Protein amyloid nanofibers provide a biocompatible platform for the development of functional nanomaterials. However, the functionalities generated up to date are still limited. Typical building ...blocks correspond to aggregation-prone proteins and peptides, which must be modified by complex and expensive reactions post-assembly. There is high interest in researching alternative strategies to tailor amyloid-based nanostructures’ functionality on demand. In the present study, the biotin-streptavidin system was exploited for this purpose. Prion-inspired heptapeptides (Ac-NYNYNYN-NH2, Ac-QYQYQYQ-NH2, and Ac-SYSYSYS-NH2) were doped with biotin-conjugated counterparts and assembled into amyloid-like fibers under mild conditions. The scaffolds’ versatile functionalization was demonstrated by decorating them with different streptavidin conjugates, including gold nanoparticles, quantum dots, and enzymes. In particular, they were functionalized with peroxidase or phosphatase activities using streptavidin conjugated with horseradish peroxidase and alkaline phosphatase, respectively. Modification of amyloid-like nanostructures has generally been restricted to the addition of a single protein moiety. We functionalized the fibrils simultaneously with glucose oxidase and horseradish peroxidase, coupling these activities to build up a nanostructured glucose biosensor. Overall, we present a simple, modular, and multivalent approach for developing amyloid-based nanomaterials functionalized with any desired combination of chemical and biological moieties.
Nature provides copious examples of self-assembling supramolecular nanofibers. Among them, amyloid structures have found amazing applications as advanced materials in fields such as biomedicine and ...nanotechnology. Prions are a singular subset of proteins able to switch between a soluble conformation and an amyloid state. The ability to transit between these two conformations is encoded in the so-called prion domains (PrDs), which are long and disordered regions of low complexity, enriched in polar and uncharged amino acids such as Gln, Asn, Tyr, Ser, and Gly. The polar nature of PrDs results in slow amyloid formation, which allows kinetic control of fiber assembly. This approach has been exploited for fabrication of multifunctional materials because in contrast to most amyloids, PrDs lack hydrophobic stretches that can nucleate their aggregation, their assembly depends on the establishment of a large number of weak interactions along the complete domain. The length and low complexity of PrDs make their chemical synthesis for applied purposed hardly affordable. Here, we designed four minimalist polar binary patterned peptides inspired in PrDs, which include the Q/N/G/S-Y-Q/N/G/S motif frequently observed in these domains: NYNYNYN, QYQYQYQ, SYSYSYS, and GYGYGYG. Despite their small size, they all recapitulate the properties of full-length PrDs, self-assembling into nontoxic amyloids under physiological conditions. Thus, they constitute small building blocks for the construction of tailored prion-inspired nanostructures. We exploited Tyr residues in these peptides to generate highly stable dityrosine cross-linked assemblies for the immobilization of metal nanoparticles in the fibrils surface and to develop an electrocatalytic amyloid scaffold. Moreover, we show that the shorter and more polar NYNNYN, QYQQYQ, and SYSSYS hexapeptides also self-assemble into amyloid-like structures, consistent with the presence of these tandem motifs in human prion-like proteins.
Enzymes typically fold into defined 3D protein structures exhibiting a high catalytic efficiency and selectivity. It has been proposed that the earliest enzymes may have arisen from the self-assembly ...of short peptides into supramolecular amyloid-like structures. Several artificial amyloids have been shown to display catalytic activity while offering advantages over natural enzymes in terms of modularity, flexibility, stability, and reusability. Hydrolases, especially esterases, are the most common artificial amyloid-like nanozymes with some reported to act as carbonic anhydrases (CA). Their hydrolytic activity is often dependent on the binding of metallic cofactors through a coordination triad composed of His residues in the β-strands, which mimic the arrangement found in natural metalloenzymes. Tyr residues contribute to the coordination of metal ions in the active center of metalloproteins; however, their use has been mostly neglected in the design of metal-containing amyloid-based nanozymes. We recently reported that four different polar prion-inspired heptapeptides spontaneously self-assembled into amyloid fibrils. Their sequences lack His but contain three alternate Tyr residues exposed to solvent. We combine experiments and simulations to demonstrate that the amyloid fibrils formed by these peptides can efficiently coordinate and retain different divalent metal cations, functioning as both metal scavengers and nanozymes. The metallized fibrils exhibit esterase and CA activities without the need for a histidine triad. These findings highlight the functional versatility of prion-inspired peptide assemblies and provide a new sequential context for the creation of artificial metalloenzymes. Furthermore, our data support amyloid-like structures acting as ancestral catalysts at the origin of life.
Amyloids are associated with human disease. However, they are also exploited by nature for functional purposes. Functional amyloids have inspired amyloid-based biomaterials for different ...nanotechnologies. Early soluble species in the fibrillation pathway seem to be the primary elicitors of cytotoxicity, instead of fibrils. Organisms have evolved dedicated mechanisms to avoid toxicity during the assembly of functional amyloids. In their absence, artificial amyloid-based nanomaterials might also produce toxic intermediates. We show here that even when the building blocks of artificial amyloids are small, polar, and compositionally simple, their early soluble assemblies are extremely cytotoxic, causing cell death through mechanisms identical to those of disease-associated proteins. Our results raise safety concerns about the use of non-natural amyloid-based materials without a rigorous characterization of their fibrillation pathway. Besides, the simple, cheap, and easy to synthesize peptides we use here might turn very useful to understand the molecular determinants behind amyloid cytotoxicity.
The aggregation propensity of each particular protein seems to be shaped by evolution according to its natural abundance in the cell. The production and downstream processing of recombinant ...polypeptides implies attaining concentrations that are orders of magnitude above their natural levels, often resulting in their aggregation; a phenomenon that precludes the marketing of many globular proteins for biomedical or biotechnological applications. Therefore, there is a huge interest in methods aimed to increase the proteins solubility above their natural limits. Here, we demonstrate that an updated version of our AGGRESCAN 3D structural aggregation predictor, that now takes into account protein stability, allows for designing mutations at specific positions in the structure that improve the solubility of proteins without compromising their conformation. Using this approach, we have designed a highly soluble variant of the green fluorescent protein and a human single-domain VH antibody displaying significantly reduced aggregation propensity. Overall, our data indicate that the solubility of unrelated proteins can be easily tuned by in silico-designed nondestabilizing amino acid changes at their surfaces.
This article provides the computational prediction of the atomistic architectures resulting from self-assembly of the polar heptapeptide sequences NYNYNYN, SYSYSYS and GYGYGYG. Using a combination of ...molecular dynamics and a newly developed tool for non-covalent interaction analysis, we uncover the properties of a new class of bionanomaterials, including hydrogen-bonded polar zippers, and the relationship between peptide composition, fibril geometry and weak interaction networks. Our results, corroborated by experimental observations, provide the basis for the rational design of prion-inspired nanomaterials.
This article provides the computational prediction of the atomistic architectures resulting from self-assembly of the polar heptapeptide sequences NYNYNYN, SYSYSYS and GYGYGYG.
Introduction/Aims
Very few studies analyzing the pattern of muscle involvement in magnetic resonance imaging (MRI) of patients with McArdle disease have been reported to date. We aimed to examine the ...pattern of muscle fat replacement in patients with McArdle disease.
Methods
We performed a retrospective study including all patients with genetically confirmed McArdle disease followed in our center from January 2010 to March 2021. Clinical data were collected from the medical record. Whole‐body MRI was performed as part of the diagnostic evaluation. The distribution of muscle fat replacement and its severity were analyzed.
Results
Nine patients were included. Median age at onset was 7 y (range, 5–58) and median age at the time when MRI was performed was 57.3 y (range, 37.2–72.8). At physical examination, four patients had permanent weakness: in three the weakness was limited to paraspinal muscles, whereas in one the weakness involved the paraspinal and proximal upper limb muscles. Muscle MRI showed abnormalities in six of the seven studied patients. In all of them, fat replacement of paravertebral muscles was found. Other muscles frequently affected were the tongue in three, subscapularis in three, and long head of biceps femoris and semimembranosus in two.
Discussion
Our findings suggest that paraspinal muscle involvement is common in McArdle disease and support the need to include this disease in the differential diagnosis of the causes of paraspinal muscle weakness. Involvement of the tongue and subscapularis are also frequent in McArdle disease.
Protein misfolding and aggregation into highly ordered fibrillar structures have been traditionally associated with pathological processes. Nevertheless, nature has taken advantage of the particular ...properties of amyloids for functional purposes, like in the protection of organisms against environmental changing conditions. Over the last decades, these fibrillar structures have inspired the design of new nanomaterials with intriguing applications in biomedicine and nanotechnology such as tissue engineering, drug delivery, adhesive materials, biodegradable nanocomposites, nanowires or biosensors. Prion and prion-like proteins, which are considered a subclass of amyloids, are becoming ideal candidates for the design of new and tunable nanomaterials. In this review, we discuss the particular properties of this kind of proteins, and the current advances on the design of new materials based on prion sequences.
Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were composed of entire or fractions of the ...infectious agent. However, challenges remain, and new vaccine technologies are mandatory. In this context, the use of mRNA for immunizing purposes has shown an enhanced performance, as demonstrated by the speedy approval of two mRNA vaccines preventing SARS-CoV-2 infection. Beyond success in preventing viral infections, mRNA vaccines can also be used for therapeutic cancer applications. Nevertheless, the instability of mRNA and its fast clearance from the body due to the presence of nucleases makes its naked delivery not possible. In this context, nanomedicines, and specifically polymeric nanoparticles, are critical mRNA delivery systems. Thus, the aim of this article is to describe the protocol for the formulation and test of an mRNA vaccine candidate based on the proprietary polymeric nanoparticles. The synthesis and chemical characterization of the poly(beta aminoesters) polymers used, their complexation with mRNA to form nanoparticles, and their lyophilization methodology will be discussed here. This is a crucial step for decreasing storage and distribution costs. Finally, the required tests to demonstrate their capacity to in vitro transfect and mature model dendritic cells will be indicated. This protocol will benefit the scientific community working on vaccination because of its high versatility that enables these vaccines to prevent or cure a wide variety of diseases.
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