Optimizing Antimicrobial Peptide Design: Integration of Cell-Penetrating Peptides, Amyloidogenic Fragments, and Amino Acid Residue Modifications.

The escalating threat of multidrug-resistant pathogens necessitates innovative approaches to combat infectious diseases. In this study, we examined peptides R23FS*, V31KS*, and R44KS*, which were engineered to include an amyloidogenic fragment sourced from the S1 protein of S. aureus, along with one or two cell-penetrating peptide (CPP) components. We assessed the antimicrobial efficacy of these peptides in a liquid medium against various strains of both Gram-positive bacteria, including S. aureus (209P and 129B strains), MRSA (SA 180 and ATCC 43300 strains), and B. cereus (strain IP 5832), and Gram-negative bacteria such as P. aeruginosa (ATCC 28753 and 2943 strains) and E. coli (MG1655 and K12 strains). Peptides R23FS*, V31KS*, and R44KS* exhibited antimicrobial activity comparable to gentamicin and meropenem against all tested bacteria at concentrations ranging from 24 to 48 µM. The peptides showed a stronger antimicrobial effect against B. cereus. Notably, peptide R44KS* displayed high efficacy compared to peptides R23FS* and V31KS*, particularly evident at lower concentrations, resulting in significant inhibition of bacterial growth. Furthermore, modified peptides V31KS* and R44KS* demonstrated enhanced inhibitory effects on bacterial growth across different strains compared to their unmodified counterparts V31KS and R44KS. These results highlight the potential of integrating cell-penetrating peptides, amyloidogenic fragments, and amino acid residue modifications to advance the innovation in the field of antimicrobial peptides, thereby increasing their effectiveness against a broad spectrum of pathogens.

Rapid aggregation of amyloid-like protein enhanced by mTGase to prepare functional wool fabrics for efficient and sustainable remove heavy metals from wastewater.

To counteract the increasing severity of water pollution and purify water sources, wastewater treatment materials are essential. In particular, it is necessary to improve the bonding strength between the adsorption material and the substrate in a long-term humid environment, and resist the invasion of microorganisms to prolong the service life. In this study, an amyloid-like aggregation method of lysozyme catalyzed by microbial transglutaminase (mTGase). Lysozyme self-assembles into an amyloid-like phase-transited lysozyme (PTL) in the presence of a reducing agent. Simultaneously, mTGase catalyzes acyl transfer reactions within lysozyme molecules or between lysozyme and keratin molecules, and driving PTL assembly on the wool fiber (TG-PTL@wool). This process enhances the grafting amount and fastness of PTL on the wool. Moreover, the tensile strength of wool fabric increased to 523 N. TG-PTL@wool achieves a 97.32 % removal rate of heavy metals, maintaining a removal rate of over 95 % after 5 cycles. TG-PTL@wool has excellent antibacterial property (99 %), and it remains above 90 % after 50 times of circulating washing. This study proved that mTGase can enhance the amyloid aggregation of lysozyme and enhance the bonding strength between PTL coating and substrate. Moreover, TG-PTL@wool provides a sustainable, efficient and cleaner solution for removing heavy metals from water.

Characterization of self-templating catalytic amyloids.

Amyloid aggregates with unique periodic structures have garnered significant attention due to their association with numerous diseases, including systemic amyloidoses and the neurodegenerative diseases Parkinson's, Alzheimer's, and Creutzfeld-Jakob. However, more recent investigations have expanded our understanding of amyloids, revealing their diverse functional biological roles. Amyloids have also been proposed to have played a significant role in prebiotic molecular evolution because of their exceptional stability, spontaneous formation in a prebiotic environment, catalytic and templating abilities, and cooperative interaction with fatty acids, polysaccharides, and nucleic acids. This chapter summarizes methods and techniques associated with studying short amyloidogenic peptides, including detailed procedures for investigating cross-templating and autocatalytic templating reactions. Since the work with amyloidogenic peptides and their aggregates present unique challenges, we have attempted to address these with essential details throughout the procedures. The lessons herein may be used in any amyloid-related research to ensure more reproducible results and reduce entrance barriers for researchers new to the field.

Atomic force microscopy 3D structural reconstruction of individual particles in the study of amyloid protein assemblies.

Recent developments in atomic force microscopy (AFM) image analysis have made three-dimensional (3D) structural reconstruction of individual particles observed on 2D AFM height images a reality. Here, we review the emerging contact point reconstruction AFM (CPR-AFM) methodology and its application in 3D reconstruction of individual helical amyloid filaments in the context of the challenges presented by the structural analysis of highly polymorphous and heterogeneous amyloid protein structures. How individual particle-level structural analysis can contribute to resolving the amyloid polymorph structure-function relationships, the environmental triggers leading to protein misfolding and aggregation into amyloid species, the influences by the conditions or minor fluctuations in the initial monomeric protein structure on the speed of amyloid fibril formation, and the extent of the different types of amyloid species that can be formed, are discussed. Future perspectives in the capabilities of AFM-based 3D structural reconstruction methodology exploiting synergies with other recent AFM technology advances are also discussed to highlight the potential of AFM as an emergent general, accessible and multimodal structural biology tool for the analysis of individual biomolecules.

Role of Amyloidogenic and Non-Amyloidogenic Protein Spaces in Neurodegenerative Diseases and their Mitigation Using Theranostic Agents.

Neurodegenerative diseases (NDDs) refer to a complex heterogeneous group of diseases which are associated with the accumulation of amyloid fibrils or plaques in the brain leading to progressive loss of neuronal functions. Alzheimer's disease is one of the major NDD responsible for 60-80 % of all dementia cases. Currently, there are no curative or disease-reversing/modifying molecules for many of the NDDs except a few such as donepezil, rivastigmine, galantamine, carbidopa and levodopa which treat the disease-associated symptoms. Similarly, there are very few FDA-approved tracers such as flortaucipir (Tauvid) for tau fibril imaging and florbetaben (Neuraceq), flutemetamol (Vizamyl), and florbetapir (Amyvid) for amyloid imaging available for diagnosis. Recent advances in the cryogenic electron microscopy reported distinctly different microstructures for tau fibrils associated with different tauopathies highlighting the possibility to develop tauopathy-specific imaging agents and therapeutics. In addition, it is important to identify the proteins that are associated with disease development and progression to know about their 3D structure to develop various diagnostics, therapeutics and theranostic agents. The current article discusses in detail the disease-associated amyloid and non-amyloid proteins along with their structural insights. We comprehensively discussed various novel proteins associated with NDDs and their implications in disease pathology. In addition, we document various emerging chemical compounds developed for diagnosis and therapy of different NDDs with special emphasis on theranostic agents for better management of NDDs.

Amyloids, amorphous aggregates and assemblies of peptides - Assessing aggregation.

Amyloid and amorphous aggregates represent the two major categories of aggregates associated with diseases, and although exhibiting distinct features, researchers often treat them as equivalent, which demonstrates the need for more thorough characterization. Here, we compare amyloid and amorphous aggregates based on their biochemical properties, kinetics, and morphological features. To further decipher this issue, we propose the use of peptide self-assemblies as minimalistic models for understanding the aggregation process. Peptide building blocks are significantly smaller than proteins that participate in aggregation, however, they make a plausible means to bridge the gap in discerning the aggregation process at the more complex, protein level. Additionally, we explore the potential use of peptide-inspired models to research the liquid-liquid phase separation as a feasible mechanism preceding amyloid formation. Connecting these concepts can help clarify our understanding of aggregation-related disorders and potentially provide novel drug targets to impede and reverse these serious illnesses.

Exploring Peptido-Nanocomposites in the Context of Amyloid Diseases.

Therapeutic peptides are a major class of pharmaceutical drugs owing to their target-binding specificity as well as their versatility in inhibiting aberrant protein-protein interactions associated with human pathologies. Within the realm of amyloid diseases, the use of peptides and peptidomimetics tailor-designed to overcome amyloidogenesis has been an active research endeavor since the late 90s. In more recent years, incorporating nanoparticles for enhancing the biocirculation and delivery of peptide drugs has emerged as a frontier in nanomedicine, and nanoparticles have further demonstrated a potency against amyloid aggregation and cellular inflammation to rival strategies employing small molecules, peptides, and antibodies. Despite these efforts, however, a fundamental understanding of the chemistry, characteristics and function of peptido-nanocomposites is lacking, and a systematic analysis of such strategy for combating a range of amyloid pathogeneses is missing. Here we review the history, principles and evolving chemistry of constructing peptido-nanocomposites from bottom up and discuss their future application against amyloid diseases that debilitate a significant portion of the global population.

Observations of amyloid breakdown by proteases over time using scanning acoustic microscopy.

Amyloid consists of insoluble beta-fibrillar proteins with stable structures. The Congo red staining method for histologically detecting amyloid is unsuitable for quantitatively assessing amyloid fibers. Scanning acoustic microscopy (SAM) detects the attenuation of sound (AOS) through sections. This study aimed to clarify whether AOS values reflected the amount of amyloid fibril degradation in tissues. Formalin-fixed paraffin-embedded unstained sections of various types of amyloidosis were digested with different endopeptidases. The AOS images after digestion were observed over time via SAM. The corresponding Congo red-stained images were followed to identify the amyloid. The amyloid and nonamyloid portions were statistically examined over time to determine the changes in the AOS values. Most of the amyloid areas showed significantly different AOS values from nonamyloid portions before digestion and significantly decreased after digestion; these findings corresponded with the disappearance and waning of the Congo red staining in the light microscopic images. Some nonamyloid areas with high AOS masked the reduction in AOS in the amyloid areas. The method used in this study may help detect the amyloid quantity and determine the appropriate treatment method for removing amyloid deposits from tissues.

Quantitative Attribution of the Protective Effects of Aminosterols against Protein Aggregates to Their Chemical Structures and Ability to Modulate Biological Membranes.

Natural aminosterols are promising drug candidates against neurodegenerative diseases, like Alzheimer and Parkinson, and one relevant protective mechanism occurs via their binding to biological membranes and displacement or binding inhibition of amyloidogenic proteins and their cytotoxic oligomers. We compared three chemically different aminosterols, finding that they exhibited different (i) binding affinities, (ii) charge neutralizations, (iii) mechanical reinforcements, and (iv) key lipid redistributions within membranes of reconstituted liposomes. They also had different potencies (EC50) in protecting cultured cell membranes against amyloid-ß oligomers. A global fitting analysis led to an analytical equation describing quantitatively the protective effects of aminosterols as a function of their concentration and relevant membrane effects. The analysis correlates aminosterol-mediated protection with well-defined chemical moieties, including the polyamine group inducing a partial membrane-neutralizing effect (79 ± 7%) and the cholestane-like tail causing lipid redistribution and bilayer mechanical resistance (21 ± 7%), linking quantitatively their chemistry to their protective effects on biological membranes.

Sequence-targeted Peptides Divert Functional Bacterial Amyloid Towards Destabilized Aggregates and Reduce Biofilm Formation.

Functional bacterial amyloid provides structural stability in biofilm, making it a promising target for anti-biofilm therapeutics. Fibrils formed by CsgA, the major amyloid component in E. coli are extremely robust and can withstand very harsh conditions. Like other functional amyloids, CsgA contains relatively short aggregation-prone regions (APR) which drive amyloid formation. Here, we demonstrate the use of aggregation-modulating peptides to knock down CsgA protein into aggregates with low stability and altered morphology. Remarkably, these CsgA-peptides also modulate fibrillation of the unrelated functional amyloid protein FapC from Pseudomonas, possibly through recognition of FapC segments with structural and sequence similarity with CsgA. The peptides also reduce the level of biofilm formation in E. coli and P. aeruginosa, demonstrating the potential for selective amyloid targeting to combat bacterial biofilm.

Solution-state NMR assignment and secondary structure propensity of the full length and minimalistic-truncated prefibrillar monomeric form of biofilm forming functional amyloid FapC from Pseudomonas aeruginosa.

Functional bacterial amyloids provide structural scaffolding to bacterial biofilms. In contrast to the pathological amyloids, they have a role in vivo and are tightly regulated. Their presence is essential to the integrity of the bacterial communities surviving in biofilms and may cause serious health complications. Targeting amyloids in biofilms could be a novel approach to prevent chronic infections. However, structural information is very scarce on them in both soluble monomeric and insoluble fibrillar forms, hindering our molecular understanding and strategies to fight biofilm related diseases. Here, we present solution-state NMR assignment of 250 amino acid long biofilm-forming functional-amyloid FapC from Pseudomonas aeruginosa. We studied full-length (FL) and shorter minimalistic-truncated (L2R3C) FapC constructs without the signal-sequence that is required for secretion. 91% and 100% backbone NH resonance assignments for FL and L2R3C constructs, respectively, indicate that soluble monomeric FapC is predominantly disordered, with sizeable secondary structural propensities mostly as PP2 helices, but also as α-helices and ß-sheets highlighting hotspots for fibrillation initiation interface. A shorter construct showing almost identical NMR chemical shifts highlights the promise of utilizing it for more demanding solid-state NMR studies that require methods to alleviate signal redundancy due to almost identical repeat units. This study provides key NMR resonance assignments for future structural studies of soluble, pre-fibrillar and fibrillar forms of FapC.

Mechanistic and biophysical insight into the inhibitory and disaggregase role of antibiotic moxifloxacin on human lysozyme amyloid formation.

Lysozyme amyloidosis is a systemic non-neuropathic disease caused by the accumulation of amyloids of mutant lysozyme. Presently, therapeutic interventions targeting lysozyme amyloidosis, remain elusive with only therapy available for lysozyme amyloidosis being supportive management. In this work, we examined the effects of moxifloxacin, a synthetic fluoroquinolone antibiotic on the amyloid formation of human lysozyme. The ability of moxifloxacin to interfere with lysozyme amyloid aggregation was examined using various biophysical methods like Rayleigh light scattering, Thioflavin T fluorescence assay, transmission electron microscopy and docking method. The reduction in scattering and ThT fluorescence along with extended lag phase in presence of moxifloxacin, suggest that the antibiotic inhibits and impedes the lysozyme fibrillation in concentration dependent manner. From ANS experiment, we deduce that moxifloxacin is able to decrease the hydrophobicity of the protein molecule thereby preventing aggregation. Our CD and DLS results show that moxifloxacin stabilizes the protein in its native monomeric structure, thus also showing retention of lytic activity upto 69% and inhibition of cytotoxicity at highest concentration of moxifloxacin. The molecular docking showed that moxifloxacin forms a stable complex of -7.6 kcal/mol binding energy and binds to the aggregation prone region of lysozyme thereby stabilising it and preventing aggregation. Moxifloxacin also showed disaggregase potential by disrupting fibrils and decreasing the ß-sheet content of the fibrils. Our current study, thus highlight the anti-amyloid and disaggregase property of an antibiotic moxifloxacin and hence sheds light on the future of antibiotics against protein aggregation, a hallmark event in many neurodegenerative diseases.

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification.

Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. The cellular energy currency ATP is known to indirectly regulate the formation, dissolution, or transmission of amyloid-like aggregates by providing energy to the molecular chaperones that maintain protein homeostasis. In this work, we demonstrate that ATP molecules, independent of any chaperones, modulate the formation and dissolution of amyloids from a yeast prion domain (NM domain of Saccharomyces cerevisiae Sup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP disaggregates preformed NM fibrils in a dose-independent manner. Our results indicate that ATP-mediated disaggregation, unlike the disaggregation by the disaggregase Hsp104, yields no oligomers that are considered one of the critical species for amyloid transmission. Furthermore, high concentrations of ATP delimited the number of seeds by giving rise to compact ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase to generate lower molecular weight amyloids. In addition, (low) pathologically relevant ATP concentrations restricted autocatalytic amplification by forming structurally distinct amyloids that are found seeding inefficient because of their reduced ß-content. Our results provide key mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions of amyloids.

Semiconductive and Biocompatible Nanofibrils from the Self-Assembly of Amyloid π-Conjugated Peptides.

Owing to their capacity to self-assemble into organized nanostructures, amyloid polypeptides can serve as scaffolds for the design of biocompatible semiconductive materials. Herein, symmetric and asymmetric amyloid π-conjugated peptides were prepared through condensation of perylene diimide (PDI) with a natural amyloidogenic sequence derived from the islet amyloid polypeptide. These PDI-bioconjugates assembled into long and linear nanofilaments in aqueous solution, which were characterized by a cross-ß-sheet quaternary organization. Current-voltage curves exhibited a clear signature of semiconductors, whereas the cellular assays revealed cytocompatibility and potential application in fluorescence microscopy. Although the incorporation of a single amyloid peptide appeared sufficient to drive the self-assembly into organized fibrils, the incorporation of two peptide sequences at the PDI's imide positions significantly enhanced the conductivity of nanofibril-based films. Overall, this study exposes a novel strategy based on amyloidogenic peptide to guide the self-assembly of π-conjugated systems into robust, biocompatible, and optoelectronic nanofilaments.

The Regulatory Mechanism of Transthyretin Irreversible Aggregation through Liquid-to-Solid Phase Transition.

Transthyretin (TTR) aggregation and amyloid formation are associated with several ATTR diseases, such as senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP). However, the mechanism that triggers the initial pathologic aggregation process of TTR remains largely elusive. Lately, increasing evidence has suggested that many proteins associated with neurodegenerative diseases undergo liquid-liquid phase separation (LLPS) and subsequent liquid-to-solid phase transition before the formation of amyloid fibrils. Here, we demonstrate that electrostatic interactions mediate LLPS of TTR, followed by a liquid-solid phase transition, and eventually the formation of amyloid fibrils under a mildly acidic pH in vitro. Furthermore, pathogenic mutations (V30M, R34T, and K35T) of TTR and heparin promote the process of phase transition and facilitate the formation of fibrillar aggregates. In addition, S-cysteinylation, which is a kind of post-translational modification of TTR, reduces the kinetic stability of TTR and increases the propensity for aggregation, while another modification, S-sulfonation, stabilizes the TTR tetramer and reduces the aggregation rate. Once TTR was S-cysteinylated or S-sulfonated, they dramatically underwent the process of phase transition, providing a foundation for post-translational modifications that could modulate TTR LLPS in the context of pathological interactions. These novel findings reveal molecular insights into the mechanism of TTR from initial LLPS and subsequent liquid-to-solid phase transition to amyloid fibrils, providing a new dimension for ATTR therapy.

Illustrating the Effect of Small Molecules Derived from Natural Resources on Amyloid Peptides.

The onset of amyloidogenic diseases is associated with the misfolding and aggregation of proteins. Despite extensive research, no effective therapeutics are yet available to treat these chronic degenerative diseases. Targeting the aggregation of disease-specific proteins is regarded as a promising new approach to treat these diseases. In the past few years, rapid progress in this field has been made in vitro, in vivo, and in silico to generate potential drug candidates, ranging from small molecules to polymers to nanoparticles. Small molecular probes, mostly those derived from natural sources, have been of particular interest among amyloid inhibitors. Here, we summarize some of the most important natural small molecular probes which can inhibit the aggregation of Aß, hIAPP, and α-syn peptides and discuss how their binding efficacy and preference for the peptides vary with their structure and conformation. This provides a comprehensive idea of the crucial factors which should be incorporated into the future design of novel drug candidates useful for the treatment of amyloid diseases.

Amyloids and Amyloid-like Protein Aggregates in Foods: Challenges and New Perspectives.

Protein misfolding and amyloid formations are associated with many neurodegenerative and systemic diseases. The discovery of Alzheimer's disease and its association with the accumulation of Amyloid-ß (Aß) peptides in the plaques uncovered the pleiotropic nature of peptides/ proteins. As of today, more than 50 proteins/ peptides are reported to form amyloids or amyloid-like protein aggregates under different conditions, establishing that amyloid formation could be a generic property of many proteins. In principle, under certain conditions, all the proteins have this property to form amyloid-like aggregates, which can be toxic or non-toxic. The extensive research in this direction led to an understanding of the ubiquitous nature of amyloids. Mounting evidences suggest that processed foods, particularly protein-rich foods, could be a plethora of amyloids or amyloid-like protein aggregates. Many are reported to be toxic, and their consumption raises health concerns. The assimilation of dietary proteins in the human body largely depends upon their conformational states and the digestive integrity of the gastrointestinal system. Amyloids or amyloid-like protein aggregates are usually protease resistant, and their presence in foods is likely to reduce nutritional value. Several biochemical and biophysical factors, commonly evident in various food processing industries, such as high temperature, the addition of acid, etc., are likely to induce the formation of protease-resistant protein aggregates. Aging significantly alters gastrointestinal health, predisposing aged individuals to be more susceptible to protein aggregation-related diseases. Consumption of foods containing such protein aggregates will lead to a poor supply of essential amino acids and might exaggerate the amyloid-related disease etiology. On the other hand, the gut microbiome plays a crucial role during pathological events leading to the development of Alzheimer's and Parkinson's diseases. The activity of gastrointestinal proteases, pH change, gut microbiome, and intestinal epithelium integrity would largely determine the outcome of consuming foods loaded with such protein aggregates. The current review outlines the recent development in this area and a new perspective for designing safe protein-rich diets for healthy nutrition.>.

Inhibition of Amyloid Protein Aggregation Using Selected Peptidomimetics.

Aberrant protein aggregation leads to the formation of amyloid fibrils. This phenomenon is linked to the development of more than 40 irremediable diseases such as Alzheimer's disease, Parkinson's disease, type 2 diabetes, and cancer. Plenty of research efforts have been given to understanding the underlying mechanism of protein aggregation, associated toxicity, and the development of amyloid inhibitors. Recently, the peptidomimetic approach has emerged as a potential tool to modulate several protein-protein interactions (PPIs). In this review, we discussed selected peptidomimetic-based approaches for the modulation of important amyloid proteins (Islet Amyloid Polypeptide, Amyloid Beta, α-synuclein, mutant p53, and insulin) aggregation. This approach holds a powerful platform for creating an essential stepping stone for the vital development of anti-amyloid therapeutic agents.

Human RAD51 Protein Forms Amyloid-like Aggregates In Vitro.

RAD51 is a central protein of homologous recombination and DNA repair processes that maintains genome stability and ensures the accurate repair of double-stranded breaks (DSBs). In this work, we assessed amyloid properties of RAD51 in vitro and in the bacterial curli-dependent amyloid generator (C-DAG) system. Resistance to ionic detergents, staining with amyloid-specific dyes, polarized microscopy, transmission electron microscopy (TEM), X-ray diffraction and other methods were used to evaluate the properties and structure of RAD51 aggregates. The purified human RAD51 protein formed detergent-resistant aggregates in vitro that had an unbranched cross-ß fibrillar structure, which is typical for amyloids, and were stained with amyloid-specific dyes. Congo-red-stained RAD51 aggregates demonstrated birefringence under polarized light. RAD51 fibrils produced sharp circular X-ray reflections at 4.7 Å and 10 Å, demonstrating that they had a cross-ß structure. Cytoplasmic aggregates of RAD51 were observed in cell cultures overexpressing RAD51. We demonstrated that a key protein that maintains genome stability, RAD51, has amyloid properties in vitro and in the C-DAG system and discussed the possible biological relevance of this observation.

Charge of Phospholipids Determines the Rate of Lysozyme Aggregation but Not the Structure and Toxicity of Amyloid Aggregates.

Biophysical properties of plasma membranes are determined by a chemical structure of phospholipids, including saturation of fatty acids and charge of polar heads of these molecules. Phospholipids not only determine fluidity and plasticity of membranes but also play an important role in abrupt aggregation of misfolded proteins. In this study, we investigate the role of the charge of the most abundant phospholipids in the plasma membrane on the aggregation properties of the lysozyme. We found that the charge of phospholipids determines the aggregation rate of lysozyme and the morphology of the protein aggregates. However, the secondary structure and toxicity of these protein specimens are determined by the chemical nature rather than the charge of phospholipids. These findings show that the charge of phospholipids can be a key factor that determines the stability and aggregation mechanism of amyloidogenic proteins.