (PPO) block and two terminal polyethylene oxide (PEO) segments, Pluronic F-127, and the antibacterial enzyme lysozyme attached to the telechelic groups of the PEO chains. Covalent conjugation of lysozyme proceeded via reductive amination of aldehyde functionalized PEO blocks (CHO-Pluronic) and the amine groups of the lysine residues in the protein. SDS-PAGE gel electrophoresis together with MALDI-TOF mass spectrometry analysis revealed formation of conjugates of one or two lysozyme molecules per Pluronic polymer chain. The conjugated lysozyme showed antibacterial activity towards Bacillus subtilis. Analysis with a quartz crystal microbalance with dissipation revealed that Pluronic-lysozyme conjugates adsorb in a brush conformation on a hydrophobic gold-coated quartz surface. X-ray photoelectron spectroscopy indicated surface coverage of 32% by lysozyme when adsorbed from a mixture of unconjugated Pluronic and Pluronic-lysozyme conjugate (ratio 99:1) and of 47% after adsorption of 100% Pluronic-lysozyme conjugates. Thus, bifunctional brushes were created, possessing both anti-adhesive activity due to the polymer brush, combined with the antibacterial activity of lysozyme. The coating having a lower degree of lysozyme coverage proved to be more bactericidal.
We describe the synthesis of a series of sialic acid-conjugated, polyglycerol-based nanoparticles with diameters in the range of 1-100 nm. Particle sizes were varied along with the degree of functionalization to match the corresponding virus size and receptor multiplicity in order to achieve maximum efficiency. To build up these architectures, we used biocompatible, hyperbranched polyglycerols as scaffolds and recently developed polyglycerol-based nanogels, the sizes of which can be varied between 2-4 nm and 40-100 nm, respectively. We demonstrate here that such multivalent nanoparticles inhibit influenza A virus cell binding and fusion and consequently infectivity. The potential of multivalency is evident from larger particles showing very efficient inhibition of viral infection up to 80?%. Indeed, both the size of the nanoparticle and the amount of ligand density are important determinants of inhibition efficiency. The inhibitory activity of the tested polymeric nanoparticles drastically increased with size. Particles with similar dimensions to the virus (50-100 nm) are exceedingly effective. We also observed a saturation point in degree of surface functionalization (i.e. ligand density), above which inhibition was not significantly improved. Our study emphasizes the importance of matching particle sizes and ligand densities to mimic biological surfaces and improve interactions; this is a vital concept underlying multivalent interactions.
In the presence of alkali metal cations, guanosine-5-hydrazide (1) forms stable supramolecular hydrogels by selective self-assembly into a G-quartet structure. Besides being physically trapped inside the gel structure, biologically active aldehydes or ketones can also reversibly react with the free hydrazide functions at the periphery of the G-quartet to form acylhydrazones. This particularity makes the hydrogels interesting as delivery systems for the slow release of bioactive carbonyl derivatives. Hydrogels formed from 1 were found to be significantly more stable than those obtained from guanosine. Both physical inclusion of bioactive volatiles and reversible hydrazone formation could be demonstrated by indirect methods. Gel stabilities were measured by oscillating disk rheology measurements, which showed that thermodynamic equilibration of the gel is slow and requires several cooling and heating cycles. Furthermore, combining the rheology data with dynamic headspace analysis of fragrance evaporation suggested that reversible hydrazone formation of some carbonyl compounds influences the release of volatiles, whereas the absolute stability of the gel seemed to have no influence on the evaporation rates.
Stat3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in multiple cancers. Inhibition of Stat3 signaling pathways suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting direct inhibition of Stat3 function is a viable therapeutic approach. Herein, we identify a small molecule, C48, as a selective Stat3-family member inhibitor. To determine its mechanism of action, we used site-directed mutagenesis and multiple biochemical techniques to show that C48 alkylates Cys468 in Stat3, a residue at the DNA-binding interface. We further demonstrate that C48 blocks accumulation of activated Stat3 in the nucleus in tumor cell lines that overexpress active Stat3, leading to impressive inhibition of tumor growth in mouse models. Collectively, these findings suggest Cys468 in Stat3 represents a novel site for therapeutic intervention and demonstrates the promise of alkylation as a potentially effective chemical approach for Stat3-dependent cancers.
Lysosomes, enveloped viruses, as well as synaptic and secretory vesicles are all examples of natural nanocontainers (diameter ? 100 nm) which specifically rely on their lipid bilayer to protect and exchange their contents with the cell. We have applied methods primarily based on atomic force microscopy and finite element modeling that allow precise investigation of the mechanical properties of the influenza virus lipid envelope. The mechanical properties of small, spherical vesicles made from PR8 influenza lipids were probed by an atomic force microscopy tip applying forces up to 0.2 nN, which led to an elastic deformation up to 20%, on average. The liposome deformation was modeled using finite element methods to extract the lipid bilayer elastic properties. We found that influenza liposomes were softer than what would be expected for a gel phase bilayer and highly deformable: Consistent with previous suggestion that influenza lipids do not undergo a major phase transition, we observe that the stiffness of influenza liposomes increases gradually and weakly (within one order of magnitude) with temperature. Surprisingly, influenza liposomes were, in most cases, able to withstand wall-to-wall deformation, and forces >1 nN were generally required to puncture the influenza envelope, which is similar to viral protein shells. Hence, the choice of a highly flexible lipid envelope may provide as efficient a protection for a viral genome as a stiff protein shell.
Cholesterol with BODIPY at carbon-24 of the side chain (BCh2) has recently been introduced as new cholesterol probe with superior fluorescence properties. We compare BCh2 with the intrinsically fluorescent dehydroergosterol (DHE), a well-established marker for cholesterol, by introducing simultaneous imaging of both sterols in model membranes and living cells. BCh2 had a lower affinity than DHE for the biologically relevant liquid-ordered phase in model membranes. Still, DHE and BCh2 trafficked from the plasma membrane to the endocytic recycling compartment (ERC) of BHK cells with identical kinetics. This transport pathway was strongly reduced after energy depletion of cells or expression of the dominant-negative clathrin heavy chain. The partitioning into lipid droplets of BHK and HeLa cells was higher for BCh2 than for DHE. Within droplets, the photodegradation of BCh2 was enhanced and followed a stretched exponential decay, while the fluorescence lifetime of BCh2 was comparable in various cellular regions. Our results indicate that BCh2 is suitable for analyzing sterol uptake pathways and inter-organelle sterol flux in living cells. The BODIPY-moiety affects lipid phase preference of the sterol probe and causes some differential targeting of BCh2 and DHE in cells with high fat content.
Ultra-high molecular weight DNA/polymer hybrid materials were prepared employing molecular biology techniques. Nucleic acid restriction and ligation enzymes were used to generate linear DNA di- and triblock copolymers that contain up to thousands of base pairs in the DNA segments.
The K-Ras4B GTPase is a major oncoprotein whose signaling activity depends on its correct localization to negatively charged subcellular membranes and nanoclustering in membrane microdomains. Selective localization and clustering are mediated by the polybasic farnesylated C-terminus of K-Ras4B, but the mechanisms and molecular determinants involved are largely unknown. In a combined chemical biological and biophysical approach we investigated the partitioning of semisynthetic fully functional lipidated K-Ras4B proteins into heterogeneous anionic model membranes and membranes composed of viral lipid extracts. Independent of GDP/GTP-loading, K-Ras4B is preferentially localized in liquid-disordered (l(d)) lipid domains and forms new protein-containing fluid domains that are recruiting multivalent acidic lipids by an effective, electrostatic lipid sorting mechanism. In addition, GDP-GTP exchange and, thereby, Ras activation results in a higher concentration of activated K-Ras4B in the nanoscale signaling platforms. Conversely, palmitoylated and farnesylated N-Ras proteins partition into the l(d) phase and concentrate at the l(d)/l(o) phase boundary of heterogeneous membranes. Next to the lipid anchor system, the results reveal an involvement of the G-domain in the membrane interaction process by determining minor but yet significant structural reorientations of the GDP/GTP-K-Ras4B proteins at lipid interfaces. A molecular mechanism for isoform-specific Ras signaling from separate membrane microdomains is postulated from the results of this study.
Adoptive cell therapy with engineered T cells to improve natural immune response and antitumor functions has shown promise for treating cancer. However, the requirement for extensive ex vivo manipulation of T cells and the immunosuppressive effects of the tumor microenvironment limit this therapeutic modality. In the present study, we investigated the possibility to circumvent these limitations by engineering Stat3 -deficient CD8(+) T cells or by targeting Stat3 in the tumor microenvironment. We show that ablating Stat3in CD8(+) T cells prior to their transfer allows their efficient tumor infiltration and robust proliferation, resulting in increased tumor antigen-specific T-cell activity and tumor growth inhibition. For potential clinical translation, we combined adoptive T-cell therapy with a Food and Drug Administration-approved tyrosine kinase inhibitor, sunitinib, in renal cell carcinoma and melanoma tumor models. Sunitinib inhibited Stat3 in dendritic cells and T cells and reduced conversion of transferred FoxP3(-) T cells to tumor-associated regulatory T cells while increasing transferred CD8(+) T-cell infiltration and activation at the tumor site, leading to inhibition of primary tumor growth. These data show that adoptively transferred T cells can be expanded and activated in vivo either by engineering Stat3-silenced T cells or by targeting Stat3 systemically with small-molecule inhibitors.
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