Synergistic Interactions Are Prevalent in Catalytic Amyloids

Interactions between multiple functional groups are key to catalysis. Previously, we reported synergistic interactions in catalytic amyloids formed by mixtures of heptameric peptides that lead to significant improvements in esterase activity. Herein, we describe the in-depth investigation of synergistic interactions within a family of amyloid fibrils, exploring the results of functional group interactions, the effects of chirality and the use of mixed enantiomers within fibrils. Remarkably, we find that synergistic interactions (either positive or negative) are found in the vast majority of binary mixtures of catalytic amyloid-forming peptides. The productive arrangements of functionalities rapidly identified by mixing different peptides will undoubtedly lead to the development of more active catalysts for a variety of different transformations.     

Covalent Linkage and Macrocylization Preserve and Enhance Synergistic Interactions in Catalytic Amyloids

The self-assembly of short peptides into catalytic amyloid-like nanomaterials has proven to be a powerful tool in both understanding the evolution of early proteins and identifying new catalysts for practically useful chemical reactions. Here we demonstrate that both parallel and antiparallel arrangements of β-sheets can accommodate metal ions in catalytically productive coordination environments. Moreover, synergistic relationships, identified in catalytic amyloid mixtures, can be captured in macrocyclic and sheet-loop-sheet species, that offer faster rates of assembly and provide more complex asymmetric arrangements of functional groups, thus paving the way for future designs of amyloid-like catalytic proteins. Our findings show how initial catalytic activity in amyloid assemblies can be propagated and improved in more-complex molecules, providing another link in a complex evolutionary chain between short, potentially abiotically produced peptides and modern-day enzymes.     

Testing Antimicrobial Properties of Selected Short Amyloids

Amyloids and antimicrobial peptides (AMPs) have many similarities, e.g., both kill microorganisms by destroying their membranes, form aggregates, and modulate the innate immune system. Given these similarities and the fact that the antimicrobial properties of short amyloids have not yet been investigated, we chose a group of potentially antimicrobial short amyloids to verify their impact on bacterial and eukaryotic cells. We used AmpGram, a best-performing AMP classification model, and selected ten amyloids with the highest AMP probability for our experimental research. Our results indicate that four tested amyloids: VQIVCK, VCIVYK, KCWCFT, and GGYLLG, formed aggregates under the conditions routinely used to evaluate peptide antimicrobial properties, but none of the tested amyloids exhibited antimicrobial or cytotoxic properties. Accordingly, they should be included in the negative datasets to train the next-generation AMP prediction models, based on experimentally confirmed AMP and non-AMP sequences. In the article, we also emphasize the importance of reporting non-AMPs, given that only a handful of such sequences have been officially confirmed.     

Amyloidogenic Peptides: New Class of Antimicrobial Peptides with the Novel Mechanism of Activity

Antibiotic-resistant bacteria are recognized as one of the leading causes of death in the world. We proposed and successfully tested peptides with a new mechanism of antimicrobial action “protein silencing” based on directed co-aggregation. The amyloidogenic antimicrobial peptide (AAMP) interacts with the target protein of model or pathogenic bacteria and forms aggregates, thereby knocking out the protein from its working condition. In this review, we consider antimicrobial effects of the designed peptides on two model organisms, E. coli and T. thermophilus, and two pathogenic organisms, P. aeruginosa and S. aureus. We compare the amino acid composition of proteomes and especially S1 ribosomal proteins. Since this protein is inherent only in bacterial cells, it is a good target for studying the process of co-aggregation. This review presents a bioinformatics analysis of these proteins. We sum up all the peptides predicted as amyloidogenic by several programs and synthesized by us. For the four organisms we studied, we show how amyloidogenicity correlates with antibacterial properties. Let us especially dwell on peptides that have demonstrated themselves as AMPs for two pathogenic organisms that cause dangerous hospital infections, and in which the minimal inhibitory concentration (MIC) turned out to be comparable to the MIC of gentamicin sulfate. All this makes our study encouraging for the further development of AAMP. The hybrid peptides may thus provide a starting point for the antibacterial application of amyloidogenic peptides.     

The Bacterial Amyloids Phenol Soluble Modulins from Staphylococcus aureus Catalyze Alpha-Synuclein Aggregation

Aggregated α-synuclein (α-syn) is the main constituent of Lewy bodies, which are a pathological hallmark of Parkinson’s disease (PD). Environmental factors are thought to be potential triggers capable of initiating the aggregation of the otherwise monomeric α-syn. Braak’s seminal work redirected attention to the intestine and recent reports of dysbiosis have highlighted the potential causative role of the microbiome in the initiation of pathology of PD. Staphylococcus aureus is a bacterium carried by 30–70% of the general population. It has been shown to produce functional amyloids, called phenol soluble modulins (PSMαs). Here, we studied the kinetics of α-syn aggregation under quiescent conditions in the presence or absence of four different PSMα peptides and observed a remarkable shortening of the lag phase in their presence. Whereas pure α-syn monomer did not aggregate up to 450 h after initiation of the experiment in neither neutral nor mildly acidic buffer, the addition of different PSMα peptides resulted in an almost immediate increase in the Thioflavin T (ThT) fluorescence. Despite similar peptide sequences, the different PSMα peptides displayed distinct effects on the kinetics of α-syn aggregation. Kinetic analyses of the data suggest that all four peptides catalyze α-syn aggregation through heterogeneous primary nucleation. The immunogold electron microscopic analyses showed that the aggregates were fibrillar and composed of α-syn. In addition of the co-aggregated materials to a cell model expressing the A53T α-syn variant fused to GFP was found to catalyze α-syn aggregation and phosphorylation in the cells. Our results provide evidence of a potential trigger of synucleinopathies and could have implications for the prevention of the diseases.     

Secondary Structure in Amyloids in Relation to Their Wild Type Forms

The amyloid structures and their wild type forms, available in the PDB database, provide the basis for comparative analyses. Globular proteins are characterised by a 3D spatial structure, while a chain in any amyloid fibril has a 2D structure. Another difference lies in the structuring of the hydrogen bond network. Amyloid forms theoretically engage all the NH and C=O groups of the peptide bonds in a chain with two hydrogen bonds each. In addition, the hydrogen bond network is highly ordered—as perpendicular to the plane of the chain. The β-structure segments provide the hydrogen bond system with an anti-parallel system. The folds appearing in the rectilinear propagation of the segment with the β-structure are caused by just by one of the residues in the sequence—residues with a Rα-helical or Lα-helical conformation. The antiparallel system of the hydrogen bonds in the β-structure sections at the site of the amino acid with a Rα- or Lα-helical conformation changes into a parallel system locally. This system also ensures that the involvement of the C=O and H-N groups in the construction of the interchain hydrogen bond, while maintaining a perpendicular orientation towards the plane of the chain. Conformational analysis at the level of the Phi and Psi angles indicates the presence of the conditions for the structures observed in the amyloids. The specificity of amyloid structures with the dominant conformation expressed as |Psi| = |Phi| reveals the system of organisation present in amyloid fibrils. The Phi, Psi angles, as present in this particular structure, transformed to form |Psi| = |Phi| appear to be ordered co-linearly. Therefore, the calculation of the correlation coefficient may express the distribution around this idealised localisation on the Ramachandran map. Additionally, when the outstanding points are eliminated, the part of amyloid chain can be classified as fulfilling the defined conditions. In addition, the presentation of the chain structure using geometric parameters, V-angle—the angle between the planes of the adjacent peptide bonds (angle versus the virtual axis Cα-Cα) and the radius of the curvature R, depending on the size of the angle V, allows for a quantitative assessment of changes during amyloid transformation.     

Cationic Amphiphilic Peptides: Synthetic Antimicrobial Agents Inspired by Nature

Antimicrobial peptides are ubiquitous in multicellular organisms and have served as defense mechanisms for their successful evolution and throughout their life cycle. These peptides are short cationic amphiphilic polypeptides of fewer than 50 amino acids containing either a few disulfide-linked cysteine residues with a characteristic β-sheet-rich structure or linear α-helical conformations with hydrophilic side chains at one side of the helix and hydrophobic side chains on the other side. Antimicrobial peptides cause bacterial cell lysis either by direct cell-surface damage via electrostatic interactions between the cationic side chains of the peptide and the negatively charged cell surface, or by indirect modulation of the host defense systems. Electrostatic interactions lead to bacterial cell membrane disruption followed by leakage of cellular components and finally bacterial cell death. Because of their unusual mechanism of cell damage, antimicrobial peptides are effective against drug-resistant bacteria and may therefore prove more effective than classical antibiotics in certain cases. Currently, around 3000 natural antimicrobial peptides from six kingdoms (bacteria, archaea, protists, fungi, plants, and animals) have been isolated and sequenced. However, only a few of them are under clinical trials and/or in the commercial development stage for the treatment of bacterial infections caused by antibiotic-resistant bacteria. Moreover, high structural complexity, poor pharmacokinetic properties, and low antibacterial activity of natural antimicrobial peptides hinder their progress in drug development. To overcome these hurdles, researchers have become increasingly interested in modification and nature-inspired synthetic antimicrobial peptides. This review discusses some of the recent studies reported on antimicrobial peptides.     

Nordihydroguaiaretic Acid (NDGA) Inhibits CsgA Polymerization,Bacterial Amyloid Biogenesis,and Biofilm Formation

Escherichia coli and other Enterobacteriaceae thrive in robust biofilm communities through the coproduction of curli amyloid fibers and phosphoethanolamine cellulose. Curli promote adhesion to abiotic surfaces and plant and human host tissues and are associated with pathogenesis in urinary tract infection and food-borne illness. The production of curli in the host has also been implicated in the pathogenesis of neurodegenerative diseases. We report that the natural product nordihydroguaiaretic acid (NDGA) is effective as a curlicide in E. coli. NDGA prevents CsgA polymerization in vitro in a dose-dependent manner. NDGA selectively inhibits cell-associated curli assembly and inhibits uropathogenic E. coli biofilm formation. More broadly, this work emphasizes the ability to evaluate and identify bioactive amyloid assembly inhibitors by using the powerful gene-directed amyloid biogenesis machinery in E. coli.     

Comparative Biochemistry of Four Polyester (PET) Hydrolases**

The potential of bioprocessing in a circular plastic economy has strongly stimulated research into the enzymatic degradation of different synthetic polymers. Particular interest has been devoted to the commonly used polyester, poly(ethylene terephthalate) (PET), and a number of PET hydrolases have been described. However, a kinetic framework for comparisons of PET hydrolases (or other plastic-degrading enzymes) acting on the insoluble substrate has not been established. Herein, we propose such a framework, which we have tested against kinetic measurements for four PET hydrolases. The analysis provided values of k_cat and K_M, as well as an apparent specificity constant in the conventional units of M⁻¹s⁻¹. These parameters, together with experimental values for the number of enzyme attack sites on the PET surface, enabled comparative analyses. A variant of the PET hydrolase from Ideonella sakaiensis was the most efficient enzyme at ambient conditions; it relied on a high k_cat rather than a low K_M. Moreover, both soluble and insoluble PET fragments were consistently hydrolyzed much faster than intact PET. This suggests that interactions between polymer strands slow down PET degradation, whereas the chemical steps of catalysis and the low accessibility associated with solid substrate were less important for the overall rate. Finally, the investigated enzymes showed a remarkable substrate affinity, and reached half the saturation rate on PET when the concentration of attack sites in the suspension was only about 50 nM. We propose that this is linked to nonspecific adsorption, which promotes the nearness of enzyme and attack sites.     

Antimicrobial and Anticancer Properties of Synthetic Peptides Derived from the Wasp Parachartergus fraternus

Agelaia-MPI and protonectin are antimicrobial peptides isolated from the wasp Parachartergus fraternus that show antimicrobial and neuroactive activities. Previously, two analogues of these peptides, neuroVAL and protonectin-F, were designed to reduce nonspecific toxicity and improve potency. Here, the three-dimensional structures of neuroVAL, protonectin and protonectin-F were determined by using circular dichroism and NMR spectroscopy. Antibacterial, antifungal, cytotoxic and hemolytic activities were tested for the parent peptides and analogues. All peptides showed moderate antimicrobial activity against Gram-positive bacteria, with agelaia-MPI being the most active. Protonectin and protonectin-F were found to be toxic to cancerous and noncancerous cell lines. Internalization experiments revealed that these peptides accumulate inside both cell types. By contrast, neuroVAL was nontoxic to all tested cells and was able to enter cells without accumulating. In summary, neuroVAL has potential as a nontoxic cell-penetrating peptide, while protonectin-F needs further modification to realize its potential as an antitumor peptide.     

Synthetic Antibody Mimics Based on Cancer-Targeting Immunostimulatory Peptides

De novo cancer-targeting immunostimulatory peptides have been designed and developed as synthetic antibody mimics. A series of bifunctional peptides incorporating NKp30-binding and NK-cell-activating domains were synthesized as linear dimers and then extended into branching trimeric peptides by the incorporation of GRP78-targeting and tumor-cell-binding sequences. A selected trimeric peptide from this small set of peptides displayed binding capabilities on GRP78⁺ HepG2 and A549 target cells. Cell binding diminished in the presence of an anti-GRP78 peptide blocker, thus suggesting GRP78-binding dependence. Similarly, the selected trimeric peptide was also found to exhibit NK cell binding in an NKp30-dependent manner, which translated into NK cell activation as indicated by cytokine secretion. In co-culture, fluorescence microscopy revealed that the target GFP-expressing A549 cells were visibly associated with the effector NK cells when pre-activated with lead trimeric peptide. Accordingly, A549 cells were found to be compromised, as evidenced by the loss of GFP signal and notable detection of early-/late-stage apoptosis. Investigation of the immunological markers related to toxicity revealed detectable secretion of pro-inflammatory cytokines and chemokines, including IFN-γ, TNF-α, and IL-8. Furthermore, administration of peptide-activated NK cells into A549-tumor-bearing mice resulted in a consistent decrease in tumor growth when compared to the untreated control group. Taken together, the identification of a lead trimeric peptide capable of targeting and activating NK cells’ immunotoxicity directly towards GRP78⁺/B7H6^- tumors provides a novel proof-of-concept for the development of cancer-targeting immunostimulatory peptide ligands that mimic antibody-targeting and -activating functions related to cancer immunotherapy applications.     

Delineation of the core aggregation sequences of TDP-43 C-terminal fragment

Ubiquitinated cytoplasmic inclusions of TDP-43 and its C-terminal cleavage products are the pathological hallmarks of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinated inclusions. The C-terminal fragments (CTFs) of TDP-43 are increasingly considered to play an important role in its aggregation and in disease. Here, we employed a set of synthetic peptides spanning the length of the TDP-43 CTF (220-414) in order to find out its core aggregation domains. Two regions, one in the RRM-2 domain (246-255) and the other in the C-terminal domain (311-320) of TDP-43, stand out as highly aggregation prone. Studies done on recombinant purified TDP-43 CTF and its three mutants, in which these sequences were deleted individually and together, suggested that the 311-320 region has a more crucial role to play than the 246-255 in its aggregation. The study helps in defining specific peptide sequences that might form the core of TDP-43 aggregation. Identification of these sequences could help in designing peptide based inhibitors of TDP-43 aggregation.     

Development of Novel Thiazolopyrimidines as CDC25B Phosphatase Inhibitors

CDC25 inhibition by thiazolopyrimidines : CDC25 is an attractive target for cancer therapy, as it is overexpressed in numerous cancers and is often associated with tumor aggressiveness and poor prognosis. Based on an in silico/in vitro screen, we developed a series of thiazolopyrimidines to further improve inhibitory activity and cytotoxic properties.

     

合成肽在不对称催化反应中的应用

综述了合成肽作为催化剂在不对称催化反应中的应用,总结了各种合成肽催化剂的结构及其在催化不同类型反应如氰醇化反应、Strecker反应、Aldol反应、Michael加成反应、Morita-Baylis-Hillman反应和Stetter反应等中的催化活性及影响因素.     

GPA合成新工艺

通过两步乙酰化反应合成了葡萄糖五乙酸酯（ＧＰＡ），获得了最佳工艺条件。产品收率达９１．２％。     

A Novel Synthetic Route to Hexanitrohexaazaisowurtzitane

A new, non‐hydrogenolytic synthesis of hexanitrohexaazaisowurtzitane (HNIW, 1 ) has been investigated. The treatment of hexabenzylhexaazaisowurtzitane (HBIW, 2 ) with potassium permanganate in acetic anhydride resulted in oxidative debenzylation and acetylation to give tetraacetyldibenzylhexaazaisowurtzitane (TADB, 3a ) and other intermediates from the oxidation. Subsequent nitrosolysis and nitrolysis of these intermediates with 65% HNO₃/NaNO₂ and 70% HNO₃/AN produced HNIW in fair yield.     

新型除草剂氟丁酰草胺的合成研究

以邻氟三氟甲苯等为初始原料,经硝化、还原、重氮化、水解得到4-氟-3-三氟甲基苯酚,然后与烷基化剂2-溴丁酸乙酯反应,得到2-(4-氟-3-三氟甲基苯氧基)丁酸乙酯,再与苄胺发生酰化反应,得到目标产物氟丁酰草胺,总收率52.3%。     

环虫酰肼的合成

乙酰乙酸乙酯与甲醛缩合,然后脱羧得到2-甲基-4-氧-2-环己烯甲酸乙酯,在四乙基溴化铵催化下与1-溴-3-氯丙烷反应,得到5-甲基-7,8-2H-6-苯并二氢吡喃甲酸乙酯,再在硫粉的催化下脱氢得到5-甲基苯并二氢吡喃-6-甲酸乙酯,然后经水解、酰肼化得到关键中间体N-5-甲基苯并二氢吡喃-6-甲酰-N'-特丁基肼,最后与3,5-二甲基苯甲酰氯反应得到环虫酰肼,总收率23.3%。