Oxidative damages are linked to several aging-related diseases and are among the chemical pathways determining protein degradation. Specifically, interplay of oxidative stress and protein aggregation ...is recognized to have a link to the loss of cellular function in pathologies like Alzheimer's and Parkinson's diseases. Interaction between protein and reactive oxygen species may indeed induce small changes in protein structure and lead to the inhibition/modification of protein aggregation process, potentially determining the formation of species with different inherent toxicity. Understanding the temperate relationship between these events can be of utmost importance in unraveling the molecular basis of neurodegeneration. In this work, we investigated the effect of hydrogen peroxide oxidation on Human Serum Albumin (HSA) structure, thermal stability and aggregation properties. In the selected conditions, HSA forms fibrillar aggregates, while the oxidized protein undergoes aggregation via new routes involving, in different extents, specific domains of the molecule. Minute variations due to oxidation of single residues affect HSA tertiary structure leading to protein compaction, increased thermal stability, and reduced association propensity.
Continuous progress has been made in the development of new molecules for therapeutic purposes. This is driven by the need to address several challenges such as molecular instability and ...biocompatibility, difficulties in crossing the plasma membrane, and the development of host resistance. In this context, cell-penetrating peptides (CPPs) constitute a promising tool for the development of new therapies due to their intrinsic ability to deliver therapeutic molecules to cells and tissues. These short peptides have gained increasing attention for applications in drug delivery as well as for their antimicrobial and anticancer activity but the general rules regulating the events involved in cellular uptake and in the following processes are still unclear. Here, we use fluorescence microscopy methods to analyze the interactions between the multifunctional peptide Transportan 10 (TP10) and the giant plasma membrane vesicles (GPMVs) derived from cancer cells. This aims to highlight the molecular mechanisms underlying functional interactions which bring its translocation across the membrane or cytotoxic mechanisms leading to membrane collapse and disruption. The Fluorescence Lifetime Imaging Microscopy (FLIM) method coupled with the phasor approach analysis proved to be the winning choice for following highly dynamic spatially heterogeneous events in real-time and highlighting aspects of such complex phenomena. Thanks to the presented approach, we were able to identify and monitor TP10 translocation into the lumen, internalization, and membrane-induced modifications depending on the peptide concentration regime.
An overview of the biophysics activity at the Department of Physics and Chemistry Emilio Segrè of the University of Palermo is given. For forty years, the focus of the research has been on the ...protein structure–dynamics–function paradigm, with the aim of understanding the molecular basis of the relevant mechanisms and the key role of solvent. At least three research lines are identified; the main results obtained in collaboration with other groups in Italy and abroad are presented. This review is dedicated to the memory of Professors Massimo Ugo Palma, Maria Beatrice Palma Vittorelli, and Lorenzo Cordone, which were the founders of the Palermo School of Biophysics. We all have been, directly or indirectly, their pupils; we miss their enthusiasm for scientific research, their deep physical insights, their suggestions, their strict but always constructive criticisms, and, most of all, their friendship. This paper is dedicated also to the memory of Prof. Hans Frauenfelder, whose pioneering works on nonexponential rebinding kinetics, protein substates, and energy landscape have inspired a large part of our work in the field of protein dynamics.
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•Inflammatory S100A9 was found in controlled cortical impact mouse model of focal TBI.•S100A9 was produced by neuronal and microglial cells.•S100A9 aggregated intracellularly into ...amyloid oligomers.•In vitro S100A9 self-assembles into amyloid oligomers within minutes.•S100A9 amyloid aggregation is dependent on environmental conditions.
Traumatic brain injury (TBI) represents a significant risk factor for development of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. The S100A9-driven amyloid-neuroinflammatory cascade occurring during primary and secondary TBI events can serve as a mechanistic link between TBI and Alzheimer’s as demonstrated recently in the human brain tissues. Here by using immunohistochemistry in the controlled cortical impact TBI mouse model we have found pro-inflammatory S100A9 in the brain tissues of all mice on the first and third post-TBI days, while 70% of mice did not show any S100A9 presence on seventh post-TBI day similar to controls. This indicates that defensive mechanisms effectively cleared S100A9 in these mouse brain tissues during post-TBI recovery. By using sequential immunohistochemistry we have shown that S100A9 was produced by both neuronal and microglial cells. However, Aβ peptide deposits characteristic for Alzheimer’s disease were not detected in any post-TBI animals. On the first and third post-TBI days S100A9 was found to aggregate intracellularly into amyloid oligomers, similar to what was previously observed in human TBI tissues. Complementary, by using Rayleigh scatting, intrinsic fluorescence and atomic force microscopy we demonstrated that in vitro S100A9 self-assembles into amyloid oligomers within minutes. Its amyloid aggregation is highly dependent on changes of environmental conditions such as variation of calcium levels, pH, temperature and reduction/oxidation, which might be relevant to perturbation of cellular and tissues homeostasis under TBI. Present results demonstrate that S100A9 induction mechanisms in TBI are similar in mice and humans, emphasizing that S100A9 is an important marker of brain injury and therefore can be a potential therapeutic target.
We report a kinetic study on thermal aggregation process of the model protein bovine serum albumin (BSA) in low concentration regime. Aim of this study is to provide information on relationship ...between conformational changes and initial step of aggregation. The experimental approach is based on steady-state fluorescence spectra of the two tryptophans located in two different domains, in way to study conformational changes in the surrounding of these residues. We also follow emission spectra of Fluorescein-5-Maleimide dye bound to the single free cysteine of BSA. Complementary information on the extent of aggregation and on the structural changes is obtained by Rayleigh scattering and circular dichroism measurements. These data contribute to clarify the connection between conformational changes at tertiary and secondary structure level during the aggregation and how the different domains are involved. We also discuss the relevant role played by cysteine 34 in the aggregation pathways.
Some marine organisms can resist to aqueous tidal environments and adhere tightly on wet surface. This behavior has raised increasing attention for potential applications in medicine, biomaterials, ...and tissue engineering. In mussels, adhesive forces to the rock are the resultant of proteinic fibrous formations called byssus. We present the solution structure of Pvfp-5β, one of the three byssal plaque proteins secreted by the Asian green mussel Perna viridis, and the component responsible for initiating interactions with the substrate. We demonstrate that Pvfp-5β has a stably folded structure in agreement with the presence in the sequence of two EGF motifs. The structure is highly rigid except for a few residues affected by slow local motions in the µs-ms time scale, and differs from the model calculated by artificial intelligence methods for the relative orientation of the EGF modules, which is something where computational methods still underperform. We also show that Pvfp-5β is able to coacervate even with no DOPA modification, giving thus insights both for understanding the adhesion mechanism of adhesive mussel proteins, and developing of biomaterials.
Oral 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).
Protein aggregation is one of the most challenging topics in life sciences, and it is implicated in several human pathologies. The nature and the role of toxic species is highly debated, with amyloid ...fibrils being among the most relevant species for their peculiar structural and functional properties. Protein dynamics and in particular the ability to fluctuate through a large number of conformational substates are closely related to protein function. This Letter focuses on amyloid fibril dynamics, and, to our knowledge, it is the first neutron scattering study on a protein (Concanavalin A) isolated in its fibril state. Our results reveal enhanced atomic fluctuations in amyloid fibrils and indicate that the protein is “softer” in the fibril state with respect to the native and amorphous aggregate states. We discuss this finding in terms of a structural interpretation and suggest that the paradigm ordered structure ↔ lower flexibility can be questioned when considering the local fast side-chain protein dynamics.
Understanding the early events during amyloid aggregation processes is crucial to single out the involved molecular mechanisms and for designing ad hoc strategies to prevent and reverse amyloidogenic ...disorders. Here, we show that, in conditions in which the protein is positively charged and its conformational flexibility is enhanced, Concanavalin A leads to fibril formation via a non-conventional aggregation pathway. Using a combination of light scattering, circular dichroism, small angle X-ray scattering, intrinsic (Tryptophan) and extrinsic (ANS) fluorescence and confocal and 2-photon fluorescence microscopy we characterize the aggregation process as a function of the temperature. We highlight a multi-step pathway with the formation of an on-pathway long-lived intermediate and a subsequent coagulation of such "crinkled" precursors into amyloid-like fibrils. The process results in a temperature-dependent aggregation-coagulation pathway, with the late phase of coagulation determined by the interplay between hydrophobic and electrostatic forces. Our data provide evidence for the complex aggregation pathway for a protein with a highly flexible native conformation. We demonstrate the possibility to generate a long-lived intermediate whose proportion and occurrence are easily tunable by experimental parameters (i.e. temperature). As a consequence, in the case of aggregation processes developing through well-defined energy barriers, our results can open the way to new strategies to induce more stable in vitro on-pathway intermediate species through a minute change in the initial conformational flexibility of the protein. This will allow isolating and experimentally studying such transient species, often indicated as relevant in neurodegenerative diseases, both in terms of structural and cytotoxic properties.
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