Biomimetic folding strategies for chemical synthesis of disulfide-bonded peptides and proteins.

Disulfide-bonded peptides and proteins, including hormones, toxins, growth factors, and others, are abundant in living organisms. These molecules play crucial physiological roles such as regulating cell and organism growth, development, and metabolism. They have also found widespread applications as drugs or tool molecules in biomedical and pharmaceutical research. However, the chemical synthesis of disulfide-bonded proteins is complicated by the challenges associated with their folding. This review focuses on the latest advancements in disulfide-bonded peptide and protein folding technologies. Particularly, it highlights biomimetic folding strategies that emulate the naturally occurring oxidative folding processes in nature. These strategies include chaperone-assisted folding, glycosylation-assisted folding, and organic-based oxidative folding methods. The review also anticipates future directions in folding technology. Such research offers innovative approaches for the chemical synthesis of complex proteins that are otherwise difficult-to-fold.

Chemical synthesis of a 28 kDa full-length PET degrading enzyme ICCG by the removable backbone modification strategy.

Chemical protein synthesis offers a powerful way to access otherwise-difficult-to-obtain proteins such as mirror-image proteins. Although a large number of proteins have been chemically synthesized to date, the acquisition to proteins containing hydrophobic peptide fragments has proven challenging. Here, we describe an approach that combines the removable backbone modification strategy and the peptide hydrazide-based native chemical ligation for the chemical synthesis of a 28 kDa full-length PET degrading enzyme IGGC (a higher depolymerization efficiency of variant leaf-branch compost cutinase (LCC)) containing hydrophobic peptide segments. The synthetic ICCG exhibits the enzymatic activity and will be useful in establishing the corresponding mirror-image version of ICCG.

L-Glycosidase-Cleavable Natural Glycans Facilitate the Chemical Synthesis of Correctly Folded Disulfide-Bonded D-Proteins.

D-peptide ligands can be screened for therapeutic potency and enzymatic stability using synthetic mirror-image proteins (D-proteins), but efficient acquisition of these D-proteins can be hampered by the need to accomplish their in vitro folding, which often requires the formation of correctly linked disulfide bonds. Here, we report the finding that temporary installation of natural O-linked-ß-N-acetyl-D-glucosamine (O-GlcNAc) groups onto selected D-serine or D-threonine residues of the synthetic disulfide-bonded D-proteins can facilitate their folding in vitro, and that the natural glycosyl groups can be completely removed from the folded D-proteins to afford the desired chirally inverted D-protein targets using naturally occurring O-GlcNAcase. This approach enabled the efficient chemical syntheses of several important but difficult-to-fold D-proteins incorporating disulfide bonds including the mirror-image tumor necrosis factor alpha (D-TNFα) homotrimer and the mirror-image receptor-binding domain of the Omicron spike protein (D-RBD). Our work establishes the use of O-GlcNAc to facilitate D-protein synthesis and folding and proves that D-proteins bearing O-GlcNAc can be good substrates for naturally occurring O-GlcNAcase.

An Enzyme-Cleavable Solubilizing-Tag Facilitates the Chemical Synthesis of Mirror-Image Proteins.

Mirror-image proteins (D-proteins) are useful in biomedical research for purposes such as mirror-image screening for D-peptide drug discovery, but the chemical synthesis of many D-proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys-C protease-cleavable solubilizing tag and its use to synthesize difficult-to-obtain D-proteins. Our tag is easily installed onto multiple amino acids such as DLys, DSer, DThr, and/or the N-terminal amino acid of hydrophobic D-peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal-mediated deprotection, and yet can be completely removed by Lys-C protease under denaturing conditions to give the desired D-protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D-proteins: D-enantiomers of programmed cell death protein 1 IgV domain and SARS-CoV-2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more D-proteins.

Dealloying-Induced Zeolite-like Metal Framework of AB2 Laves Phase Intermetallic Electrocatalysts.

Exploring an efficient and robust electrocatalyst for hydrogen evolution reaction (HER) at high pH and temperature holds the key to the industrial application of alkaline water electrolysis (AWE). Herein, we design an open tunnel structure by dealloying a series of Laves phase intermetallics, i.e., MCo2 and MRu0.25Co1.75 (M = Sc and Zr). The dealloying process can induce a zeolite-like metal framework for ScCo2 and ScRu0.25Co1.75 by stripping Sc metal from the center of a tunnel structure. This structural engineering significantly lowers their overpotentials at a current density of 500 mA/cm2 (η500) ca. 80 mV in 1.0 M KOH. Through a simple process, ScRu0.25Co1.75 can be easily decorated on a carbon cloth substrate and only requires 132 mV to reach 500 mA/cm2. More importantly it can maintain activity over 1000 h in industrial conditions (6.0 M KOH at 333 K), showing its potential for practical industrial applications.

Dual Function of Hypo-d-electronic Transition Metals in the Brewer Intermetallic Phase for the Highly Efficient Electrocatalytic Hydrogen Evolution Reaction in Alkaline Electrolytes.

Reported are the synthesis, material characterization, and electrocatalytic hydrogen evolution reaction (HER) in acid and alkaline electrolytes for the Brewer intermetallic phase, Nb6Co7 and Mo6Co7. It was realized that the overpotential at a current density of 10 mA/cm2 (η10) for Nb6Co7 (η10 = 62 mV) and Mo6Co7 (η10 = 143 mV) are both much lower than that of using a single Co metal (η10 = 253 mV) in alkaline electrolytes. The enhancement of electrocatalytic HER activity of Nb6Co7 and Mo6Co7 can be attributed to the hypo-hyper-d-electronic interaction between Nb/Mo and Co elements. Based on the result of density functional theory calculation, alloying between Nb/Mo and Co elements will increase the antibonding state population of the Co-Co bond near the Fermi level (EF), which induces the synergistic effect to influence the adsorption energy of the H atom (ΔGH) on the surface of Nb6Co7 and Mo6Co7. Moreover, the role of the Nb element is not only a simple electron donor but is also an anchor position for the OH molecule (i.e., dual function) due to the bonding character of the Nb-Co bond near EF. It can reduce the OH position effect as well as the activation energy for water dissociation, which rationalizes the high and robust HER performance of Nb6Co7 to that of commercial Pt/C (η10 = 67 mV) in alkaline electrolytes.

MRTCM: A comprehensive dataset for probabilistic risk assessment of metals and metalloids in traditional Chinese medicine.

Traditional Chinese medicine (TCM) is still considered a global complementary or alternative medical system, but exogenous hazardous contaminants remain in TCM even after decocting. Besides, it is time-consuming to conduct a risk assessment of trace elements in TCMs with a non-automatic approach due to the wide variety of TCMs. Here, we present MRTCM, a cloud-computing infrastructure for automating the probabilistic risk assessment of metals and metalloids in TCM. MRTCM includes a consumption database and a pollutant database involving forty million rows of consumption data and fourteen types of TCM potentially toxic elements concentrations. The algorithm of probabilistic risk assessment was also packaged in MRTCM to assess the risks of eight elements with Monte Carlo simulation. The results demonstrated that 96.64% and 99.46% had no non-carcinogenic risk (hazard indices (HI) were < 1.0) for animal and herbal medicines consumers, respectively. After twenty years of exposure, less than 1% of the total carcinogenic risk (CRt) was > 10-4 for TCM consumers, indicating that they are at potential risk for carcinogenicity. Sensitivity analysis revealed that annual consumption and concentration were the main variables affecting the assessment results. Ultimately, a priority management list of TCMs was also generated, indicating that more attention should be paid to the non-carcinogenic risks of As, Mn, and Hg and the carcinogenic risks of As and Cr in Pheretima and Cr in Arcae Conch. In general, MRTCM could significantly enhance the efficiency of risk assessment in TCM and provide reasonable guidance for policymakers to optimize risk management.

Low-Temperature Cleanup Followed by Dispersive Solid-Phase Extraction for Determination of Nine Polar Plant Growth Regulators in Herbal Matrices Using Liquid Chromatography-Tandem Mass Spectrometry.

Polar plant growth regulators, used alone or doped in fertilizers, are most effective and widely utilized plant growth regulators (PGRs) in agriculture, which play important roles in mediating the yield and quality of crops and foodstuffs. The application scope has been extended to herbal medicines in the past 2 decades and relevant study is inadequate. The aim of this study is to establish a QuPPe-based extraction method containing low-temperature and d-SPE cleanup procedure followed by the detection on a selective multiresidue ultrahigh-performance liquid chromatography - triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) in three herbal matrices. This simple, accurate, versatile and robust method was verified according to the validation criteria of the SANTE/12682/2019 guideline document. The analytical range was from 2.5 to 200 µg/L, and the average recoveries were in the range of 64.6-117.8% (n = 6). The optimized method was applied to 135 herbal medicines thereof. Result showed that the detection frequency of chlormequat was the highest in the investigated PGRs, with the positive rate of 15.6%. Improvement of the detection method for polar PGRs will enrich the coverage of PGRs, which is conducive to safeguard public health and ensure drug safety. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-023-04254-3.

Backbone-Installed Split Intein-Assisted Ligation for the Chemical Synthesis of Mirror-Image Proteins.

Membrane-associated D-proteins are an important class of synthetic molecules needed for D-peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a Backbone-Installed Split Intein-Assisted Ligation (BISIAL) method for the synthesis of these proteins, wherein the native L-forms of the N- and C-intein fragments of the unique consensus-fast (Cfa) (i.e. L-CfaN and L-CfaC ) are separately installed onto the two D-peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (µM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D-proteins without leaving any "ligation scar" on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D-enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D-protein targets.

Total Chemical Synthesis of Correctly Folded Disulfide-Rich Proteins Using a Removable O-Linked ß-N-Acetylglucosamine Strategy.

Disulfide-rich proteins are useful as drugs or tool molecules in biomedical studies, but their synthesis is complicated by the difficulties associated with their folding. Here, we describe a removable glycosylation modification (RGM) strategy that expedites the chemical synthesis of correctly folded proteins with multiple or even interchain disulfide bonds. Our strategy comprises the introduction of simple O-linked ß-N-acetylglucosamine (O-GlcNAc) groups at the Ser/Thr sites that effectively improve the folding of disulfide-rich proteins by stabilization of their folding intermediates. After folding, the O-GlcNAc groups can be efficiently removed using O-GlcNAcase (OGA) to afford the correctly folded proteins. Using this strategy, we completed the synthesis of correctly folded hepcidin, an iron-regulating hormone bearing four pairs of disulfide-bonds, and the first total synthesis of correctly folded interleukin-5 (IL-5), a 26 kDa homodimer cytokine responsible for eosinophil growth and differentiation.

Function of Internal and External Fe in a Ni-Based Precatalyst System Toward Oxygen Evolution Reaction.

It is well known that the "iron" impurity will influence the oxygen evolution reaction (OER) in an alkaline electrolyte, especially for the Ni-based electrocatalyst. Many research studies have investigated the function of Fe in the OER active phase, such as M(OH)2/MOOH (M = Ni and/or Fe), while, surprisingly, very few studies have examined the function of Fe in the "precatalyst" system. Accordingly, in this work, the Ni3-xFexP (x = 0, 0.5, 1) series as an Ni-based precatalyst was employed to inspect the function of internal and external Fe in the Ni-based precatalyst system. It was realized that the sample with internal Fe (i.e., Ni2.5Fe0.5P and Ni2FeP) exhibits efficient OER activity compared to that of the Fe-free one (i.e., Ni3P) owing to the large amount of active M(OH)2/MOOH formed on the surface. This indicates that the internal Fe in the present system may have the ability to facilitate the phase transformation; it was later rationalized from electronic structural calculations that the d band center of the internal Fe (middle transition metal) and Ni (late transition metal) holds the key for this observation. Adding excessive ferrous chloride tetrahydrate (FeCl2·4H2O) as the external Fe in the electrolyte will greatly improve the OER performances for Ni3P; nevertheless, that the OER activity of Ni2FeP is still much superior than that of Ni3P corroborates the fact that the Fe impurity is not the only reason for the elevated OER activity of Ni2FeP and that internal Fe is also critical to the phase transformation as well as OER performance.

Realgar-induced nephrotoxicity via ferroptosis in mice.

Ferroptosis is a novel form of iron-dependent cell death that is involved in arsenic-induced toxicity. Realgar is an arsenic-containing Chinese medicine, which can result in nephrotoxicity because of long-term exposure. However, it remains scientifically unknown whether Realgar is an inducer of ferroptosis in the kidney. This study investigated the role of ferroptosis in Realgar-induced kidney toxicity in mice. ICR mice were exposed to Realgar for 28 days, and HK2 cells were exposed to Realgar in the presence or absence of treatment with ferrostatin-1, a ferroptosis inhibitor. The ferroptosis-related indicators were further evaluated. Realgar can cause nephrotoxicity in mice by continuous gavage for 28 days, accompanied by an increase in iron accumulation and reactive oxygen species (ROS). The reduced expression of Slc7A11 and Gpx4 further confirmed the ferroptosis mediated by Realgar. Meanwhile, Realgar disrupted the antioxidant system as evidenced by the formation of ROS leading to the inactivation of antioxidant enzymes. Realgar caused ferroptosis in a dose-dependent manner, which was significantly reduced by ferrostatin-1 in HK2 cells. This study revealed that Realgar-induced ferroptosis triggered nephrotoxicity in mice and provided new clues to elucidate the mechanism of Realgar-induced nephrotoxicity.

Exposure assessment and risk-based limit levels evaluation of ochratoxin A in Astragali Radix in China.

Ochratoxin A (OTA) is a mycotoxin found in a variety of foods and herbal medicines, and several governmental bodies around the world have set maximum allowable levels of OTA in different foods and herbal medicines. This study aims to evaluate the health risk of OTA in Astragali Radix (AR) in China, and to evaluate the effects of different limit levels on the risk control of OTA in AR. The concentrations of OTA in 187 samples of AR were investigated, and 61 (32.6%) samples were positive. The mean, 50th and 95th percentile values of OTA in positive samples were 56.2, 5.1 and 304.5 µg/kg, respectively. A margin of exposure (MOE) approach was applied to assess the risk. Considering other food sources, long-term consumers have a relatively high risk of OTA exposure due to the ingestion of AR. Theoretical limit levels of OTA in AR were evaluated from two dimensions by weighing the costs and the benefits. The results indicated that the limit levels that might be applied to the management of OTA contamination in AR in China could be screened out through risk-based evaluation of limit levels.

Integrated Metabolome and Lipidome Strategy to Reveal the Action Pattern of Paclobutrazol, a Plant Growth Retardant, in Varying the Chemical Constituents of Platycodon Root.

Platycodon root, a medicinal food homology species which has been used in Asian countries for hundreds of years, is now widely cultivated in China. Treatment with paclobutrazol, a typical plant growth retardant, has raised uncertainties regarding the quality of Platycodon root, which have been rarely investigated. In the present study, metabolomic and lipidomic differences were revealed by ultra-high performance liquid chromatography coupled to ion mobility-quadrupole time of flight mass spectrometry (UPLC-IM-QTOF-MS). A significant decrease of platycodigenin-type saponins was observed in the paclobutrazol-treated sample. Carrying out a comprehensive quantitative analysis, the contents of total saponins and saccharides were determined to illustrate the mode of action of paclobutrazol on Platycodon root. This study demonstrated an exemplary research model in explaining how the exogenous matter influences the chemical properties of medicinal plants, and therefore might provide insights into the reasonable application of plant growth regulators.

A Strategy for Rapid Discovery of Marker Peptides Associated with Fibrinolytic Efficacy of Pheretima aspergillum Based on Bioinformatics Combined with Parallel Reaction Monitoring.

Quality control of animal-derived traditional Chinese medicines has improved dramatically as proteomics research advanced in the past few decades. However, it remains challenging to identify quality attributes with routine proteomics approaches since protein with fibrinolytic activity is rarely reported in pheretima, a typical animal-derived traditional medicine. A novel strategy based on bioinformatics combined with parallel reaction monitoring (PRM) was developed here to rapidly discover the marker peptides associated with a fibrinolytic effect. Potential marker peptides were found by lumbrokinase sequences' alignment and in silico digestion. The fibrinogen zymography was used to visually identify fibrinolytic proteins in pheretima. As a result, it was found that the fibrinolytic activity varied among different portions of pheretima. Fibrinolytic proteins were distributed regionally in the anterior and anterior-mid portion and there was no significant fibrinogenolytic activity observed in the mid-posterior and posterior portion. Finally, PRM experiments were deployed to validate and quantify selected marker peptides and a total of 11 peptides were identified as marker peptides, which could be potentially used in quality control of pheretima. This strategy provides a robust workflow to benefit the quality control of other animal-derived traditional medicines.

Chemical Synthesis of a Full-Length G-Protein-Coupled Receptor ß2-Adrenergic Receptor with Defined Modification Patterns at the C-Terminus.

The ß2-adrenergic receptor (ß2AR) is a G-protein-coupled receptor (GPCR) that responds to the hormone adrenaline and is an important drug target in the context of respiratory diseases, including asthma. ß2AR function can be regulated by post-translational modifications such as phosphorylation and ubiquitination at the C-terminus, but access to the full-length ß2AR with well-defined and homogeneous modification patterns critical for biochemical and biophysical studies remains challenging. Here, we report a practical synthesis of differentially modified, full-length ß2AR based on a combined native chemical ligation (NCL) and sortase ligation strategy. An array of homogeneous samples of full-length ß2ARs with distinct modification patterns, including a full-length ß2AR bearing both monoubiquitination and octaphosphorylation modifications, were successfully prepared for the first time. Using these homogeneously modified full-length ß2AR receptors, we found that different phosphorylation patterns mediate different interactions with ß-arrestin1 as reflected in different agonist binding affinities. Our experiments also indicated that ubiquitination can further modulate interactions between ß2AR and ß-arrestin1. Access to full-length ß2AR with well-defined and homogeneous modification patterns at the C-terminus opens a door to further in-depth mechanistic studies into the structure and dynamics of ß2AR complexes with downstream transducer proteins, including G proteins, arrestins, and GPCR kinases.

Function of Doping Ru Element in the Hydrogen Evolution Reaction in Rare-Earth Transition-Metal Intermetallics.

Transition metal-based intermetallics are promising electrocatalysts for replacing the commercial Pt metal in the hydrogen evolution reaction (HER). In this work, RENi2 and RERu0.25Ni1.75 (RE = Pr, Tb, and Er) were synthesized and their electrocatalytic HER activities were explored. Among undoped compounds, PrNi2 exhibits the best performance and requires an overpotential of 55 mV, while partially replacing Ni with Ru element (PrRu0.25Ni1.75) can greatly reduce the overpotential to 20 mV at a current density of 10 mA/cm2. Such enhancement was recognized that belongs to their extrinsic property, and their intrinsic HER activities were similar after normalizing the electrocatalytic surface area. Further investigation on ScM2 and ScRu0.25M1.75 (M = Co and Ni) suggests that doping Ru element in ScCo2 will significantly enhance antibonding character around the Fermi level (EF) and weaken hydrogen adsorption energy. On the other hand, the antibonding population for ScNi2 and ScRu0.25Ni1.75 is similar at EF, which accounts for their close intrinsic HER activities.

Synthesis, Crystal Structure, Electronic Structure, and Electrocatalytic Hydrogen Evolution Reaction of Synthetic Perryite Mineral.

Recently, it has been reported that the enstatite chondrite (EC) meteorite may contain enough hydrogen to provide a plausible explanation for water's initial existence on Earth. Perryite mineral is one of the key components of EC, but its detailed chemical composition and phase width remain elusive compared with other minerals found in EC. Therefore, we embark on a series of investigations of the synthesis, crystal structure, and electronic structure of the synthetic perryite mineral (NixFe1-x)8(TyP1-y)3 (T = Si and Ge; 1 ≥ x, y ≥ 0). Its crystal structures were established based on single-crystal and powder X-ray diffraction techniques. It is realized that its structural and phase stabilities are highly dependent on the nature of the doping element (i.e., Fe and Si). The inclusion of Si and Fe elements can greatly alter the bonding scheme near the Fermi level (Ef), which is vital to the phase stability and accounts for the chemical composition of the natural perryite mineral (quaternary compound) in EC meteorites. Furthermore, this phase exhibits good electrocatalytic activity toward the hydrogen evolution reaction (HER). The best and the worst HER performances are for the Ni8Ge2P and Ni8Si2P samples, respectively, which suggests that the long bond length and high polarity of the covalent bond are the preferred criteria to enhance the electrocatalytic HER in this series.

Marker peptide screening and species-specific authentication of Pheretima using proteomics.

Pheretima is a common and valuable animal-derived medication used in traditional Chinese medicine. There are four species of Pheretima specified in the Chinese Pharmacopoeia (2015 edition), i.e. Pheretima aspergillum, P. vulgaris, P. guillelmi, and P. pectinifera. A recent report revealed ~ 55% of Pheretima in the commercial marketplace may be adulterated by other species, contrary to the Pharmacopoeia standard. The safety, efficacy, and authenticity of Pheretima is an important issue. Currently, the availability of specific quality-markers for the various species and effective identification methods are still limited. In this study, label-free quantification proteomics of species from Pheretima and Amynthas was carried out using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS), and marker peptides were identified based on their ion intensities using multivariate data analysis (principal component analysis and supervised partial least-squares discriminant analysis). A total of 48,476 peptides with high confidence corresponding to 13,397 proteins were identified from all samples. The marker peptides were validated by comparison with synthetic peptide reference standards using LC-MS/MS operating in a multiple-reaction monitoring mode. A multiple-peptide identification strategy was proposed for the authentication of Pheretima and subsequently applied to samples obtained from retail outlets in various regions of China. The results showed that eight out of the 15 samples tested were deemed authentic Pheretima.

Experimental and numerical study on the transport of droplet aerosols generated by occupants in a fever clinic.

The outbreak of the corona virus disease 2019 (COVID-19) infection has spread to a large number of countries worldwide. The early diagnosis of COVID-19 is complicated by its strong transmission characteristics and no obvious symptoms in the incubation period. Due to the relatively sealed indoor environment and the existing ventilation system, the patients and doctors in the fever clinics of the major hospitals are faced with a huge risk of infection. This study aims to investigate the transport of droplet aerosols generated by both doctors and patients to seek measures to reduce the risk of infection. Taking a typical fever clinic as an object of study, two links in the actual diagnosis and treatment process are selected in this manuscript for investigation by experimental and numerical methods. The effects of different cases of coughing and talking, as well as different contact distances, on the inhalation rate of human droplet aerosols are studied. The purification capacity of the ventilation is evaluated by the analysis results of the particle diffusion track and regional concentration of the entire indoor area and breathing zones. The results show that purification of the same number of droplet aerosols and purification by ventilation work better for coughing than for talking. The best ventilation performance appeared for the case of a patient sitting and coughing (PSC), while the case of a patient lying and talking (PLT) was the worst. Corresponding measures are suggested to improve the air purification effect and reduce the risk of cross infection.