In this study, we induced a 6% protein concentration of whey protein (WP) to form fibrous and granular whey protein self-assembly (FWS and GWS respectively) by a pH-temperature gradient. The fibrous ...and granular whey protein self-assembly were used as raw material to explore the mechanism of gel formation and their potential a new delivery system loaded with Epigallocatechin gallate (EGCG) was evaluated. Compared with native WP, the α-helix decreased, and the β-sheet increased in FWS, while the opposite was found in GWS. The hydrophobic interaction played a crucial role in the formation process of FWS and GWS, while disulfide bonds played a slightly more vital role in the formation process of GWS. The gels prepared by FWS and GWS had denser microstructure and stronger mechanical strength than native WP gel. Both the water holding capacity and hardness of the gels prepared by FWS and GWS were significantly higher than those prepared by native WP. The water holding capacity of the gel prepared by FWS was higher than that of the gel prepared by GWS at the same protein concentration, but the hardness results were the opposite. Hydrophobic interaction and hydrogen bonds played a dominant role during the gel formation process of FWS and GWS, while disulfide bonds only played a role in the gel formation process of GWS. In addition, the gel formed by FWS and GWS had higher epigallocatechin gallate encapsulation efficiency (94.40% and 95.09%, respectively) and a slow control effect during digestion. Therefore, gel prepared by FWS and GWS can provide essential ideas for building new protein gel technology and is expected to be an ideal carrier for bioactive substances.
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•Disulfide bonds had a role only in granular whey protein self-assembled gels.•Self-assembled gels had denser microstructure than native whey protein gel.•Self-assembled gels had better ability to load bioactive substances.•Chitosan-coated self-assembled gels enhance the slow release performance.
Misfolded proteins associated with diverse aggregation disorders assemble not only into a single toxic conformer but rather into a suite of aggregated conformers with unique biochemical properties ...and toxicities. To what extent small molecules can target and neutralize specific aggregated conformers is poorly understood. Therefore, we have investigated the capacity of resveratrol to recognize and remodel five conformers (monomers, soluble oligomers, non-toxic oligomers, fibrillar intermediates, and amyloid fibrils) of the Aβ1–42 peptide associated with Alzheimer disease. We find that resveratrol selectively remodels three of these conformers (soluble oligomers, fibrillar intermediates, and amyloid fibrils) into an alternative aggregated species that is non-toxic, high molecular weight, and unstructured. Surprisingly, resveratrol does not remodel non-toxic oligomers or accelerate Aβ monomer aggregation despite that both conformers possess random coil secondary structures indistinguishable from soluble oligomers and significantly different from their β-sheet rich, fibrillar counterparts. We expect that resveratrol and other small molecules with similar conformational specificity will aid in illuminating the conformational epitopes responsible for Aβ-mediated toxicity.
Tau aggregation underlies neurodegenerative tauopathies, and transcellular propagation of tau assemblies of unique structure, i.e., strains, may underlie the diversity of these disorders. Polyanions ...have been reported to induce tau aggregation in vitro, but the precise trigger to convert tau from an inert to a seed-competent form in disease states is unknown. RNA triggers tau fibril formation in vitro and has been observed to associate with neurofibrillary tangles in human brain. Here, we have tested whether RNA exerts sequence-specific effects on tau assembly and strain formation. We found that three RNA homopolymers, polyA, polyU, and polyC, all bound tau, but only polyA RNA triggered seed and fibril formation. In addition, polyA:tau seeds and fibrils were sensitive to RNase. We also observed that the origin of the RNA influenced the ability of tau to adopt a structure that would form stable strains. Human RNA potently induced tau seed formation and created tau conformations that preferentially formed stable strains in a HEK293T cell model, whereas RNA from other sources, or heparin, produced strains that were not stably maintained in cultured cells. Finally, we found that soluble, but not insoluble seeds from Alzheimer’s disease brain were also sensitive to RNase. We conclude that human RNA specifically induces formation of stable tau strains and may trigger the formation of dominant pathological assemblies that propagate in Alzheimer’s disease and possibly other tauopathies.
The identification of toxic Aβ species and/or the process of their formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in Alzheimer disease and also for the development of ...effective diagnostic and therapeutic interventions. To elucidate the structural basis of Aβ toxicity, we developed different procedures to isolate Aβ species of defined size and morphology distribution, and we investigated their toxicity in different cell lines and primary neurons. We observed that crude Aβ42 preparations, containing a monomeric and heterogeneous mixture of Aβ42 oligomers, were more toxic than purified monomeric, protofibrillar fractions, or fibrils. The toxicity of protofibrils was directly linked to their interactions with monomeric Aβ42 and strongly dependent on their ability to convert into amyloid fibrils. Subfractionation of protofibrils diminished their fibrillization and toxicity, whereas reintroduction of monomeric Aβ42 into purified protofibril fractions restored amyloid formation and enhanced their toxicity. Selective removal of monomeric Aβ42 from these preparations, using insulin-degrading enzyme, reversed the toxicity of Aβ42 protofibrils. Together, our findings demonstrate that Aβ42 toxicity is not linked to specific prefibrillar aggregate(s) but rather to the ability of these species to grow and undergo fibril formation, which depends on the presence of monomeric Aβ42. These findings contribute significantly to the understanding of amyloid formation and toxicity in Alzheimer disease, provide novel insight into mechanisms of Aβ protofibril toxicity, and important implications for designing anti-amyloid therapies.
Protein assembly is the structural basis for the collaboration between proteins to accomplish life activities due to its realization of the domain‐limited and precise spatial arrangement of proteins. ...Therefore, artificial manipulation of protein self‐assembly has profound implications in areas such as exploring the mysteries of life and developing biomaterials. In this review, we not only summarize the classical assembly strategies and the structures of exquisite protein assemblies, but also aim to generalize the flexible and controllable assembly tools and the “interaction” between the assembled protein‐based materials and the external environment. On the basis of this, the application, challenges and further development of protein assembly in biology are reviewed.
Artificial control of protein self‐assembly can help to explain the information exchange and cooperation between proteins, and opens the way for the development of novel biomaterials. In this paper, the classical protein assembly construction tools and the derived controllable design strategies are summarized and analyzed. In addition, the applications of protein assemblies in biology are also reviewed.
Protein phase separation is thought to be a primary driving force for the formation of membrane-less organelles, which control a wide range of biological functions from stress response to ribosome ...biogenesis. Among phase-separating (PS) proteins, many have intrinsically disordered regions (IDRs) that are needed for phase separation to occur. Accurate identification of IDRs that drive phase separation is important for testing the underlying mechanisms of phase separation, identifying biological processes that rely on phase separation, and designing sequences that modulate phase separation. To identify IDRs that drive phase separation, we first curated datasets of folded, ID, and PS ID sequences. We then used these sequence sets to examine how broadly existing amino acid property scales can be used to distinguish between the three classes of protein regions. We found that there are robust property differences between the classes and, consequently, that numerous combinations of amino acid property scales can be used to make robust predictions of protein phase separation. This result indicates that multiple, redundant mechanisms contribute to the formation of phase-separated droplets from IDRs. The top-performing scales were used to further optimize our previously developed predictor of PS IDRs, ParSe. We then modified ParSe to account for interactions between amino acids and obtained reasonable predictive power for mutations that have been designed to test the role of amino acid interactions in driving protein phase separation. Collectively, our findings provide further insight into the classification of IDRs and the elements involved in protein phase separation.
The development of extracellular matrix mimetics that imitate niche stem cell microenvironments and support cell growth for technological applications is intensely pursued. Specifically, mimetics are ...sought that can enact control over the self‐renewal and directed differentiation of human pluripotent stem cells (hPSCs) for clinical use. Despite considerable progress in the field, a major impediment to the clinical translation of hPSCs is the difficulty and high cost of large‐scale cell production under xeno‐free culture conditions using current matrices. Here, a bioactive, recombinant, protein‐based polymer, termed ZTFn, is presented that closely mimics human plasma fibronectin and serves as an economical, xeno‐free, biodegradable, and functionally adaptable cell substrate. The ZTFn substrate supports with high performance the propagation and long‐term self‐renewal of human embryonic stem cells while preserving their pluripotency. The ZTFn polymer can, therefore, be proposed as an efficient and affordable replacement for fibronectin in clinical grade cell culturing. Further, it can be postulated that the ZT polymer has significant engineering potential for further orthogonal functionalization in complex cell applications.
A self‐assembling protein scaffold is functionalized using a bottom‐up approach to support stem cell propagation and self‐renewal. The scaffold effectively replaces native fibronectin in the culture of pluripotent human embryonic stem cells and multipotent murine stromal cells. The simplicity and economy of production of the reported scaffold can allow for an affordable expansion of stem cells for therapeutic applications.
Amyloid fibrils from plant-based food protein sources bear a large unexploited potential for applications in food and other biomaterials due to their techno-functional features. However, their low ...solubility and highly complex, inhomogeneous protein composition often hamper fibrillization. The objective of this study was to evaluate the feasibility of amyloid fibril production from hemp seed protein, known as a sustainable and low-allergenic protein source. Hemp protein concentrate (HPC), primarily constituted of the 11 S globulin edestin, with 89.0% protein solubility (0.25% w/w HPC, pH 2) was extracted using gentle micellization. Fibrillization of HPC (2% w/w, pH 2, 90 °C, 300 rpm) was monitored over 5 h by ThT fluorescence, exhibiting a steep increase in fluorescence signal after a lag phase of 180 min. SDS-PAGE analysis indicated progressive polypeptide hydrolysis upon heating and the formation of large proteinaceous aggregates after 160 min. Conformational changes towards increased β-sheet content were demonstrated by CD and FTIR. The morphology of the formed fibrillar aggregates was characterized by TEM and AFM. While essentially linear, branching effects of the fibrils became visible and kept increasing with incubation time. After a relatively short incubation time of 4 h, fibrils had an average height of 7.8 nm, a contour length of 1.8 μm, and a persistence length of ∼2.7 μm. These results suggest, that under the chosen conditions for protein extraction and incubation, HPC forms relatively flexible amyloid fibrils with a high aspect ratio and tendency to form branches. By revealing the potential of hemp seed proteins for amyloid fibril formation, these results contribute to expand the understanding of plant protein fibrillization.
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•Hemp protein concentrate (HPC) was produced by micellization.•HPC was highly soluble at pH < 4 and primarily consisted of 11 S globulin edestin.•HPC assembled into amyloid fibrils with a high aspect ratio upon heating at pH 2.•Amyloid fibrils were linear, flexible, with a tendency to branching.
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•The strategies for constructing self-assembled proteins architectures.•Developing bioinspired materials based on protein assemblies.•Protein assemblies based artificial light ...harvesting systems, intelligent protein nanocarriers, and biomimetic systems.
Sophisticated protein self-assemblies have attracted great scientific interests in recent few decades due to their various potential applications in substance/signal transmission, biosensors, or disease diagnosis and treatment. The design and construction of proteins into hierarchical nanostructures via self-assembly strategies offer unique advantages in understanding the mechanism of naturally occurring protein assemblies and/or creating various functional biomaterials with advanced properties. This review covers the recent progress and trends in the self-assembled hierarchical protein structures and their bio-inspired applications. We initially discuss the design and development of sophisticated protein nanostructures through the preciously designed protein–protein interactions. Many intricate protein nanostructures from quasi-zero dimensional (0D) polyhedral cages, one-dimensional (1D) strings/rings/tubules, two-dimensional (2D) crystal sheets/cambered surfaces, and three-dimensional (3D) crystalline frameworks/hydrogels, have been constructed through self-assembly of rationally designed proteins. In addition, we also show the representative achievements in the study of the structure–function relationship for selected protein self-assemblies and highlight the latest research progress in developing artificial light harvesting systems, biological nanoenzyme mimics, intelligent protein nanocarriers, biomimetic protocells, and so on. As expected, protein self-assembly has become a powerful tool for development of multifarious bioinspired materials with advanced structures and properties.
Formation of biomolecular condensates through liquid–liquid phase separation (LLPS) has been described for several pathogenic proteins linked to neurodegenerative diseases and is discussed as an ...early step in the formation of protein aggregates with neurotoxic properties. In prion diseases, neurodegeneration and formation of infectious prions is caused by aberrant folding of the cellular prion protein (PrPC). PrPC is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrPC is proteolytically processed in vivo into an entirely unstructured fragment, designated N1, and the corresponding C-terminal fragment C1 harboring the globular domain. Notably, N1 contains a polybasic motif that serves as a binding site for neurotoxic Aβ oligomers. PrP can undergo LLPS; however, nothing is known how phase separation of PrP is triggered on a molecular scale. Here, we show that the intrinsically disordered N1 domain is necessary and sufficient for LLPS of PrP. Similar to full-length PrP, the N1 fragment formed highly dynamic liquid-like droplets. Remarkably, a slightly shorter unstructured fragment, designated N2, which lacks the Aβ-binding domain and is generated under stress conditions, failed to form liquid-like droplets and instead formed amorphous assemblies of irregular structures. Through a mutational analysis, we identified three positively charged lysines in the postoctarepeat region as essential drivers of condensate formation, presumably largely via cation–π interactions. These findings provide insights into the molecular basis of LLPS of the mammalian prion protein and reveal a crucial role of the Aβ-binding domain in this process.
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