PTMint database of experimentally verified PTM regulation on protein-protein interaction.

MOTIVATION: Post-translational modification (PTM) is an important biochemical process. which includes six most well-studied types: phosphorylation, acetylation, methylation, sumoylation, ubiquitylation and glycosylation. PTM is involved in various cell signaling pathways and biological processes. Abnormal PTM status is closely associated with severe diseases (such as cancer and neurologic diseases) by regulating protein functions, such as protein-protein interactions (PPIs). A set of databases was constructed separately for PTM sites and PPI; however, the resource of regulation for PTM on PPI is still unsolved. RESULTS: Here, we firstly constructed a public accessible database of PTMint (PTMs that are associated with PPIs) (https://ptmint.sjtu.edu.cn/) that contains manually curated complete experimental evidence of the PTM regulation on PPIs in multiple organisms, including Homo sapiens, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae and Schizosaccharomyces pombe. Currently, the first version of PTMint encompassed 2477 non-redundant PTM sites in 1169 proteins affecting 2371 protein-protein pairs involving 357 diseases. Various annotations were systematically integrated, such as protein sequence, structure properties and protein complex analysis. PTMint database can help to insight into disease mechanism, disease diagnosis and drug discovery associated with PTM and PPI. AVAILABILITY AND IMPLEMENTATION: PTMint is freely available at: https://ptmint.sjtu.edu.cn/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Early Aggregation Mechanism of SOD128-38 Based on Force Field Parameter of 5-Cyano-Tryptophan.

The aggregation of superoxide dismutase 1 (SOD1) results in amyloid deposition and is involved in familial amyotrophic lateral sclerosis, a fatal motor neuron disease. There have been extensive studies of its aggregation mechanism. Noncanonical amino acid 5-cyano-tryptophan (5-CN-Trp), which has been incorporated into the amyloid segments of SOD1 as infrared probes to increase the structural sensitivity of IR spectroscopy, is found to accelerate the overall aggregation rate and potentially modulate the aggregation process. Despite these observations, the underlying mechanism remains elusive. Here, we optimized the force field parameters of 5-CN-Trp and then used molecular dynamics simulation along with the Markov state model on the SOD128-38 dimer to explore the kinetics of key intermediates in the presence and absence of 5-CN-Trp. Our findings indicate a significantly increased probability of protein aggregate formation in 5CN-Trp-modified ensembles compared to wildtype. Dimeric ß-sheets of different natures were observed exclusively in the 5CN-Trp-modified peptides, contrasting with wildtype simulations. Free-energy calculations and detailed analyses of the dimer structure revealed augmented interstrand interactions attributed to 5-CN-Trp, which contributed more to peptide affinity than any other residues. These results explored the key events critical for the early nucleation of amyloid-prone proteins and also shed light on the practice of using noncanonical derivatives to study the aggregation mechanism.

Phosphorylation Regulation Mechanism of ß2 Integrin for the Binding of Filamin Revealed by Markov State Model.

Leukocyte adhesion deficiency-1 (LAD-1) disorder is a severe immunodeficiency syndrome caused by deficiency or mutation of ß2 integrin. The phosphorylation on threonine 758 of ß2 integrin acts as a molecular switch inhibiting the binding of filamin. However, the switch mechanism of site-specific phosphorylation at the atom level is still poorly understood. To resolve the regulation mechanism, all-atom molecular dynamics simulation and Markov state model were used to study the dynamic regulation pathway of phosphorylation. Wild type system possessed lower binding free energy and fewer number of states than the phosphorylated system. Both systems underwent local disorder-to-order conformation conversion when achieving steady states. To reach steady states, wild type adopted less number of transition paths/shortest path according to the transition path theory than the phosphorylated system. The underlying phosphorylated regulation pathway was from P1 to P0 and then P4 state, and the main driving force should be hydrogen bond and hydrophobic interaction disturbing the secondary structure of phosphorylated states. These studies will shed light on the pathogenesis of LAD-1 disease and lay a foundation for drug development.

Multiplex metabolic pathway engineering of Monascus pilosus enhances lovastatin production.

Monascus sp. is an important food microbial resource with the production of cholesterol-lowering agent lovastatin and other healthy metabolites. However, the mycotoxin citrinin naturally produced by Monascus sp. and the insufficient productivity of lovastatin limit its large-scale use in food industry. The aim of this paper is to modify a lovastatin-producing strain Monascus pilosus GN-01 through metabolic engineering to obtain a citrinin-free M. pilosus strain with higher yield of lovastatin. The citrinin synthesis regulator gene ctnR was firstly disrupted to obtain GN-02 without citrinin production. Based on that, the lovastatin biosynthesis genes (mokC, mokD, mokE, mokF, mokH, mokI, and LaeA) were, respectively, overexpressed, and pigment-regulatory gene (pigR) was knocked out to improve lovastatin production. The results indicated ctnR inactivation effectively disrupted the citrinin release by M. pilosus GN-01. The overexpression of lovastatin biosynthesis genes and pigR knockout could lead higher contents of lovastatin, of which pigR knockout strain achieved 76.60% increase in the yield of lovastatin compared to GN-02. These studies suggest that such multiplex metabolic pathway engineering in M. pilosus GN-01 is promising for high lovastatin production by a safe strain for application in Monascus-related food. KEY POINTS: • Disruption of the regulator gene ctnR inhibited citrinin production of M. pilosus. • Synchronous overexpression of biosynthesis gene enhanced lovastatin production. • pigR knockout enhanced lovastatin of ΔctnR strain of M. pilosus.

Identification and validation of new MADS-box homologous genes in 3010 rice pan-genome.

KEY MESSAGE: Identification and validation of ten new MADS-box homologous genes in 3010 rice pan-genome for rice breeding. The functional genome is significant for rice breeding. MADS-box genes encode transcription factors that are indispensable for rice growth and development. The reported 15,362 novel genes in the rice pan-genome (RPAN) of Asian cultivated rice accessions provided a useful gene reservoir for the identification of more MADS-box candidates to overcome the limitation for the usage of only 75 MADS-box genes identified in Nipponbare for rice breeding. Here, we report the identification and validation of ten MADS-box homologous genes in RPAN. Origin and identity analysis indicated that they are originated from different wild rice accessions and structure of motif analysis revealed high variations in their amino acid sequences. Phylogenetic results with 277 MADS-box genes in 41 species showed that all these ten MADS-box homologous genes belong to type I (SRF-like, M-type). Gene expression analysis confirmed the existence of these ten MADS-box genes in IRIS_313-10,394, all of them were expressed in flower tissues, and six of them were highly expressed during seed development. Altogether, we identified and validated experimentally, for the first time, ten novel MADS-box genes in RPAN, which provides new genetic sources for rice improvement.

Databases and Bioinformatic Tools for Glycobiology and Glycoproteomics.

Glycosylation plays critical roles in various biological processes and is closely related to diseases. Deciphering the glycocode in diverse cells and tissues offers opportunities to develop new disease biomarkers and more effective recombinant therapeutics. In the past few decades, with the development of glycobiology, glycomics, and glycoproteomics technologies, a large amount of glycoscience data has been generated. Subsequently, a number of glycobiology databases covering glycan structure, the glycosylation sites, the protein scaffolds, and related glycogenes have been developed to store, analyze, and integrate these data. However, these databases and tools are not well known or widely used by the public, including clinicians and other researchers who are not in the field of glycobiology, but are interested in glycoproteins. In this study, the representative databases of glycan structure, glycoprotein, glycan-protein interactions, glycogenes, and the newly developed bioinformatic tools and integrated portal for glycoproteomics are reviewed. We hope this overview could assist readers in searching for information on glycoproteins of interest, and promote further clinical application of glycobiology.

The effect of normal human serum on the mouse trypanosome Trypanosoma musculi in vitro and in vivo.

Trypanosoma musculi, a common blood flagellate found in mice, is similar in morphology and life cycle to the rat trypanosome T. lewisi. Both species belong to the subgenus Herpetosoma, and as T. lewisi has recently been shown to be a zoonotic pathogen, there is concern that T. musculi could also be potentially infective to humans. To test this hypothesis, a well-established method, the normal human serum (NHS) incubation test, was carried out which distinguishes human and non-human infective trypanosomes. We found that T. musculi could grow in 0.31% NHS in vitro, and even kept their infectivity to mice after incubation with 10% NHS for 24 h. In in vivo experiments, T. musculi were only slightly affected by NHS injection, confirming that it was less sensitive to the NHS than T. b. brucei, but more sensitive than T. lewisi. This resistance probably does not rely on a restricted uptake of ApoL-1. Due to this partial resistance, we cannot definitively confirm that T. musculi has the potential for infection to humans. As resistance is less than that of T. lewisi, our data suggest that it is unlikely to be a zoonotic pathogen although we would advise caution in the case of immunocompromised people such as AIDS and cancer patients.

[Pathogen Identification in an Imported Case of Cutaneous Leishmaniasis].

In this study, immune and molecular biological methods were used to identify the pathogen in a blood sample from a patient with dermatosis. Venous blood was collected and tested with Leish rK39 dipsticks. The lesion sample was collected and fixed in 75% ethanol, and DNA was extracted. The internal transcribed spacer 1 of rDNA and N-acetylglucosamine-1-phosphate transferase of Leishmania were amplified with PCR using primers LITSR-L5.8S and NAGTL1s-NAGTL4, respectively. The amplified products were sequenced and analyzed by BLAST. Weakly positive results were obtained for the gold-labeled Leish rK39 dipstick serological test. PCR resulted in products of 404 bp and 1 405 bp with primers LITSR-L5.8S and NAGTL1-NAGTL4, respectively. Both were 99.7% homologous to the corresponding sequence of Leishmania major. The accession number of the two sequences were KU975160 and KX150476. The case of dermatosis is diagnosed as imported cutaneous leishmaniasis and the pathogen is L. major.

Single-cell enzymatic cascade synthesis of testolactone enabled by engineering of polycyclic ketone monooxygenase and multi-gene expression fine-tuning.

The synthesis of steroids is challenging through multistep steroidal core modifications with high site-selectivity and productivity. In this work, a novel enzymatic cascade system was constructed for synthesis of testolactone by specific C17 lactonization/Δ1-dehydrogenation from inexpensive androstenedione using an engineered polycyclic ketone monooxygenase (PockeMO) and an appropriate 3-ketosteroid-Δ1-dehydrogenase (ReKstD). The focused saturation mutagenesis in the substrate binding pocket was implemented for evolution of PockeMO to eliminate the bottleneck effect. A best mutant MU3 (I225L/L226V/L532Y) was obtained with 20-fold higher specific activity compared to PockeMO. The catalytic efficiency (kcat/Km) of MU3 was 171-fold higher and the substrate scope shifted to polycyclic ketones. Molecular dynamic simulations suggested that the activity was improved by stabilization of the pre-lactonization state and generation of productive orientation of 4-AD mediated by distal L532Y mutation. Based on that, the three genes, MU3, ReKstD and a ketoreductase for NADPH regeneration, were rationally integrated in one cell via expression fine-tuning to form the efficient single cell catalyst E. coli S9. The single whole-cell biocatalytic process was scaled up and could generate 9.0 g/L testolactone with the high space time yield of 1 g/L/h without steroidal by-product, indicating the potential for site-specific and one-pot synthesis of steroid.

Reproductive chemical database: a curated database of chemicals that modulate protein targets regulating important reproductive biological processes.

Recent studies have shifted the spotlight from adult disease to gametogenesis and embryo developmental events, and these are greatly affected by various environmental chemicals, such as drugs, metabolites, pollutants, and others. Growing research has highlighted the critical importance of identifying and understanding the roles of chemicals in reproductive biology. However, the functions and mechanisms of chemicals in reproductive processes remain incomplete. We developed a comprehensive database called the Reproductive Chemical Database (RCDB) ( https://yu.life.sjtu.edu.cn/ChenLab/RCDB ) to facilitate research on chemicals in reproductive biology. This resource is founded on rigorous manual literature extraction and precise protein target prediction methodologies. This database focuses on the delineation of chemicals associated with phenotypes, diseases, or endpoints intricately associated with four important reproductive processes: female and male gamete generation, fertilization, and embryo development in human and mouse. The RCDB encompasses 93 sub-GO processes, and it revealed 1447 intricate chemical-biological process interactions. To date, the RCDB has meticulously cataloged and annotated 830 distinct chemicals, while also predicting 614 target proteins from a selection of 3800 potential candidates. Additionally, the RCDB offers an online predictive tool that empowers researchers to ascertain whether specific chemicals play discernible functional roles in these reproductive processes. The RCDB is an exhaustive, cross-platform, manually curated database, which provides a user-friendly interface to search, browse, and use reproductive processes modulators and their comprehensive related information. The RCDB will help researchers to understand the whole reproductive process and related diseases and it has the potential to promote reproduction research in the pharmacological and pathophysiological areas.

Improved Antimicrobial Activity of Bovine Lactoferrin Peptide (LFcinB) Based on Rational Design.

Bovine lactoferrin peptide (LFcinB), as an antimicrobial peptide, is expected to be an alternative of antibiotics owing to its broad-spectrum antimicrobial activity and specific mechanism. However, the weak antimicrobial activity, high hemolysis, and poor stability of LFcinB limited its applications in the field of biomedicine, food and agriculture. In order to improve the antimicrobial activity of LFcinB, five mutants were designed rationally, of which mutant LF4 (M10W/P16R/A24L) showed highest antimicrobial activity. The bioinformatics analysis indicated that the improved antimicrobial activity of LF4 was related to its increased cations, higher amphiphilicity and the extension of the ß-sheet in the structure. These studies will highlight the important role of bioinformatic tools in designing ideal biopeptides and lay a foundation for further development of antimicrobial peptides.

Sequence-based machine learning method for predicting the effects of phosphorylation on protein-protein interactions.

Protein phosphorylation, catalyzed by kinases, is an important biochemical process, which plays an essential role in multiple cell signaling pathways. Meanwhile, protein-protein interactions (PPI) constitute the signaling pathways. Abnormal phosphorylation status on protein can regulate protein functions through PPI to evoke severe diseases, such as Cancer and Alzheimer's disease. Due to the limited experimental evidence and high costs to experimentally identify novel evidence of phosphorylation regulation on PPI, it is necessary to develop a high-accuracy and user-friendly artificial intelligence method to predict phosphorylation effect on PPI. Here, we proposed a novel sequence-based machine learning method named PhosPPI, which achieved better identification performance (Accuracy and AUC) than other competing predictive methods of Betts, HawkDock and FoldX. PhosPPI is now freely available in web server (https://phosppi.sjtu.edu.cn/). This tool can help the user to identify functional phosphorylation sites affecting PPI and explore phosphorylation-associated disease mechanism and drug development.

PCR-based identification of Trypanosoma lewisi and Trypanosoma musculi using maxicircle kinetoplast DNA.

Trypanosoma lewisi, transmitted by rat fleas, is a widespread pathogen specific to rats with records of human infection cases. Its closely related species with global distribution, Trypanosoma musculi, is transmitted between mice by ingestion of infected fleas. These trypanosomes are of similar morphology, making it difficult to distinguish them by microscopy. In this study, we have developed a rapid, sensitive and reliable PCR method for the diagnosis of T. lewisi and T. musculi. The T. lewisi-specific amplicons were not produced by other Trypanosoma, such as T. musculi, T. brucei complex or T. cruzi, neither by an outgroup of Leishmania amazonensis. The detection limits of the three pairs of T. lewisi-specific primers were 50ng, 1ng and 10ng of total DNA, respectively. The primers designed for T. musculi primers showed specifically that amplicon strictly in T. musculi and their detection limits were 10ng and 1ng of total DNA. To simplify the detection process, we managed to apply our method directly on tail blood samples without complicated DNA purification. In conclusion, PCR with our primers could be a highly sensitive, specific protocol to detect and distinguish T. lewisi and T. musculi from other trypanosomes.

Current status of Clonorchis sinensis and clonorchiasis in China.

The oriental liver fluke, Clonorchis sinensis, a pathogen causing clonorchiasis, is of major socio-economic importance in East Asia, including China, Korea and Vietnam. This parasite is now recognized as a biocarcinogen strongly linked to cholangiocarcinoma in humans. Here, we describe the status of clonorchiasis in China, where it has been estimated that more than 15 million patients are affected. This paper also summarizes the major advances in the field of clonorchiasis research during last decade, including diagnosis techniques, pathogenesis and genome/transcriptome/proteome studies in the last years. We strongly hope that our work can stimulate the governments of the countries or regions where clonorchiasis is endemic to pay more attention to this disease and establish related guidelines to prevent and control it.

Molecular species analysis of phosphatidylcholine by reversed-phase ion-pair high-performance liquid chromatography.

The retention behavior of molecular species of phosphatidylcholine (PC) is studied by reversed-phase (RP) ion-pair high-performance liquid chromatography (HPLC). Mobile phases contain tetraalkyl ammonium phosphates (TAAPs) in methano-acetonitrile-water. The stationary phase is alkyl-bonded silica. Competitive interactions of TAAPs, analyte solutes, and an RP-HPLC column result in reduced retention of PC molecular species. PC molecular species are eluted at longer retention times with a larger size of TAAP in the mobile phase, and an increase in the TAAP concentration invariably causes a decrease in PC molecular species retention times. There is a linear correlation between the logarithmic retention factors (k) of PC molecular species and the total number of carbon atoms of TAAP, and the logarithm of k values of PC molecular species can be approximated as a linear function of the logarithm of the counter-ion concentration. There is found to be no distinct dependence between k values of PC molecular species and the mobile phase pH.

[Separation of bile acids by capillary zone electrophoresis].

AIM: To develop a method for separating the major bile acids by capillary zone electrophoresis (CZE). METHODS: The effect of different separations, such as the compose, pH and the concentration of buffer, on the electro-osmotic flow (EOF), the migration time and resolution of 8 bile acids in this system were studied. The general trends in migration time could be correlated to the pH and concentration of the buffer. The effect of organic reagent on EOF and migration time were also investigated. By addition of methanol, the EOF went smaller than before, and better resolution was achieved. The experimental results showed that optimum separation was achieved under the following condition: buffer composition of 126 mmol.L-1 disodium tetraborate, 43 mmol.L-1 disodium hydrogenphosphate, 18% methanol; temperature 30 degrees C; voltage 30 kV; total length of capillary 570 mm and 500 mm from injection end; ultraviolet detection at 200 nm; pressure injection 5 kPa for 8 s. RESULTS: Eight kinds of bile acid had been separated by CZE with only one injection. The method was used to analyse the contents of bile acids from different kinds of bear biles, the recovery was 89%-107%. CONCLUSION: This method is simple and rapid, and can be used to determine the content of bile acids in bear biles. The calibration curve showed good linearity for eight bile acids in the concentration range of 4-60 mg.mL-1 (gamma > 0.9954). The total time for seperation and determination was within 25 min.

[Separation of different phosphatidyl cholines by reversed-phase ion-pair high performance liquid chromatography].

A reversed-phase ion-pair high performance liquid chromatographic (RP-IP-HPLC) method for the separation of different phosphatidyl cholines (PC) was established. The optimized conditions were a PERKIN-ELMER/HS-5 C18 column (150 mm x 4.6 mm i.d., 5 microns), isocratic elution with mobile phase of methanol-acetonitrile-water (70:22:8, volume ratio) containing 15 mmol/L tetramethyl ammonium phosphate (TMAP) at pH 7.0, flow rate of 2.0 mL/min and UV detection at 208 nm. Seven kinds of PC could be separated with this method.