Biology subjects

Wang, X.-B.

Publications and source records attributed to Wang, X.-B..

6 recordsLinked to original sources

bioRxiv2024

Peptide Information Compression Technology (PICT): A New Frontier in Therapeutic Peptide Discovery

Therapeutic peptides hold tremendous market potential, yet the discovery methods for active peptides lag considerably behind those for small molecules and biological macromolecule drugs. Due to their respective limitations, traditional peptide compound synthesis and display libraries fail to meet the growing demands of the peptide drug market. Here, we introduce a novel Peptide Information Compression Technology (PICT) and, based on this technology, a large-scale physical peptide library and its accompanying high-throughput screening (HTS) platform. By screening for inhibitors of TSLP/TSLPR/IL7R and antagonists of MC3R at both the molecular and cellular levels, we have identified active molecules in the single-digital micromolar and nanomolar ranges, respectively. This demonstrates the immense application value of Peptide Information Compression Technology in the efficient discovery of peptide drugs.

molecular biology↗

bioRxiv2023

An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase

In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full-length AEP-type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self-activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolysis activity. After site-directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To solve this problem, we developed a novel protease-assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via an ion-exchange chromatography. After the non-covalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolysis activity, and could efficiently catalyze inter-molecular protein ligation and intra-molecular peptide cyclization. Thus, the engineered bamboo-derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis.

biochemistry↗

bioRxiv2022

Barley stripe mosaic virus γb protein targets thioredoxin h-type 1 to dampen SA-mediated antiviral defenses

Salicylic acid (SA) acts as a signaling molecule to perceive and defend against pathogen infections. Accordingly, pathogens evolve versatile strategies to disrupt the SA-mediated signal transduction. However, it is necessary to further characterize how plant viruses manipulate the SA-dependent defense responses. Here, we show that Barley stripe mosaic virus (BSMV) infection activates SA-mediated defense signaling pathway and upregulates the expression of Nicotiana benthamiana thioredoxin h-type 1 (NbTRXh1). The {gamma}b protein interacts directly with NbTRXh1 in vivo and in vitro. Overexpression of NbTRXh1, but not a reductase-defective mutant, impedes BSMV infection, whereas low NbTRXh1 expression level results in increased viral accumulation. Similar with its orthologues in Arabidopsis, NbTRXh1 also plays an essential role in SA signaling transduction in N. benthamiana. To counteract NbTRXh1-mediated defenses, the BSMV {gamma}b protein targets NbTRXh1 to dampen its reductase activity and thereby impairing downstream SA defense genes expression to optimize viral cell-to-cell movement. We also found that NbTRXh1-mediated resistance defends against Lychnis ringspot virus, Beet black scorch virus, and Beet necrotic yellow vein virus. Taken together, our results reveal a novel role for the multifunctional {gamma}b protein in counteracting plant defense responses, and broadens the broad-spectrum antibiotic role of SA signaling pathway. One sentence summaryBSMV {gamma}b protein impairs NbTRXh1 reductase activity and dampen downstream SA-related genes expression to facilitate viral cell-to-cell movement.

microbiology↗

bioRxiv2021

Host casein kinase 1-mediated phosphorylation modulates phase separation of a rhabdovirus phosphoprotein and virus infection

Liquid-liquid phase separation (LLPS) plays important roles in forming cellular membraneless organelles. However, how host factors regulate LLPS of viral proteins during negative-sense RNA (NSR) virus infections is largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV) as a model to demonstrate regulation of host casein kinase 1 in phase separation and infection of NSR viruses. We first found that the BYSMV phosphoprotein (P) formed spherical granules with liquid properties and recruited viral nucleotide (N) and polymerase (L) proteins in vivo. Moreover, the P-formed granules were tethered to the ER/actin network for trafficking and fusion. BYSMV P alone formed droplets and incorporated the N protein and genomic RNA in vitro. Interestingly, phase separation of BYSMV P was inhibited by host casein kinase 1 (CK1)-dependent phosphorylation of an intrinsically disordered P protein region. Genetic assays demonstrated that the unphosphorylated mutant of BYSMV P exhibited condensed phase, which promoted virus replication through concentrating the N, L proteins, and genome RNA into viroplasms. Whereas, the phosphorylation-mimic mutant existed in diffuse phase state leading to enhanced virus transcription. Collectively, our results demonstrate that host CK1 modulates phase separation of viral P protein and virus infection.

microbiology↗

bioRxiv2021

Development of a novel bioluminescent activity assay for peptide ligases

In recent years, some peptide ligases have been identified, such as bacterial sortases and certain plant asparaginyl or prolyl endopeptidases. Peptide ligases have wide applications in protein labeling and cyclic peptide synthesis. To characterize known peptide ligases or identify new ones, we propose a novel bioluminescent activity assay via the genetic fusion of a recognition motif of a peptide ligase to the C-terminus of an inactive large NanoLuc fragment (LgBiT) and the chemical introduction of a nucleophilic motif preferred by the peptide ligase to the N-terminus of the low-affinity SmBiT complementation tag. When the inactive ligation version LgBiT protein was ligated with the low-affinity ligation version SmBiT tag by the expected peptide ligase, its luciferase activity would be restored and could be quantified sensitively according to the measured bioluminescence. In the present study, we first validated the novel bioluminescent activity assay using bacterial sortase A and plant butelase-1. Subsequently, we screened novel peptide ligases from crude extracts of selected plants using two LgBiT-SmBiT ligation pairs. Among 80 common higher plants, we identified that five of them likely express asparaginyl endopeptidase-type peptide ligase and four of them likely express prolyl endopeptidase-type peptide ligase, suggesting that peptide ligases are not so rare in higher plants and more of them await discovery. The novel bioluminescent activity assay is ultrasensitive, convenient for use, and resistant to protease interference, and thus would have wide applications for characterizing known peptide ligases or screening new ones from various sources in future studies.

biochemistry↗

bioRxiv2020

Nitrapyrin has far reaching effects on the soil microbial community structure, composition, diversity and functions

Nitrapyrin is one of the most common nitrification inhibitors that are used to retain N in the ammonia form in soil to improve crop yields and quality. Whereas the inhibitory effect of nitrapyrin is supposedly specific to ammonia oxidizers, in view of the keystone role of this group in soils, nitrapyrin could have far-reaching impacts. Here, we tested the hypothesis that nitrapyrin leads to large shifts in soil microbial community structure, composition, diversity and functions, beyond its effect on ammonia-oxidizers. To test this hypothesis, we set-up a field experiment where wheat (Triticum aestivum cv. AC Walton) was fertilized with ammonium nitrate (NH4NO3) and supplemented or not with nitrapyrin. Rhizosphere and bulk soils were sampled twice, DNA was extracted, the 16S rRNA gene and ITS region were amplified and sequenced to follow shifts in archaeal, bacterial and fungal community structure, composition and diversity. To assess microbial functions, several genes involved in the nitrogen cycle were quantified by real-time qPCR and volatile organic compounds (VOCs) were trapped in the rhizosphere at the moment of sampling. As expected, sampling date and plant compartment had overwhelming effects on the microbial communities. However, within these strong effects, we found statistically significant effects of nitrapyrin on the relative abundance of Thaumarchaeota, Proteobacteria, Nitrospirae and Basidiomycota, and on several genera. Nitrapyrin also significantly affected bacterial and fungal community structure, and the abundance of all the N-cycle gene tested, but always in interaction with sampling date. In contrast, nitrapyrin had no significant effect on the emission of VOCs, where only sampling date significantly influenced the profiles observed. Our results point out far-reaching effects of nitrapyrin on soil and plant associated microbial communities, well beyond its predicted direct effect on ammonia-oxidizers. In the longer term, these shifts might counteract the positive effect of nitrapyrin on crop nutrition and greenhouse gas emissions.

microbiology↗