Peptide Scaffold-Based Discovery of Nonpeptide Natural Medicines to Target PI3K p85 SH2 Domain

Targeting phosphoinositide 3-kinase (PI3K) has been recognized as an attractive strategy for anticancer therapy. The PI3K is a heterodimer composed of a catalytic subunit p110 and a regulatory subunit p85. Here, instead of targeting the catalytic p110 that has been considered previously, we purposed targeting the peptide-recognition domain SH2 of regulatory p85 with natural medicines obtained by using a peptide scaffold-based screening scheme. In the procedure, a core binding motif was extracted from the cocrystallized complex of a cognate phosphopeptide with the domain, which was considered as basic scaffold to perform high-through virtual screening against a structurally diverse, nonredundant library of natural products. A number of hit compounds with high binding potency to the domain and significant conformational similarity with the peptide scaffold were identified; in vitro affinity assay confirmed that five hits have moderate or high affinity for the domain with measured dissociation constants K_d range between 25 and 360 μM, which are comparable to or even better than that of the cognate phosphopeptide SDpYMNMTP and its core motif peptide pYMNM (K_d?=?15 and 32 μM, respectively). Structural analysis and nonbonded comparison of SH2 interactions with phosphopeptides and potent hit compounds revealed that only negatively charged phosphate and, sometime, sulfate can confer domain-binding capability to small-molecule compounds, but carboxylate cannot. A similar binding mode of compounds with phosphopeptide is important for the compounds to have high affinity and specificity.     

Rice stripe virus coat protein induces the accumulation of jasmonic acid,activating plant defence against the virus while also attracting its vector to feed

The jasmonic acid (JA) pathway plays crucial roles in plant defence against pathogens and herbivores. Rice stripe virus (RSV) is the type member of the genus Tenuivirus. It is transmitted by the small brown planthopper (SBPH) and causes damaging epidemics in East Asia. The role(s) that JA may play in the tripartite interaction against RSV, its host, and vector are poorly understood. Here, we found that the JA pathway was induced by RSV infection and played a defence role against RSV. The coat protein (CP) was the major viral component responsible for inducing the JA pathway. Methyl jasmonate treatment attracted SBPHs to feed on rice plants while a JA-deficient mutant was less attractive than wild-type rice. SBPHs showed an obvious preference for feeding on transgenic rice lines expressing RSV CP. Our results demonstrate that CP is an inducer of the JA pathway that activates plant defence against RSV while also attracting SBPHs to feed and benefitting viral transmission. This is the first report of the function of JA in the tripartite interaction between RSV, its host, and its vector.     

Physiological and genetic convergence supports hypoxia resistance in high-altitude songbirds

Skeletal muscle plays a central role in regulating glucose uptake and body metabolism; however, highland hypoxia is a severe challenge to aerobic metabolism in small endotherms. Therefore, understanding the physiological and genetic convergence of muscle hypoxia tolerance has a potential broad range of medical implications. Here we report and experimentally validate a common physiological mechanism across multiple high-altitude songbirds that improvement in insulin sensitivity contributes to glucose homeostasis, low oxygen consumption, and relative activity, and thus increases body weight. By contrast, low-altitude songbirds exhibit muscle loss, glucose intolerance, and increase energy expenditures under hypoxia. This adaptive mechanism is attributable to convergent missense mutations in the BNIP3L gene, and METTL8 gene that activates MEF2C expression in highlanders, which in turn increases hypoxia tolerance. Together, our findings from wild high-altitude songbirds suggest convergent physiological and genetic mechanisms of skeletal muscle in hypoxia resistance, which highlights the potentially medical implications of hypoxia-related metabolic diseases.     

Comparative study on the replication of HCV1b genome between wild-type and cell culture-adaptive mutant in regard to sensitivities against anti-HCV drugs

The replicon system, which mimics viral genome replication in culture cells, has been widely used to analyze the genome replication of the hepatitis C virus (HCV). However, most HCV genomes used in the system include adaptive mutations (AMs) that are vital for replication in culture cells despite the nonexistence of such mutations in the genome of wild-type (WT) HCV in patients. In order to study the genome replications of WT HCV, new HCV subgenomic replicon (SGR) systems were established using Huh-7.5-derived cells producing Sec14-like protein 2 constitutively and SGR of KT9 (one of the HCV genotype 1b clones) with WT genome (SGR KT9WT) in this study. The replication efficiency and sensitivities of SGR KT9WT to anti-HCV drugs in the cloned cells permanently bearing replicon RNA, HS55-4 cells, were similar to those of reports using SGR, including AM. The SGR transient transfection system using SGR KT9WT and SGR KT9AM encoding secreted Nano-luciferase and HS55-4C cells established by the elimination of SGR KT9 RNA from HS55-4 cells, however, showed that the replication efficiency of SGR KT9WT was much lower than that of SGR KT9AM under a same condition. Furthermore, the sensitivities of SGR KT9WT to almost all tested anti-HCV reagents, except the inhibitor of miR-122, a cellular factor important for HCV replication, were quite low compared with SGR KT9AM. These results suggested that the new replicon systems might not only provide information about precise responses against new anti-HCV drugs but also reveal novel molecular mechanisms supporting negligent proliferation of HCV.     

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple‐mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two‐orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross‐reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr‐containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.     

Effects of stocking density during live transportation on haematological parameters of Siberian sturgeon (Acipenser baerii,Brandt, 1869)

In this study, we investigated the effects of different stocking densities water quality and blood parameters during the transportation of the Siberian sturgeon (Acipenser baerii). Experiments were carried out in high-density polyethylene tank with three different stocking densities (50, 100 and 150 kg/m³) for 20 h. Ammonium nitrogen (NH₄-N) and nitrite nitrogen (NO₂-N) were measured in the water samples. The erythrocyte, leucocyte, haematocrit (HCT), haemoglobin (Hb), cortisol, and sodium ion (Na⁺) were measured in blood samples. Ammonium nitrogen and nitrite nitrogen concentrations in the water have increased in parallel with the stocking density depending on the time. In the highest stocking density, the maximum levels of NH₄-N and NO₂-N at the 16^th h reached 2.64 mg/L and 4.74 mg/L, respectively. Erythrocyte, leucocyte, HCT, and Hb values did not differ significantly between the experimental groups (p > .05). The results showed that fish could be transported safely for 20 h in 15℃ water temperature at 50 kg/m³ stocking density; however, stocking density of 100 kg/m³ and over could threaten fish welfare and health as from 16^th h.     

Histone H3K27 dimethylation landscapes contribute to genome stability and genetic recombination during wheat polyploidization

Bread wheat (Triticum aestivum) is an allohexaploid that was formed via two allopolyploidization events. Growing evidence suggests histone modifications are involved in the response to ‘genomic shock’ and environmental adaptation during polyploid formation and evolution. However, the role of histone modifications, especially histone H3 lysine-27 dimethylation (H3K27me2), in genome evolution remains elusive. Here we analyzed H3K27me2 and H3K27me3 profiles in hexaploid wheat and its tetraploid and diploid relatives. Although H3K27me3 levels were relatively stable among wheat species with different ploidy levels, H3K27me2 intensities increased concurrent with increased ploidy levels, and H3K27me2 peaks were colocalized with massively amplified DTC transposons (CACTA family) in euchromatin, which may silence euchromatic transposons to maintain genome stability during polyploid wheat evolution. Consistently, the distribution of H3K27me2 is mutually exclusive with another repressive histone mark, H3K9me2, that mainly silences transposons in heterochromatic regions. Remarkably, the regions with low H3K27me2 levels (named H3K27me2 valleys) were associated with the formation of DNA double-strand breaks in genomes of wheat, maize (Zea mays) and Arabidopsis. Our results provide a comprehensive view of H3K27me2 and H3K27me3 distributions during wheat evolution, which support roles for H3K27me2 in silencing euchromatic transposons to maintain genome stability and in modifying genetic recombination landscapes. These genomic insights may empower breeding improvement of crops.