MarR family proteins sense sulfane sulfur in bacteria.

Members of the multiple antibiotic resistance regulator (MarR) protein family are ubiquitous in bacteria and play critical roles in regulating cellular metabolism and antibiotic resistance. MarR family proteins function as repressors, and their interactions with modulators induce the expression of controlled genes. The previously characterized modulators are insufficient to explain the activities of certain MarR family proteins. However, recently, several MarR family proteins have been reported to sense sulfane sulfur, including zero-valent sulfur, persulfide (R-SSH), and polysulfide (R-SnH, n ≥ 2). Sulfane sulfur is a common cellular component in bacteria whose levels vary during bacterial growth. The changing levels of sulfane sulfur affect the expression of many MarR-controlled genes. Sulfane sulfur reacts with the cysteine thiols of MarR family proteins, causing the formation of protein thiol persulfide, disulfide bonds, and other modifications. Several MarR family proteins that respond to reactive oxygen species (ROS) also sense sulfane sulfur, as both sulfane sulfur and ROS induce the formation of disulfide bonds. This review focused on MarR family proteins that sense sulfane sulfur. However, the sensing mechanisms reviewed here may also apply to other proteins that detect sulfane sulfur, which is emerging as a modulator of gene regulation.

Disulfidptosis: A new type of cell death.

Disulfidptosis is a novel form of cell death that is distinguishable from established programmed cell death pathways such as apoptosis, pyroptosis, autophagy, ferroptosis, and oxeiptosis. This process is characterized by the rapid depletion of nicotinamide adenine dinucleotide phosphate (NADPH) in cells and high expression of solute carrier family 7 member 11 (SLC7A11) during glucose starvation, resulting in abnormal cystine accumulation, which subsequently induces andabnormal disulfide bond formation in actin cytoskeleton proteins, culminating in actin network collapse and disulfidptosis. This review aimed to summarize the underlying mechanisms, influencing factors, comparisons with traditional cell death pathways, associations with related diseases, application prospects, and future research directions related to disulfidptosis.

Glutathione-responsive CD-MOFs co-loading honokiol and indocyanine green biomimetic active targeting to enhance photochemotherapy for breast cancer.

Breast cancer has now replaced lung cancer as the most prevalent malignant tumor worldwide, posing a serious health risk to women. We have recently designed a promising option strategy for the treatment of breast cancer. In this work, cyclodextrin metal-organic frameworks with high drug-carrying properties were endo-crosslinked by 3,3'dithiodipropionyl chloride to form cubic phase gel nanoparticles, which were drug-loaded and then coated by MCF-7 cell membranes. After intravenous injection, this multifunctional nanomedicine achieved dramatically homologous targeting co-delivery of honokiol and indocyanine green to the breast tumor. Further, the disulfide bonds in the nanostructures achieved glutathione-responsive drug release, induced tumor cells to produce reactive oxygen species and promoted apoptosis, resulting in tumor necrosis, and at the same time, inhibited Ki67 protein expression, which enhanced photochemotherapy, and resulted in a 94.08 % in vivo tumor suppression rate in transplanted tumor-bearing mice. Thereby, this nanomimetic co-delivery system may have a place in breast cancer therapy due to its simple fabrication process, excellent biocompatibility, efficient targeted delivery of insoluble drugs, and enhanced photochemotherapy.

Rational Design of Loop Dynamics for a Barrel-Shaped Enzyme by Introducing Disulfide Bonds.

Loop dynamics redesign is an important strategy to manipulate protein function. Cellobiose 2-epimerase (CE) and other members of its superfamily are widely used for diverse industrial applications. The structural feature of the loops connecting barrel helices contributes greatly to the differences in their functional characteristics. Inspired by the in-silico mutation with molecular dynamics (MD) simulation analysis, we propose a strategy for identifying disulfide bond mutation candidates based on the prediction of protein flexibility and residue-residue interaction. The most beneficial mutant with the newly introduced disulfide bond would simultaneously improve both its thermostability and its reaction propensity to the targeting isomerization product. The ratio of the isomerization/epimerization catalytic rate was improved from 4:103 to 9:22. MD simulation and binding free energy calculations were applied to provide insights into molecular recognition upon mutations. The comparative analysis of enzyme/substrate binding modes indicates that the altered catalytic reaction pathway is due to less efficient binding of the native product. The key residue responsible for the observed phenotype was identified by energy decomposition and was further confirmed by the mutation experiment. The rational design of the key loop region might be a promising strategy to alter the catalytic behavior of all (α/α)6-barrel-like proteins.

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus.

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acid cycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.

Iron (II)-based metal-organic framework nanozyme for boosting tumor ferroptosis through inhibiting DNA damage repair and system Xc.

Development of ferroptosis-inducible nanoplatforms with high efficiency and specificity is highly needed and challenging in tumor ferrotherapy. Here, we demonstrate highly effective tumor ferrotherapy using iron (II)-based metal-organic framework (FessMOF) nanoparticles, assembled from disulfide bonds and ferrous ions. The as-prepared FessMOF nanoparticles exhibit peroxidase-like activity and pH/glutathione-dependent degradability, which enables tumor-responsive catalytic therapy and glutathione depletion by the thiol/disulfide exchange to suppress glutathione peroxidase 4, respectively. Upon PEGylation and Actinomycin D (ActD) loading, the resulting FessMOF/ActD-PEG nanoplatform induces marked DNA damage and lipid peroxidation. Concurrently, we found that ActD can inhibit Xc- system and elicit ferritinophagy, which further boosts the ferrotherapeutic efficacy of the FessMOF/ActD-PEG. In vivo experiments demonstrate that our fabricated nanoplatform presents excellent biocompatibility and a high tumor inhibition rate of 91.89%.

Crystal structure of the iron-sulfur cluster transfer protein ApbC from Escherichia coli.

Iron-sulfur (Fe-S) clusters are ubiquitous and are necessary to sustain basic life processes. The intracellular Fe-S clusters do not form spontaneously and many proteins are required for their biosynthesis and delivery. The bacterial P-loop NTPase family protein ApbC participates in Fe-S cluster assembly and transfers the cluster into apoproteins, with the Walker A motif and CxxC motif being essential for functionality of ApbC in Fe-S protein biogenesis. However, the structural basis underlying the ApbC activity and the motifs' role remains unclear. Here, we report the crystal structure of Escherichia coli ApbC at 2.8 Å resolution. The dimeric structure is in a W shape and the active site is located in the 2-fold center. The function of the motifs can be annotated by structural analyses. ApbC has an additional N-terminal domain that differs from other P-loop NTPases, possibly conferring its inherent specificity in vivo.

Warfarin analogs target disulfide bond-forming enzymes and suggest a residue important for quinone and coumarin binding.

Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes. In bacteria, disulfide bonds are catalyzed by DsbA and DsbB/VKOR enzymes. First, DsbA, a periplasmic disulfide oxidoreductase, introduces disulfide bonds into substrate proteins. Then, the membrane enzyme, either DsbB or VKOR, regenerate DsbA's activity by the formation of de novo disulfide bonds which reduce quinone. We have previously performed a high-throughput chemical screen and identified a family of warfarin analogs that target either bacterial DsbB or VKOR. In this work, we expressed functional human VKORc1 in Escherichia coli and performed a structure-activity-relationship analysis to study drug selectivity between bacterial and mammalian enzymes. We found that human VKORc1 can function in E. coli by removing two positive residues, allowing the search for novel anticoagulants using bacteria. We also found one warfarin analog capable of inhibiting both bacterial DsbB and VKOR and a second one antagonized only the mammalian enzymes when expressed in E. coli. The difference in the warfarin structure suggests that substituents at positions three and six in the coumarin ring can provide selectivity between the bacterial and mammalian enzymes. Finally, we identified the two amino acid residues responsible for drug binding. One of these is also essential for de novo disulfide bond formation in both DsbB and VKOR enzymes. Our studies highlight a conserved role of this residue in de novo disulfide-generating enzymes and enable the design of novel anticoagulants or antibacterials using coumarin as a scaffold.

In situ autophagy regulation in synergy with phototherapy for breast cancer treatment.

Autophagy is an important factor in reducing the efficacy of tumor phototherapy (including PTT and PDT). Accurate regulation of autophagy in tumor cells is a new strategy to improve the anti-tumor efficiency of PTT/PDT. This project intended to construct a tumor-activated autophagy regulator to efficiently block PTT/PDT-induced autophagy and realize synergistic sensitization to tumor phototherapy. To achieve this goal, we first synthesized TRANSFERRIN (Tf) biomimetic mineralized nano-tellurium (Tf-Te) as photosensitizer and then used disulfide bond reconstruction technology to induce Tf-Te self-assembly. The autophagy inhibitor hydroxychloroquine (HCQ) and iron ions carried by Tf were simultaneously loaded to prepare a tumor-responsive drug reservoir Tf-Te/HCQ. After entering breast cancer cells through the "self-guidance system", Tf-Te/HCQ can generate hyperpyrexia and ROS under NIR laser irradiation, to efficiently induce PTT/PDT effect. Meanwhile, the disulfide bond broke down in response to GSH, and the nanoparticles disintegrated to release Fe2+ and HCQ at fixed points. They simultaneously induce lysosomal alkalinization and increased osmotic pressure, effectively inhibit autophagy, and synergistically enhance the therapeutic effect of phototherapy. In vivo anti-tumor results have proved that the tumor inhibition rate of Tf-Te/HCQ can be as high as 88.6% on 4T1 tumor-bearing mice. This multifunctional drug delivery system might provide a new alternative for more precise and effective tumor phototherapy.

Effect and mechanism of C-terminal cysteine on the properties of HEV p222 protein.

Preliminary investigations have demonstrated that the cysteines located at the C-terminus of HEV ORF2 protein exhibits disulfide bonding capability during virus-like particles (VLPs) assembly. However, the effect and mechanism underlying the pairing of disulfide bonds formed by C627, C630, and C638 remains unclear. The p222 protein encompasses C-terminus and serves as a representative of HEV ORF2 to investigate the specific impacts of C627, C630, and C638. The three cysteines were subjected to site-directed mutagenesis and expressed in prokaryotes; Both the mutated proteins and p222 underwent polymerization except for p222A; Surprisingly, only p222 was observed as abundant spherical particles under transmission electron microscope (TEM); Stability and immunogenicity of the p222 exhibited higher than other mutated proteins; LC/MS/MS analysis identified four disulfide bonds in the p222. The novel findings suggest that the three cysteines contribute to structural and functional properties of ORF2 protein, highlighting the indispensability of each cysteine.

Unraveling redox pathways of the disulfide bond in dimethyl disulfide: Ab initio modeling.

CONTEXT: In cellular environments, the reduction of disulfide bonds is pivotal for protein folding and synthesis. However, the intricate enzymatic mechanisms governing this process remain poorly understood. This study addresses this gap by investigating a disulfide bridge reduction reaction, serving as a model for comprehending electron and proton transfer in biological systems. Six potential mechanisms for reducing the dimethyl disulfide (DMDS) bridge through electron and proton capture were explored. Thermodynamic and kinetic analyses elucidated the sequence of proton and electron addition. MD-PMM, a method that combines molecular dynamics simulations and quantum-chemical calculations, was employed to compute the redox potential of the mechanism. This research provides valuable insights into the mechanisms and redox potentials involved in disulfide bridge reduction within proteins, offering an understanding of phenomena that are challenging to explore experimentally. METHODS: All calculations used the Gaussian 09 software package at the MP2/6-311 + g(d,p) theory level. Visualization of the molecular orbitals and electron densities was conducted using Gaussview6. Molecular dynamics simulations were performed using GROMACS with the CHARMM36 force field. The PyMM program (Python Program for QM/MM Simulations Based on the Perturbed Matrix Method) is used to apply the Perturbed Matrix Method to MD simulations.

Enhanced Surface Accessibility of SARS-CoV-2 Omicron Spike Protein Due to an Altered Glycosylation Profile.

SARS-CoV-2 spike (S) proteins undergo extensive glycosylation, aiding in proper folding, enhancing stability, and evading host immune surveillance. In this study, we used mass spectrometric analysis to elucidate the N-glycosylation characteristics and disulfide bonding of recombinant spike proteins derived from the SARS-CoV-2 Omicron variant (B.1.1.529) in comparison with the D614G spike variant. Furthermore, we conducted microsecond-long molecular dynamics simulations on spike proteins to resolve how the different N-glycans impact spike conformational sampling in the two variants. Our findings reveal that the Omicron spike protein maintains an overall resemblance to the D614G spike variant in terms of site-specific glycan processing and disulfide bond formation. Nonetheless, alterations in glycans were observed at certain N-glycosylation sites. These changes, in synergy with mutations within the Omicron spike protein, result in increased surface accessibility of the macromolecule, including the ectodomain, receptor-binding domain, and N-terminal domain. Additionally, mutagenesis and pull-down assays reveal the role of glycosylation of a specific sequon (N149); furthermore, the correlation of MD simulation and HDX-MS identified several high-dynamic areas of the spike proteins. These insights contribute to our understanding of the interplay between structure and function, thereby advancing effective vaccination and therapeutic strategies.

Solvent-Free Reaction for Unsymmetrical Organodisulfides with High Purity and Application as Cathode-Active Materials.

Unsymmetrical disulfides, in which different organic groups are bonded to disulfide bonds, have been synthesized by cross-coupling reactions using thiols as substrates. However, due to the low-binding energy of unsymmetrical disulfides, its disproportionation occurs based on the side reactions with nucleophilic thiols, resulting in the impurity of symmetric disulfides. In this study, we developed a solvent-free synthesis method for unsymmetrical disulfides using thiosulfonates, thiols, and a base. This synthetic method enabled us to obtain highly pure diaryl-substituted unsymmetrical disulfides with particularly low-binding energy without control over the nucleophilicity and elimination properties of the substrate. Furthermore, it was observed that the disproportionation of unsymmetrical disulfides occurred in the solvent. This means that solvent-free condition is one of the factors to obtain unsymmetrical disulfides. As a new application of unsymmetrical disulfides, we applied unsymmetrical disulfides to cathode active materials of lithium batteries based on the reversible multi-electron redox activity of S-S bonds. The batteries using unsymmetrical disulfide cathode-active materials with a carbon nanotube exhibited initial capacities of 127 and 158 Ah/kg, equal to 42 and 53% of their theoretical ones. We demonstrated that unsymmetrical disulfides could be used as cathode-active materials for rechargeable batteries.

Fine-tuning the activation behaviors of ternary modular cabazitaxel prodrugs for efficient and on-target oral anti-cancer therapy.

The disulfide bond plays a crucial role in the design of anti-tumor prodrugs due to its exceptional tumor-specific redox responsiveness. However, premature breaking of disulfide bonds is triggered by small amounts of reducing substances (e.g., ascorbic acid, glutathione, uric acid and tea polyphenols) in the systemic circulation. This may lead to toxicity, particularly in oral prodrugs that require more frequent and high-dose treatments. Fine-tuning the activation kinetics of these prodrugs is a promising prospect for more efficient on-target cancer therapies. In this study, disulfide, steric disulfide, and ester bonds were used to bridge cabazitaxel (CTX) to an intestinal lymph vessel-directed triglyceride (TG) module. Then, synthetic prodrugs were efficiently incorporated into self-nanoemulsifying drug delivery system (corn oil and Maisine CC were used as the oil phase and Cremophor EL as the surfactant). All three prodrugs had excellent gastric stability and intestinal permeability. The oral bioavailability of the disulfide bond-based prodrugs (CTX-(C)S-(C)S-TG and CTX-S-S-TG) was 11.5- and 19.1-fold higher than that of the CTX solution, respectively, demonstrating good oral delivery efficiency. However, the excessive reduction sensitivity of the disulfide bond resulted in lower plasma stability and safety of CTX-S-S-TG than that of CTX-(C)S-(C)S-TG. Moreover, introducing steric hindrance into disulfide bonds could also modulate drug release and cytotoxicity, significantly improving the anti-tumor activity even compared to that of intravenous CTX solution at half dosage while minimizing off-target adverse effects. Our findings provide insights into the design and fine-tuning of different disulfide bond-based linkers, which may help identify oral prodrugs with more potent therapeutic efficacy and safety for cancer therapy.

m-nitrobenzyl alcohol supercharging reagent enhances the chromatographic separation and the charging of disulfide bond linked and His-tag peptides.

The linkages of disulfide bond (DSB) play important roles in protein stability and activity. Mass spectrometry-based (MS-based) techniques become accepted tools for DSB analysis in the recent decade. In the bottom-up approach, after enzyme digestion, the neighbouring amino acids of cysteines have great impacts on the physicochemical properties of resulting disulfide bond peptides, determining their retention behaviour on liquid chromatography (LC) and their MS ionization efficiency. In this study, the addition of supercharging reagent in LC mobile phase was used to examine the impact of supercharging reagent on the charge states of disulfide-bond peptides. The results showed that 0.1 % m-nitrobenzyl alcohol (m-NBA) in LC mobile phase increased the sensitivity and charge states of DSB peptides from our model protein, equine Interleukin-5 (eIL5), as well as the resolution of reversed-phase chromatography. Notably, also the sensitivity of C-terminal peptide with His-tag significantly improved. Our findings highlight the effectiveness of employing m-NBA as a supercharging reagent when investigating disulfide-linked peptides and the C-terminal peptide with a His-tag through nano-liquid chromatography mass spectrometry.

Reconstructing dynamics correlation network to simultaneously improve activity and stability of 2,3-butanediol dehydrogenase by design of distal interchain disulfide bonds.

The complete enzyme catalytic cycle includes substrate binding, chemical reaction and product release, in which different dynamic conformations are adopted. Due to the complex relationship among enzyme activity, stability and dynamics, the directed evolution of enzymes for improved activity or stability commonly leads to a trade-off in stability or activity. It hence remains a challenge to engineer an enzyme to have both enhanced activity and stability. Here, we have attempted to reconstruct the dynamics correlation network involved with active center to improve both activity and stability of a 2,3-butanediol dehydrogenase (2,3-BDH) by introducing inter-chain disulfide bonds. A computational strategy was first applied to evaluate the effect of introducing inter-chain disulfide bond on activity and stability of three 2,3-BDHs, and the N258C mutation of 2,3-BDH from Corynebacterium glutamicum (CgBDH) was proved to be effective in improving both activity and stability. In the results, CgBDH-N258C showed a different unfolding curve from the wild type, with two melting temperatures (Tm) of 68.3 °C and 50.8 °C, 19.7 °C and 2 °C higher than 48.6 °C of the wild type. Its half-life was also improved by 14.8-fold compared to the wild type. Catalytic efficiency (kcat/Km) of the mutant was increased by 7.9-fold toward native substrate diacetyl and 8.8-fold toward non-native substrate 2,5-hexanedione compared to the wild type. Molecular dynamics simulations revealed that an interaction network formed by Cys258, Arg162, Ala144 and the catalytic residues was reconstructed in the mutant and the dynamics change caused by the disulfide bond could be propagated through the interactions network. This improved the enzyme stability and activity by decreasing the flexibility and locking more "reactive" pose, respectively. Further construction of mutations including A144G showing a 44-fold improvement in catalytic efficiency toward meso-2,3-BD confirmed the role of modifying dynamics correlation network in tunning enzyme activity and selectivity. This study provided important insights into the relationship among dynamics, enzyme catalysis and stability, and will be useful in the designing new enzymes with co-evolution of stability, activity and selectivity.

A responsive disulfide bond switch aptamer prodrug exhibiting enhanced stability and anticancer efficacy.

Aptamers have shown significant potential in treating diverse diseases. However, challenges such as stability and drug delivery limited their clinical application. In this paper, the development of AS1411 prodrug-type aptamers for tumor treatment was introduced. A Short oligonucleotide was introduced at the end of the AS1411 sequence with a disulfide bond as responsive switch. The results indicated that the aptamer prodrugs not only enhanced the stability of the aptamer against nuclease activity but also facilitated binding to serum albumin. Furthermore, in the reducing microenvironment of tumor cells, disulfide bonds triggered drug release, resulting in superior therapeutic effects in vitro and in vivo compared to original drugs. This paper proposes a novel approach for optimizing the structure of nucleic acid drugs, that promises to protect other oligonucleotides or secondary structures, thus opening up new possibilities for nucleic acid drug design.

Evaluating Novel SP-B and SP-C Synthetic Analogues for Pulmonary Surfactant Efficacy.

Pulmonary surfactants, a complex assembly of phospholipids and surfactant proteins such as SP-B and SP-C, are critical for maintaining respiratory system functionality by lowering surface tension (ST) and preventing alveolar collapse. Our study introduced five synthetic SP-B peptides and one SP-C peptide, leading to the synthesis of CHAsurf candidates (CHAsurf-1 to CHAsurf-5) for evaluation. We utilized a modified Wilhelmy balance test to assess the surface tension properties of the surfactants, measuring spreading rate, surface adsorption, and ST-area diagrams to comprehensively evaluate their performance. Animal experiments were performed on New Zealand white rabbits to test the efficacy of CHAsurf-4B, a variant chosen for its economic viability and promising ST reduction properties, comparable to Curosurf®. The study confirmed that higher doses of SP-B in CHAsurf-4 are associated with improved ST reduction. However, due to cost constraints, CHAsurf-4B was selected for in vivo assessment. The animal model revealed that CHAsurf-4B could restore alveolar structure and improve lung elasticity, akin to Curosurf®. Our research highlights the significance of cysteine residues and disulfide bonds in the structural integrity and function of synthetic SP-B analogues, offering a foundation for future surfactant therapy in respiratory disorders. This study's findings support the potential of CHAsurf-4B as a therapeutic agent, meriting further investigation to solidify its role in clinical applications.

Disulfide Bonds of Thyroid Peroxidase Are Critical Elements for Subcellular Localization, Proteasome-Dependent Degradation, and Enzyme Activity.

Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Methods: Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) (p < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.

Engineering circular bacteriocins: structural and functional effects of α-helix exchanges and disulfide introductions in circularin A.

Circular bacteriocins form a distinct group of antimicrobial peptides (AMPs) characterized by their unique head-to-tail ligated circular structure and functional properties. They belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family. The ribosomal origin of these peptides facilitates rapid diversification through mutations in the precursor genes combined with specific modification enzymes. In this study, we primarily explored the bacteriocin engineering potential of circularin A, a circular bacteriocin produced by Clostridium beijerinckii ATCC 25752. Specifically, we employed strategies involving α-helix replacements and disulfide bond introductions to investigate their effects on both biosynthesis and bioactivity of the bacteriocin. The results show the feasibility of peptide engineering to introduce certain structural properties into circularin A through carefully designed approaches. The introduction of cysteines for potential disulfide bonds resulted in a substantial reduction in bacteriocin biosynthesis and/or bioactivity, indicating the importance of maintaining dynamic flexibility of α-helices in circularin A, while reduction of the potential disulfide in one case increased the activity. The 5 α-helices of circularin A were respectively replaced by corresponding helices from another circular peptide, enterocin AS-48, and modestly active peptides were obtained in a few cases. Overall, this study provides valuable insights into the engineering potential of circular bacteriocins as antimicrobial agents, including their structural and functional restrictions and their suitability as peptide engineering scaffolds. This helps to pave the way for the development of novel antimicrobial peptides with tailored properties based on circular bacteriocins.