A microbial transporter of the dietary antioxidant ergothioneine.

Low-molecular-weight (LMW) thiols are small-molecule antioxidants required for the maintenance of intracellular redox homeostasis. However, many host-associated microbes, including the gastric pathogen Helicobacter pylori, unexpectedly lack LMW-thiol biosynthetic pathways. Using reactivity-guided metabolomics, we identified the unusual LMW thiol ergothioneine (EGT) in H. pylori. Dietary EGT accumulates to millimolar levels in human tissues and has been broadly implicated in mitigating disease risk. Although certain microorganisms synthesize EGT, we discovered that H. pylori acquires this LMW thiol from the host environment using a highly selective ATP-binding cassette transporter-EgtUV. EgtUV confers a competitive colonization advantage in vivo and is widely conserved in gastrointestinal microbes. Furthermore, we found that human fecal bacteria metabolize EGT, which may contribute to production of the disease-associated metabolite trimethylamine N-oxide. Collectively, our findings illustrate a previously unappreciated mechanism of microbial redox regulation in the gut and suggest that inter-kingdom competition for dietary EGT may broadly impact human health.

Multiple Functions and Regulation of Mammalian Peroxiredoxins.

Peroxiredoxins (Prxs) constitute a major family of peroxidases, with mammalian cells expressing six Prx isoforms (PrxI to PrxVI). Cells produce hydrogen peroxide (H2O2) at various intracellular locations where it can serve as a signaling molecule. Given that Prxs are abundant and possess a structure that renders the cysteine (Cys) residue at the active site highly sensitive to oxidation by H2O2, the signaling function of this oxidant requires extensive and highly localized regulation. Recent findings on the reversible regulation of PrxI through phosphorylation at the centrosome and on the hyperoxidation of the Cys at the active site of PrxIII in mitochondria are described in this review as examples of such local regulation of H2O2 signaling. Moreover, their high affinity for and sensitivity to oxidation by H2O2 confer on Prxs the ability to serve as sensors and transducers of H2O2 signaling through transfer of their oxidation state to bound effector proteins.

A peroxiredoxin-P38 MAPK scaffold increases MAPK activity by MAP3K-independent mechanisms.

Peroxiredoxins (Prdxs) utilize reversibly oxidized cysteine residues to reduce peroxides and promote H2O2 signal transduction, including H2O2-induced activation of P38 MAPK. Prdxs form H2O2-induced disulfide complexes with many proteins, including multiple kinases involved in P38 MAPK signaling. Here, we show that a genetically encoded fusion between a Prdx and P38 MAPK is sufficient to hyperactivate the kinase in yeast and human cells by a mechanism that does not require the H2O2-sensing cysteine of the Prdx. We demonstrate that a P38-Prdx fusion protein compensates for loss of the yeast scaffold protein Mcs4 and MAP3K activity, driving yeast into mitosis. Based on our findings, we propose that the H2O2-induced formation of Prdx-MAPK disulfide complexes provides an alternative scaffold and signaling platform for MAPKK-MAPK signaling. The demonstration that formation of a complex with a Prdx is sufficient to modify the activity of a kinase has broad implications for peroxide-based signal transduction in eukaryotes.

Evidence against Stable Protein S-Nitrosylation as a Widespread Mechanism of Post-translational Regulation.

S-nitrosation, commonly referred to as S-nitrosylation, is widely regarded as a ubiquitous, stable post-translational modification that directly regulates many proteins. Such a widespread role would appear to be incompatible with the inherent lability of the S-nitroso bond, especially its propensity to rapidly react with thiols to generate disulfide bonds. As anticipated, we observed robust and widespread protein S-nitrosation after exposing cells to nitrosocysteine or lipopolysaccharide. Proteins detected using the ascorbate-dependent biotin switch method are typically interpreted to be directly regulated by S-nitrosation. However, these S-nitrosated proteins are shown to predominantly comprise transient intermediates leading to disulfide bond formation. These disulfides are likely to be the dominant end effectors resulting from elevations in nitrosating cellular nitric oxide species. We propose that S-nitrosation primarily serves as a transient intermediate leading to disulfide formation. Overall, we conclude that the current widely held perception that stable S-nitrosation directly regulates the function of many proteins is significantly incorrect.

Carbon dioxide/bicarbonate is required for sensitive inactivation of mammalian glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contains an active site Cys and is one of the most sensitive cellular enzymes to oxidative inactivation and redox regulation. Here, we show that inactivation by hydrogen peroxide is strongly enhanced in the presence of carbon dioxide/bicarbonate. Inactivation of isolated mammalian GAPDH by H2O2 increased with increasing bicarbonate concentration and was sevenfold faster in 25 mM (physiological) bicarbonate compared with bicarbonate-free buffer of the same pH. H2O2 reacts reversibly with CO2 to form a more reactive oxidant, peroxymonocarbonate (HCO4-), which is most likely responsible for the enhanced inactivation. However, to account for the extent of enhancement, we propose that GAPDH must facilitate formation and/or targeting of HCO4- to promote its own inactivation. Inactivation of intracellular GAPDH was also strongly enhanced by bicarbonate: treatment of Jurkat cells with 20 µM H2O2 in 25 mM bicarbonate buffer for 5 min caused almost complete GAPDH inactivation, but no loss of activity when bicarbonate was not present. H2O2-dependent GAPDH inhibition in bicarbonate buffer was observed even in the presence of reduced peroxiredoxin 2 and there was a significant increase in cellular glyceraldehyde-3-phosphate/dihydroxyacetone phosphate. Our results identify an unrecognized role for bicarbonate in enabling H2O2 to influence inactivation of GAPDH and potentially reroute glucose metabolism from glycolysis to the pentose phosphate pathway and NAPDH production. They also demonstrate what could be wider interplay between CO2 and H2O2 in redox biology and the potential for variations in CO2 metabolism to influence oxidative responses and redox signaling.

Acidity of persulfides and its modulation by the protein environments in sulfide quinone oxidoreductase and thiosulfate sulfurtransferase.

Persulfides (RSSH/RSS-) participate in sulfur metabolism and are proposed to transduce hydrogen sulfide (H2S) signaling. Their biochemical properties are poorly understood. Herein, we studied the acidity and nucleophilicity of several low molecular weight persulfides using the alkylating agent, monobromobimane. The different persulfides presented similar pKa values (4.6-6.3) and pH-independent rate constants (3.2-9.0 × 103 M-1 s-1), indicating that the substituents in persulfides affect properties to a lesser extent than in thiols because of the larger distance to the outer sulfur. The persulfides had higher reactivity with monobromobimane than analogous thiols and putative thiols with the same pKa, providing evidence for the alpha effect (enhanced nucleophilicity by the presence of a contiguous atom with high electron density). Additionally, we investigated two enzymes from the human mitochondrial H2S oxidation pathway that form catalytic persulfide intermediates, sulfide quinone oxidoreductase and thiosulfate sulfurtransferase (TST, rhodanese). The pH dependence of the activities of both enzymes was measured using sulfite and/or cyanide as sulfur acceptors. The TST half-reactions were also studied by stopped-flow fluorescence spectroscopy. Both persulfidated enzymes relied on protonated groups for reaction with the acceptors. Persulfidated sulfide quinone oxidoreductase appeared to have a pKa of 7.8 ± 0.2. Persulfidated TST presented a pKa of 9.38 ± 0.04, probably due to a critical active site residue rather than the persulfide itself. The TST thiol reacted in the anionic state with thiosulfate, with an apparent pKa of 6.5 ± 0.1. Overall, our study contributes to a fundamental understanding of persulfide properties and their modulation by protein environments.

Scs system links copper and redox homeostasis in bacterial pathogens.

The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when homeostasis is challenged during host-pathogen interactions. In particular, the presence of free radical groups and excess copper in the periplasm causes noxious reactions, such as sulfhydryl group oxidation leading to enzymatic inactivation and protein denaturation. In response to this, canonical and accessory oxidoreductase systems are induced, performing quality control of thiol groups, and therefore contributing to restoring homeostasis and preserving survival under these conditions. Here, we examine recent advances in the characterization of the Dsb-like, Salmonella-specific Scs system. This system includes the ScsC/ScsB pair of Cu+-binding proteins with thiol-oxidoreductase activity, an alternative ScsB-partner, the membrane-linked ScsD, and a likely associated protein, ScsA, with a role in peroxide resistance. We discuss the acquisition of the scsABCD locus and its integration into a global regulatory pathway directing envelope response to Cu stress during the evolution of pathogens that also harbor the canonical Dsb systems. The evidence suggests that the canonical Dsb systems cannot satisfy the extra demands that the host-pathogen interface imposes to preserve functional thiol groups. This resulted in the acquisition of the Scs system by Salmonella. We propose that the ScsABCD complex evolved to connect Cu and redox stress responses in this pathogen as well as in other bacterial pathogens.

Improved resolution of 3-mercaptopropionate dioxygenase active site provided by ENDOR spectroscopy offers insight into catalytic mechanism.

3-mercaptopropionate (3MPA) dioxygenase (MDO) is a mononuclear nonheme iron enzyme that catalyzes the O2-dependent oxidation of thiol-bearing substrates to yield the corresponding sulfinic acid. MDO is a member of the cysteine dioxygenase family of small molecule thiol dioxygenases and thus shares a conserved sequence of active site residues (Serine-155, Histidine-157, and Tyrosine-159), collectively referred to as the SHY-motif. It has been demonstrated that these amino acids directly interact with the mononuclear Fe-site, influencing steady-state catalysis, catalytic efficiency, O2-binding, and substrate coordination. However, the underlying mechanism by which this is accomplished is poorly understood. Here, pulsed electron paramagnetic resonance spectroscopy [1H Mims electron nuclear double resonance spectroscopy] is applied to validate density functional theory computational models for the MDO Fe-site simultaneously coordinated by substrate and nitric oxide (NO), (3MPA/NO)-MDO. The enhanced resolution provided by electron nuclear double resonance spectroscopy allows for direct observation of Fe-bound substrate conformations and H-bond donation from Tyr159 to the Fe-bound NO ligand. Further inclusion of SHY-motif residues within the validated model reveals a distinct channel restricting movement of the Fe-bound NO-ligand. It has been argued that the iron-nitrosyl emulates the structure of potential Fe(III)-superoxide intermediates within the MDO catalytic cycle. While the merit of this assumption remains unconfirmed, the model reported here offers a framework to evaluate oxygen binding at the substrate-bound Fe-site and possible reaction mechanisms. It also underscores the significance of hydrogen bonding interactions within the enzymatic active site.

Structural and biochemical characterization of Arabidopsis alcohol dehydrogenases reveals distinct functional properties but similar redox sensitivity.

Alcohol dehydrogenases (ADHs) are a group of zinc-binding enzymes belonging to the medium-length dehydrogenase/reductase (MDR) protein superfamily. In plants, these enzymes fulfill important functions involving the reduction of toxic aldehydes to the corresponding alcohols (as well as catalyzing the reverse reaction, i.e., alcohol oxidation; ADH1) and the reduction of nitrosoglutathione (GSNO; ADH2/GSNOR). We investigated and compared the structural and biochemical properties of ADH1 and GSNOR from Arabidopsis thaliana. We expressed and purified ADH1 and GSNOR and determined two new structures, NADH-ADH1 and apo-GSNOR, thus completing the structural landscape of Arabidopsis ADHs in both apo- and holo-forms. A structural comparison of these Arabidopsis ADHs revealed a high sequence conservation (59% identity) and a similar fold. In contrast, a striking dissimilarity was observed in the catalytic cavity supporting substrate specificity and accommodation. Consistently, ADH1 and GSNOR showed strict specificity for their substrates (ethanol and GSNO, respectively), although both enzymes had the ability to oxidize long-chain alcohols, with ADH1 performing better than GSNOR. Both enzymes contain a high number of cysteines (12 and 15 out of 379 residues for ADH1 and GSNOR, respectively) and showed a significant and similar responsivity to thiol-oxidizing agents, indicating that redox modifications may constitute a mechanism for controlling enzyme activity under both optimal growth and stress conditions.

Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein.

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.

Solvent-Mediated Modulation of the Au-S Bond in Dithiol Molecular Junctions.

Gold-dithiol molecular junctions have been studied both experimentally and theoretically. However, the nature of the gold-thiolate bond as it relates to the solvent has seldom been investigated. It is known that solvents can impact the electronic structure of single-molecule junctions, but the correlation between the solvent and dithiol-linked single-molecule junction conductance is not well understood. We study molecular junctions formed with thiol-terminated phenylenes from both 1-chloronaphthalene and 1-bromonaphthalene solutions. We find that the most probable conductance and the distribution of conductances are both affected by the solvent. First-principles calculations show that junction conductance depends on the binding configurations (adatom, atop, and bridge) of the thiolate on the Au surface, as has been shown previously. More importantly, we find that brominated solvents can restrict the binding of thiols to specific Au sites. This mechanism offers new insight into the effects of the solvent environment on covalent bonding in molecular junctions.

Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.

Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation.

Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) in vitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.

Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in Vibrio cholerae.

The vertebrate host's immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae, sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass spectrometry-based profiling, metabolomics, expression assays, and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular forms of sulfur with sulfur-sulfur bonds, termed reactive sulfur species (RSS). We first present a comprehensive sequence similarity network analysis of the arsenic repressor superfamily of transcriptional regulators, where RSS and hydrogen peroxide sensors segregate into distinct clusters of sequences. We show that HlyU, transcriptional activator of hlyA in V. cholerae, belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity or DNA dissociation following treatment with glutathione disulfide or hydrogen peroxide. Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA. However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.

Excess copper catalyzes protein disulfide bond formation in the bacterial periplasm but not in the cytoplasm.

Copper avidly binds thiols and is redox active, and it follows that one element of copper toxicity may be the generation of undesirable disulfide bonds in proteins. In the present study, copper oxidized the model thiol N-acetylcysteine in vitro. Alkaline phosphatase (AP) requires disulfide bonds for activity, and copper activated reduced AP both in vitro and when it was expressed in the periplasm of mutants lacking their native disulfide-generating system. However, AP was not activated when it was expressed in the cytoplasm of copper-overloaded cells. Similarly, this copper stress failed to activate OxyR, a transcription factor that responds to the creation of a disulfide bond. The elimination of cellular disulfide-reducing systems did not change these results. Nevertheless, in these cells, the cytoplasmic copper concentration was high enough to impair growth and completely inactivate enzymes with solvent-exposed [4Fe-4S] clusters. Experiments with N-acetylcysteine determined that the efficiency of thiol oxidation is limited by the sluggish pace at which oxygen regenerates copper(II) through oxidation of the thiyl radical-Cu(I) complex. We conclude that this slow step makes copper too inefficient a catalyst to create disulfide stress in the thiol-rich cytoplasm, but it can still impact the few thiol-containing proteins in the periplasm. It also ensures that copper accumulates intracellularly in the Cu(I) valence.

The MerR-family regulator NmlR is involved in the defense against oxidative stress in Streptococcus pneumoniae.

Streptococcus pneumoniae has to cope with the strong oxidant hypochlorous acid (HOCl), during host-pathogen interactions. Thus, we analyzed the global gene expression profile of S. pneumoniae D39 towards HOCl stress. In the RNA-seq transcriptome, the NmlR, SifR, CtsR, HrcA, SczA and CopY regulons and the etrx1-ccdA1-msrAB2 operon were most strongly induced under HOCl stress, which participate in the oxidative, electrophile and metal stress response in S. pneumoniae. The MerR-family regulator NmlR harbors a conserved Cys52 and controls the alcohol dehydrogenase-encoding adhC gene under carbonyl and NO stress. We demonstrated that NmlR senses also HOCl stress to activate transcription of the nmlR-adhC operon. HOCl-induced transcription of adhC required Cys52 of NmlR in vivo. Using mass spectrometry, NmlR was shown to be oxidized to intersubunit disulfides or S-glutathionylated under oxidative stress in vitro. A broccoli-FLAP-based assay further showed that both NmlR disulfides significantly increased transcription initiation at the nmlR promoter by RNAP in vitro, which depends on Cys52. Phenotype analyses revealed that NmlR functions in the defense against oxidative stress and promotes survival of S. pneumoniae during macrophage infections. In conclusion, NmlR was characterized as HOCl-sensing transcriptional regulator, which activates transcription of adhC under oxidative stress by thiol switches in S. pneumoniae.

Inhibition of Pseudomonas aeruginosa quorum sensing by chemical induction of the MexEF-oprN efflux pump.

The cell-to-cell communication system quorum sensing (QS), used by various pathogenic bacteria to synchronize gene expression and increase host invasion potentials, is studied as a potential target for persistent infection control. To search for novel molecules targeting the QS system in the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, a chemical library consisting of 3,280 small compounds from LifeArc was screened. A series of 10 conjugated phenones that have not previously been reported to target bacteria were identified as inhibitors of QS in P. aeruginosa. Two lead compounds (ethylthio enynone and propylthio enynone) were re-synthesized for verification of activity and further elucidation of the mode of action. The isomeric pure Z-ethylthio enynone was used for RNA sequencing, revealing a strong inhibitor of QS-regulated genes, and the QS-regulated virulence factors rhamnolipid and pyocyanin were significantly decreased by treatment with the compounds. A transposon mutagenesis screen performed in a newly constructed lasB-gfp monitor strain identified the target of Z-ethylthio enynone in P. aeruginosa to be the MexEF-OprN efflux pump, which was further established using defined mex knockout mutants. Our data indicate that the QS inhibitory capabilities of Z-ethylthio enynone were caused by the drainage of intracellular signal molecules as a response to chemical-induced stimulation of the MexEF-oprN efflux pump, thereby inhibiting the autogenerated positive feedback and its enhanced signal-molecule synthesis.

Durable Superhydrophobic Surfaces with Self-Generated Wenzel Sites for Efficient Fog Collection.

Harvesting freshwater from fog is one of the possible solutions to the global water scarcity crisis. Surfaces with both hydrophobic and hydrophilic regions are extensively employed for this purpose. Nevertheless, the longevity of these surfaces is still constrained by their delicate surface structures. The hydrophilic zones may become damaged or contaminated after repeated use, thereby compromising their effectiveness in fog collection. The preparation of generally applicable durable superhydrophobic coatings with self-generated Wenzel sites is reported here for long-term efficient and stable fog collection. The coatings are prepared by depositing the poly(tannic acid) coating as the primer layer on various substrates, self-assembly of trichlorovinylsilane into staggered silicone nanofilaments, and then thiol-ene click reaction with 1H,1H,2H,2H-perfluorodecanethiol. The coatings demonstrate remarkable static superhydrophobicity, robust impalement resistance, and stable self-generated Wenzel sites for water droplets. Therefore, the fog collection rate (FCR) of the coatings reaches 2.13 g cm-2 h-1 during 192 h continuous fog collection, which is triple that of bare substrate and outperforms most previous studies. Moreover, the systematic experiments and models have revealed that the key factors for achieving high FCR on superhydrophobic coatings are forming condensed droplets ≈1 mm in critical radius and a Wenzel site proportion of 0.3-0.4.

A Versatile Platform to Generate Prodrugs with Rapid and Precise Albumin Hitchhiking and High Cargo Loading for Tumor-Targeted Chemotherapy.

Due to its tumor homing and long serum half-life, albumin is an ideal drug carrier for chemotherapy. For endogenous albumin hitchhiking with high cargo loading, a trimeric albumin-binding domain (ABD), i.e., ABD-Tri is designed by fusing an ABD with high specificity and affinity for albumin to a self-trimerizing domain (Tri) with an additional cysteine residue. ABD-Tri is highly (40 mg L-1) expressed as soluble and trimeric proteins in Escherichia coli (E. coli). Once mixed together, ABD-Tri rapidly and specifically forms a stable complex with albumin under physiological conditions without obviously changing its receptor- and cell-binding and tumor-homing properties. Maleimide-modified prodrugs are highly effectively conjugated to ABD-Tri to produce homogenous ABD-Tri-prodrugs with triple cargo loading under physiological conditions by thiol-maleimide click chemistry. Unlike the maleimide moiety, which can only mediate time- and concentration-dependent albumin binding, ABD-Tri mediated fast (within several minutes) albumin binding of drugs even at extremely low concentrations (µg mL-1). Compared to maleimide-modified prodrugs, ABD-Tri-prodrugs exhibit better tumor homing and greater in vivo antitumor effect, indicating that conjugation of chemical drug to ABD-Tri outperforms maleimide modification for endogenous albumin hitchhiking. The results demonstrate that ABD-Tri may serve as a novel platform to produce albumin-binding prodrugs with high cargo-loading capacity for tumor-targeted chemotherapy.

Efficient Polycrystalline Single-Cation Perovskite Light-Emitting Diodes by Simultaneous Intracrystal and Interfacial Defect Passivation.

Polycrystalline perovskite light-emitting diodes (PeLEDs) have shown great promise with high efficiency and easy processability. However, PeLEDs using single-cation polycrystalline perovskite emitters have demonstrated low efficiency due to defects within the grains and at the interfaces between the perovskite layer and the charge injection contact. Thus, simultaneous defect engineering of perovskites to suppress exciton loss within the grains and at the interfaces is crucial for achieving high efficiency in PeLEDs. Here, 1,8-octanedithiol which is a strong nucleophile, is used to increase the luminescence efficiency of a single-cation perovskite by suppressing non-radiative recombination within the grains of their polycrystalline emitter film as well as at their interface with an anode. The dithiol additive performs a multifunctional role in defect passivation, spatial confinement of excitons, and prevention of exciton quenching at the interface between the perovskite layer and the underlying hole-injection layer. Photoluminescence studies demonstrate that incorporating the dithiol additive significantly enhances the charge carrier dynamics in perovskites, resulting in an external quantum efficiency (EQE) of up to 23.46% even in a simplified PeLED that does not use a hole-injection layer. This represents the highest level of EQE achieved among devices utilizing polycrystalline single-cation perovskites.