Amyloid-like Assemblies Research Articles

Clearance of an amyloid-like translational repressor is governed by 14-3-3 proteins.

SUMMARYAmyloids are fibrous protein aggregates associated with age-related diseases. While these aggregates are typically described as irreversible and pathogenic, some cells use reversible amyloid-like structures that serve important functions. The RNA-binding protein Rim4 forms amyloid-like assemblies that are essential for translational control during Saccharomyces cerevisiae meiosis. Rim4 amyloid-like assemblies are disassembled in a phosphorylation-dependent manner at meiosis II onset. By investigating Rim4 clearance, we elucidate co-factors that mediate clearance of amyloid-like assemblies in a physiological setting. We demonstrate that yeast 14-3-3 proteins bind to Rim4 assemblies and facilitate their subsequent phosphorylation and timely clearance. Furthermore, distinct 14-3-3 proteins play non-redundant roles in facilitating phosphorylation and clearance of amyloid-like Rim4. Additionally, we find that 14-3-3 proteins contribute to global protein aggregate homeostasis. Based on the role of 14-3-3 proteins in aggregate homeostasis and their interactions with disease-associated assemblies, we propose that these proteins may protect against pathological protein aggregates.

Amyloid-like assembly converting commercial proteins to water-insoluble adsorbents with ultrahigh adsorption capacity and excellent antifouling property for uranium extraction

Uranium extraction from seawater is regarded as the most promising strategy for solving the concerns regarding the increasing demands of the nuclear industry.

Modeling and Designing Particle-Regulated Amyloid-like Assembly of Synthetic Polypeptides in Aqueous Solution.

In cells, actin and tubulin polymerization is regulated by nucleation factors, which promote the nucleation and subsequent growth of protein filaments in a controlled manner. Mimicking this natural mechanism to control the supramolecular polymerization of macromolecular monomers by artificially created nucleation factors remains a largely unmet challenge. Biological nucleation factors act as molecular scaffolds to boost the local concentrations of protein monomers and facilitate the required conformational changes to accelerate the nucleation and subsequent polymerization. An accelerated assembly of synthetic poly(l-glutamic acid) into amyloid fibrils catalyzed by cationic silica nanoparticle clusters (NPCs) as artificial nucleation factors is demonstrated here and modeled as supramolecular polymerization with a surface-induced heterogeneous nucleation pathway. Kinetic studies of fibril growth coupled with mechanistic analysis demonstrate that the artificial nucleators predictably accelerate the supramolecular polymerization process by orders of magnitude (e.g., shortening the assembly time by more than 10 times) when compared to the uncatalyzed reaction, under otherwise identical conditions. Amyloid-like fibrillation was supported by a variety of standard characterization methods. Nucleation followed a Michaelis-Menten-like scheme for the cationic silica NPCs, while the corresponding anionic or neutral nanoparticles had no effect on fibrillation. This approach shows the effectiveness of charge-charge interactions and surface functionalities in facilitating the conformational change of macromolecular monomers and controlling the rates of nucleation for fibril growth. Molecular design approaches like these inspire the development of novel materials via biomimetic supramolecular polymerizations.

Protonation-Deprotonation Switch Controls the Amyloid-like Misfolding of Nucleic-Acid-Binding Domains of TDP-43.

Nutrient starvation stress acidifies the cytosol and leads to the formation of large protein assemblies and misfolded aggregates. However, how starvation stress is sensed at the molecular level and leads to protein misfolding is poorly understood. TDP-43 is a vital protein, which, under stress-like conditions, associates with stress granule proteins via its functional nucleic-acid-binding domains (TDP-43tRRM) and misfolds to form aberrant aggregates. Here, we show that the monomeric N form of TDP-43tRRM forms a misfolded amyloid-like protein assembly, β form, in a pH-dependent manner and identified the critical protein side-chain residue whose protonation triggers its misfolding. We systematically mutated the three buried ionizable residues, D105, H166, and H256, to neutral amino acids to block the pH-dependent protonation-deprotonation titration of their side chain and studied their effect on the N-to-β transition. We observed that D105A and H256Q resembled TDP-43tRRM in their pH-dependent misfolding behavior. However, H166Q retains the N-like secondary structure under low-pH conditions and does not show pH-dependent misfolding to the β form. These results indicate that H166 is the critical side-chain residue whose protonation triggers the misfolding of TDP-43tRRM and shed light on how stress-induced misfolding of proteins during neurodegeneration could begin from site-specific triggers.

The intrinsic amyloidogenic propensity of cofilin-1 is aggravated by Cys-80 oxidation: A possible link with neurodegenerative diseases

Cofilin-1, an actin dynamizing protein, forms actin-cofilin rods, which is one of the major events that exacerbates the pathophysiology of amyloidogenic diseases. Cysteine oxidation in cofilin-1 under oxidative stress plays a crucial role in the formation of these rods. Others and we have reported that cofilin-1 possesses a self-oligomerization property in vitro and in vivo under physiological conditions. However, it remains elusive if cofilin-1 itself forms amyloid-like structures. We, therefore, hypothesized that cofilin-1 might form amyloid-like assemblies, with a potential to intensify the pathophysiology of amyloid-linked diseases. We used various in silico and in vitro techniques and examined the amyloid-forming propensity of cofilin-1. The study confirms that cofilin-1 possesses an intrinsic tendency of aggregation and forms amyloid-like structures in vitro.Further, we studied the effect of cysteine oxidation on the stability and structural features of cofilin-1. Our data show that oxidation at Cys-80 renders cofilin-1 unstable, leading to a partial loss of protein structure. The results substantiate our hypothesis and establish a strong possibility that cofilin-1 aggregation might play a role in cofilin-mediated pathology and the progression of several amyloid-linked diseases.

Thermo-Responsive self-assembly of a dual glucagon-like peptide and glucagon receptor agonist

The human peptide hormone Oxyntomodulin (Oxm) is known to induce satiety, increase energy expenditure, and control blood glucose in humans, making it a promising candidate for treatment of obesity and/or type 2 diabetes mellitus. However, a pharmaceutical exploitation has thus far been impeded by fast in vivo clearance and the molecule‘s sensitivity to half-life extending structural modifications. We recently showed that Oxm self-assembles into amyloid-like nanofibrils that continuously release active, soluble Oxm in a peptide-deprived environment. S.c. injected Oxm nanofibrils extended plasma exposure from a few hours to five days in rodents, compared to s.c. applied soluble Oxm. Here we show that Oxm fibril elongation kinetics and thermodynamics display a uniquely low temperature optimum compared to previously reported amyloid-like peptide and protein assemblies. Elongation rate is optimal at room temperature, with association rates 2–3 times higher at 25 °C than at ≥37 °C or ≤20 °C. We deduce from a combination of Cryo electron microscopy and spectroscopic methods that Oxm fibrils have a double-layered, triangular cross-section composed of arch-shaped monomers. We suggest a thermodynamic model that links the necessary molecular rearrangements during fibrillation and peptide release to the unique temperature effects in Oxm self-assembly and disassembly.

Amyloid-Like Aggregation in Diseases and Biomaterials: Osmosis of Structural Information.

The discovery that the polypeptide chain has a remarkable and intrinsic propensity to form amyloid-like aggregates endowed with an extraordinary stability is one of the most relevant breakthroughs of the last decades in both protein/peptide chemistry and structural biology. This observation has fundamental implications, as the formation of these assemblies is systematically associated with the insurgence of severe neurodegenerative diseases. Although the ability of proteins to form aggregates rich in cross-β structure has been highlighted by recent studies of structural biology, the determination of the underlying atomic models has required immense efforts and inventiveness. Interestingly, the progressive molecular and structural characterization of these assemblies has opened new perspectives in apparently unrelated fields. Indeed, the self-assembling through the cross-β structure has been exploited to generate innovative biomaterials endowed with promising mechanical and spectroscopic properties. Therefore, this structural motif has become the fil rouge connecting these diversified research areas. In the present review, we report a chronological recapitulation, also performing a survey of the structural content of the Protein Data Bank, of the milestones achieved over the years in the characterization of cross-β assemblies involved in the insurgence of neurodegenerative diseases. A particular emphasis is given to the very recent successful elucidation of amyloid-like aggregates characterized by remarkable molecular and structural complexities. We also review the state of the art of the structural characterization of cross-β based biomaterials by highlighting the benefits of the osmosis of information between these two research areas. Finally, we underline the new promising perspectives that recent successful characterizations of disease-related amyloid-like assemblies can open in the biomaterial field.

Clearance of an Amyloid-Like Translational Repressor is Governed by 14-3-3 Proteins

Amyloids are fibrous protein aggregates associated with age-related diseases. While these aggregates are typically described as irreversible and pathogenic, some cells utilize reversible amyloid-like structures that serve important functions. The RNA-binding protein Rim4 forms amyloid-like assemblies that are essential for translational control during S. cerevisiae meiosis. Rim4 amyloid-like assemblies are disassembled in a phosphorylation-dependent manner. By investigating Rim4 clearance, we elucidate how phosphorylation contributes to reversal and clearance of amyloid-like assemblies in a physiological setting. We demonstrate that yeast 14-3-3 proteins link Rim4 to its kinase, thus facilitating phosphorylation and timely clearance of Rim4 assemblies. Furthermore, distinct 14-3-3 proteins play non-redundant roles in facilitating phosphorylation and clearance of amyloid-like Rim4. Additionally, we find that 14-3-3 proteins contribute to global protein aggregate homeostasis. Based on the role of 14-3- 3 proteins in aggregate homeostasis and their interactions with disease-associated assemblies, we propose that these proteins may protect against pathological protein aggregates.

All-natural superhydrophobic coating for packaging and blood-repelling materials

The design of a bio-safe superhydrophobic coating to repel water adsorption, food and blood adherence is significantly important to attenuate food spoilage, food waste and thrombi. Herein, we synthesize an all-natural superhydrophobic interfacial material that has strong interfacial stability on a variety of substrates, and has good repellence towater, highly-viscous liquid foods and biofluids (blood, urine, etc.). The base material is simply prepared in an aqueous solution of lysozyme and cysteine, in which the rapid phase transition of lysozyme is triggered by a cysteine-induced thiol-disulfide exchange reaction. The resulting phase-transited lysozyme (PTL) aggregates into microparticles with a nanoscale roughness, which are further packed into a dense layer with a micro-/nano-topography. The PTL is composed of an inner amyloid-like structure and shows strong interfacial adhesion with the underlying substrate due to amyloid-inspired adhesion. The PTL layer is finally converted to a robust coating to effectively repel liquid foods and biofluids by being decorated with a natural hydrophobic carnauba wax. This coating noticeably enhances the water repellence of cellulose-based packaging materials, and the resulting superhydrophobic paper is completely non-wetted by water without weakening the mechanical stability and usability of the modified paper. The coating further resists the adhesion of milk, yogurt, honey and various other beverages and edible liquids, which leaves no residue on the coated surface. This coating also significantly resists wetting by blood and other human biofluids with a noticeable decrease in platelet adhesion on the surface. This work underlines the importance of an amyloid-like protein assembly in the preparation of an all-natural green interfacial coating towards the robust repellence of water, edible liquids and blood.

Meiotic Cells Counteract Programmed Retrotransposon Activation via RNA-Binding Translational Repressor Assemblies.

Retrotransposon proliferation poses a threat to germline integrity. While retrotransposons must be activated in developing germ cells in order to survive and propagate, how they are selectively activated in the context of meiosis is unclear. We demonstrate that the transcriptional activation of Ty3/Gypsy retrotransposons and host defense are controlled by master meiotic regulators. We show that budding yeast Ty3/Gypsy co-opts binding sites of the essential meiotic transcription factor Ndt80 upstream of the integration site, thereby tightly linking its transcriptional activation to meiotic progression. We also elucidate how yeast cells thwart Ty3/Gypsy proliferation by blocking translation of the retrotransposon mRNA using amyloid-like assemblies of the RNA-binding protein Rim4. In mammals, several inactive Ty3/Gypsy elements are undergoing domestication. We show that mammals utilize equivalent master meiotic regulators (Stra8, Mybl1, Dazl) to regulate Ty3/Gypsy-derived genes in developing gametes. Our findings inform how genes that are evolving from retrotransposons can build upon existing regulatory networks during domestication.

Meiotic Cells Counteract Programmed Retrotransposon Activation Via RNA-Binding Translational Repressor Assemblies

While retrotransposons must activate in developing gametes in order to survive and propagate, how a retrotransposon selectively activates in the context of meiosis is unclear. These mobile genetic elements can promote evolutionary innovations, but their unchecked proliferation is catastrophic for their host. Consequently, organisms have developed elegant mechanisms to counteract retrotransposon proliferation. In this manuscript, we demonstrate that both transcriptional activation of Ty3/Gypsy retrotransposons and host defense are controlled by master meiotic regulators. We show that Ty3 retrotransposons in budding yeast co-opt binding sites of the essential meiotic transcription factor Ndt80 upstream of the integration site, thereby tightly linking their transcriptional activation to meiotic progression. Our results raise the question of how yeast protects the genomes of its developing gametes from retrotransposon proliferation during meiosis. We elucidate how yeast cells thwart the Ty3/Gypsy retrotransposition life cycle by blocking translation of the retrotransposon mRNA using amyloid-like assemblies of the RNA-binding protein Rim4. In mammals, Gypsy elements have lost retrotransposition capacity. Building from our findings in yeast, we show that developing gametes in mammals also utilize essential meiotic transcription factors and assembly-forming RNA-binding proteins to regulate Gypsy-derived genes. Our findings inform how novel genes that are evolving from retrotransposons can build upon existing regulatory networks during domestication.

Proteostasis unbalance of nucleophosmin 1 in Acute Myeloid Leukemia: An aggregomic perspective

The role exerted by the nucleus in the regulation of proteostasis in both health and disease is recognized of outmost importance, even though not fully understood. Many recent investigations are focused on its ability to modulate and coordinate protein quality control machineries in mammalian cells. Nucleophosmin 1 (NPM1) is one of the most abundant nucleolar proteins and its gene is mutated in ~30% of Acute Myeloid Leukemia (AML) patients.Mutations are localized in the C-terminal domain of the protein and cause cytoplasmatically delocalized and possibly aggregated forms of NPM1 (NPM1c+). Therapeutic interventions targeted on NPM1c+ are in demand and, to this end, deeper knowledge of NPM1c+ behavior in the blasts' cytosol is required.Here by means of complementary biophysical techniques we compared the conformational and aggregative behavior of the entire C-terminal domains of NPM1wt and type A NPM1c+ (bearing the most common mutation). Overall data show that only Cterm_mutA is able to form amyloid-like assemblies with fibrillar morphology and that the oligomers are toxic in human neuroblastoma SHSY cells. This study adds a novel piece of knowledge to the comprehension of the molecular roles exerted by cytoplasmatic NPM1c+ and suggests the exploitation of the amyloidogenic propensity of NPM1c+ as a new strategy for targeting AML with NPM1 mutations.

Hefty structures of IgA and IgM complexes

Immunology Immunoglobulin M (IgM) and IgA are antibody isotypes that can form higher-order secretory complexes (sIgM and sIgA), which allows them to effectively bind and neutralize antigens with low-affinity repetitive epitopes, such as those found on the surface of many bacteria and viruses. The assembly and transport of these molecules is also dependent on the joining chain (J-chain) and the polymeric immunoglobulin receptor (pIgR) secretory component (SC). The architecture of these complex, multimeric structures has remained elusive. Li et al. resolved cryo–electron microscopy structures of the sIgM-Fc pentamer in complex with the J-chain and SC. Using similar techniques, Kumar et al. visualized dimeric, tetrameric, and pentameric structures of secretory sIgA-Fc interacting with the J-chain and SC. Both groups report highly similar mechanisms wherein the J-chain serves as a template for antibody oligomerization. An unanticipated, amyloid-like assembly of the oligomerized structure is present in both cases, with the J-chain conferring asymmetry for pIgR binding and transcytosis. These studies may inform structure-based engineering of these molecules for future therapeutic purposes. Science , this issue p. [1014][1], p. [1008][2] [1]: /lookup/doi/10.1126/science.aaz5425 [2]: /lookup/doi/10.1126/science.aaz5807

Structure of the secretory immunoglobulin A core.

Secretory immunoglobulin A (sIgA) represents the immune system's first line of defense against mucosal pathogens. IgAs are transported across the epithelium, as dimers and higher-order polymers, by the polymeric immunoglobulin receptor (pIgR). Upon reaching the luminal side, sIgAs mediate host protection and pathogen neutralization. In recent years, an increasing amount of attention has been given to IgA as a novel therapeutic antibody. However, despite extensive studies, sIgA structures have remained elusive. Here, we determine the atomic resolution structures of dimeric, tetrameric, and pentameric IgA-Fc linked by the joining chain (JC) and in complex with the secretory component of the pIgR. We suggest a mechanism in which the JC templates IgA oligomerization and imparts asymmetry for pIgR binding and transcytosis. This framework will inform the design of future IgA-based therapeutics.

Amyloid-like Assembly Activates a Phosphatase in the Developing Drosophila Embryo.

Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. Here, we report the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely being involved in TGF-β/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog is enzymatically inactive and becomes active upon amyloid-like assembly. The prion-like domain of Herzog is necessary for both its assembly and membrane targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane-targeting motif can restore phosphatase activity. This study provides an example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development.

Sn-Triggered Two-Dimensional Fast Protein Assembly with Emergent Functions.

The discovery of a general strategy for organizing functional proteins into stable nanostructures with the desired dimension, shape, and function is an important focus in developing protein-based self-assembled materials, but the scalable synthesis of such materials and transfer to other substrates remain great challenges. We herein tackle this issue by creating a two-dimensional metal-protein hybrid nanofilm that is flexible and cost-effective with reliable self-recovery, stability, and multifunctionality. As it differs from traditional metal ions, we discover the capability of Sn2+ to initiate fast amyloid-like protein assembly (occurring in seconds) by effectively reducing the disulfide bonds of native globular proteins. The Sn2+-initiated lysozyme aggregation at the air/water interface leads to droplet flattening, a result never before reported in a protein system, which finally affords a multifunctional 2D Sn-doped hybrid lysozyme nanofilm with an ultralarge area (e.g., 0.2 m2) within a few minutes. The hybrid film is distinctive in its ease of coating on versatile material surfaces with endurable chemical and mechanical stability, optical transparency, and diverse end uses in antimicrobial and photo-/electrocatalytic scaffolds. Our approach provides not only insights into the effect of tin ions on macroscopic self-assembly of proteins but also a controllable and scalable synthesis of a potential biomimic framework for biomedical and biocatalytic applications.

Zinc binding regulates amyloid-like aggregation of GAPR-1.

Members of the CAP superfamily (Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins) are characterized by the presence of a CAP domain that is defined by four sequence motifs and a highly conserved tertiary structure. A common structure–function relationship for this domain is hitherto unknown. A characteristic of several CAP proteins is their formation of amyloid-like structures in the presence of lipids. Here we investigate the structural modulation of Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) by known interactors of the CAP domain, preceding amyloid-like aggregation. Using isothermal titration calorimetry (ITC), we demonstrate that GAPR-1 binds zinc ions. Zn2+ binding causes a slight but significant conformational change as revealed by CD, tryptophan fluorescence, and trypsin digestion. The Zn2+-induced conformational change was required for the formation of GAPR-1 oligomers and amyloid-like assemblies in the presence of heparin, as shown by ThT fluorescence and TEM. Molecular dynamics simulations show binding of Zn2+ to His54 and His103. Mutation of these two highly conserved residues resulted in strongly diminished amyloid-like aggregation. Finally, we show that proteins from the cysteine-rich secretory protein (CRISP) subfamily are also able to form ThT-positive structures in vitro in a heparin- and Zn2+-dependent manner, suggesting that oligomerization regulated by metal ions could be a common structural property of the CAP domain.

Amyloid-Like Assembly During Embryogenesis Activates Herzog, a Novel Prion-Like Phosphatase

Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests prion-like proteins are prevalent in various species, however it remains unclear what functional space they occupy in multicellular organisms. Here, we report a novel prion-like protein, Herzog (CG5830), identified through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning by acting on components of TGF-β/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog is enzymatically inactive and becomes active upon amyloid-like assembly during gastrulation. The prion-like domain of Herzog is necessary for both its assembly and membrane-targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane targeting motif can restore phosphatase activity. This study provides a definitive example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development.

TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle.

SummaryA dominant histopathological feature in neuromuscular diseases including amyotrophic lateral sclerosis and inclusion body myopathy is cytoplasmic aggregation of the RNA-binding protein TDP-43. Although rare protein-misfolding mutations in TDP-43 often cause protein aggregation, most patients do not have a TDP-43 mutation suggesting aggregates of wild-type TDP-43 arise by an unknown mechanism. Here we show TDP-43 is an essential protein for normal skeletal muscle formation that unexpectedly forms cytoplasmic, amyloid-like oligomeric assemblies, termed myo-granules, during skeletal muscle regeneration in mice and humans. Myo-granules bind mRNAs encoding sarcomeric proteins and are cleared as myofibers mature. Although myo-granules occur during normal skeletal muscle regeneration, myo-granules can seed TDP-43 amyloid fibrils in vitro, and are increased in a mouse model of inclusion body myopathy. Therefore, heightened assembly or decreased clearance of functionally normal myo-granules could be the source of cytoplasmic TDP-43 aggregates common to neuromuscular disease.

X-ray Crystallographic Structure of a Teixobactin Derivative Reveals Amyloid-like Assembly.

This paper describes the X-ray crystallographic structure of a derivative of the antibiotic teixobactin and shows that its supramolecular assembly through the formation of antiparallel β-sheets creates binding sites for oxyanions. An active derivative of teixobactin containing lysine in place of allo-enduracididine assembles to form amyloid-like fibrils, which are observed through a thioflavin T fluorescence assay and by transmission electron microscopy. A homologue, bearing an N-methyl substituent, to attenuate fibril formation, and an iodine atom, to facilitate X-ray crystallographic phase determination, crystallizes as double helices of β-sheets that bind sulfate anions. β-Sheet dimers are key subunits of these assemblies, with the N-terminal methylammonium group of one monomer and the C-terminal macrocycle of the other monomer binding each anion. These observations suggest a working model for the mechanism of action of teixobactin, in which the antibiotic assembles and the assemblies bind lipid II and related bacterial cell wall precursors on the surface of Gram-positive bacteria.