Influences of protein-corona on stability and aggregation kinetics of Ti3C2Tx nanosheets in aquatic environment.

Proteins existed in aquatic environments strongly influence the transport, fate of nanomaterials due to the formation of protein-corona surrounding nanomaterials. To date, how do proteins affect the aggregation behaviors of MXene, a new family of two-dimensional materials, in aquatic environment remains unknown. Here the aggregation kinetics of MXene Ti3C2Tx nanosheets in various electrolytes (NaCl, CaCl2 and Na2SO4) was investigated by time-resolved dynamic light scattering in absence or presence of bovine serum albumin (BSA). Results showed that BSA affected the aggregation of Ti3C2Tx in a concentration-dependent manner. Addition of 3 mg/L BSA decreased the critical coagulation concentrations (CCCs) of Ti3C2Tx about 1.6-2.1 times, showing obvious destabilization effect; while BSA greater than 30 mg/L created a high-protein environment covering Ti3C2Tx, producing high spatial repulsion and enhancing the dispersibility of Ti3C2Tx. Ca2+ ions have greater effect on the aggregation of Ti3C2Tx due to the larger surface charge and bridging effect. The interaction between Ti3C2Tx and BSA followed Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, and mainly attributed to hydrogen bonding and van der Waals forces, while positively charged lysine and arginine in BSA might attract onto Ti3C2Tx through electrostatic attraction. The interaction decreased the content of α-helix structure in BSA from 74.7% to 53.1%. Ti3C2Tx easily suffered from aggregation and their long-distance transport seemed impossible in synthetic or natural waters. The present findings provided new insights for understanding the transfer and fate of this nanomaterial in aquatic environments.

RNF20/RNF40 ameliorates streptozotocin-induced type 1 diabetes by activating vitamin D receptors in vivo.

BACKGROUND: Type 1 diabetes is one of the chronic autoimmune diseases. Its features include the immune-triggered pancreatic beta-cells destruction. Ubiquitin ligases RNF20 and RNF40 have been discovered to participate into beta cells gene expression, insulin secretion, and expression of vitamin D receptors (VDRs). However, no reports about the role of RNF20/RNF40 in type 1 diabetes are known till now. The aim of this study was to clarify the role of RNF20/RNF40 in type 1 diabetes and explore the mechanism. METHODS: In this study, streptozotocin (STZ)-induced mice type 1 diabetes model was used. The protein expressions of genes were examined through Western blot analysis. Fasting blood glucose was detected through glucose meter. The plasma insulin was tested through the commercial kit. Hematoxylin and eosin staining was utilized to observe pathological changes of pancreatic tissues. Immunofluorescence assay was performed to evaluate the level of insulin. The levels of pro-inflammatory cytokines in serum were assessed by enzyme-linked-immunosorbent serologic assay. The cell apoptosis was measured through terminal deoxynucleotidyl transferase dUTP nick end labelling assay. RESULTS: STZ was used to stimulate mice model for type 1 diabetes. At first, both RNF20 and RNF40 expressions were down-regulated in STZ-mediated type 1 diabetes. Additionally, RNF20/RNF40 improved hyperglycemia in STZ-stimulated mice. Moreover, RNF20/RNF40 relieved pancreatic tissue injury in STZ-induced mice. Further experiments found that RNF20/RNF40 rescued the strengthened inflammation mediated by STZ treatment. The cell apoptosis was enhanced in the pancreatic tissues of STZ-triggered mice, but this effect was weakened by overexpression of RNF20/RNF40. Besides, the VDR expression was positively regulated by RNF20/RNF40. Finally, VDR knockdown reversed improved hyperglycemia, inflammation, and cell apoptosis stimulated by overexpression of RNF20/RNF40. CONCLUSION: Our findings proved that RNF20/RNF40 activated VDR to relieve type 1 diabetes. This work might highlight the functioning of RNF20/RNF40 in the treatment of type 1 diabetes.

Inhibiting Phase Transfer of Protein Nanoparticles by Surface Camouflage-A Versatile and Efficient Protein Encapsulation Strategy.

Engineering a system with a high mass fraction of active ingredients, especially water-soluble proteins, is still an ongoing challenge. In this work, we developed a versatile surface camouflage strategy that can engineer systems with an ultrahigh mass fraction of proteins. By formulating protein molecules into nanoparticles, the demand of molecular modification was transformed into a surface camouflage of protein nanoparticles. Thanks to electrostatic attractions and van der Waals interactions, we camouflaged the surface of protein nanoparticles through the adsorption of carrier materials. The adsorption of carrier materials successfully inhibited the phase transfer of insulin, albumin, ß-lactoglobulin, and ovalbumin nanoparticles. As a result, the obtained microcomposites featured with a record of protein encapsulation efficiencies near 100% and a record of protein mass fraction of 77%. After the encapsulation in microcomposites, the insulin revealed a hypoglycemic effect for at least 14 d with one single injection, while that of insulin solution was only ∼4 h.

Recombinant Reg3α Prevents Islet ß-Cell Apoptosis and Promotes ß-Cell Regeneration.

Progressive loss and dysfunction of islet ß-cells has not yet been solved in the treatment of diabetes. Regenerating protein (Reg) has been identified as a trophic factor which is demonstrated to be associated with pancreatic tissue regeneration. We previously produced recombinant Reg3α protein (rReg3α) and proved that it protects against acute pancreatitis in mice. Whether rReg3α protects islet ß-cells in diabetes has been elusive. In the present study, rReg3α stimulated MIN6 cell proliferation and resisted STZ-caused cell death. The protective effect of rReg3α was also found in mouse primary islets. In BALB/c mice, rReg3α administration largely alleviated STZ-induced diabetes by the preservation of ß-cell mass. The protective mechanism could be attributed to Akt/Bcl-2/-xL activation and GRP78 upregulation. Scattered insulin-expressing cells and clusters with small size, low insulin density, and exocrine distribution were observed and considered to be neogenic. In isolated acinar cells with wheat germ agglutinin (WGA) labeling, rReg3α treatment generated insulin-producing cells through Stat3/Ngn3 signaling, but these cells were not fully functional in response to glucose stimulation. Our results demonstrated that rReg3α resists STZ-induced ß-cell death and promotes ß-cell regeneration. rReg3α could serve as a potential drug for ß-cell maintenance in anti-diabetic treatment.

Riboflavin Is Directly Involved in N-Dealkylation Catalyzed by Bacterial Cytochrome P450 Monooxygenases.

Like a vast number of enzymes in nature, bacterial cytochrome P450 monooxygenases require an activated form of flavin as a cofactor for catalytic activity. Riboflavin is the precursor of FAD and FMN that serves as indispensable cofactor for flavoenzymes. In contrast to previous notions, herein we describe the identification of an electron-transfer process that is directly mediated by riboflavin for N-dealkylation by bacterial P450 monooxygenases. The electron relay from NADPH to riboflavin and then via activated oxygen to heme was proposed based on a combination of X-ray crystallography, molecular modeling and molecular dynamics simulation, site-directed mutagenesis and biochemical analysis of representative bacterial P450 monooxygenases. This study provides new insights into the electron transfer mechanism in bacterial P450 enzyme catalysis and likely in yeasts, fungi, plants and mammals.

Identification of truncated form of NosP as a transcription factor to regulate the biosynthesis of nosiheptide.

Nosiheptide (NOS), a typical member of the thiopeptides, possesses strong activities against multidrug-resistant, gram-positive bacterial pathogens. Similar to other thiopeptides, the biosynthetic pathway of NOS belongs to a ribosomally synthesized and posttranslationally modified peptide system. Bioinformatics analysis of the NOS gene cluster suggests that nosP gene encodes a homologous protein of the Streptomyces antibiotic regulatory protein (SARP) family. In the present study, the actual initiation codon of nosP was identified by comparison of potential initiation codons GUG and AUG. In contrast to previous predictions of starting with GUG, AUG, corresponding to methionine residue as the 53rd residue in the original sequence, is actually the initiation codon of nosP, indicating that a truncated form of NosP (NosP53-323) is a functional protein. For better understanding of the transcriptional regulation for NOS biosynthesis, the binding region was subsequently investigated with NosP53-323, demonstrating that NosP53-323 specifically binds the bidirectional nosL-nosM promoter region. Additionally, NosP53-323 was confirmed to serve as a transcription factor to activate the transcription of all 15 structural genes in the gene cluster. The present study provides new insights into pathway-specific regulation of the biosynthesis of NOS, which would be beneficial to the investigation of the regulatory function of similar SARP proteins in the gene clusters of other thiopeptides.-Wu, X., Jin, L., Zhang, H., Tong, R., Ma, M., Chen, Y. Identification of truncated form of NosP as a transcription factor to regulate the biosynthesis of nosiheptide.

Mutagenesis of NosM Leader Peptide Reveals Important Elements in Nosiheptide Biosynthesis.

Nosiheptide, a typical member of the ribosomally synthesized and posttranslationally modified peptides (RiPPs), exhibits potent activity against multidrug-resistant Gram-positive bacterial pathogens. The precursor peptide of nosiheptide (NosM) is comprised of a leader peptide with 37 amino acids and a core peptide containing 13 amino acids. To pinpoint elements in the leader peptide that are essential for nosiheptide biosynthesis, a collection of mutants with unique sequence features, including N- and C-terminal motifs, peptide length, and specific sites in the leader peptide, was generated by mutagenesis in vivo The effects of various mutants on nosiheptide biosynthesis were evaluated. In addition to the necessity of a conserved motif LEIS box, native length and the N-terminal 12 amino acid residues were indispensable, and single-site substitutions of these 12 amino acid residues resulted in changes ranging from a greater-than-5-fold decrease to a 2-fold increase of nosiheptide production, depending on the sites and substituted residues. Moreover, although the C-terminal motif is not conservative, significant effects of this portion on nosiheptide production were also evident. Taken together, the present results further highlight the importance of the leader peptide in nosiheptide biosynthesis, and provide new insights into the diversity and specificity of leader peptides in the biosynthesis of various RiPPs. IMPORTANCE: As a representative thiopeptide, nosiheptide exhibits excellent antibacterial activity. Although the biosynthetic gene cluster and several modification steps have been revealed, the presence and roles of the leader peptide within the precursor peptide of the nosiheptide gene cluster remain elusive. Thus, identification of specific elements in the leader peptide can significantly facilitate the genetic manipulation of the gene cluster for increasing nosiheptide production or generating diverse analogues. Given the complexity of the biosynthetic process, the instability of the leader peptide, and the unavailability of intermediates, cocrystallization of intermediates, leader peptide, and modification enzymes is currently not feasible. Therefore, a mutagenesis approach was used to construct a series of leader peptide mutants to uncover a number of crucial and characteristic elements affecting nosiheptide biosynthesis, which moves a considerable distance toward a thorough understanding of the biosynthetic machinery for thiopeptides.

Expanding the Catalytic Promiscuity of Heparinase†III from Pedobacter heparinus.

Glycosaminoglycans (GAG) lyases are useful biocatalysts for the preparation of oligosaccharides, but their substrate spectra are limited to the same family. Thus, the degradation activity across families of GAG lyases is advantageous and desirable for various applications. In this study, residue Lys130 at the substrate entrance of monomeric heparinase III from Pedobacter heparinus ATCC 13125 was replaced by cysteine, and the resulting mutant K130C showed novel catalytic activity in degrading hyaluronic acid without affecting its native activity toward heparin and heparan sulfate. The broadened catalytic promiscuity by mutant K130C was the result of dimerization through a disulfide bond to expand the substrate binding pocket. This bifunctional enzyme is potentially valuable in the degradation of different types of GAGs.

Highly specific quantification of microRNA by coupling probe-rolling circle amplification and Förster resonance energy transfer.

MicroRNA (miRNA) plays vital roles in various biological processes. In general, sensitivity and specificity are the major parameters for the quantification of miRNA. In this study, padlock probe-rolling circle amplification and Förster resonance energy transfer (pRCA-FRET) were coupled for specific and quantitative detection of miRNA. pRCA-FRET showed superior specificity to differentiate single-base mismatch and excellent sensitivity with a detection limit of 103 aM. The current method has the potential to quantify low amounts of miRNA in the same family for studies on their biological functions.

Enzymatic synthesis of L-norephedrine by coupling recombinant pyruvate decarboxylase and ω-transaminase.

L-Norephedrine, a natural plant alkaloid, possesses similar activity as ephedrine and can be used as a vicinal amino alcohol for the asymmetric synthesis of a variety of optically pure compounds, including pharmaceuticals, fine chemicals, and agrochemicals. Because of the existence of two asymmetric centers, efficient synthesis of L-norephedrine has been challenging. In the present study, an R-selective pyruvate decarboxylase from Saccharomyces cerevisiae and an S-selective ω-transaminase from Vibrio fluvialis JS17 were coupled to develop a sequential process for the stereoselective biosynthesis of L-norephedrine. After systematic optimization of the reaction conditions, a green, economic, and practical biocatalytic method to prepare L-norephedrine was established to achieve de and ee values of greater than 99.5 % and a molar yield over 60 %. The present coupling approach can facilitate the development of sequential reactions by various biocatalysts.

A machine learning-based QSAR model reveals important molecular features for understanding the potential inhibition mechanism of ionic liquids to acetylcholinesterase.

The broad application of ionic liquids (ILs) has been hindered by uncertainties surrounding their ecotoxicity. In this work, a Quantitative Structure-Activity Relationship (QSAR) model was devised to predict the inhibition of ILs towards the activity of AChE, employing both Random Forest (RF) and eXtreme Gradient Boosting (XGBoost) machine learning approaches. Fourteen kings of essential molecular feature descriptors were screened from an initial roster of 244 descriptors through the application of a feature importance index and they showed a significant impact on the activity of AChE activity. The two models based solely on the 14 most critical molecular descriptors could maintain model's robustness and reliability. The correlation analysis between these 14 descriptors and the inhibition of AChE activity revealed the potential impact of the molecular characteristics on ILs toxicity. The results underscored the main influence of cations in ILs on the inhibitory activity towards the AChE enzyme. Specifically, cations exhibiting hydrophobicity properties were found to exert more potent inhibitory effects on the AChE enzyme. In addition, some other properties of the cations, such as the degree of branching, atomic weight and partial charge also modulated their inhibition potential. This study enhances the comprehension of the structure-activity relationship between ILs and AChE inhibition, providing a reference for designing safer and greener ILs.

Cellular uptake and cytotoxicity of PEGylated MXene nanomaterials mediated by protein corona.

A stringent analysis of the biocompatibility of MXene is a necessary condition for assessing the biological risk of MXene. Owing to high surface free energy, MXene is capable of adsorbing a large amount of blood proteins to form MXene-protein corona complexes, however, a comprehensive understanding of the relationship between MXene and cellular physiological systems remains limited. Therefore, we investigated the cellular uptake and cytotoxicity effect of MXene Ti3C2Tx and PEGylation Ti3C2Tx mediated by human serum protein corona in THP-1 cells. It was found that PEGylation can alter the interaction between Ti3C2Tx and serum proteins, inducing a significant transformation in the fingerprint of the protein corona. Following protein corona formation, both Ti3C2Tx and PEGylated Ti3C2Tx predominantly accumulated at lysosomal sites within THP-1 cells. Further analysis revealed that clathrin-mediated endocytosis was the primary mechanism of Ti3C2Tx internalization by THP-1 cells. There was no significant effect on cell viability. However, we found that Ti3C2Tx plays a dual role as both a stimulus and scavenger of ROS within THP-1 cells, influenced by its PEGylation and the formation of a protein corona. This study provides important insights for biocompatibility evaluation and rational design of nanoproducts based on Ti3C2Tx in the future.

Selective biosynthesis of a rhamnosyl nosiheptide by a novel bacterial rhamnosyltransferase.

Nosiheptide (NOS) is a thiopeptide antibiotic produced by the bacterium Streptomyces actuosus. The hydroxyl group of 3-hydroxypyridine in NOS has been identified as a promising site for modification, which we therefore aimed to rhamnosylate. After screening, Streptomyces sp. 147326 was found to regioselectively attach a rhamnosyl unit to the 3-hydroxypyridine site in NOS, resulting in the formation of a derivative named NOS-R at a productivity of 24.6%. In comparison with NOS, NOS-R exhibited a 17.6-fold increase in aqueous solubility and a new protective effect against MRSA infection in mice, while maintaining a similar in vitro activity. Subsequently, SrGT822 was identified as the rhamnosyltransferase in Streptomyces sp. 147326 responsible for the biosynthesis of NOS-R using dTDP-L-rhamnose. SrGT822 demonstrated an optimal reaction pH of 10.0 and temperature of 55°C, which resulted in a NOS-R yield of 74.9%. Based on the catalytic properties and evolutionary analysis, SrGT822 is anticipated to be a potential rhamnosyltransferase for use in the modification of various complex scaffolds.

Enhancement of biocatalytic efficiency by increasing substrate loading: enzymatic preparation of L-homophenylalanine.

Enantiomerically pure L-homophenylalanine (L-HPA) is a key building block for the synthesis of angiotensin-converting enzyme inhibitors and other chiral pharmaceuticals. Among the processes developed for the L-HPA production, biocatalytic synthesis employing phenylalanine dehydrogenase has been proven as the most promising route. However, similar to other dehydrogenase-catalyzed reactions, the viability of this process is markedly affected by insufficient substrate loading and high costs of the indispensable cofactors. In the present work, a highly efficient and economic biocatalytic process for L-HPA was established by coupling genetically modified phenylalanine dehydrogenase and formate dehydrogenase. Combination of fed-batch substrate addition and a continuous product removal greatly increased substrate loading and cofactor utilization. After systemic optimization, 40 g (0.22 mol) of keto acid substrate was transformed to L-HPA within 24 h and a total of 0.2 mM NAD(+) was reused effectively in eight cycles of fed-batch operation, consequently giving an average substrate concentration of 510 mM and a productivity of 84.1 g l(-1) day(-1) for L-HPA. The present study provides an efficient and feasible enzymatic process for the production of L-HPA and a general solution for the increase of substrate loading.

Dicarbonyl reduction by single enzyme for the preparation of chiral diols.

Chiral diols are a group of key building blocks useful for preparing a variety of important chiral chemicals. While the preparation of optically pure diols is generally not an easy task in synthetic organic chemistry, three classes of enzymes, namely dicarbonyl reductase, dioxygenase and epoxide hydrolase, display remarkable ability to stereoselectively introduce two hydroxyl groups in a single-step enzymatic conversion. In this tutorial review, we pay special attention to dicarbonyl reductases that directly produce chiral diols through the bio-reduction of two carbonyl groups. The dicarbonyl reductases include diketoreductase, α-acetoxy ketone reductase and sepiapterin reductase. We present these exceptional enzymes in the context of source and properties, structure and catalytic mechanism as well as biocatalytic application. In addition to the broad substrate specificity, the excellent stereoselectivity and high catalytic efficiency of these enzymes have positioned them as valuable biocatalysts. With more sophisticated understanding of the structure-function relationship, the practical utilities of these enzymes associated with their interesting chemistry will be considerably appreciated over time. Moreover, rational redesign and molecular evolution of these unusual biocatalysts will truly enable their broader applications in the synthesis of chiral diols in the future.

Efficient snailase-based production of mogrol from Luo Han Guo extract in an aqueous-organic system.

To solve the insufficient availability of mogrol, an 11α-hydroxy aglycone of mogrosides in Siraitia grosvenorii, snailase was employed as the enzyme to completely deglycosylate LHG extract containing 50% mogroside V. Other commonly used glycosidases performed less efficiently. Response surface methodology was conducted to optimize the productivity of mogrol, which peaked at 74.7% in an aqueous reaction. In view of the differences in water-solubility between mogrol and LHG extract, we employed an aqueous-organic system for the snailase-catalyzed reaction. Of five tested organic solvents, toluene performed best and was relatively well tolerated by snailase. After optimization, biphasic medium containing 30% toluene (v/v) could produce a high-quality mogrol (98.1% purity) at a 0.5 L scale with a production rate of 93.2% within 20 h. This toluene-aqueous biphasic system would not only provide sufficient mogrol to construct future synthetic biology systems for the preparation of mogrosides, but also facilitate the development of mogrol-based medicines.

A Type I/Type III PKS Hybrid Generates Cinnamomycin A-D.

3,5-Dihydroxybenzoic acid (3,5-DHBA), biosynthesized by type III PKS and tailoring enzymes, is an unconventional starter unit for bacterial type I PKS. Genome mining of 3,5-DHBA-specific biosynthetic gene clusters could lead to discovering new type I/type III PKS hybrids. Herein, we report the discovery and characterization of atypical compounds, namely cinnamomycin A-D, exhibiting selective antiproliferative activity. The biosynthetic pathway of cinnamomycins was proposed based on genetic manipulation, enzymatic reaction, and precursor feeding.

Design of a chimeric glycosyltransferase OleD for the site-specific O-monoglycosylation of 3-hydroxypyridine in nosiheptide.

To identify the potential role of the 3-hydroxyl group of the pyridine ring in nosiheptide (NOS) for its antibacterial activity against Gram-positive pathogens, enzymatic glycosylation was utilized to regio-selectively create a monoglycosyl NOS derivative, NOS-G. For this purpose, we selected OleD, a UDP glycosyltransferase from Streptomyces antibioticus that has a low productivity for NOS-G. Activity of the enzyme was increased by swapping domains derived from OleI, both single and in combination. Activity enhancement was best in mutant OleD-10 that contained four OleI domains. This chimer was engineered by site-directed mutagenesis (single and in combination) to increase its activity further, whereby variants were screened using a newly-established colorimetric assay. OleD-10 with I117F and T118G substitutions (FG) had an increased NOS-G productivity of 56%, approximately 70 times higher than that of wild-type OleD. The reason for improved activity of FG towards NOS was structurally attributed to a closer distance (<3 Å) between NOS/sugar donor and the catalytic amino acid H25. The engineered enzyme allowed sufficient activity to demonstrate that the produced NOS-G had enhanced stability and aqueous solubility compared to NOS. Using a murine MRSA infection model, it was established that NOS-G resulted in partial protection within 20 h of administration and delayed the death of infected mice. We conclude that 3-hydroxypyridine is a promising site for structural modification of NOS, which may pave the way for producing nosiheptide derivatives as a potential antibiotic for application in clinical treatment.

Engineering of a UDP-Glycosyltransferase for the Efficient Whole-Cell Biosynthesis of Siamenoside I in Escherichia coli.

The combination of the insufficient availability and the complex structure of siamenoside I (SI), the sweetest glucoside isolated from Siraitia grosvenorii to date, limited its use as a natural sweetener. To solve this problem, an improved biocatalyst, UGT-M2, was semi-rationally created by engineering the uridine diphosphate glycosyltransferase UGT94-289-2 from S. grosvenorii for the monoglucosylation of mogroside IIIE (MG IIIE) to SI. Subsequently, an engineered Escherichia coli cell was constructed, which combined UGT-M2 with a UDP-glucose regeneration system to circumvent the need for expensive UDP-glucose to produce SI. After optimization, high-purity SI (>96.4%) was efficiently prepared from MG IIIE at a 1 L scale with a productivity of 29.78 g/(L day) and a molar yield of 76.5% and without using exogenous UDP-glucose. This study not only developed a whole-cell approach for the preparation of SI but also provided an alternative glycosyltransferase variant for SI biosynthesis with synthetic biology in the future.

Enzymatic hydrolyzation of mogrosides in Luo Han Guo extract by NKA-adsorbed snailase improves its sensory profile.

Extracts of Siraitia grosvenorii (Swingle), in Chinese known as Luo Han Guo (LHG), is authorized for use as a natural sweetener. LHG is rich in mogroside V that contains five glucoses, but also contains mogroside IIIE and analogues with fewer than three glucose units that cause an unpleasant aftertaste, limiting the use of the extract. Snailase was applied here to convert mogroside V in LHG extract in favor of siamenoside I formation, the sweetest mogroside with a taste similar to sucrose. For application, snailase was immobilized by adsorption to NKA (a macroporous resin), resulting in 10.9 U per g of adsorbed protein. Reuse of the NKA-adsorbed snailase was demonstrated for four cycles, and a continuous production of improved LHG extract at a 0.5 L scale had a productivity of 68.4 g/(L⋅day). The resulting product containing over 50% siamenoside I displayed an improved taste profile with satisfying safety toward HEK293T cells.