Search Results (18)

Despite its ecological and economic importance, many aspects of Varroa destructor’s biology remain poorly understood, particularly its defense mechanisms against pathogens. The limited knowledge of Varroa’s immunity has hindered the development of RNA interference (RNAi)-based strategies targeting immune-related genes. In this study, we investigated the immune gene repertoire of V. destructor by querying its NCBI nr protein database and comparing it to model species of ticks (Ixodes scapularis) and mites (Galendromus occidentalis and Tetranychus urticae). Transcription of candidate immune genes was confirmed by analyzing a de novo assembled transcriptome of V. destructor. Our findings reveal that V. destructor shares key immunological traits with ticks, including lysozymes, chitinases, and thioester-containing proteins (TEPs), but also shares the absence of transmembrane peptidoglycan recognition proteins (PGRPs), Gram-negative binding proteins, and several lectin families involved in pathogen recognition. Additionally, Varroa mites, like ticks, lack homologs of crucial immune signaling components, such as the unpaired ligand (JAK/STAT), Eiger (JNK), and multiple elements of the IMD pathway. They also do not encode canonical antimicrobial peptides (AMPs) like defensins but possess putative homologs of ctenidins, AMPs previously identified in spiders and ticks, which may be adopted as a novel genetic readout for immune response in mites. Our findings lay the groundwork for future functional studies on mite immunity and open new avenues for RNAi-based biocontrol strategies targeting immune pathways to enhance Varroa management. Full article

(1) Background: Since the discovery of antibiotics in the first half of the 20th century, humans have abused this privilege, giving rise to antibiotic-resistant pathogens. Recent research has brought to light the use of antimicrobial peptides in polymers, hydrogels, and nanoparticles (NPs) as a newer and safer alternative to traditional antibiotics. (2) Methods: This review article is a synthesis of the scientific works published in the last 15 years, focusing on the synthesis of polymers with proven antimicrobial properties. (3) Results: After a critical review of the literature was made, information and data about the synthesis and antimicrobial activity of antibacterial polymers and NPs functionalized with antibiotics were extracted. Fluorinated surfactants such as the Quaterfluo^® series presented significant antimicrobial effects and could be modulated to contain thioesters to boost this characteristic. Biopolymers like chitosan and starch were also doped with iodine and used as iodophors to deliver iodine atoms directly to pathogens, as well as being antimicrobial on their own. Quaternary phosphonium salts are known for their increased antimicrobial activity compared to ammonium-containing polymers and are more thermally stable. (4) Conclusions: In summary, polymers and polymeric NPs seem like future alternatives to traditional antibiotics. Future research is needed to determine functional doses for clinical use and their toxicity. Full article

Bioconjugation reactions are critical to the modification of peptides and proteins, permitting the introduction of biophysical probes onto proteins as well as drugs for use in antibody-targeted medicines. A diverse set of chemical reagents can be employed in these circumstances to covalently label protein side chains, such as the amine moiety in the side chain of lysine and the thiol functionality in cysteine residues, two of the more frequently employed sites for modification. To provide researchers with a thermodynamic survey of the reaction of these residues with frequently employed chemical modification reagents as well as reactive cellular intermediates also known to modify proteins non-enzymatically, a theoretical investigation of the overall thermodynamics of models of these reactions was undertaken at the T1 and G3(MP2) thermochemical recipe levels (gas phase), the M06-2X/6-311+G(2df,2p)/B3LYP/6-31G(d) (gas and water phase), and the M06-2X/cc-PVTZ(-f)++ density functional levels of theory (water phase). Discussions of the relationship between the reagent structure and the overall thermodynamics of amine or thiol modification are presented. Of additional interest are the observations that routine cellular intermediates such as certain thioesters, acyl phosphates, and acetyl-L-carnitine can contribute to non-enzymatic protein modifications. These reactions and representative click chemistry reactions were also investigated. The computational survey presented herein (>320 reaction computations were undertaken) should serve as a valuable resource for researchers undertaking protein bioconjugation. A concluding section addresses the ability of computation to provide predictions as to the potential for protein modification by new chemical entities, with a cautionary note on protein modification side reactions that may occur when employing synthetic substrates to measure enzyme kinetic activities. Full article

Biomimetic N-acetylcysteamine thioesters are essential for the study of polyketide synthases, non-ribosomal peptide synthetases and fatty acid synthases. The chemistry for their preparation is, however, limited by their specific functionalization and their susceptibility to undesired side reactions. Here we report a method for the rapid preparation of N-acetylcysteamine (SNAC) 7-hydroxy-2-enethioates, which are suitable for the study of various enzymatic domains of megasynthase enzymes. The method is based on a one-pot sequence of hydroboration and the Suzuki–Miyaura reaction. The optimization of the reaction conditions made it possible to suppress potential side reactions and to introduce the highly functionalized SNAC methacrylate unit in a high yield. The versatility of the sequence was demonstrated by the synthesis of the complex polyketide-SNAC thioesters 12 and 33. Brown crotylation followed by the hydroboration to Suzuki–Miyaura reaction sequence enabled the introduction of the target motif in significantly fewer steps and with a higher overall yield and stereoselectivity than previously described approaches. This is the first report of a transition-metal-catalyzed cross-coupling reaction in the presence of an SNAC thioester. Full article

Thiol catalysts are essential in native chemical ligation (NCL) to increase the reaction efficiency. In this paper, we report the use of thiocholine in chemical protein synthesis, including NCL-based peptide ligation and metal-free desulfurization. Evaluation of thiocholine peptide thioester in terms of NCL and hydrolysis kinetics revealed its practical utility, which was comparable to that of other alkyl thioesters. Importantly, thiocholine showed better reactivity as a thiol additive in desulfurization, which is often used in chemical protein synthesis to convert Cys residues to more abundant Ala residues. Finally, we achieved chemical synthesis of two differently methylated histone H3 proteins via one-pot NCL and desulfurization with thiocholine. Full article

The multiplicity of simple molecules available on the primitive Earth probably made possible the development of extremely diverse prebiotic chemistry. The importance of thiols is widely recognized in the community studying the origin of life. De Duve’s “thioester world” has been considered a major contribution in this regard, where thioester bonds have high energies and thus can contribute to several chemical reactions. Herein, we propose specific models of thiols that exhibit unique activities toward several chemical reactions. Thanks to aminothiol and aminonitrile behaviors, we were able to obtain thiol-rich peptides with interesting catalytic activities leading to the formation of structurally diverse molecules. In a broader context, such chemistry could be introduced into systems chemistry scenarios in which it would be associated with the chemistry of nucleic acids or their precursors, as well as that of fatty acids. Full article

Microbial infections remain a global health concern, calling for the urgent need to implement effective prevention measures. Antimicrobial peptides (AMPs) have been extensively studied as potential antimicrobial coating agents. However, an efficient and economical method for AMP production is lacking. Here, we synthesized the direct coating adhesive AMP, NKC-DOPA₅, composed of NKC, a potent AMP, and repeats of the adhesive amino acid 3,4-dihydroxyphenylalanine (DOPA) via an intein-mediated protein ligation strategy. NKC was expressed as a soluble fusion protein His-NKC-GyrA (HNG) in Escherichia coli, comprising an N-terminal 6× His-tag and a C-terminal Mxe GyrA intein. The HNG protein was efficiently produced in a 500-L fermenter, with a titer of 1.63 g/L. The NKC-thioester was released from the purified HNG fusion protein by thiol attack and subsequently ligated with chemically synthesized Cys-DOPA₅. The ligated peptide His-NKC-Cys-DOPA₅ was obtained at a yield of 88.7%. The purified His-NKC-Cys-DOPA₅ possessed surface-binding and antimicrobial properties identical to those of the peptide obtained via solid-phase peptide synthesis. His-NKC-Cys-DOPA₅ can be applied as a practical and functional antimicrobial coating to various materials, such as medical devices and home appliances. Full article

In this review, we discuss various methods of reproducing life dynamics using a constructive approach. An increase in the structural complexity of a model protocell is accompanied by an increase in the stage of reproduction of a compartment (giant vesicle; GV) from simple reproduction to linked reproduction with the replication of information molecules (DNA), and eventually to recursive proliferation of a model protocell. An encounter between a plural protic catalyst (C) and DNA within a GV membrane containing a plural cationic lipid (V) spontaneously forms a supramolecular catalyst (C@DNA) that catalyzes the production of cationic membrane lipid V. The local formation of V causes budding deformation of the GV and equivolume divisions. The length of the DNA strand influences the frequency of proliferation, associated with the emergence of a primitive information flow that induces phenotypic plasticity in response to environmental conditions. A predominant protocell appears from the competitive proliferation of protocells containing DNA with different strand lengths, leading to an evolvable model protocell. Recently, peptides of amino acid thioesters have been used to construct peptide droplets through liquid–liquid phase separation. These droplets grew, owing to the supply of nutrients, and were divided repeatedly under a physical stimulus. This proposed chemical system demonstrates a new perspective of the origins of membraneless protocells, i.e., the “droplet world” hypothesis. Proliferative model protocells can be regarded as autonomous supramolecular machines. This concept of this review may open new horizons of “evolution” for intelligent supramolecular machines and robotics. Full article

Complex lasso proteins are a recently identified class of biological compounds that are present in considerable fraction of proteins with disulfide bridges. In this work, we look at complex lasso proteins as a generalization of well-known cysteine knots and miniproteins (lasso peptides). In particular, we show that complex lasso proteins with the same crucial topological features—cysteine knots and lasso peptides—are antimicrobial proteins, which suggests that they act as a molecular plug. Based on an analysis of the stability of the lasso piercing residue, we also introduce a method to determine which lasso motif is potentially functional. Using this method, we show that the lasso motif in antimicrobial proteins, as well in that in cytokines, is functionally relevant. We also study the evolution of lasso motifs, their conservation, and the usefulness of the lasso fingerprint, which extracts all topologically non-triviality concerning covalent loops. The work is completed by the presentation of extensive statistics on complex lasso proteins to analyze, in particular, the strange propensity for “negative” piercings. We also identify 21 previously unknown complex lasso proteins with an ester and a thioester bridge. Full article

While thiol-based catalysts are widely employed for chemical protein synthesis relying on peptide thioester chemistry, this is less true for selenol-based catalysts whose development is in its infancy. In this study, we compared different selenols derived from the selenocysteamine scaffold for their capacity to promote thiol–thioester exchanges in water at mildly acidic pH and the production of peptide thioesters from bis(2-sulfanylethyl)amido (SEA) peptides. The usefulness of a selected selenol compound is illustrated by the total synthesis of a biologically active human chemotactic protein, which plays an important role in innate and adaptive immunity. Full article

The early metabolism arising in a Thioester world gave rise to amino acids and their simple peptides. The catalytic activity of these early simple peptides became instrumental in the transition from Thioester World to a Phosphate World. This transition involved the appearances of sugar phosphates, nucleotides, and polynucleotides. The coupling of the amino acids and peptides to nucleotides and polynucleotides is the origin for the genetic code. Many of the key steps in this transition are seen in the catalytic cores of the nucleotidyltransferases, the class II tRNA synthetases (aaRSs) and the CCA adding enzyme. These catalytic cores are dominated by simple beta hairpin structures formed in the Thioester World. The code evolved from a proto-tRNA, a tetramer XCCA interacting with a proto-aminoacyl-tRNA synthetase (aaRS) activating Glycine and Proline. The initial expanded code is found in the acceptor arm of the tRNA, the operational code. It is the coevolution of the tRNA with the aaRSs that is at the heart of the origin and evolution of the genetic code. There is also a close relationship between the accretion models of the evolving tRNA and that of the ribosome. Full article

Amide bonds are the most prevalent structures found in organic molecules and various biomolecules such as peptides, proteins, DNA, and RNA. The unique feature of amide bonds is their ability to form resonating structures, thus, they are highly stable and adopt particular three-dimensional structures, which, in turn, are responsible for their functions. The main focus of this review article is to report the methodologies for the activation of the unactivated amide bonds present in biomolecules, which includes the enzymatic approach, metal complexes, and non-metal based methods. This article also discusses some of the applications of amide bond activation approaches in the sequencing of proteins and the synthesis of peptide acids, esters, amides, and thioesters. Full article

Selenoprotein K (SELENOK) is a selenocysteine (Sec)-containing protein localized in the endoplasmic reticulum (ER) membrane where it interacts with the DHHC6 (where single letter symbols represent Asp-His-His-Cys amino acids) enzyme to promote protein acyl transferase (PAT) reactions. PAT reactions involve the DHHC enzymatic capture of palmitate via a thioester bond to cysteine (Cys) residues that form an unstable palmitoyl-DHHC intermediate, followed by transfer of palmitate to Cys residues of target proteins. How SELENOK facilitates this reaction has not been determined. Splenocyte microsomal preparations from wild-type mice versus SELENOK knockout mice were used to establish PAT assays and showed decreased PAT activity (~50%) under conditions of SELENOK deficiency. Using recombinant, soluble versions of DHHC6 along with SELENOK containing Sec₉₂, Cys₉₂, or alanine (Ala₉₂), we evaluated the stability of the acyl-DHHC6 intermediate and its capacity to transfer the palmitate residue to Cys residues on target peptides. Versions of SELENOK containing either Ala or Cys residues in place of Sec were equivalently less effective than Sec at stabilizing the acyl-DHHC6 intermediate or promoting PAT activity. These data suggest that Sec₉₂ in SELENOK serves to stabilize the palmitoyl-DHHC6 intermediate by reducing hydrolyzation of the thioester bond until transfer of the palmitoyl group to the Cys residue on the target protein can occur. Full article

The chemical synthesis of an 85 residue analogue of the pore-forming protein, Equinatoxin II (EqtII), was achieved. Peptide precursors with over 40 residues were assembled by solid phase synthesis. The EqtII(1–46) fragment was modified to the reactive C-terminal thioester and native chemical ligation was performed with the A47C mutated EqtII(47–85) peptide to form the EqtII(1–85) analogue. Circular dichroism spectroscopy indicated that the N-terminal domain of EqtII(1–46) and EqtII(1–85) maintains predominantly an α-helical structure in solution and also in the presence of lipid micelles. This demonstrates the feasibility of assembling the full 179 residue protein EqtII via chemical means. Site-specific isotopic labels could be incorporated for structural studies in membranes by solid-state NMR spectroscopy. Full article

Aminoacyl-tRNA synthetases (AARSs) have evolved “quality control” mechanisms which prevent tRNA aminoacylation with non-protein amino acids, such as homocysteine, homoserine, and ornithine, and thus their access to the Genetic Code. Of the ten AARSs that possess editing function, five edit homocysteine: Class I MetRS, ValRS, IleRS, LeuRS, and Class II LysRS. Studies of their editing function reveal that catalytic modules of these AARSs have a thiol-binding site that confers the ability to catalyze the aminoacylation of coenzyme A, pantetheine, and other thiols. Other AARSs also catalyze aminoacyl-thioester synthesis. Amino acid selectivity of AARSs in the aminoacyl thioesters formation reaction is relaxed, characteristic of primitive amino acid activation systems that may have originated in the Thioester World. With homocysteine and cysteine as thiol substrates, AARSs support peptide bond synthesis. Evolutionary origin of these activities is revealed by genomic comparisons, which show that AARSs are structurally related to proteins involved in coenzyme A/sulfur metabolism and non-coded peptide bond synthesis. These findings suggest that the extant AARSs descended from ancestral forms that were involved in non-coded Thioester-dependent peptide synthesis, functionally similar to the present-day non-ribosomal peptide synthetases. Full article