Comparative transcriptome analysis of the hyperaccumulator plant Phytolacca americana in response to cadmium stress.

To study the molecular mechanism of the hyperaccumulator plant Phytolacca americana against cadmium (Cd) stress, the leaves of P. americana treated with 400 µM Cd for 0, 2, 12, and 24 h were harvested for comparative transcriptome analysis. In total, 110.07 Gb of clean data were obtained, and 63,957 unigenes were acquired after being assembled. Due to the lack of P. americana genome information, only 24,517 unigenes were annotated by public databases. After Cd treatment, 5054 differentially expressed genes (DEGs) were identified. KEGG pathway enrichment analysis of DEGs showed that genes involved in the flavonoid biosynthesis and antenna proteins of photosynthesis were significantly down-regulated, while genes related to the lignin biosynthesis pathway were remarkably up-regulated, indicating that P. americana could synthesize more lignin to cope with Cd stress. Moreover, genes related to heavy metal accumulation, sulfur metabolism and glutathione metabolism were also significantly up-regulated. The gene expression pattern of several key genes related to distinct metabolic pathways was verified by qRT-PCR. The results indicated that the immobilization of lignin in cell wall, chelation, vacuolar compartmentalization, as well as the increase of thiol compounds content may be the important mechanisms of Cd detoxification in hyperaccumulator plant P. americana. Accession numbers: the raw data of P. americana transcriptome presented in this study are openly available in NCBI SRA database, under the BioProject of PRJNA649785. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02865-x.

Insight into the structural stability of wild type and mutants of the tobacco etch virus protease with molecular dynamics simulations.

The efficiency and high specificity of tobacco etch virus protease (TEVp) has made it widely used for cleavage of recombinant fusion proteins. However, TEVp suffers from a few intrinsic defects such as self-cleavage, poorly expressed in E. coli and less soluble. So some mutants were designed to improve it, such as S219V, T17S/N68D/I77V and L56V/S135G etc. MD simulations for the WT TEVp and its mutants were performed to explore the underlying dynamic effects of mutations on TEVp. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of TEVp, including the elongation of ß-sheet, conversion of loop to helix and the flexibility of active core. Our present study indicates that the three mutants for TEVp can change their secondary structure and tend to form more helixes and sheets to improve stability. The study also helps us to understand the effects of some mutations on TEVp, provides us insights into the change of them at the atomic level and gives a potential rational method to design an improved protein.

Aluminum(III) interferes with the structure and the activity of the peptidyl-prolyl cis-trans isomerase (Pin1): a new mechanism contributing to the pathogenesis of Alzheimer's disease and cancers?

The enzyme peptidyl-prolyl cis-trans isomerase (Pin1) may play an important role in preventing the development of Alzheimer's disease (AD). The structural and functional stability of Pin1 is extremely important. Previously, we have determined the stability of Pin1 under stressed conditions, such as thermal treatment and acidic-pH. Considering that aluminum (Al(III)) is well known for its potential neurotoxicity in the pathogenesis of AD, we examined whether Al(III) affects the structure and function of Pin1, by means of a PPIase activity assay, intrinsic fluorescence, circular dichroism (CD) spectroscopy, FTIR, and differential scanning calorimetry (DSC). The intrinsic tryptophan fluorescence measurements mainly show that Al(III) may bind to the clusters nearby W11 and W34 in the WW domain of Pin1, quenching the intrinsic fluorescence of the two tryptophan residues, which possibly results in the decreased binding affinity of Pin1 to substrates. The secondary structural analysis as revealed by FTIR and CD measurements indicate that Al(III) induces the increase in ß-sheet and the decrease in α-helix in Pin1. Furthermore, the changes of the thermodynamic parameters for Pin1 as monitored by DSC confirm that the thermal stability of Pin1 significantly increases in the presence of Al(III). The Al(III)-induced structural changes of Pin1 result in a sharp decrease of the PPIase activity of Pin1. To some extent, our research is suggestive that Al(III) may inhibit the isomerization activity of Pin1 in vivo, which may contribute to the pathogenesis of AD.

The acidic pH-induced structural changes in Pin1 as revealed by spectral methodologies.

Pin1 is closely associated with the pathogenesis of cancers and Alzheimer's disease (AD). Previously, we have shown the characteristics of the thermal denaturation of Pin1. Herein, the acid-induced denaturation of Pin1 was determined by means of fluorescence emission, synchronous fluorescence, far-UV CD, ANS fluorescence and RLS spectroscopies. The fluorescence emission spectra and the synchronous fluorescence spectra suggested the partially reversible unfolding (approximately from pH 7.0 to 4.0) and refolding (approximately from pH 4.0 to 1.0) of the structures around the chromophores in Pin1, apparently with an intermediate state at about pH 4.0-4.5. The far-UV CD spectra indicated that acidic pH (below pH 4.0) induced the structural transition from α-helix and random coils to ß-sheet in Pin1. The ANS fluorescence and the RLS spectra further suggested the exposure of the hydrophobic side-chains of Pin1 and the aggregation of it especially below pH 2.3, and the aggregation possibly resulted in the formation of extra intermolecular ß-sheet. The present work primarily shows that acidic pH can induce kinds of irreversible structural changes in Pin1, such as the exposure of the hydrophobic side-chains, the transition from α-helix to ß-sheet and the aggregation of Pin1, and also explains why Pin1 loses most of its activity below pH 5.0. The results emphasize the important role of decreased pH in the pathogenesis of some Pin1-related diseases, and support the therapeutic approach for them by targeting acidosis and modifying the intracellular pH gradients.