1,720,973 research outputs found
Protein-membrane interaction: insights from advanced microscopy
Full text linkThe interaction between proteins and membranes is sub ject of renewed interest in biomedical and biotechnologi cal research for its implication in many functional and dys functional processes and for its pharmaceutical applications.
It has been shown that the interaction between amyloido genic proteins and membranes results in mutually destruc tive structural perturbations. The study we present is focused on the interaction between synthetic model membranes
and alpha-lactalbumin (α-La), widely studied for its biolog ical function since it can induce apoptosis in tumor cells.
Upon α-La addition to giant vesicles (GVs) samples, the sys tem has been characterized by means of spectroscopy meth ods and advanced microscopy techniques. Using Raster Image Correlation Spectroscopy (RICS) and Fluorescence Life time Imaging Microscopy (FLIM), the interaction has been investigated at different protein:lipid ratios. Starting from the molten globule conformation, a quick insertion of α-La into the lipid bilayer takes place, with evident changes in
GVs morphology as well as in protein structure. The pro cess, ruled by a combination of electrostatic and hydrophobic interactions, ends with the formation of heterogeneous struc tures containing both protein and lipids.
Eur Biophys J (2017) 46 (Suppl 1):S43–S402 S351 1
Protein materials as sustainable non- and minimally invasive strategies for biomedical applications
Full text linkProtein-based materials have found applications in a wide range of biomedical fields because of their biocompatibility, biodegradability and great versatility. Materials of different physical forms including particles, hydrogels, films, fibers and microneedles have been fabricated e.g. as carriers for drug delivery, factors to promote wound healing and as structural support for the generation of new tissue. This review aims at providing an overview of the current scientific knowledge on protein-based materials, and selected preclinical and clinical studies will be reviewed in depth as examples of the latest progress within the field of protein-based materials, specifically focusing on non- and minimally invasive strategies mainly for topical application
α-casein micelles-membranes interaction: Flower-like lipid protein coaggregates formation
No full textBackground: Environmental conditions regulate the association/aggregation states of proteins and their action in cellular compartments. Analysing protein behaviour in presence of lipid membranes is fundamental for the comprehension of many functional and dysfunctional processes. Here, we present an experimental study on the interaction between model membranes and α-casein. α-casein is the major component of milk proteins and it is recognised to play a key role in performing biological functions. The conformational properties of this protein and its capability to form supramolecular structures, like micelles or irreversible aggregates, are key effectors in functional and pathological effects. Methods: By means of quantitative fluorescence imaging and complementary spectroscopic methods, we were able to characterise α-casein association state and the course of events induced by pH changes, which regulate the interaction of this molecule with membranes. Results: The study of these complex dynamic events revealed that the initial conformation of the protein critically regulates the fate of α-casein, size and structure of the newly formed aggregates and their effect on membrane structures. Disassembly of micelles due to modification in electrostatic interactions results in increased membrane structure rigidity which accompanies the formation of protein lipid flower-like co-aggregates with protein molecules localised in the external part. General significance: These results may contribute to the comprehension of how the initial state of a protein establishes the course of events that occur upon changes in the molecular environment. These events which may occur in cells may be essential to functional, pathological or therapeutical properties specifically associated to casein proteins
Increased carrier peptide stability through ph adjustment improves insulin and pth(1-34) delivery in vitro and in vivo rather than by enforced carrier peptide-cargo complexation
Full text linkOral delivery of therapeutic peptides is hampered by their large molecular size and labile nature, thus limiting their permeation across the intestinal epithelium. Promising approaches to overcome the latter include co-administration with carrier peptides. In this study, the cell-penetrating peptide penetratin was employed to investigate effects of co-administration with insulin and the pharmacologically active part of parathyroid hormone (PTH(1-34)) at pH 5, 6.5, and 7.4 with respect to complexation, enzymatic stability, and transepithelial permeation of the therapeutic peptide in vitro and in vivo. Complex formation between insulin or PTH(1-34) and penetratin was pH-dependent. Micron-sized complexes dominated in the samples prepared at pH-values at which penetratin interacts electrostatically with the therapeutic peptide. The association efficiency was more pronounced between insulin and penetratin than between PTH(1-34) and penetratin. Despite the high degree of complexation, penetratin retained its membrane activity when applied to liposomal structures. The enzymatic stability of penetratin during incubation on polarized Caco-2 cell monolayers was pH-dependent with a prolonged half-live determined at pH 5 when compared to pH 6.5 and 7.4. Also, the penetratin-mediated transepithelial permeation of insulin and PTH(1-34) was increased in vitro and in vivo upon lowering the sample pH from 7.4 or 6.5 to 5. Thus, the formation of penetratin-cargo complexes with several molecular entities is not prerequisite for penetratin-mediated transepithelial permeation a therapeutic peptide. Rather, a sample pH, which improves the penetratin stability, appears to optimize the penetratin-mediated transepithelial permeation of insulin and PTH(1-34)
Pulsatile protein release and protection using radiation-crosslinked polypeptide hydrogel delivery devices
Full text linkIn the recent years recombinant technology has identified numerous protein based therapeutics. Their
effective delivery, though, can be challenging due to the poor stability of most proteins along their pathway
to the target site in the body. Hydrogels have been identified as good candidates for protein encapsulation
and delivery thanks to both material and manufacturing process advantages. In this work we
propose high energy irradiation as a synthetic methodology of choice to engineer hydrogel-based
delivery devices for encapsulation and pulsatile release of proteins, triggered by pH, and for prevention
of their denaturation when encapsulated. In particular, maleic anhydride functionalised poly(N-2-
HydroxyEthyl)-DL-Aspartamide (PHEA-MA) hydrogels have been crosslinked without the use of toxic
reagents or catalysts and in mild conditions via gamma irradiation. At the variance of the irradiation conditions,
hydrogels with dramatically different crosslinked structure, thus rheological properties and
swelling behaviour, have been obtained. The ability to swell and shrink cyclically upon repeated pH
jumps and the absence of cytotoxicity have been demonstrated for all the hydrogels produced. Moreover
some of the variants exhibited full degradability at 37 C with degradation products that are not-toxic for
the cell. Depending on the networks average mesh size, as derived by the treatment of rheological data
with simple rubber elasticity equations, with respect to the characteristic dimension of the chosen model
protein, substantial loading of the protein and its retention or release, controlled by pH, have been
achieved. These results, coupled with the versatility of the synthetic platform, suggest the possibility
to use these materials as components of intelligent/programmable devices specifically designed as to
release theoretically any protein based therapeutic
Concanavalin A fibrils formation from Coagulation of Long-lived" Crinkled" Intermediates
No full textPeptide-Membrane Interactions Monitored by Fluorescence Lifetime Imaging: A Study Case of Transportan 10
Full text linkThe interest on detailed analysis of peptide-membrane interactions is of great interest in both fundamental and applied sciences as these may relate to both functional and pathogenic events. Such interactions are highly dynamic and spatially heterogeneous, making the investigation of the associated phenomena highly complex. The specific properties of membranes and peptide structural details, together with environmental conditions, may determine different events at the membrane interface, which will drive the fate of the peptide-membrane system. Here, we use an experimental approach based on the combination of spectroscopy and fluorescence microscopy methods to characterize the interactions of the multifunctional amphiphilic peptide transportan 10 with model membranes. Our approach, based on the use of suitable fluorescence reporters, exploits the advantages of phasor plot analysis of fluorescence lifetime imaging microscopy measurements to highlight the molecular details of occurring membrane alterations in terms of rigidity and hydration. Simultaneously, it allows following dynamic events in real time without sample manipulation distinguishing, with high spatial resolution, whether the peptide is adsorbed to or inserted in the membrane
Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly
Full text linkEnsembles of protein aggregates are characterized by a nano- and micro-scale heterogeneity of the species. This diversity translates into a variety of effects that protein aggregates may have in biological systems, both in connection to neurodegenerative diseases and immunogenic risk of protein drug products. Moreover, this naturally occurring variety offers unique opportunities in the field of protein-based biomaterials. In the above-mentioned fields, the isolation and structural analysis of the different amyloid types within the same ensemble remain a priority, still representing a significant experimental challenge. Here we address such complexity in the case of insulin for its relevance as biopharmaceutical and its involvement in insulin-derived amyloidosis. By combining Fourier Transform Infrared Microscopy (micro-FTIR) and fluorescence lifetime imaging microscopy (FLIM) we show the occurrence, within the same ensemble of insulin protein aggregates, of a variable β-structure architecture and content not only dependent on the species analyzed (spherulites or fibrils), but also on the position within a single spherulite at submicron scale. We unambiguously reveal that the surface of the spherulites are characterized by β-structures with an enhanced H-bond coupling compared to the core. This information, inaccessible via bulk methods, allows us to relate the aggregate structure at molecular level to the overall morphology of the aggregates. Our findings robustly solve the problem of probing the ensemble and single particle heterogeneity of amyloid samples. Furthermore, we offer a unique, scalable and ready-to-use screening methodology for in-depth characterization of self-assembled structures, being this translatable to material sciences, drug quality control and clinical imaging of amyloid-affected tissues
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