Formation of self-assembled fibril aggregates of trypsin-controllably hydrolyzed soy protein and its regulation on stability of high internal phase Pickering emulsions.

The mechanisms of trypsin hydrolysis time on the structure of soy protein hydrolysate fibril aggregates (SPHFAs) and the stability of SPHFAs-high internal phase Pickering emulsions (HIPPEs) were investigated. SPHFAs were prepared using soy protein hydrolysate (SPH) with different trypsin hydrolysis time (0 min-120 min) to stabilize SPHFAs-HIPPEs. The results showed that moderate trypsin hydrolysis (30 min, hydrolysis degree of 2.31 %) induced SPH unfolding and increased the surface hydrophobicity of SPH, thereby promoting the formation of flexible SPHFAs with maximal thioflavin T intensity and ζ-potential. Moreover, moderate trypsin hydrolysis improved the viscoelasticity of SPHFAs-HIPPEs, and SPHFAs-HIPPEs remained stable after storage at 25 °C for 80 d and heating at 100 °C for 1 h. Excessive trypsin hydrolysis (> 30 min) decreased the stability of SPHFAs-HIPPEs. In conclusion, moderate trypsin hydrolysis promoted the formation of flexible SPHFAs with high surface charge by inducing SPH unfolding, thereby promoting the stability of SPHFAs-HIPPEs.

Highly ordered aggregation of soy protein isolate particles for enhanced gel-related properties through konjac glucomannan addition.

This study assessed the effect of konjac glucomannan (KGM) on the aggregation of soy protein isolate (SPI) and its gel-related structure and properties. Raman results showed that KGM promoted the rearrangement of SPI to form more ß-sheets, contributing to the formation of an ordered structure. Atomic force microscopy, confocal laser scanning microscopy, and small-angle X-ray scattering results indicated that KGM reduced the size of SPI particles, narrowed their size distribution, and loosened the large aggregates formed by the stacking of SPI particles, improving the uniformity of gel system. As the hydrogen bonding between the KGM and SPI molecules enhanced, a well-developed network structure was obtained, further reducing the immobilized water's content (T22) and increasing the water-holding capacity (WHC) of SPI gel. Furthermore, this gel structure showed improved gel hardness and resistance to both small and large deformations. These findings facilitate the design and production of SPI-based gels with desired performance.

Multivalency drives interactions of alpha-synuclein fibrils with tau.

Age-related neurodegenerative disorders like Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by deposits of protein aggregates, or amyloid, in various regions of the brain. Historically, aggregation of a single protein was observed to be correlated with these different pathologies: tau in AD and α-synuclein (αS) in PD. However, there is increasing evidence that the pathologies of these two diseases overlap, and the individual proteins may even promote each other's aggregation. Both tau and αS are intrinsically disordered proteins (IDPs), lacking stable secondary and tertiary structure under physiological conditions. In this study we used a combination of biochemical and biophysical techniques to interrogate the interaction of tau with both soluble and fibrillar αS. Fluorescence correlation spectroscopy (FCS) was used to assess the interactions of specific domains of fluorescently labeled tau with full length and C-terminally truncated αS in both monomer and fibrillar forms. We found that full-length tau as well as individual tau domains interact with monomer αS weakly, but this interaction is much more pronounced with αS aggregates. αS aggregates also mildly slow the rate of tau aggregation, although not the final degree of aggregation. Our findings suggest that co-occurrence of tau and αS in disease are more likely to occur through monomer-fiber binding interactions, rather than monomer-monomer or co-aggregation.

Engineered NLS-chimera downregulates expression of aggregation-prone endogenous FUS.

Importin ß-superfamily nuclear import receptors (NIRs) mitigate mislocalization and aggregation of RNA-binding proteins (RBPs), like FUS and TDP-43, which are implicated in neurodegenerative diseases. NIRs potently disaggregate RBPs by recognizing their nuclear localization signal (NLS). However, disease-causing mutations in NLS compromise NIR binding and activity. Here, we define features that characterize the anti-aggregation activity of NIR and NLS. We find that high binding affinity between NIR and NLS, and optimal NLS location relative to the aggregating domain plays a role in determining NIR disaggregation activity. A designed FUS chimera (FUSIBB), carrying the importin ß binding (IBB) domain, is solubilized by importin ß in vitro, translocated to the nucleus in cultured cells, and downregulates the expression of endogenous FUS. In this study, we posit that guiding the mutual recognition of NLSs and NIRs will aid the development of therapeutics, illustrated by the highly soluble FUSIBB replacing the aggregation-prone endogenous FUS.

GALNT9 enrichment attenuates MPP+-induced cytotoxicity by ameliorating protein aggregations containing α-synuclein and mitochondrial dysfunction.

BACKGROUND: GALNTs (UDP-GalNAc; polypeptide N-acetylgalactosaminyltransferases) initiate mucin-type O-GalNAc glycosylation by adding N-GalNAc to protein serine/threonine residues. Abnormalities in O-GalNAc glycosylation are involved in various disorders such as Parkinson's disease (PD), a neurodegenerative disorder. GALNT9 is potentially downregulated in PD patients. METHODS: To determine whether GALNT9 enrichment ameliorates cytotoxicity related to PD-like variations, a pcDNA3.1-GALNT9 plasmid was constructed and transfected into SH-SY5Y cells to establish a GALNT9-overexpressing cell model. RESULTS: Downregulation of GALNT9 and O-GalNAc glycosylation was confirmed in our animal and cellular models of PD-like variations. GALNT9 supplementation greatly attenuated cytotoxicity induced by MPP+ (1-Methyl-4-phenylpyridinium iodide) since it led to increased levels of tyrosine hydroxylase and dopamine, reduced rates of apoptosis, and significantly ameliorated MPP+-induced mitochondrial dysfunction by alleviating abnormal levels of mitochondrial membrane potential and reactive oxygen species. A long-lasting mPTP (mitochondrial permeability transition pores) opening and calcium efflux resulted in significantly lower activity in the cytochrome C-associated apoptotic pathway and mitophagy process, signifying that GALNT9 supplementation maintained neuronal cell health under MPP+ exposure. Additionally, it was found that glycans linked to proteins influenced the formation of protein aggregates containing α-synuclein, and GALNT9 supplement dramatically reduced such insoluble protein aggregations under MPP+ treatment. Glial GALNT9 predominantly appears under pathological conditions like PD-like variations. CONCLUSIONS: GALNT9 enrichment improved cell survival, and glial GALNT9 potentially represents a pathogenic index for PD patients. This study provides insights into the development of therapeutic strategies for the treatment of PD.

Two novel DnaJ chaperone proteins CG5001 and P58IPK regulate the pathogenicity of Huntington's disease related aggregates.

Huntington's disease (HD) is a rare neurodegenerative disease caused due to aggregation of Huntingtin (HTT) protein. This study involves the cloning of 40 DnaJ chaperones from Drosophila, and overexpressing them in yeasts and fly models of HD. Accordingly, DnaJ chaperones were catalogued as enhancers or suppressors based on their growth phenotypes and aggregation properties. 2 of the chaperones that came up as targets were CG5001 and P58IPK. Protein aggregation and slow growth phenotype was rescued in yeasts, S2 cells, and Drosophila transgenic lines of HTT103Q with these overexpressed chaperones. Since DnaJ chaperones have protein sequence similarity across species, they can be used as possible tools to combat the effects of neurodegenerative diseases.

Controlled preparation of tannic acid-derived carbonized dots and their use to inhibit amyloid aggregation and promote aggregate disaggregation.

Tannic acid (TA)-derived carbon dots (TACDs) were synthesized for the first time via a solvothermal method using TA as one of the raw materials, which may effectively inhibit amyloid fibril aggregation and disaggregate mature fibril. The fluorescent property of TACDs were modulated by adjusting the ratio of TA to o-phenylenediamine (oPD), and TACDs fabricated with the precursor ratio as 1:1 showed the best fluorescent property. Circular dichroism spectra (CD) showed that the structure of ß-sheet decreased as the concentration of TACDs increased. The inhibition efficiency, as confirmed by thioflavin T (ThT) and transmission electron microscopy (TEM), is extraordinary at 98.16%, whereas disaggregation efficiency is noteworthy at 97.97%, and the disaggregated lysozyme fibrils did not reaggregate after 7 days. More critically, TACDs can also alleviate the cellular toxicity caused by Aß fibrils and improve cell viability. This work offers a new perspective on the design of scavengers for amyloid plaques.

Mispacking of the F87 sidechain drives aggregation-promoting conformational fluctuations in the subunit interfaces of the transthyretin tetramer.

Aberrant formation and deposition of human transthyretin (TTR) aggregates causes transthyretin amyloidosis. To initialize aggregation, transthyretin tetramers must first dissociate into monomers that partially unfold to promote entry into the aggregation pathway. The native TTR tetramer (T) is stabilized by docking of the F87 sidechain into an interfacial cavity enclosed by several hydrophobic residues including A120. We have previously shown that an alternative tetramer (T*) with mispacked F87 sidechains is more prone to dissociation and aggregation than the native T state. However, the molecular basis for the reduced stability in T* remains unclear. Here we report characterization of the A120L mutant, where steric hindrance is introduced into the F87 binding site. The x-ray structure of A120L shows that the F87 sidechain is displaced from its docking site across the subunit interface. In A120S, a naturally occurring pathogenic mutant that is less aggregation-prone than A120L, the F87 sidechain is correctly docked, as in the native TTR tetramer. Nevertheless, 19F-NMR aggregation assays show an elevated population of a monomeric aggregation intermediate in A120S relative to a control containing the native A120, due to accelerated tetramer dissociation and slowed monomer tetramerization. The mispacking of the F87 sidechain is associated with enhanced exchange dynamics for interfacial residues. At 298 K, the T* populations of various naturally occurring mutants fall between 4% and 7% (ΔG ~ 1.5-1.9 kcal/mol), consistent with the free energy change expected for undocking and solvent exposure of one of the four F87 sidechains in the tetramer (ΔG ~ 1.6 kcal/mol). Our data provide a molecular-level picture of the likely universal F87 sidechain mispacking in tetrameric TTR that promotes interfacial conformational dynamics and increases aggregation propensity.

α-Synuclein oligomers form by secondary nucleation.

Oligomeric species arising during the aggregation of α-synuclein are implicated as a major source of toxicity in Parkinson's disease, and thus a major potential drug target. However, both their mechanism of formation and role in aggregation are largely unresolved. Here we show that, at physiological pH and in the absence of lipid membranes, α-synuclein aggregates form by secondary nucleation, rather than simple primary nucleation, and that this process is enhanced by agitation. Moreover, using a combination of single molecule and bulk level techniques, we identify secondary nucleation on the surfaces of existing fibrils, rather than formation directly from monomers, as the dominant source of oligomers. Our results highlight secondary nucleation as not only the key source of oligomers, but also the main mechanism of aggregate formation, and show that these processes take place under conditions which recapitulate the neutral pH and ionic strength of the cytosol.

Mild alkalinity preheating treatment regulates the heat and ionic strength co-tolerance of whey protein aggregates.

A major obstacle to the use of whey protein in protein-enriched sports beverages is the heat-induced gelation of the protein in the presence of salt. In this study, whey protein soluble aggregates (WPSAs) with high tolerance to NaCl and heat were successfully generated by preheating whey protein isolate (WPI) at a low concentration (1 % w/v) and pH 8.5. The suspension of WPSAs (5 % w/v) with 100 mM NaCl maintained clarity, transparency, and good flowability even after 30 min of heating at 100 °C. However, suspensions prepared by untreated WPI turned into milky white gels. WPSAs had a reduced Zeta potential at pH 7 compared to WPI, making them more resistant to the electrostatic screening caused by NaCl. Additionally, WPSAs exhibited reduced sensitivity to heat treatment due to a more compact structure achieved through preheating modification. In light of these findings, a straightforward and effective method was presented for regulating the heat and ionic strength tolerance of whey protein aggregates.

Impact of NaCl on Monoclonal Antibody Aggregation Induced by Agitation.

Monoclonal antibodies (mAbs) are a successful class of therapeutics, but their development can be challenging due to the risk of degradation that could happen during manufacturing, storage, and clinical use. One of the common causes of degradation is agitation stress from transportation and clinical handling, which increases interfacial stresses to mAbs. For example, the preparation of the dose solution prior to administration often requires diluting therapeutic mAbs in intravenous (IV) infusion bags containing normal saline, which can substantially reduce the level of protective surfactant and increase the level of salt in mAb solutions. Then the interfacial stress in the subsequent transportation of IV bags can cause mAb aggregation or even particle formation. To better understand the complex interplay between dilution, interfacial stress, and salt, we studied the impact of sodium chloride (NaCl) on the aggregation of two mAbs under agitation stress. We found that the presence of NaCl accelerates the aggregation of both mAbs, but the aggregation mechanism, morphology, and reversibility are very different. Our results clearly highlight the impact of salt on mAb stability at the clinical in-use condition. We believe this study further increases our understanding of protein aggregation mediated by interfacial stresses and brings valuable insights to support development of mAb formulations for patients.

Overexpression of Toxic Poly(Glycine-Alanine) Aggregates in Primary Neuronal Cultures Induces Time-Dependent Autophagic and Synaptic Alterations but Subtle Activity Impairments.

The pathogenic expansion of the intronic GGGGCC hexanucleotide located in the non-coding region of the C9orf72 gene represents the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This mutation leads to the accumulation of toxic RNA foci and dipeptide repeats (DPRs), as well as reduced levels of the C9orf72 protein. Thus, both gain and loss of function are coexisting pathogenic aspects linked to C9orf72-ALS/FTD. Synaptic alterations have been largely described in C9orf72 models, but it is still not clear which aspect of the pathology mostly contributes to these impairments. To address this question, we investigated the dynamic changes occurring over time at the synapse upon accumulation of poly(GA), the most abundant DPR. Overexpression of this toxic form induced a drastic loss of synaptic proteins in primary neuron cultures, anticipating autophagic defects. Surprisingly, the dramatic impairment characterizing the synaptic proteome was not fully matched by changes in network properties. In fact, high-density multi-electrode array analysis highlighted only minor reductions in the spike number and firing rate of poly(GA) neurons. Our data show that the toxic gain of function linked to C9orf72 affects the synaptic proteome but exerts only minor effects on the network activity.

Studying Selective Autophagy of Protein Aggregates Using Particles Induced by Multimerization (PIMs).

Selective autophagy of protein aggregates, called aggrephagy, is vital for maintaining cellular homeostasis. Classically, studying aggrephagy has been challenging due to the infrequent occurrence of autophagic events and the lack of control over the specificity and timing of protein aggregation. We previously reported two variants of a PIM (particles induced by multimerization) assay that enable the formation of chemically induced, fluorescently labeled protein aggregates in cells. PIMs are recognized by the selective autophagy machinery and are subsequently degraded in the lysosome. By making use of pH-sensitive fluorescent proteins, such as GFP or mKeima, the PIM assay allows for direct visualization of aggregate clearance in cells. Here, we describe a protocol for the use of the PIM assay to study aggrephagy in live and fixed cells.

Investigation of the Aggregation of Aß Peptide (1-40) in the Presence of κ-Carrageenan-Stabilised Liposomes Loaded with Homotaurine.

The kinetics of amyloid aggregation was studied indirectly by monitoring the changes in the polydispersity of mixed dispersion of amyloid ß peptide (1-40) and composite liposomes. The liposomes were prepared from the 1,2-dioleoyl-sn-glicero-3-phoshocholine (DOPC) phospholipid and stabilised by the electrostatic adsorption of κ-carrageenan. The produced homotaurine-loaded and unloaded liposomes had a highly negative electrokinetic potential and remarkable stability in phosphate buffer (pH 4 and 7.4). For the first time, the appearance and evolution of the aggregation of Aß were presented through the variation in the standard percentile readings (D10, D50, and D90) obtained from the particle size distribution analysis. The kinetic experiments indicated the appearance of the first aggregates almost 30 min after mixing the liposomes and peptide solution. It was observed that by adding unloaded liposomes, the size of 90% of the particles in the dispersion (D90) increased. In contrast, the addition of homotaurine-loaded liposomes had almost minimal impact on the size of the fractions of larger particles during the kinetic experiments. Despite the specific bioactivity of homotaurine in the presence of natural cell membranes, this study reported an additional inhibitory effect of the compound on the amyloid peptide aggregation due to the charge effects and 'molecular crowding'.

Synthesis and Evaluation of Chloride-Substituted Ramalin Derivatives for Alzheimer's Disease Treatment.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by the accumulation of amyloid-beta plaques and hyperphosphorylated tau proteins, leading to cognitive decline and neuronal death. However, despite extensive research, there are still no effective treatments for this condition. In this study, a series of chloride-substituted Ramalin derivatives is synthesized to optimize their antioxidant, anti-inflammatory, and their potential to target key pathological features of Alzheimer's disease. The effect of the chloride position on these properties is investigated, specifically examining the potential of these derivatives to inhibit tau aggregation and beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1) activity. Our findings demonstrate that several derivatives, particularly RA-3Cl, RA-4Cl, RA-26Cl, RA-34Cl, and RA-35Cl, significantly inhibit tau aggregation with inhibition rates of approximately 50%. For BACE-1 inhibition, Ramalin and RA-4Cl also significantly decrease BACE-1 expression in N2a cells by 40% and 38%, respectively, while RA-23Cl and RA-24Cl showed inhibition rates of 30% and 35% in SH-SY5Y cells. These results suggest that chloride-substituted Ramalin derivatives possess promising multifunctional properties for AD treatment, warranting further investigation and optimization for clinical applications.

Chemiexcitation-Triggered Photosensitizer Activation for Photooxidation of Aß1-42 Aggregates.

Oxidative degradation of the pathogenic amyloid-ß-peptide (Aß) aggregation is an effective and promising method to treat Alzheimer's disease under light irradiation. However, the limited penetration of external light sources into deep tissues has hindered the development of this treatment. Therefore, we have designed an unprecedented chemiluminescence-initiated photodynamic therapy system to replace external laser irradiation, primarily composed of d-glucose-based polyoxalate (G-poly(oxalate)), the novel photosensitizer (BD-Se-QM), and bis [2,4,5-trichloro-6-(pentoxy-carbonyl) phenyl] ester. BD-Se-QM possesses excellent singlet oxygen (1O2) generation efficiency and the ability to photooxidize Aß1-42 aggregates under white light. G-poly(oxalate) not only helps the nanosystem to cross the blood-brain barrier but also has sufficient oxalate ester groups to significantly enhance the efficiency of chemiluminescence resonance energy transfer. The oxalate ester groups in BD-Se-QM/NPs can chemically react with H2O2 to produce high-energy intermediates that activate BD-Se-QM, which can generate 1O2 to inhibit Aß1-42 aggregates and also promote microglial uptake of Aß1-42, reducing the Aß1-42-induced neurotoxicity. The chemically stimulated nanoplatform not only solves the drug delivery problem but also eliminates the need for external light sources. We anticipate that this chemically excited nanosystem could also be used for targeted delivery of other small molecule drugs.

Misfolding and aggregation in neurodegenerative diseases: protein quality control machinery as potential therapeutic clearance pathways.

The primary challenge in today's world of neuroscience is the search for new therapeutic possibilities for neurodegenerative disease. Central to these disorders lies among other factors, the aberrant folding, aggregation, and accumulation of proteins, resulting in the formation of toxic entities that contribute to neuronal degeneration. This review concentrates on the key proteins such as ß-amyloid (Aß), tau, and α-synuclein, elucidating the intricate molecular events underlying their misfolding and aggregation. We critically evaluate the molecular mechanisms governing the elimination of misfolded proteins, shedding light on potential therapeutic strategies. We specifically examine pathways such as the endoplasmic reticulum (ER) and unfolded protein response (UPR), chaperones, chaperone-mediated autophagy (CMA), and the intersecting signaling of Keap1-Nrf2-ARE, along with autophagy connected through p62. Above all, we emphasize the significance of these pathways as protein quality control mechanisms, encompassing interventions targeting protein aggregation, regulation of post-translational modifications, and enhancement of molecular chaperones and clearance. Additionally, we focus on current therapeutic possibilities and new, multi-target approaches. In conclusion, this review systematically consolidates insights into emerging therapeutic strategies predicated on protein aggregates clearance.

Aggregate-selective removal of pathological tau by clustering-activated degraders.

Selective degradation of pathological protein aggregates while sparing monomeric forms is of major therapeutic interest. The E3 ligase tripartite motif-containing protein 21 (TRIM21) degrades antibody-bound proteins in an assembly state-specific manner due to the requirement of TRIM21 RING domain clustering for activation, yet effective targeting of intracellular assemblies remains challenging. Here, we fused the RING domain of TRIM21 to a target-specific nanobody to create intracellularly expressed constructs capable of selectively degrading assembled proteins. We evaluated this approach against green fluorescent protein-tagged histone 2B (H2B-GFP) and tau, a protein that undergoes pathological aggregation in Alzheimer's and other neurodegenerative diseases. RING-nanobody degraders prevented or reversed tau aggregation in culture and in vivo, with minimal impact on monomeric tau. This approach may have therapeutic potential for the many disorders driven by intracellular protein aggregation.

Desmodium intortum (Mill.) Urb. Protein Isolate Aggregates as Pickering Stabilizers: Physicochemical Characteristics and Emulsifying Properties.

This work aimed to investigate the feasibility of fabricating Pickering emulsions stabilized by Desmodium intortum protein isolate (DIPI) aggregates. The DIPI aggregates were formed using heat treatment, and the effects of ionic strength and pH on their properties were investigated. The heat-treated protein exposes its hydrophobic groups due to structural damage, resulting in rapid aggregation of the protein into aggregates with a size of 236 nm. The results showed that the aggregates induced by ionic strength had larger particle size and higher surface hydrophobicity and partial wettability. Moreover, this study explored effective strategies for bolstering Pickering emulsion stability through optimized DIPI aggregate concentration (c) and oil fraction (ø). The DIPI Pickering emulsion (DIPIPE) formed at c = 5% and ø = 0.7 was still highly stable after 30 days of storage. As confirmed by laser confocal microscopy, DIPI aggregates could be adsorbed onto the oil-water interface to form a network structure that could trap oil droplets in the network. Collectively, the Pickering emulsion stabilized by DIPI aggregates exhibited excellent stability, which not only deeply utilizes the low-value protein resources in the Desmodium intortum for the first time, but also demonstrates the potential of DIPI for the bio-based field.

Tailoring the pKa of Fluorescence Lifetime Imaging Probes to Visualize Aggrephagy and Resolve Its Microenvironmental Viscosity.

Aggrephagy describes lysosomal transport and degradation of protein aggregates via cellular macroautophagy, a key mechanism to prevent neurodegenerative diseases. Here, we develop a dual-probe method to visualize the aggrephagy process and resolve its viscosity heterogeneity using fluorescence lifetime imaging (FLIM). The dual-probe system consists of (1) a near-infrared lysosomal targeting FLIM probe (Lyso-P1) that is derived from a rhodamine scaffold with a tailored pKa value to accommodate an acidic lysosomal environment and (2) a green BODIPY-based FLIM probe (Agg-P2) that reports on degradation of cellular aggregates via HaloTag. Both probes exhibit acid-resistant, viscosity-dependent fluorescence intensity and lifetime (τ) responses, which are ready for intensity- and FLIM-based imaging. Photochemical, theoretical, and biochemical characterizations reveal the probes' mechanism-of-actions. In cells, we exploit Lyso-P1 and Agg-P2 to simultaneously quantify both lysosomal and protein aggegates' viscosity changes upon the aggrephagy process via FLIM. We reveal orthogonal changes in microenvironmental viscosities and morphological heterogeneity upon various cellular stresses. Overall, we provide an imaging toolset to quantitatively study aggrephay, which may benefit screening of aggrephay modulators for disease intervention.