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Neurodegenerative diseases (NDs) represent an unsolved problem to date with an ever-increasing population incidence. Particularly, Alzheimer's disease (AD) is the most widespread ND characterized by an accumulation of amyloid aggregates of beta-amyloid (Aß) and Tau proteins that lead to neuronal death and subsequent cognitive decline. Although neuroimaging techniques are needed to diagnose AD, the investigation of biomarkers within body fluids could provide important information on neurodegeneration. Indeed, as there is no definitive solution for AD, the monitoring of these biomarkers is of strategic importance as they are useful for both diagnosing AD and assessing the progression of the neurodegenerative state. In this context, exercise is known to be an effective non-pharmacological management strategy for AD that can counteract cognitive decline and neurodegeneration. However, investigation of the concentration of fluid biomarkers in AD patients undergoing exercise protocols has led to unclear and often conflicting results, suggesting the need to clarify the role of exercise in modulating fluid biomarkers in AD. Therefore, this critical literature review aims to gather evidence on the main fluid biomarkers of AD and the modulatory effects of exercise to clarify the efficacy and usefulness of this non-pharmacological strategy in counteracting neurodegeneration in AD.

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Amyloid aggregates have attracted significant interest in regard to diverse biomedical applications, particularly in the field of drug delivery. Here, we report novel amyloid aggregates based on a 12-amino-acid peptide from the amyloidogenic region of the receptor-interacting kinase 3 (RIP3) protein and a thermoresponsive triblock copolymer, namely, Pluronic F127 (RIP3/F127). Physicochemical characterization was performed to determine the aggregation size, morphology, and stimuli-responsive properties. The potential of the aggregates as a drug depot was assessed in lung cancer cells, using Doxorubicin (Dox) as a model drug. The results show that RIP3 and RIP3/F127 exhibit amyloidogenic properties. Further, the RIP3/F127 amyloids exhibited significant ultrasound-responsive properties compared to amyloid aggregates without Pluronic F127. Moreover, the RIP3/F127/Dox amyloid formulations that were subjected to ultrasound treatment exhibited greater toxicity to lung cancer cells compared to that of Dox alone at equal concentrations. Overall, the results from this proof-of-concept study show that amyloidogenic peptide aggregates with stimuli-responsive properties can be utilized as efficient drug delivery depots.

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The aggregation of amyloid peptides and proteins into toxic oligomers is a hallmark of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Machado-Joseph's disease, and transmissible spongiform encephalopathies. Inhibition of amyloid oligomers formation and interactions with biological counterparts, as well as the triggering of non-toxic amorphous aggregates, are strategies towards preventive interventions against these pathologies. NMR spectroscopy addresses the need for structural characterization of amyloid proteins and their aggregates, their binding to inhibitors, and rapid screening of compound libraries for ligand identification. Here we briefly discuss the solution experiments constituting the NMR spectroscopist's toolkit and provide examples of their application.

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This paper presents new structural data about mitochondria using correlative light and electron microscopy (CLEM) and cryo-electron tomography. These state-of-the-art structural biology methods allow studying biological objects at nanometer scales under natural conditions. Non-invasiveness of these methods makes them comparable to observing animals in their natural environment on a safari. The paper highlights two areas of research that can only be accomplished using these methods. The study visualized location of the Aß42 amyloid aggregates in relation to mitochondria to test a hypothesis of development of mitochondrial dysfunction in Alzheimer's disease. The results showed that the Aß42 aggregates do not interact with mitochondria, although some of them are closely located. Therefore, the study demonstrated that mitochondrial dysfunction is not directly associated with the effects of aggregates on mitochondrial structure. Other processes should be considered as sources of mitochondrial dysfunction. Second unique area presented in this work is high-resolution visualization of the mitochondrial membranes and proteins in them. Analysis of the cryo-ET data reveals toroidal holes in the lamellar structures of cardiac mitochondrial cristae, where ATP synthases are located. The study proposes a new mechanism for sorting and clustering protein complexes in the membrane based on topology. According to this suggestion, position of the OXPHOS system proteins in the membrane is determined by its curvature. High-resolution tomography expands and complements existing ideas about the structural and functional organization of mitochondria. This makes it possible to study the previously inaccessible structural interactions of proteins with each other and with membranes in vivo.

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PURPOSE: Ionizing radiation is a harsh environmental factor that could induce plant senescence. We hypothesized that radiation-related senescence remodels proteome, particularly by triggering the accumulation of prion-like proteins in plant tissues. The object of this study, pea (Pisum sativum L.), is an agriculturally important legume. Research on the functional importance of amyloidogenic proteins was never performed on this species. MATERIALS AND METHODS: Pea seeds were irradiated in the dose range 5-50 Gy of X-rays. Afterward, Fourier-transform infrared spectroscopy (FTIR) was used to investigate changes in the secondary structure of proteins in germinated 3-day-old seedlings. Specifically, we evaluated the ratio between the amide I and II peaks. Next, we performed protein staining with Congo red to compare the presence of amyloids in the samples. In parallel, we profiled the detergent-resistant proteome fraction by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS). Differentially accumulated proteins were functionally analyzed in MapMan software, and the PLAAC tool was used to predict putative prion-like proteins. RESULTS: We showed a reduced germination rate but higher plant height and faster appearance of reproductive organs in the irradiated at dose of 50 Gy group compared with the control; furthermore, we demonstrated more ß-sheets and amyloid aggregates in the roots of stressed plants. We detected 531 proteins in detergent-resistant fraction extracted from roots, and 45 were annotated as putative prion-like proteins. Notably, 29 proteins were significantly differentially abundant between the irradiated and the control groups. These proteins belong to several functional categories: amino acid metabolism, carbohydrate metabolism, cytoskeleton organization, regulatory processes, protein biosynthesis, and RNA processing. Thus, the discovery proteomics provided deep data on novel aspects of plant stress biology. CONCLUSION: Our data hinted that protein accumulation stimulated seedlings' growth as well as accelerated ontogenesis and, eventually, senescence, primarily through translation and RNA processing. The increased abundance of primary metabolism-related proteins indicates more intensive metabolic processes triggered in germinating pea seeds upon X-ray exposure. The functional role of detected putative amyloidogenic proteins should be validated in overexpression or knockout follow-up studies.

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Cerebral soluble ß-amyloid aggregates (sAßs) accumulation is one of the most important causes in Alzheimer's disease (AD) progression. In order to mitigate the neurotoxicity induced by sAßs and achieve enhanced AD therapeutic outcomes, robust sAßs clearance become an emerging task. Herein, a self-destructive nanoscavenger (SDNS) is reported based on multifunctional peptide-polymer complexes that can capture extracellular sAßs via hydrogen-bonding interactions and deliver them into microglial lysosomes. The internalized SDNS then occurs self-destruction within lysosomes and upregulates autophagy, thereby promoting the degradation of neurotoxic sAßs. Importantly, the enhanced autophagy also significantly suppresses the secretion of inflammatory factors by microglia, which is induced by internalized sAßs. Given that cerebral persistent inflammatory environment disturbs microglia-mediated phagocytosis and degradation, it is believed that this synergistic approach has valuable potential as a therapeutic strategy for AD.

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The development of an accurate and sensitive sensor for detecting amyloid plaques, which are responsible for many protein disorders like Alzheimer's disease, is crucial for early diagnosis. Recently, there has been a notable increase in the development of fluorescence probes that exhibit emission in the red region (>600 nm), aiming to effectively tackle the challenges encountered when working with complex biological matrices. In the current investigation, a hemicyanine-based probe, called LDS730, has been used for the sensing of amyloid fibrils, which belong to the Near-Infrared Fluorescence (NIRF) family of dyes. NIRF probes provide higher precision in detection, prevent photo-damage, and minimize the autofluorescence of biological specimens. The LDS730 sensor emits in the near-infrared region and shows a 110-fold increase in fluorescence turn-on emission when bound to insulin fibrils, making it a highly sensitive sensor. The sensor has an emission maximum of ~710 nm in a fibril-bound state, which shows a significant red shift along with a Stokes' shift of ~50 nm. The LDS730 sensor also displays excellent performance in the complicated human serum matrix, with a limit of detection (LOD) of 103 nM. Molecular docking calculations suggest that the most likely binding location of LDS730 in the fibrillar structure is the inner channels of amyloid fibrils along its long axis, and the sensor engages in several types of hydrophobic interactions with neighboring amino acid residues of the fibrillar structure. Overall, this new amyloid sensor has great potential for the early detection of amyloid plaques and for improving diagnostic accuracy.

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Interactions between amyloid protein structures and nanomaterials have been extensively studied to develop effective inhibitors of amyloid aggregation. Limited investigations are reported on the impact of nanoparticles on mature fibrils. In this work, gold nanoparticles are used as photothermal agents to alter insulin fibrils. To this end, gold colloids bearing a negatively charged capping shell, with an average diameter of 14 nm and a plasmon resonance maximum at 520 nm are synthesized. The effects on mature insulin fibril morphology and structure upon plasmonic excitation of the nanoparticles-fibril samples have been monitored by spectroscopic and microscopic methods. The obtained data indicate that an effective destruction of the amyloid aggregates occur upon irradiation of the plasmonic nanoparticles, allowing the development of emerging strategies to alter the structure of amyloid fibrils.

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INTRODUCTION: Protein misfolding diseases, including Alzheimer's and Parkinson's diseases, are characterized by the aberrant aggregation of proteins. These conditions are still largely untreatable, despite having a major impact on our healthcare systems and societies. AREAS COVERED: We describe drug discovery strategies to target protein misfolding and aggregation. We compare thermodynamic approaches, which are based on the stabilization of the native states of proteins, with kinetic approaches, which are based on the slowing down of the aggregation process. This comparison is carried out in terms of the current knowledge of the process of protein misfolding and aggregation, the mechanisms of disease and the therapeutic targets. EXPERT OPINION: There is an unmet need for disease-modifying treatments that target protein misfolding and aggregation for the over 50 human disorders known to be associated with this phenomenon. With the approval of the first drugs that can prevent misfolding or inhibit aggregation, future efforts will be focused on the discovery of effective compounds with these mechanisms of action for a wide range of conditions.

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Molecular insights on the ß-lactoglobulin thermal unfolding and aggregation are derived from FTIR and UV Resonance Raman (UVRR) investigations. We propose an in situ and in real-time approach that thanks to the identification of specific spectroscopic markers can distinguish the two different unfolding pathways pursued by ß-lactoglobulin during the conformational transition from the folded to the molten globule state, as triggered by the pH conditions. For both the investigated pH values (1.4 and 7.5) the greatest conformational variation of ß-lactoglobulin occurs at 80 °C and a high degree of structural reversibility after cooling is observed. In acidic condition ß-lactoglobulin exposes to the solvent its hydrophobic moieties in a much higher extent than in neutral solution, resulting on a highly open conformation. Moving from the diluted to the self-crowded regime, the solution pH and consequently the different molten globule conformation select the amyloid or non-amyloid aggregation pathway. At acidic condition the amyloid aggregates form during the heating cycle leading to the formation of transparent hydrogel. On the contrary, in neutral condition the amyloid aggregates never form. Information on the secondary structure conformational change of ß-lactoglobulin and the formation of amyloid aggregates are obtained by FTIR spectroscopy and are related to the information of the structural changes localized around the aromatic amino acid sites by UVRR technique. Our results highlight a strong involvement of the chain portions where tryptophan is located on the formation of amyloid aggregates.

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Soybean 7S storage protein (ß-conglycinin) is the most important allergen, exhibits resistance in gastrointestinal (GI) digestion, and causes allergies in humans and animals. A previous study has demonstrated that 7S proteins contained innate amyloid aggregates, but the fate of these specific protein aggregates in intestinal digestion and correlation to allergenicity are unclear. In this study, via a modified INFOGEST static in vitro digestion and IgE binding test, we illustrate that the survived amyloid aggregates of soybean 7S protein in GI digestion might be dominant IgE epitopes of soybean protein in humans. The impact of conjugated primary bile acid salt (BS) profile on digestion resistance and immunogenicity of soybean protein is assessed, regarding the binding affinity of BS to protein aggregates with consideration of the BS composition and the physiologically relevant colloidal structure. The results show that chenodeoxycholate-containing colloidal structures exhibit high affinity and unfolding capacity to protein amyloid aggregates, promoting proteolysis by pancreatic enzymes and thus mitigating the antigenicity of soybean protein. This study presents a novel understanding of bile acid profile and colloidal structure influence on the digestibility and antigenicity of dietary proteins. It should be helpful to design in vitro digestion protocol and accurately replicate physiologically relevant digestion conditions.

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Essential trace metals like zinc (Zn), iron (Fe), and copper (Cu) play an important physiological role in the metabolomics and healthy functioning of body organs, including the brain. However, abnormal accumulation of trace metals in the brain and dyshomeostasis in the different regions of the brain have emerged as contributing factors in neuronal degeneration, Aß aggregation, and Tau formation. The link between these essential trace metal ions and the risk of AD has been widely studied, although the conclusions have been ambiguous. Despite the absence of evidence for any clinical benefit, therapeutic chelation is still hypothesized to be a therapeutic option for AD. Furthermore, the parameters like bioavailability, ability to cross the BBB, and chelation specificity must be taken into consideration while selecting a suitable chelation therapy. The data in this review summarizes that the primary intervention in AD is brain metal homeostasis along with brain metal scavenging. This review evaluates the impact of different trace metals (Cu, Zn, Fe) on normal brain functioning and their association with neurodegeneration in AD. Also, it investigates the therapeutic potential of metal chelators in the management of AD. An extensive literature search was carried out on the "Web of Science, PubMed, Science Direct, and Google Scholar" to investigate the effect of trace elements in neurological impairment and the role of metal chelators in AD. In addition, the current review highlights the advantages and limitations of chelation therapies and the difficulties involved in developing selective metal chelation therapy in AD patients.

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Fructosylation of proteins results in the formation of advanced glycation end-products (AGEs). A diet rich in fructose along with hyperglycemia can cause fructose mediated glycation (fructosylation) of proteins, which results in AGEs formation. Insulin is a peptide hormone that is glycated when exposed to carbohydrates such as glucose. In this study, we have analysed the interaction of insulin with fructose and biophysically characterized fructose modified insulin. In silico studies performed through molecular docking and molecular dynamics simulation revealed that fructose binds to insulin with strong affinity resulting in the formation of insulin-fructose complex. Fructosylation of insulin caused hyperchromicity, loss of intrinsic fluorescence, gain in AGEs specific fluorescence and elevated the carbonyl and fructosamine content. Enhanced thioflavin T fluorescence suggested the presence of fibrillar structures at higher concentrations of fructose. Electron microscopy revealed the formation of characteristic amorphous and amyloid like aggregates at lower and higher concentrations of fructose, respectively. These findings show that fructosylation of insulin causes AGEs production, aggregation and alters its gross structural integrity. These changes may reduce the biological activity of insulin that can aggravate conditions like type II diabetes mellitus.Communicated by Ramaswamy H. Sarma.

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The formation of droplets of bio-molecular condensates through liquid-liquid phase separation (LLPS) of their component proteins is a key factor in the maintenance of cellular homeostasis. Different protein properties were shown to be important in LLPS onset, making it possible to develop predictors, which try to discriminate a positive set of proteins involved in LLPS against a negative set of proteins not involved in LLPS. On the other hand, the redundancy and multivalency of the interactions driving LLPS led to the suggestion that the large conformational entropy associated with non specific side-chain interactions is also a key factor in LLPS. In this work we build a LLPS predictor which combines the ability to form pi-pi interactions, with an unrelated feature, the propensity to stabilize the ß-pairing interaction mode. The cross-ß structure is formed in the amyloid aggregates, which are involved in degenerative diseases and may be the final thermodynamically stable state of protein condensates. Our results show that the combination of pi-pi and ß-pairing propensity yields an improved performance. They also suggest that protein sequences are more likely to be involved in phase separation if the main chain conformational entropy of the ß-pairing maintained droplet state is increased. This would stabilize the droplet state against the more ordered amyloid state. Interestingly, the entropic stabilization of the droplet state appears to proceed according to different mechanisms, depending on the fraction of "droplet-driving" proteins present in the positive set.

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Amyloid aggregation, microbial infection, and the blood-brain barrier (BBB) are considered critical obstructions for the treatment of Alzheimer's disease (AD). At present, existing treatment strategies are rarely able to overcome these critical factors. Herein, we propose an innovative treatment strategy and design multifunctional nanoassemblies (yCDs-Ce6) from coassembling photosensitizers (chlorine e6) and yellow fluorescent carbon dots, which endow yCDs-Ce6 with the functions for photodynamic and photothermal therapy (PDT and PTT). Compared with reported inhibitors, yCDs-Ce6 can suppress amyloid aggregation for 7 days, disaggregate aggregates, reduce amyloid aggregation-induced cytotoxicity, and prevent microbial growth by PDT and PTT. Moreover, yCDs-Ce6 can specifically target amyloid aggregates and visually label amyloid aggregates. yCDs-Ce6 can also cross the BBB upon near-infrared light irradiation and clear amyloid deposition in APP/PS1 mice by PDT and PTT. Meanwhile, yCDs-Ce6 did not cause significant negative effects on normal cells or tissues. Based on the methods of PPT and PTT treatment, the research deeply explores the effect of the novel nanoassemblies on two hypotheses of AD, opening a novel therapeutic paradigm for research amyloid-related diseases.

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Abrupt aggregation of misfolded proteins is a hallmark of the large group of amyloid pathologies that include diabetes type 2, Alzheimer and Parkinson's diseases. Protein aggregation yields oligomers and fibrils, ß-sheet-rich structures that exert cell toxicity. Microscopic examination of amyloid deposits reveals the presence of lipids membranes, which suggests that lipids can be involved in the process of pathogenic protein assembly. In this study, we show that lipids can uniquely alter the aggregation rates of lysozyme, a protein that is associated with systemic amyloidosis. Specifically, cardiolipin (CL), ceramide (CER), and sphingomyelin (SM) accelerate, phosphatidylcholine (PC) strongly inhibits, whereas phosphatidylserine (PS) has no effect on the rate of protein aggregation. Furthermore, lipids uniquely alter the secondary structure of lysozyme aggregates. Furthermore, we found that lysozyme aggregates grown in the presence of CL, CER, SM, PS, and CL:PC mixtures exert significantly lower production of reactive oxygen species and mitochondrial dysfunction compared to lysozyme:PC aggregates and lysozyme fibrils grown in the lipid-free environment. These findings suggest that a change in the lipid composition of cell membranes, which is taken place upon neurodegeneration, may trigger the formation of toxic protein species that otherwise would not be formed.

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Previous studies have found that soybean protein, especially soybean 7S protein (ß-conglycinin), exhibits digestion resistance, but the mechanism of digestion resistance and its implications for human health are still unclear. Here, we show that the extracted soybean 7S protein contains both oligomer globulins and amyloid aggregates, while the gastric digested soybean 7S protein only contains amyloid aggregates and thus exhibits digestion resistance. An animal experiment shows that un-digestible soybean 7S protein effectively prevents aspirin-induced acute gastric mucosa damage. The impacts of un-digestible soybean 7S protein on gastric mucus barrier properties are investigated using quartz crystal microbalance with dissipation (QCM-D), Langmuir monolayer, and multiple particle tracking (MPT). Results show that these un-digestible protein aggregates can penetrate into gastric mucus, increase the viscosity and compactness of the mucin layer, and reinforce the gastric mucus barrier properties. The findings are helpful to understand that high consumption of non-fermented soybean foods is associated with a decreased risk of gastric cancer.

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Flaviviruses replicate in membrane factories associated with the endoplasmic reticulum (ER). Significant levels of flavivirus viral protein accumulation contribute to ER stress. As a consequence, the host cell exhibits an Unfolded Protein Response (UPR), subsequently stimulating appropriate cellular responses such as adaptation, autophagy or apoptosis. The correct redox conditions of this compartment are essential to forming native disulfide bonds in proteins. Zika virus (ZIKV) has the ability to induce persistent ER stress leading to the activation of UPR pathways. In this study, we wondered whether ZIKV affects the redox balance and consequently the oxidative protein folding in the ER. We found that ZIKV replication influences the redox state, leading to the aggregation of the viral envelope protein as amyloid-like structures in the infected cells.

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Photodynamic therapy (PDT) has been explored as a therapeutic strategy to clear toxic amyloid aggregates involved in neurodegenerative disorders such as Alzheimer's disease. A major limitation of PDT is off-target oxidation, which can be lethal for the surrounding cells. We have shown that a novel class of oligo-p-phenylene ethynylenes (OPEs) exhibit selective binding and fluorescence turn-on in the presence of prefibrillar and fibrillar aggregates of disease-relevant proteins such as amyloid-ß (Aß) and α-synuclein. Concomitant with fluorescence turn-on, OPE also photosensitizes singlet oxygen under illumination through the generation of a triplet state, pointing to the potential application of OPEs as photosensitizers in PDT. Herein, we investigated the photosensitizing activity of an anionic OPE for the photo-oxidation of Aß fibrils and compared its efficacy to the well-known but nonselective photosensitizer methylene blue (MB). Our results show that, while MB photo-oxidized both monomeric and fibrillar conformers of Aß40, OPE oxidized only Aß40 fibrils, targeting two histidine residues on the fibril surface and a methionine residue located in the fibril core. Oxidized fibrils were shorter and more dispersed but retained the characteristic ß-sheet rich fibrillar structure and the ability to seed further fibril growth. Importantly, the oxidized fibrils displayed low toxicity. We have thus discovered a class of novel theranostics for the simultaneous detection and oxidization of amyloid aggregates. Importantly, the selectivity of OPE's photosensitizing activity overcomes the limitation of off-target oxidation of traditional photosensitizers and represents an advancement of PDT as a viable strategy to treat neurodegenerative disorders.

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BACE1 and BACE2 belong to a class of proteases called ß-secretases involved in ectodomain shedding of different transmembrane substrates. These enzymes have been extensively studied in Alzheimer's disease as they are responsible for the processing of APP in neurotoxic Aß peptides. These proteases, especially BACE2, are overexpressed in tumors and correlate with poor prognosis. Recently, different research groups tried to address the role of BACE1 and 2 in cancer development and progression. In this review, we summarize the latest findings on ß-secretases in cancer, highlighting the mechanisms that build the rationale to propose inhibitors of these proteins as a new line of treatment for different tumor types.

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