Alanine substitutions of noncysteine residues in the cysteine‐stabilized αβ motif

The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine‐stabilized αβ motif (CSαβ) consists of an α‐helix and an antiparallel triple‐stranded β‐sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSαβ motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty‐two of 46 residue positions of VrD1 are altered by site‐directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering.     

Population genetic structure and migration patterns of Dendrothrips minowai (Thysanoptera: Thripidae) in Guizhou,China

Dendrothrips minowai Priesner (Thysanoptera: Thripidae) is one of the most destructive insect pests on tea plants. Although outbreaks of this pest occur annually in South China, especially in Guizhou Province, little is known about its population genetics, such as genetic diversity and gene flow. To investigate its population genetic structure and migration routes in Guizhou Province, we analyzed 24 D. minowai populations across Guizhou using six microsatellite loci. We detected the moderate genetic diversity and the population genetic structure of this thrip species. Neighbor‐joining (NJ) phylogenetic tree and STRUCTURE analyses recognized two clusters within the studied populations. No correlation between genetic and geographical distances (r = 0.0139, P = 0.5830) was detected and more than 89% of the variation occurred among samples within populations. Gene flow analysis revealed high migration rates (74.0 – 894.1) among D. minowai populations. Overall, the trend of asymmetrical gene flow was from northeast to southwest. Our analyses demonstrated that D. minowai derived or originated from multiple sites and could be eventually divided into two groups in Guizhou.     

Single-cell transcriptomics reveals heterogeneous progression and EGFR activation in pancreatic adenosquamous carcinoma

Pancreatic adenosquamous carcinoma (PASC) — a rare pathological pancreatic cancer (PC) type — has a poor prognosis due to high malignancy. To examine the heterogeneity of PASC, we performed single-cell RNA sequencing (scRNA-seq) profiling with sample tissues from a healthy donor pancreas, an intraductal papillary mucinous neoplasm, and a patient with PASC. Of 9,887 individual cells, ten cell subpopulations were identified, including myeloid, immune, ductal, fibroblast, acinar, stellate, endothelial, and cancer cells. Cancer cells were divided into five clusters. Notably, cluster 1 exhibited stem-like phenotypes expressing UBE2C, ASPM, and TOP2A. We found that S100A2 is a potential biomarker for cancer cells. LGALS1, NPM1, RACK1, and PERP were upregulated from ductal to cancer cells. Furthermore, the copy number variations in ductal and cancer cells were greater than in the reference cells. The expression of EREG, FCGR2A, CCL4L2, and CTSC increased in myeloid cells from the normal pancreas to PASC. The gene sets expressed by cancer-associated fibroblasts were enriched in the immunosuppressive pathways. We demonstrate that EGFR-associated ligand-receptor pairs are activated in ductal-stromal cell communications. Hence, this study revealed the heterogeneous variations of ductal and stromal cells, defined cancer-associated signaling pathways, and deciphered intercellular interactions following PASC progression.     

Crystal structure and biophysical characterisation of Helicobacter pylori phosphopantetheine adenylyltransferase

CKLF1, a human cytokine that is a functional ligand for CCR4, is upregulated in various inflammation and autoimmune diseases. CKLF1 contains at least two secreted forms, the C-terminal peptides C19 and C27. Chemically synthesized C19 and C27 can interact with CCR4 and attenuate allergic inflammation. In this study, we found C19 and C27 could inhibit SDF-1-induced CXCR4-mediated chemotaxis and promote CXCR4 internalization. The inhibitory effect was due to desensitization of CXCR4, which was mediated by CCR4. Further experiments confirmed that CXCR4 desensitization required activation of PI3K/PKC pathway. Altogether our data elucidate the mechanism of C19- and C27-induced CXCR4 desensitization.     

Dietary isothiocyanate-induced apoptosis via thiol modification of DNA topoisomerase IIα

Studies in animal models have indicated that dietary isothiocyanates (ITCs) exhibit cancer preventive activities through carcinogen detoxification-dependent and -independent mechanisms. The carcinogen detoxification-independent mechanism of cancer prevention by ITCs has been attributed at least in part to their ability to induce apoptosis of transformed (initiated) cells (e.g. through suppression of IκB kinase and nuclear factor κB as well as other proposed mechanisms). In the current studies we show that ITC-induced apoptosis of oncogene-transformed cells involves thiol modification of DNA topoisomerase II (Top2) based on the following observations. 1) siRNA-mediated knockdown of Top2α in both SV40-transformed MEFs and Ras-transformed human mammary epithelial MCF-10A cells resulted in reduced ITC sensitivity. 2) ITCs, like some anticancer drugs and cancer-preventive dietary components, were shown to induce reversible Top2α cleavage complexes in vitro. 3) ITC-induced Top2α cleavage complexes were abolished by co-incubation with excess glutathione. In addition, proteomic analysis revealed that several cysteine residues on human Top2α were covalently modified by benzyl-ITC, suggesting that ITC-induced Top2α cleavage complexes may involve cysteine modification. Interestingly, consistent with the thiol modification mechanism for Top2α cleavage complex induction, the thiol-reactive selenocysteine, but not the non-thiol-reactive selenomethionine, was shown to induce Top2α cleavage complexes. In the aggregate, our results suggest that thiol modification of Top2α may contribute to apoptosis induction in transformed cells by ITCs.     

NMR assignments of a hypothetical pseudo-knotted protein HP0242 from Helicobacter pylori

Many knotted proteins have been discovered recently, but the folding process of which remains elusive. HP0242 is a hypothetical protein from Helicobacter pylori, which is a model system for studying the folding pathway of a knotted protein. In this study, we report the ¹H, ¹³C, and ¹⁵N chemical shift assignments of HP0242. The results will enable us to further investigate HP0242 by NMR experiments.     

Impact of alanyl-tRNA synthetase editing deficiency in yeast

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that provide the ribosome with aminoacyl-tRNA substrates for protein synthesis. Mutations in aaRSs lead to various neurological disorders in humans. Many aaRSs utilize editing to prevent error propagation during translation. Editing defects in alanyl-tRNA synthetase (AlaRS) cause neurodegeneration and cardioproteinopathy in mice and are associated with microcephaly in human patients. The cellular impact of AlaRS editing deficiency in eukaryotes remains unclear. Here we use yeast as a model organism to systematically investigate the physiological role of AlaRS editing. Our RNA sequencing and quantitative proteomics results reveal that AlaRS editing defects surprisingly activate the general amino acid control pathway and attenuate the heatshock response. We have confirmed these results with reporter and growth assays. In addition, AlaRS editing defects downregulate carbon metabolism and attenuate protein synthesis. Supplying yeast cells with extra carbon source partially rescues the heat sensitivity caused by AlaRS editing deficiency. These findings are in stark contrast with the cellular effects caused by editing deficiency in other aaRSs. Our study therefore highlights the idiosyncratic role of AlaRS editing compared with other aaRSs and provides a model for the physiological impact caused by the lack of AlaRS editing.