RESUMO
Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Plantas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Microscopia Crioeletrônica , Processamento de Imagem Assistida por Computador , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Mutação , Mutação Puntual , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/metabolismo , Espalhamento de Radiação , Raios XRESUMO
Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.
Assuntos
Colesterol , Sistemas de Liberação de Medicamentos/métodos , Ácido Fólico , Bicamadas Lipídicas , Peptídeos , Fosfolipídeos , Células CACO-2 , Colesterol/química , Colesterol/farmacologia , Ácido Fólico/química , Ácido Fólico/farmacologia , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/farmacologia , Lipossomos , Peptídeos/química , Peptídeos/farmacologia , Fosfolipídeos/química , Fosfolipídeos/farmacologiaRESUMO
Polymer-shelled magnetic microbubbles have great potential as hybrid contrast agents for ultrasound and magnetic resonance imaging. In this work, we studied US/MRI contrast agents based on air-filled poly(vinyl alcohol)-shelled microbubbles combined with superparamagnetic iron oxide nanoparticles (SPIONs). The SPIONs are integrated either physically or chemically into the polymeric shell of the microbubbles (MBs). As a result, two different designs of a hybrid contrast agent are obtained. With the physical approach, SPIONs are embedded inside the polymeric shell and with the chemical approach SPIONs are covalently linked to the shell surface. The structural design of hybrid probes is important, because it strongly determines the contrast agent's response in the considered imaging methods. In particular, we were interested how structural differences affect the shell's mechanical properties, which play a key role for the MBs' US imaging performance. Therefore, we thoroughly characterized the MBs' geometric features and investigated low-frequency mechanics by using atomic force microscopy (AFM) and high-frequency mechanics by using acoustic tests. Thus, we were able to quantify the impact of the used SPIONs integration method on the shell's elastic modulus, shear modulus and shear viscosity. In summary, the suggested approach contributes to an improved understanding of structure-property relations in US-active hybrid contrast agents and thus provides the basis for their sustainable development and optimization.
RESUMO
Microbubbles (MBs) are commonly used as injectable ultrasound contrast agent (UCA) in modern ultrasonography. Polymer-shelled UCAs present additional potentialities with respect to marketed lipid-shelled UCAs. They are more robust; that is, they have longer shelf and circulation life, and surface modifications are quite easily accomplished to obtain enhanced targeting and local drug delivery. The next generation of UCAs will be required to support not only ultrasound-based imaging methods but also other complementary diagnostic approaches such as magnetic resonance imaging or computer tomography. This work addresses the features of MBs that could function as contrast agents for both ultrasound and magnetic resonance imaging. The results indicate that the introduction of iron oxide nanoparticles (SPIONs) in the poly(vinyl alcohol) shell or on the external surface of the MBs does not greatly decrease the echogenicity of the host MBs compared with the unmodified one. The presence of SPIONs provides enough magnetic susceptibility to the MBs to accomplish good detectability both in vitro and in vivo. The distribution of SPIONs on the shell and their aggregation state seem to be key factors for the optimization of the transverse relaxation rate.
Assuntos
Meios de Contraste , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita , Microbolhas , Tomografia Computadorizada por Raios X , Meios de Contraste/química , Imageamento por Ressonância Magnética/instrumentação , Nanopartículas de Magnetita/química , Tamanho da Partícula , Tomografia Computadorizada por Raios X/instrumentaçãoRESUMO
Nano-drugs based on nanoparticles (NP) or on nano-assemblies as carriers of the active pharmaceutical ingredient (API) are often expected to perform better compared to conventional dosage forms. Maximum realization of this potential though requires optimization of multiple physico-chemical, including structural and morphological, parameters. Meaningful distributions of these parameters derived from sufficient populations of individual NPs rather than ensemble distributions are desirable for this task, provided that relevant high-resolution data is available. In this study we demonstrate powerful capabilities of the up-to-date cryogenic transmission electron-microscopy (cryo-TEM) as well as correlations with other techniques abundant in the nano-research milieu. We explored Doxil®-like (an anticancer drug and the first FDA-approved nano-drug) (75-100 nm) PEGylated liposomes encapsulating single doxorubicin-sulfate nano-rod-crystals (PLD). These crystals induce liposome sphere-to-ellipsoid deformation. Doxil® was characterized by a multitude of physicochemical methods. We demonstrate, that accompanied by advanced image-analysis means, cryo-TEM can successfully enable the determination of multiple structural parameters of such complex liposomal nano-drugs with an added value of statistically-sound distributions. The latter could not be achieved by most other physicochemical approaches. It seems that cryo-TEM is capable of quantitative description of individual liposome morphological features, including meaningful distributions of all structural elements, with averages that correlate with other physical methods. Here it is demonstrated that such quantitative cryo-TEM analysis is a powerful tool in determining what is the optimal drug to lipid ratio in PLD, which is found to be the drug to lipid ratio existing in Doxil®.
RESUMO
The remarkably efficient suppression of amyloid fibril formation by the DNAJB6 chaperone is dependent on a set of conserved S/T-residues and an oligomeric structure, features unusual among DNAJ chaperones. We explored the structure of DNAJB6 using a combination of structural methods. Lysine-specific crosslinking mass spectrometry provided distance constraints to select a homology model of the DNAJB6 monomer, which was subsequently used in crosslink-assisted docking to generate a dimer model. A peptide-binding cleft lined with S/T-residues is formed at the monomer-monomer interface. Mixed isotope crosslinking showed that the oligomers are dynamic entities that exchange subunits. The purified protein is well folded, soluble and composed of oligomers with a varying number of subunits according to small-angle X-ray scattering (SAXS). Elongated particles (160 × 120 Å) were detected by electron microscopy and single particle reconstruction resulted in a density map of 20 Å resolution into which the DNAJB6 dimers fit. The structure of the oligomer and the S/T-rich region is of great importance for the understanding of the function of DNAJB6 and how it can bind aggregation-prone peptides and prevent amyloid diseases.
Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Proteínas de Choque Térmico HSP40/química , Chaperonas Moleculares/química , Proteínas do Tecido Nervoso/química , Conformação Proteica , Amiloide/genética , Peptídeos beta-Amiloides/genética , Fenômenos Biofísicos , Proteínas de Choque Térmico HSP40/genética , Humanos , Lisina/química , Espectrometria de Massas , Modelos Estruturais , Chaperonas Moleculares/genética , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/genética , Ligação Proteica/genética , Multimerização Proteica , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
INTRODUCTION: Both diagnostic ultrasound (US) and magnetic resonance imaging (MRI) accuracy can be improved by using contrast enhancement. For US gas-filled microbubbles (MBs) or silica nanoparticles (SiNPs), and for MRI superparamagnetic or paramagnetic agents, contribute to this. However, interactions of MBs with the vascular wall and cells are not fully known for all contrast media. METHODS: We studied the in vitro interactions between three types of non-targeted air-filled MBs with a polyvinyl-alcohol shell and murine macrophages or endothelial cells. The three MB types were plain MBs and two types that were labelled (internally and externally) with superparamagnetic iron oxide nanoparticles (SPIONs) for US/MRI bimodality. Cells were incubated with MBs and imaged by microscopy to evaluate uptake and adhesion. Interactions were quantified and the MB internalization was confirmed by fluorescence quenching of non-internalized MBs. RESULTS: Macrophages internalized each MB type within different time frames: plain MBs 6 h, externally labelled MBs 25 min and internally labelled MBs 2 h. An average of 0.14 externally labelled MBs per cell were internalized after 30 min and 1.34 after 2 h; which was 113% more MBs than the number of internalized internally labelled MBs. The macrophages engulfed these three differently modified new MBs at various rate, whereas endothelial cells did not engulf MBs. CONCLUSIONS: Polyvinyl-alcohol MBs are not taken up by endothelial cells. The MB uptake by macrophages is promoted by SPION labelling, in particular external such, which may be important for macrophage targeting.
RESUMO
Intravenously injected microbubbles (MBs) can be utilized as ultrasound contrast agent (CA) resulting in enhanced image quality. A novel CA, consisting of air filled MBs stabilized with a shell of polyvinyl alcohol (PVA) has been developed. These spherical MBs have been decorated with superparamagnetic iron oxide nanoparticles (SPIONs) in order to serve as both ultrasound and magnetic resonance imaging (MRI) CA. In this study, a mathematical model was introduced that determined the shell thickness of two types of SPIONs decorated MBs (Type A and Type B). The shell thickness of MBs is important to determine, as it affects the acoustical properties. In order to investigate the shell thickness, thin sections of plastic embedded MBs were prepared and imaged using transmission electron microscopy (TEM). However, the sections were cut at random distances from the MB center, which affected the observed shell thickness. Hence, the model determined the average shell thickness of the MBs from corrected mean values of the outer and inner radii observed in the TEM sections. The model was validated using simulated slices of MBs with known shell thickness and radius. The average shell thickness of Type A and Type B MBs were 651nm and 637nm, respectively.
RESUMO
The current study describes novel multifunctional polymer-shelled microbubbles (MBs) loaded with nitric oxide (NO) for integrated therapeutic and diagnostic applications (ie, theranostics) of myocardial ischemia. We used gas-filled MBs with an average diameter of 4 µm stabilized by a biocompatible shell of polyvinyl alcohol. In vitro acoustic tests showed sufficient enhancement of the backscattered power (20 dB) acquired from the MBs' suspension. The values of attenuation coefficient (0.8 dB/cm MHz) and phase velocities (1,517 m/s) were comparable with those reported for the soft tissue. Moreover, polymer MBs demonstrate increased stability compared with clinically approved contrast agents with a fracture threshold of about 900 kPa. In vitro chemiluminescence measurements demonstrated that dry powder of NO-loaded MBs releases its gas content in about 2 hours following an exponential decay profile with an exponential time constant equal to 36 minutes. The application of high-power ultrasound pulse (mechanical index =1.2) on the MBs resuspended in saline decreases the exponential time constant from 55 to 4 minutes in air-saturated solution and from 17 to 10 minutes in degassed solution. Thus, ultrasound-triggered release of NO is achieved. Cytotoxicity tests indicate that phagocytosis of the MBs by macrophages starts within 6-8 hours. This is a suitable time for initial diagnostics, treatment, and monitoring of the therapeutic effect using a single injection of the proposed multifunctional MBs.
Assuntos
Meios de Contraste/química , Óxido Nítrico/farmacologia , Nanomedicina Teranóstica , Ultrassom/métodos , Química Farmacêutica , Meios de Contraste/efeitos adversos , Equipamentos e Provisões , Gases , Humanos , Técnicas In Vitro , Macrófagos/efeitos dos fármacos , Microbolhas , Isquemia Miocárdica/diagnóstico , Isquemia Miocárdica/tratamento farmacológico , Óxido Nítrico/química , Tamanho da Partícula , Fagocitose , PósRESUMO
Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific folate receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy.
Assuntos
Receptores de Folato com Âncoras de GPI/efeitos dos fármacos , Nanopartículas , Proteínas/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linhagem Celular , Sistemas de Liberação de Medicamentos , Emulsões , Ácido Fólico/metabolismo , Humanos , Tamanho da Partícula , Poloxâmero , Polietilenoglicóis , Polímeros , Propilenoglicóis , Proteínas/farmacologia , Soroalbumina Bovina/químicaRESUMO
Expanded polyglutamine (polyQ) stretches lead to protein aggregation and severe neurodegenerative diseases. A highly efficient suppressor of polyQ aggregation was identified, the DNAJB6, when molecular chaperones from the HSPH, HSPA, and DNAJ families were screened for huntingtin exon 1 aggregation in cells (Hageman et al. in Mol Cell 37(3):355-369, 2010). Furthermore, also aggregation of polyQ peptides expressed in cells was recently found to be efficiently suppressed by co-expression of DNAJB6 (Gillis et al. in J Biol Chem 288:17225-17237, 2013). These suppression effects can be due to an indirect effect of DNAJB6 on other cellular components or to a direct interaction between DNAJB6 and polyQ peptides that may depend on other cellular components. Here, we have purified the DNAJB6 protein to investigate the suppression mechanism. The purified DNAJB6 protein formed large heterogeneous oligomers, in contrast to the more canonical family member DNAJB1 which is dimeric. Purified DNAJB6 protein, at substoichiometric molar ratios, efficiently suppressed fibrillation of polyQ peptides with 45°Q in a thioflavin T fibrillation. No suppression was obtained with DNAJB1, but with the closest homologue to DNAJB6, DNAJB8. The suppression effect was independent of HSPA1 and ATP. These data, based on purified proteins and controlled fibrillation in vitro, strongly suggest that the fibrillation suppression is due to a direct protein-protein interaction between the polyQ peptides and DNAJB6 and that the DNAJB6 has unique fibrillation suppression properties lacking in DNAJB1. Together, the data obtained in cells and in vitro support the view that DNAJB6 is a peptide-binding chaperone that can interact with polyQ peptides that are incompletely degraded by and released from the proteasome.
Assuntos
Amiloide/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Amiloide/ultraestrutura , Proteínas de Choque Térmico HSP40/química , Humanos , Luz , Chaperonas Moleculares/química , Dados de Sequência Molecular , Peso Molecular , Proteínas do Tecido Nervoso/química , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espalhamento de Radiação , Dodecilsulfato de Sódio/farmacologia , SolubilidadeRESUMO
Liposomes and protein based nanoparticles were tuned with different polymers and glycolipids to improve stealth and thus decrease their clearance by macrophages. Liposomes were coated with polyethylene glycol (PEG) and brain-tissue-derived monosialoganglioside (GM1). Bovine serum albumin (BSA) nanoparticles were produced incorporating a PEGylated surfactant (PEG-surfactant). All obtained nanoparticles were monodisperse, with sizes ranging from 80 to 120 nm, with a zeta-potential close to zero. The presented stealth strategies lead to a decrease of internalization levels by macrophages. These surface modified nanoparticles could be used for production of new drug delivery nanosystems for systemic administration (e.g. intravenous application).