Homoharringtonine as a PHGDH inhibitor: Unraveling metabolic dependencies and developing a potent therapeutic strategy for high-risk neuroblastoma.

Neuroblastoma, a childhood cancer affecting the sympathetic nervous system, continues to challenge the development of potent treatments due to the limited availability of druggable targets for this aggressive illness. Recent investigations have uncovered that phosphoglycerate dehydrogenase (PHGDH), an essential enzyme for de novo serine synthesis, serves as a non-oncogene dependency in high-risk neuroblastoma. In this study, we show that homoharringtonine (HHT) acts as a PHGDH inhibitor, inducing intricate alterations in cellular metabolism, and thus providing an efficient treatment for neuroblastoma. We have experimentally verified the reliance of neuroblastoma on PHGDH and employed molecular docking, thermodynamic evaluations, and X-ray crystallography techniques to determine the bond interactions between HHT and PHGDH. Administering HHT to treat neuroblastoma resulted in effective cell elimination in vitro and tumor reduction in vivo. Metabolite and functional assessments additionally disclosed that HHT treatment suppressed de novo serine synthesis, initiating intricate metabolic reconfiguration and oxidative stress in neuroblastoma. Collectively, these discoveries highlight the potential of targeting PHGDH using HHT as a potent approach for managing high-risk neuroblastoma.

Arabidopsis histone H3 lysine 9 methyltransferases KYP/SUVH5/6 are involved in leaf development by interacting with AS1-AS2 to repress KNAT1 and KNAT2.

The Arabidopsis H3K9 methyltransferases KRYPTONITE/SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG 4 (KYP/SUVH4), SUVH5 and SUVH6 are redundantly involved in silencing of transposable elements (TEs). Our recent study indicated that KYP/SUVH5/6 can directly interact with the histone deacetylase HDA6 to synergistically regulate TE expression. However, the function of KYP/SUVH5/6 in plant development is still unclear. The transcriptional factors ASYMMETRIC LEAVES1 (AS1) and AS2 form a transcription complex, which is involved in leaf development by repressing the homeobox genes KNOTTED-LIKE FROM ARABIDOPSIS THALIANA 1 (KNAT1) and KNAT2. In this study, we found that KYP and SUVH5/6 directly interact with AS1-AS2 to repress KNAT1 and KNAT2 by altering histone H3 acetylation and H3K9 dimethylation levels. In addition, KYP can directly target the promoters of KNAT1 and KNAT2, and the binding of KYP depends on AS1. Furthermore, the genome-wide occupancy profile of KYP indicated that KYP is enriched in the promoter regions of coding genes, and the binding of KYP is positively correlated with that of AS1 and HDA6. Together, these results indicate that Arabidopsis H3K9 methyltransferases KYP/SUVH5/6 are involved in leaf development by interacting with AS1-AS2 to alter histone H3 acetylation and H3K9 dimethylation from KNAT1 and KNAT2 loci.

Allopatric Lineage Divergence of the East Asian Endemic Herb Conandron ramondioides Inferred from Low-Copy Nuclear and Plastid Markers.

The evolutionary histories of ornamental plants have been receiving only limited attention. We examined the origin and divergence processes of an East Asian endemic ornamental plant, Conandron ramondioides. C. ramondioides is an understory herb occurring in primary forests, which has been grouped into two varieties. We reconstructed the evolutionary and population demography history of C. ramondioides to infer its divergence process. Nuclear and chloroplast DNA sequences were obtained from 21 Conandron populations on both sides of the East China Sea (ECS) to explore its genetic diversity, structure, and population differentiation. Interestingly, the reconstructed phylogeny indicated that the populations should be classified into three clades corresponding to geographical regions: the Japan (Honshu+Shikoku) clade, the Taiwan-Iriomote clade, and the Southeast China clade. Lineage divergence between the Japan clade and the Taiwan-Iriomote and Southeast China clades occured 1.14 MYA (95% HPD: 0.82-3.86), followed by divergence between the Taiwan-Iriomote and Southeast China clades approximately 0.75 MYA (95% HPD: 0.45-1.3). Furthermore, corolla traits (floral lobe length to tube length ratios) correlated with geographical distributions. Moreover, restricted gene flow was detected among clades. Lastly, the lack of potential dispersal routes across an exposed ECS seafloor during the last glacial maximum suggests that migration among the Conandron clades was unlikely. In summary, the extant Conandron exhibits a disjunct distribution pattern as a result of vicariance rather than long-distance dispersal. We propose that allopatric divergence has occurred in C. ramondioides since the Pleistocene. Our findings highlight the critical influence of species' biological characteristics on shaping lineage diversification of East Asian relic herb species during climate oscillations since the Quaternary.

Insight into the Phylogeny and Binding Ability of WRKY Transcription Factors.

WRKY transcription factors (TFs), which make up one of the largest families of TFs in the plant kingdom, are key players in modulating gene expression relating to embryogenesis, senescence, pathogen resistance, and abiotic stress responses. However, the phylogeny and grouping of WRKY TFs and how their binding ability is affected by the flanking regions of W-box sequences remain unclear. In this study, we reconstructed the phylogeny of WRKY across the plant kingdom and characterized the DNA-binding profile of Arabidopsis thaliana WRKY (WRKY54) based on its W-box recognition sequence. We found that WRKY TFs could be separated into five clades, and that the functional zinc-finger motif at the C-terminal of WRKY appeared after several nucleotide substitutions had occurred at the 3'-end of the zinc-finger region in chlorophytes. In addition, we found that W-box flanking regions affect the binding ability of WRKY54 based on the results of a fluorescence-based electrophoretic mobility shift assay (fEMSA) and quartz crystal microbalance (QCM) analysis. The great abundance of WRKY TFs in plants implicates their involvement in diverse molecular regulatory networks, and the flanking regions of W-box sequences may contribute to their molecular recognition mechanism. This phylogeny and our findings on the molecular recognition mechanism of WRKY TFs should be helpful for further research in this area.

Phylogenetic and Structural Analysis of NIN-Like Proteins With a Type I/II PB1 Domain That Regulates Oligomerization for Nitrate Response.

Absorption of macronutrients such as nitrogen is a critical process for land plants. There is little information available on the correlation between the root evolution of land plants and the protein regulation of nitrogen absorption and responses. NIN-like protein (NLP) transcription factors contain a Phox and Bem1 (PB1) domain, which may regulate nitrate-response genes and seem to be involved in the adaptation to growing on land in terms of plant root development. In this report, we reveal the NLP phylogeny in land plants and the origin of NLP genes that may be involved in the nitrate-signaling pathway. Our NLP phylogeny showed that duplication of NLP genes occurred before divergence of chlorophyte and land plants. Duplicated NLP genes may lost in most chlorophyte lineages. The NLP genes of bryophytes were initially monophyletic, but this was followed by divergence of lycophyte NLP genes and then angiosperm NLP genes. Among those identified NLP genes, PB1, a protein-protein interaction domain was identified across our phylogeny. To understand how protein-protein interaction mediate via PB1 domain, we examined the PB1 domain of Arabidopsis thaliana NLP7 (AtNLP7) in terms of its molecular oligomerization and function as representative. Based on the structure of the PB1 domain, determined using small-angle x-ray scattering (SAXS) and site-directed mutagenesis, we found that the NLP7 PB1 protein forms oligomers and that several key residues (K867 and D909/D911/E913/D922 in the OPCA motif) play a pivotal role in the oligomerization of NLP7 proteins. The fact that these residues are all conserved across land plant lineages means that this oligomerization may have evolved after the common ancestor of extant land plants colonized the land. It would then have rapidly become established across land-plant lineages in order to mediate protein-protein interactions in the nitrate-signaling pathway.

Inhibitor development of MTH1 via high-throughput screening with fragment based library and MTH1 substrate binding cavity.

MutT Homolog 1 (MTH1) has been proven to hydrolyze oxidized nucleotide triphosphates during DNA repair. It can prevent the incorporation of wrong nucleotides during DNA replication and mitigate cell apoptosis. In a cancer cell, abundant reactive oxygen species can lead to substantial DNA damage and DNA mutations by base-pairing mismatch. MTH1 could eliminate oxidized dNTP and prevent cancer cells from entering cell death. Therefore, inhibition of MTH1 activity is considered to be an anti-cancer therapeutic target. In this study, high-throughput screening techniques were combined with a fragment-based library containing 2,313 compounds, which were used to screen for lead compounds with MTH1 inhibitor activity. Four compounds with MTH1 inhibitor ability were selected, and compound MI0639 was found to have the highest effective inhibition. To discover the selectivity and specificity of this action, several derivatives based on the MTH1 and MI0639 complex structure were synthesized. We compared 14 complex structures of MTH1 and the various compounds in combination with enzymatic inhibition and thermodynamic analysis. Nanomolar-range IC50 inhibition abilities by enzyme kinetics and Kd values by thermodynamic analysis were obtained for two compounds, named MI1020 and MI1024. Based on structural information and compound optimization, we aim to provide a strategy for the development of MTH1 inhibitors with high selectivity and specificity.

An effective computational-screening strategy for simultaneously improving both catalytic activity and thermostability of α-l-rhamnosidase.

Catalytic efficiency and thermostability are the two most important characteristics of enzymes. However, it is always tough to improve both catalytic efficiency and thermostability of enzymes simultaneously. In the present study, a computational strategy with double-screening steps was proposed to simultaneously improve both catalysis efficiency and thermostability of enzymes; and a fungal α-l-rhamnosidase was used to validate the strategy. As the result, by molecular docking and sequence alignment analysis within the binding pocket, seven mutant candidates were predicted with better catalytic efficiency. By energy variety analysis, A355N, S356Y, and D525N among the seven mutant candidates were predicted with better thermostability. The expression and characterization results showed the mutant D525N had significant improvements in both enzyme activity and thermostability. Molecular dynamics simulations indicated that the mutations located within the 5 Å range of the catalytic domain, which could improve root mean squared deviation, electrostatic, Van der Waal interaction, and polar salvation values, and formed water bridge between the substrate and the enzyme. The study indicated that the computational strategy based on the binding energy, conservation degree and mutation energy analyses was effective to develop enzymes with better catalysis and thermostability, providing practical approach for developing industrial enzymes.

Sensitive and Specific Cadmium Biosensor Developed by Reconfiguring Metal Transport and Leveraging Natural Gene Repositories.

Whole-cell biosensors are useful for monitoring heavy metal toxicity in public health and ecosystems, but their development has been hindered by intrinsic trade-offs between sensitivity and specificity. Here, we demonstrated an effective engineering solution by building a sensitive, specific, and high-response biosensor for carcinogenic cadmium ions. We genetically programmed the metal transport system of Escherichia coli to enrich intracellular cadmium ions and deprive interfering metal species. We then selected 16 cadmium-sensing transcription factors from the GenBank database and tested their reactivity to 14 metal ions in the engineered E. coli using the expression of the green fluorescent protein as the readout. The resulting cadmium biosensor was highly specific and showed a detection limit of 3 nM, a linear increase in fluorescent intensities from 0 to 200 nM, and a maximal 777-fold signal change. Using this whole-cell biosensor, a smartphone, and low-tech equipment, we developed a simple assay capable of measuring cadmium ions at the same concentration range in irrigation water and human urine. This method is user-friendly and cost-effective, making it affordable to screen large amounts of samples for cadmium toxicity in agriculture and medicine. Moreover, our work highlights natural gene repositories as a treasure chest for bioengineering.

Structure of Arabidopsis HISTONE DEACETYLASE15.

Mammalian histone deacetylases (HDACs) undergo phosphorylation to regulate their localization, activity, and function. However, little is known about the regulation of plant HDAC function and activity by phosphorylation. Here, we report the crystal structure of the Reduced Potassium Dependency3/Histone Deacetylase1 (RPD3/HDA1) type class II histone deacetylase HDA15 in Arabidopsis (Arabidopsis thaliana). The histone deacetylase domain of HDA15 (HDA15HD) assembles as tetrameric forms with each monomer composed of 12 α-helices and 9 ß-sheets. The L1 loop and ß2 sheet of HDA15HD are the essential interfaces for the tetramer formation. The N-terminal zinc finger domain enhances HDA15HD dimerization and increases its enzymatic activity. Furthermore, HDA15 can also be phosphorylated at Ser-448 and Ser-452 in etiolated seedlings. The HDA15 phosphorylation status determines its subnuclear localization and oligomerization. Phosphomimetics of HDA15 partially disrupt its oligomerization and cause loss of enzymatic activity and translocation from the nucleolus into nucleoplasm. Together, these data indicate that phosphorylation plays a critical role in regulating the structure and function of HDA15.

Structural insights into Arabidopsis ethylene response factor 96 with an extended N-terminal binding to GCC box.

The phytohormone ethylene is widely involved in many developmental processes and is a crucial regulator of defense responses against biotic and abiotic stresses in plants. Ethylene-responsive element binding protein, a member of the APETALA2/ethylene response factor (AP2/ERF) superfamily, is a transcription factor that regulates stress-responsive genes by recognizing a specific cis-acting element of target DNA. A previous study showed only the NMR structure of the AP2/ERF domain of AtERF100 in complex with a GCC box DNA motif. In this report, we determined the crystal structure of AtERF96 in complex with a GCC box at atomic resolution. We analyzed the binding residues of the conserved AP2/ERF domain in the DNA recognition sequence. In addition to the AP2/ERF domain, an N-terminal α-helix of AtERF96 participates in DNA interaction in the flanking region. We also demonstrated the structure of AtERF96 EDLL motif, a unique conserved motif in the group IX of AP2/ERF family, might involve in the transactivation of defense-related genes. Our study establishes the structural basis of the AtERF96 transcription factor in complex with the GCC box, as well as the DNA binding mechanisms of the N-terminal α-helix and AP2/ERF domain.

Citrus Taste Modification Potentials by Genetic Engineering.

Citrus fruits are mainly consumed as fresh fruit and processed juice products. They serve as nutritional and a tasty diet in our daily life. However, the formidable bitterness and delayed bitterness significantly impact the citrus industry attributable to the two major bitter compounds naringin and limonin. The extremely sour and acidic also negatively affects the sensory quality of citrus products. Citrus breeding programs have developed different strategies to improve citrus quality and a wealth of studies have aimed to uncover the genetic and biochemical basis of citrus flavor. In this minireview, we outline the major genes characterized to be involved in pathways shaping the sweet, bitter, or sour taste in citrus, and discuss briefly about the possible approaches to modify citrus taste by genetic engineering.

Heterologous overexpression, purification and functional analysis of plant cellulose synthase from green bamboo.

BACKGROUND: The cellulose synthase complex (CSC), composed of cellulose synthase (CesA) proteins, is a catalytic enzyme complex involved in cellulose synthesis in the plant cell. CesA proteins synthesize cellulose microfibrils corresponding to the microtubule direction and export linear products across the plasma membrane. However, the CSC arrangement and the mechanism of cellulose synthesis in plant cells remain unclear. Purified CesA proteins are required to determine biochemical and biophysical characteristics. RESULTS: In this study, we constructed, expressed, and purified six heterologously expressed cellulose synthases from Bambusa oldhamii (BoCesA) and analyzed the associated enzyme activity. The conjugating sequences of the maltose-binding protein (MBP) gene and the BoCesA genes were constructed into the expression vector pYES2/CT and were further transformed into yeast cells (BCY123) for fermentation culturing. Purified BoCesA recombinant proteins were obtained by a two-step purification procedure, consisting of immobilized metal affinity chromatography to purify MBP-BoCesAs and size-exclusion chromatography (Superdex-200) to isolate BoCesAs in oligomeric form. The enzymatic activity of oligomeric BoCesAs with 80% purity was determined by partially methylated alditol acetate (PMAA)-coupled gas chromatography-mass spectrometry (GC-MS) analysis. Furthermore, the long fiber-like products synthesized by oligomeric BoCesAs were observed under a transmission electron microscope (TEM) and were further confirmed as cellulose microfibril products. CONCLUSIONS: In this study, we successfully established a heterologous expression and purification system for BoCesAs. The purified recombinant BoCesA proteins display enzyme activity and can produce protein in milligram quantities for further studies on molecular composition and structure.

Heterologous Expression and Characterization of a New Clade of Aspergillus α-L-Rhamnosidase Suitable for Citrus Juice Processing.

α-L-Rhamnosidase is a glycoside hydrolase capable of removing naringin from citrus juice. However, α-L-rhamnosidases always have broad substrate spectra, causing negative effects on citrus juice. In this study, a α-L-rhamnosidase-expressing fungal strain, JMU-TS529, was identified, and its α-L-rhamnosidase was characterized. As a result, JMU-TS529 was identified as Aspergillus tubingensis via morphological and molecular characteristics. The predicted protein sequence shared an amino acid identity of less than 30% with previously characterized α-L-rhamnosidases. The optimal pH and temperature were 4.0 and 50-60 °C, respectively. Most importantly, the α-L-rhamnosidase showed a strong ability to hydrolyze naringin but scarcely acted on other substrates. Furthermore, the enzyme could efficiently remove naringin from pomelo juice without changing its attractive aroma. These results indicate that the present enzyme represents a new clade of Aspergillus α-L-rhamnosidase that is desirable for debittering citrus juice, providing a better alternative for improving the quality of citrus juice.

Quinazolin-2,4-dione-Based Hydroxamic Acids as Selective Histone Deacetylase-6 Inhibitors for Treatment of Non-Small Cell Lung Cancer.

We designed and synthesized quinazolin-2,4-dione-based hydroxamic acids to serve as selective competitive inhibitors of histone deacetylase-6 (HDAC6). The most potent and selective compound, 3d (IC50, 4 nM, HDAC6; IC50 > 10 µM, HDAC1), substantially increased acetylation of α-tubulin instead of histones in the lung cancer cell line, LL2. Paclitaxel in combination with 3d had a synergistic anticancer effect on reduction of programmed death-ligand 1 expression in LL/2 cells. When given orally, 3d was mainly found to locate in the liver and lungs, at a concentration 18- to 70-fold greater, respectively, than in plasma. As an orally active HDAC6 inhibitor, 3d (20 mg/kg) potentiated paclitaxel antitumor activity (percentage tumor growth inhibition, 67.5%) in a xenograft syngeneic non-small cell lung cancer mouse model.

A highly HDAC6-selective inhibitor acts as a fluorescent probe.

HDAC6 receives great attention because of its therapeutic potential for the treatment of various diseases. Selective fluorescence imaging for HDAC6 is important for its pathological and biological studies. However, specific detection of HDAC6 by using a fluorescent small molecule probe remains a great challenge. Herein, a series of fluorescent HDAC6-selective inhibitors incorporating a naphthalimide skeleton were designed and synthesized. A structure-activity relationship study identified that compound JW-1 had the greatest inhibitory activity and superior specificity against HDAC6. JW-1 could substantially increase α-tubulin acetylation and was active against a panel of six cancer cell lines. Photophysical characterization and cellular imaging of MDA-MB-231 cells demonstrated that JW-1 is a highly fluorescent, cell penetrable, small-molecule inhibitor of HDAC6 that can be used for the detection of HDAC6 in complex cellular environments.

HISTONE DEACETYLASE6 Acts in Concert with Histone Methyltransferases SUVH4, SUVH5, and SUVH6 to Regulate Transposon Silencing.

Histone deacetylases (HDACs) play important roles in regulating gene expression. In yeast and animals, HDACs act as components of multiprotein complexes that modulate transcription during various biological processes. However, little is known about the interacting proteins of plant HDACs. To identify the plant HDAC complexes and interacting proteins, we developed an optimized workflow using immunopurification coupled to mass spectrometry-based proteomics in Arabidopsis thaliana We found that the histone deacetylase HDA6 can interact with the histone methyltransferases SUVH4, SUVH5, and SUVH6 (SUVH4/5/6). Domain analysis revealed that the C-terminal regions of HDA6 and SUVH5 are important for their interaction. Furthermore, HDA6 interacts with SUVH4/5/6 and coregulates a subset of transposons through histone H3K9 methylation and H3 deacetylation. In addition, two phosphorylated serine residues, S427 and S429, were unambiguously identified in the C-terminal region of HDA6. Phosphomimetics (amino acid substitutions that mimic a phosphorylated protein) of HDA6 resulted in increased enzymatic activity, whereas the mutation of S427 to alanine in HDA6 abolished its interaction with SUVH5 and SUVH6, suggesting that the phosphorylation of HDA6 is important for its activity and function.

Structural basis of jasmonate-amido synthetase FIN219 in complex with glutathione S-transferase FIP1 during the JA signal regulation.

Far-red (FR) light-coupled jasmonate (JA) signaling is necessary for plant defense and development. FR insensitive 219 (FIN219) is a member of the Gretchen Hagen 3 (GH3) family of proteins in Arabidopsis and belongs to the adenylate-forming family of enzymes. It directly controls biosynthesis of jasmonoyl-isoleucine in JA-mediated defense responses and interacts with FIN219-interacting protein 1 (FIP1) under FR light conditions. FIN219 and FIP1 are involved in FR light signaling and are regulators of the interplay between light and JA signaling. However, how their interactions affect plant physiological functions remains unclear. Here, we demonstrate the crystal structures of FIN219-FIP1 while binding with substrates at atomic resolution. Our results show an unexpected FIN219 conformation and demonstrate various differences between this protein and other members of the GH3 family. We show that the rotated C-terminal domain of FIN219 alters ATP binding and the core structure of the active site. We further demonstrate that this unique FIN219-FIP1 structure is crucial for increasing FIN219 activity and determines the priority of substrate binding. We suggest that the increased FIN219 activity resulting from the complex form, a conformation for domain switching, allows FIN219 to switch to its high-affinity mode and thereby enhances JA signaling under continuous FR light conditions.

Expression patterns and structural modelling of Hsp70 and Hsp90 in a fish-borne zoonotic nematode Anisakis pegreffii.

Heat shock proteins (HSPs) are essential molecular chaperones that are highly conserved across organisms. They have a pivotal function in responding to thermal stress and are responsible for many cellular functions. Here, we aimed to elucidate the possible roles of Hsp70 and Hsp90 in the life cycle of the parasitic nematode Anisakis, particularly third- and fourth-stage larvae, from cold-blooded fish to warm-blooded marine mammals or accidentally to human hosts. We examined the expression profiles of Hsp70 and Hsp90 in different developmental stages of Anisakis pegreffii. The open reading frame of Hsp70 of A. pegreffii was 1950 bp, and deduced amino acid sequence showed high homology with those of other nematodes. Heatmap analysis revealed sequence identity of Hsp70 and Hsp90 in 13 important parasitic species, human and yeast. On heatmap and phylogenetic analysis, ApHsp70 and ApHsp90 shared the highest amino acid sequence identity with other nematodes and formed a monophyletic clade. The three-dimensional (3D) structure prediction of the newly characterized ApHsp70 and known ApHsp90 gene showed highly conserved motifs between A. pegreffii and other species. Quantitative real-time PCR and western blot analysis revealed higher mRNA and protein expression for ApHsp70 and ApHsp90 in fourth- than third-stage larvae, with higher mRNA and protein expression for ApHsp70 than ApHsp90. ApHsp70 and ApHsp90 may play important roles in Anisakis in response to thermal stress and might be important molecules in the development of A. pegreffii, which has implications for its control.

Effect of different temperatures on the expression of the newly characterized heat shock protein 90 (Hsp90) in L3 of Anisakis spp. isolated from Scomber australasicus.

Anisakid nematodes are distributed worldwide in a wide variety of marine fishes and they are known to cause the zoonotic disease, anisakiasis. The temperature control is commonly applied for prevention and control of anisakiasis. To analyze the cellular response to temperature stress in Anisakis, the heat shock protein 90 (Hsp90) was chosen in the present study, as it plays a key role in many cellular processes and responds to stress conditions such as heat or cold shock. Anisakids were sampled from spotted mackerel Scomber australasicus caught from the coastal waters of Yilan, in northeastern Taiwan (25 °N, 121 °E). Anisakid nematodes were pre-identified morphologically and later molecularly by PCR-RFLP. In total, we obtained six species of the genus Anisakis, A. typica, A. pegreffii, A. paggiae, A. brevispiculata, A. physeteris, and a recombinant genotype between A. pegreffii and A. simplex sensu stricto. Thereby we provide new host and locality records for A. paggiae, A. brevispiculata and A. physeteris. The Hsp90 genes of five species (except the recombinant genotype) were cloned by rapid amplification of cDNA ends (RACE) and their deduced amino acid sequences were further characterized. Quantitative real-time PCR and Western blot analysis were used to examine the expression levels of the Hsp90 in A. pegreffii under different temperature conditions. Quantitative RT-PCR showed that Hsp90 transcript levels increased slightly under heat shock (50 °C) treatment, and increased gradually during the first 3h, and thereafter, returned to its baseline value at 37 °C. Under cold shock (4 °C) treatment, the mRNA expression of Hsp90 did not change significantly. In addition, we found a clear time-dependent Hsp90 protein expression pattern of A. pegreffii exposed to high temperature. Our results suggest that the mRNA and protein expression patterns of Hsp90 are related to the temperature, and are especially significantly increased under heat stress.

Combining secondary-structure and protein solvent-accessibility predictions in methionine substitution for anomalous dispersion.

In X-ray crystallographic analysis, the single-wavelength and multi-wavelength anomalous diffraction (SAD and MAD) methods have been widely used in order to solve the phase problem. Selenium-labelled methionine has been shown to be very effective for anomalous dispersion phasing, and at least one selenomethionine is required for every 100 amino acids. Some proteins, such as the Arabidopsis thaliana thylakoid lumen protein AtTLP18.3, can be overexpressed in an Escherichia coli system and high-quality protein crystals can be obtained. However, AtTLP18.3 contains no methionine residues, and site-directed mutagenesis was required in order to introduce methionine residues into the protein. A criterion for the mutated residues is that they should avoid affecting the structure and function. In this study, several leucine and isoleucine residues were selected for methionine substitution by combining secondary-structure and solvent-accessibility predictions. From the secondary-structure prediction, mutated residues were first determined in the coil or loop regions at the junction of two secondary structures. Since leucine and isoleucine residues are hydrophobic and are normally buried within the protein core, these residues should have a higher solvent-accessibility prediction so that they would be partially buried or exposed in the protein. In addition, five residues (Leu107, Leu202, Ile133, Leu128 and Ile159) of AtTLP18.3 were mutated to methionine residues. After overexpression and purification, only two single-mutant lines, L128M and I159M, could be crystallized. Finally, a double-mutation line of truncated AtTLP18.3 with L128M and I159M mutations was constructed. The structure of the double mutant AtTLP18.3 protein was resolved using the single-wavelength anomalous diffraction method at 2.6 Šresolution. The results indicated that a combination of secondary-structure and solvent-accessibility prediction for methionine substitution is a useful method in SAD and MAD phasing.