Using Protein Design and Directed Evolution to Monomerize a Bright Near-Infrared Fluorescent Protein.

The small ultrared fluorescent protein (smURFP) is a bright near-infrared (NIR) fluorescent protein (FP) that forms a dimer and binds its fluorescence chromophore, biliverdin, at its dimer interface. To engineer a monomeric NIR FP based on smURFP potentially more suitable for bioimaging, we employed protein design to extend the protein backbone with a new segment of two helices that shield the original dimer interface while covering the biliverdin binding pocket in place of the second chain in the original dimer. We experimentally characterized 13 designs and obtained a monomeric protein with a weak fluorescence. We enhanced the fluorescence of this designed protein through two rounds of directed evolution and obtained designed monomeric smURFP (DMsmURFP), a bright, stable, and monomeric NIR FP with a molecular weight of 19.6 kDa. We determined the crystal structures of DMsmURFP both in the apo state and in complex with biliverdin, which confirmed the designed structure. The use of DMsmURFP in in vivo imaging of mammalian systems was demonstrated. The backbone design-based strategy used here can also be applied to monomerize other naturally multimeric proteins with intersubunit functional sites.

De novo design of cavity-containing proteins with a backbone-centered neural network energy function.

The design of small-molecule-binding proteins requires protein backbones that contain cavities. Previous design efforts were based on naturally occurring cavity-containing backbone architectures. Here, we designed diverse cavity-containing backbones without predefined architectures by introducing tailored restraints into the backbone sampling driven by SCUBA (Side Chain-Unknown Backbone Arrangement), a neural network statistical energy function. For 521 out of 5816 designs, the root-mean-square deviations (RMSDs) of the Cα atoms for the AlphaFold2-predicted structures and our designed structures are within 2.0 Å. We experimentally tested 10 designed proteins and determined the crystal structures of two of them. One closely agrees with the designed model, while the other forms a domain-swapped dimer, where the partial structures are in agreement with the designed structures. Our results indicate that data-driven methods such as SCUBA hold great potential for designing de novo proteins with tailored small-molecule-binding function.

Combined prediction and design reveals the target recognition mechanism of an intrinsically disordered protein interaction domain.

An increasing number of protein interaction domains and their targets are being found to be intrinsically disordered proteins (IDPs). The corresponding target recognition mechanisms are mostly elusive because of challenges in performing detailed structural analysis of highly dynamic IDP-IDP complexes. Here, we show that by combining recently developed computational approaches with experiments, the structure of the complex between the intrinsically disordered C-terminal domain (CTD) of protein 4.1G and its target IDP region in NuMA can be dissected at high resolution. First, we carry out systematic mutational scanning using dihydrofolate reductase-based protein complementarity analysis to identify essential interaction regions and key residues. The results are found to be highly consistent with an α/ß-type complex structure predicted by AlphaFold2 (AF2). We then design mutants based on the predicted structure using a deep learning protein sequence design method. The solved crystal structure of one mutant presents the same core structure as predicted by AF2. Further computational prediction and experimental assessment indicate that the well-defined core structure is conserved across complexes of 4.1G CTD with other potential targets. Thus, we reveal that an intrinsically disordered protein interaction domain uses an α/ß-type structure module formed through synergistic folding to recognize broad IDP targets. Moreover, we show that computational prediction and experiment can be jointly applied to segregate true IDP regions from the core structural domains of IDP-IDP complexes and to uncover the structure-dependent mechanisms of some otherwise elusive IDP-IDP interactions.

Design, synthesis and antifungal activity of novel 1,4-benzoxazin-3-one derivatives containing an acylhydrazone moiety.

A series of 1,4-benzoxazin-3-one derivatives containing an acylhydrazone moiety were designed, synthesized and evaluated for their in vitro antifungal activities against Gibberella zeae, Pellicularia sasakii, Phytophthora infestans, Capsicum wilt, and Phytophthora capsica. The structures of target compounds were characterized by 1H NMR, 13H NMR, 19F NMR and HRMS. The preliminary antifungal evaluation of all target compounds showed that some target compounds possessed moderate to good activities against G. zeae, P. sasakii, P. infestans and C. wilt. Among them, compounds 5L and 5o exhibited noticeable inhibition effects against G. zeae with the EC50 values (effective concentration for 50% activity) of 20.06 and 23.17 µg/ml, respectively, which were even nearly double effective than that of hymexazol (40.51 µg/ml). Meanwhile, compound 5q displayed a notable inhibitory effect toward P. sasakii, with the EC50 value of 26.66 µg/ml, which was better than that of hymexazol (32.77 µg/ml). In addition, compound 5r yielded the EC50 value of 15.37 µg/ml against P. infestans, which was less than those of hymexazol (18.35 µg/ml) and carbendazim (34.41 µg/ml). Eventually, compound 5p showed higher inhibitory effect against C. wilt, with EC50 value of 26.76 µg/ml, which was better than that of hymexazol (>50 µg/ml).

Untargeted metabolomics reveals alternations in metabolism of bovine mammary epithelial cells upon IFN-γ treatment.

BACKGROUND: IFN-γ is a pleiotropic cytokine that has been shown to affect multiple cellular functions of bovine mammary epithelial cells (BMECs) including impaired milk fat synthesis and induction of malignant transformation via depletion of arginine, one of host conditionally essential amino acids. But the molecular mechanisms of these IFN-γ induced phenotypes are still unknown. METHODS: BMECs were treated with IFN-γ for 6 h, 12 h, and 24 h. The metabolomic profiling in BMECs upon IFN-γ induction were assessed using untargeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) metabolomic analysis. Key differentially expressed metabolites (DEMs) were quantified by targeted metabolomics. RESULTS: IFN-γ induction resulted in significant differences in the contents of metabolites. Untargeted analysis identified 221 significantly DEMs, most of which are lipids and lipid-like molecules, organic acids and derivatives, organ heterocyclic compounds and benzenoids. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, DEMs were enriched in fructose and mannose metabolism, phosphotransferase system (PTS), ß-alanine metabolism, arginine and proline metabolism, methane metabolism, phenylalanine metabolism, and glycolysis/gluconeogenesis. Quantification of selected key DEMs by targeted metabolomics showed significantly decreased levels of D-(-)-mannitol, argininosuccinate, and phenylacetylglycine (PAG), while increased levels in S-hydroxymethylglutathione (S-HMG) and 2,3-bisphospho-D-glyceric acid (2,3-BPG). CONCLUSIONS: These results provide insights into the metabolic alterations in BMECs upon IFN-γ induction and indicate potential theoretical basis for clarifying IFN-γ-induced diseases in mammary gland.

LAP3 contributes to IFN-γ-induced arginine depletion and malignant transformation of bovine mammary epithelial cells.

BACKGROUND: IFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown. METHODS: In this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry. RESULTS: The targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control. CONCLUSIONS: These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

Carnosine Synthase 1 Contributes to Interferon Gamma-Induced Arginine Depletion via Mitogen-activated Protein Kinase 11 Signaling in Bovine Mammary Epithelial Cells.

Arginine is one of the host semiessential amino acids with diverse biological activities, and arginine depletion is associated with the incidence of many diseases. Arginine depletion induced by diet-derived interferon gamma (IFN-γ) leads to malignant transformation and impaired milk quality in healthy lactating bovine mammary epithelial cells (BMECs). However, the molecular mechanism of IFN-γ-induced arginine depletion is unclear. In this study, the BMEC cell line, mammary alveolar cells-large T antigen cells (MAC-T), was stimulated with IFN-γ (10 ng/mL) for 24 h, and cellular arginine and ornithine quantified by liquid chromatography-tandem mass spectrometry. Carnosine synthase 1 (CARNS1) was identified from RNA-seq data, CARNS1 knockdown was achieved using an shRNA interfering plasmid. The expression levels of CARNS1, argininosuccinate synthetase 1 (ASS1), mitogen-activated protein kinase 11 (p38 MAPK), and phosphorylated (p)-p38, and their cognate genes, were analyzed by Western blotting and real-time quantitative polymerase chain reaction. The results showed that IFN-γ inhibited the biosynthesis of arginine, but enhanced its catalysis via disruption of key enzymes involved in arginine metabolism. IFN-γ also inhibited the expression of CARNS1, ASS1, and cationic amino acid transporter 1, while activating the expression and phosphorylation of p38. However, knockdown of CARNS1 reduced arginine level and ASS1 expression and block of either the IFN-γ receptor IFN-γ receptor 2 or p38 relieved both the expression of Carnosine synthase 1 (CARNS1) and ASS1. In summary, these results indicate that IFN-γ induced arginine depletion through inhibition of CARNS1 signaling via activation of p38 in BMECs. These findings provide a novel insight for IFN-γ-related disease control strategies in dairy cows.

Suppression of lncRNA GAS6-AS2 alleviates sepsis-related acute kidney injury through regulating the miR-136-5p/OXSR1 axis in vitro and in vivo.

Acute kidney injury (AKI) is a common complication of sepsis and increase morbidity and mortality. Long non-coding RNA (LncRNA) GAS6-AS2 was related to inflammation and apoptosis in different diseases by regulating miRNAs and downstream genes, but its role in AKI remains unclear. Thus, we speculated that GAS6-AS2 might function in sepsis-related AKI via regulating target genes. Here, LPS or CLP was used to establish in vitro or in vivo sepsis-related AKI model. The interactions between GAS6-AS2 and miR-136-5p, and miR-136-5p and OXSR1, were validated by luciferase reporter assay, RNA pull-down, or RIP assay. Cell apoptosis was determined by flow cytometry, Western blotting, or IHC. The kidney injury was evaluated by H&E staining. The expression of GAS6-AS2, miR-136-5p, and OXSR1 was determined by qRT-PCR or Western blotting. We found that GAS6-AS2 was up-regulated in LPS-treated HK2 cells and the CLP-induced rat model. In vitro, GAS6-AS2 knockdown decreased cleaved caspase-3 and bax expression and increased bcl-2 expression. The levels of TNF-α, IL-1ß, and IL-6 were reduced by GAS6-AS2 down-regulation. GAS6-AS2 knockdown ameliorated oxidative stress in the cells, as indicated by the reduced ROS and MDA levels and the elevated SOD level. In vivo, GAS6-AS2 down-regulation decreased urinary NGAL and Kim-1 levels and serum sCr and BUN levels, and H&E proved that the kidney injury was alleviated. GAS6-AS2 knockdown also reduced apoptosis, inflammation, and oxidation induced by CLP in vivo. Mechanically, GAS6-AS2 sponged miR-136-5p which targeted OXSR1. Overall, lncRNA GAS6-AS2 knockdown has the potential to ameliorate sepsis-related AKI, and the mechanism is related to miR-136-5p/OXSR1 axis.

A backbone-centred energy function of neural networks for protein design.

A protein backbone structure is designable if a substantial number of amino acid sequences exist that autonomously fold into it1,2. It has been suggested that the designability of backbones is governed mainly by side chain-independent or side chain type-insensitive molecular interactions3-5, indicating an approach for designing new backbones (ready for amino acid selection) based on continuous sampling and optimization of the backbone-centred energy surface. However, a sufficiently comprehensive and precise energy function has yet to be established for this purpose. Here we show that this goal is met by a statistical model named SCUBA (for Side Chain-Unknown Backbone Arrangement) that uses neural network-form energy terms. These terms are learned with a two-step approach that comprises kernel density estimation followed by neural network training and can analytically represent multidimensional, high-order correlations in known protein structures. We report the crystal structures of nine de novo proteins whose backbones were designed to high precision using SCUBA, four of which have novel, non-natural overall architectures. By eschewing use of fragments from existing protein structures, SCUBA-driven structure design facilitates far-reaching exploration of the designable backbone space, thus extending the novelty and diversity of the proteins amenable to de novo design.

Development and application of sequential window acquisition of all theoretical mass spectra data acquisition modes on ultra-high-performance liquid chromatography triple-quadrupole/time-of-flight mass spectrometry for metabolic profiling of amino acids in human plasma.

Accumulating evidence suggests that amino acids are important indicators of nutritional and metabolic status. A high-resolution mass spectrometry method based on sequential window acquisition of all theoretical mass spectra acquisition was developed for the simultaneous determination of 16 amino acids in human plasma. Sample preparation by protein precipitation using a mixture of acetonitrile and formic acid was followed by a BEH Amide column. The superiority of this method was investigated by comparing it to time-of-flight scan and multiple reaction monitoring modes. The limit of detection in sequential window acquisition of all theoretical mass spectra mode for threonine, methionine, histidine, citrulline, and tryptophan is 0.1 ng on the column; for lysine and asparagine is 0.2 ng; for alanine, pyroglutamic acid, leucine, ornithine, and aspartate is 0.5 ng, for arginine is 1.0 ng; for glutamate and serine is 2.0 ng; for glutamine is 10.0 ng. This method was linear in the range 0.8-40 µg/mL for arginine, citrulline, glutamate, histidine, leucine, methionine, pyroglutamic acid, threonine, tryptophan; 2-100 µg/mL for asparagine, aspartate, lysine, ornithine, serine; and 4-200 µg/mL for alanine, glutamine with good accuracy and precision. Significantly different levels in 11 amino acids were observed between childhood and adulthood, representing the growth and development of individuals relating to the level of amino acids.

Bailing Capsule combined with α-ketoacid tablets for stage 3 chronic kidney disease: Protocol of a double-blinded, randomized, controlled trial.

BACKGROUND: Chronic kidney disease (CKD) is a progressive and irreversible loss of kidney function. After stage 3, there will be increased risks of hypertension, heart failure, bone disease, anemia, gastrointestinal symptoms, and progression to end-stage kidney failure without proper intervention and treatment. Compound α-ketoacid tablets (KA) administration plays an important role in clinical CKD adjunctive therapy for patients with restricted protein intake. Bailing Capsule (BC), a commonly used Chinese patent medicine for renal diseases, could regulate human immune function, repair renal tubular epithelial cells, prevent renal tubular atrophy, and reduce kidney damage to improve renal function. In this study, we try to conduct a double-blinded, randomized, controlled trial to observe the efficacy and safety of BC combined with KA in treating patients with stage 3 CKD. METHODS: This is a double-blinded, randomized, controlled trial. Patients will be randomly divided into treatment group (BC and KT) and control group (BC-simulation and KT) in a 1:1 ratio according to random number table. The treatment course will be 8 weeks, and the changes of subjective symptoms, patient global assessment (PGA) scale, serum creatinine, cystatin C, and estimated glomerular filtration rate, all related adverse events, vital sign measurements, and physical examinations will be recorded. SPSS 21.0 will be used for data analysis. CONCLUSIONS: The results will show whether BC combined with KA could alleviate the symptoms of fatigue, anorexia, halitosis, nausea, itching, and edema, improve kidney function in patients with CKD at stage 3. TRIAL REGISTRATION: OSF Registration number: DOI 10.17605/OSF.IO/24AJ7.

Increasing the efficiency and accuracy of the ABACUS protein sequence design method.

MOTIVATION: The ABACUS (a backbone-based amino acid usage survey) method uses unique statistical energy functions to carry out protein sequence design. Although some of its results have been experimentally verified, its accuracy remains improvable because several important components of the method have not been specifically optimized for sequence design or in contexts of other parts of the method. The computational efficiency also needs to be improved to support interactive online applications or the consideration of a large number of alternative backbone structures. RESULTS: We derived a model to measure solvent accessibility with larger mutual information with residue types than previous models, optimized a set of rotamers which can approximate the sidechain atomic positions more accurately, and devised an empirical function to treat inter-atomic packing with parameters fitted to native structures and optimized in consistence with the rotamer set. Energy calculations have been accelerated by interpolation between pre-determined representative points in high-dimensional structural feature spaces. Sidechain repacking tests showed that ABACUS2 can accurately reproduce the conformation of native sidechains. In sequence design tests, the native residue type recovery rate reached 37.7%, exceeding the value of 32.7% for ABACUS1. Applying ABACUS2 to designed sequences on three native backbones produced proteins shown to be well-folded by experiments. AVAILABILITY AND IMPLEMENTATION: The ABACUS2 sequence design server can be visited at http://biocomp.ustc.edu.cn/servers/abacus-design.php. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pre-dialysis renal clinic visits and patients' outcomes on peritoneal dialysis.

PURPOSE: To investigate the effect of pre-dialysis renal care on peritoneal dialysis (PD) patients' outcomes in China. METHODS: In this retrospective cohort study, patients who started PD during January 1, 2006, to December 31, 2014, were included. Patients' medical charts were reviewed to extract the information. To explore the effect of pre-dialysis renal care on patients' outcomes, patient were divided into two groups according to whether or not they had frequent renal clinic visits: Group A (with frequent visits) and Group B (without frequent visits). RESULTS: A total of 668 patients were included. Patients who admitted to emergency room before PD initiations were significantly higher in Group B than in Group A (42.7 vs. 33 %, p = 0.01). However, there was no significant difference in the proportion of patients requiring emergency hemodialysis prior to PD commencement (20.5 vs. 24.6 %, p = 0.21), acute heart failure (30.2 vs. 35.4 %, p = 0.16) and pulmonary infection (15.4 vs. 12.1 %, p = 0.23) between groups. Both the mortality and technical failure rate in Group A were significantly lower as compared to Group B (p = 0.003 and p < 0.01, respectively). Multivariable Cox regression analysis showed frequent pre-dialysis renal clinic visits were associated with both lower mortality rate (HR 0.62, 95 % CI 0.46-0.85, p = 0.003) and technical failure on PD (HR 0.58, 95 % CI 0.36-0.92, p = 0.022). CONCLUSION: Pre-dialysis frequent clinic visits were associated with better PD outcomes. Pre-dialysis renal clinic management was suboptimal in the present cohort. More organized system to ensure people with established chronic kidney disease are well managed is necessary in China.