Genome-Wide Analysis and Expression Profiling of the Glutathione Peroxidase-like Enzyme Gene Family in Solanum tuberosum.

Glutathione peroxidase-like enzyme is an important enzymatic antioxidant in plants. It is involved in scavenging reactive oxygen species, which can effectively prevent oxidative damage and improve resistance. GPXL has been studied in many plants but has not been reported in potatoes, the world's fourth-largest food crop. This study identified eight StGPXL genes in potatoes for the first time through genome-wide bioinformatics analysis and further studied the expression patterns of these genes using qRT-PCR. The results showed that the expression of StGPXL1 was significantly upregulated under high-temperature stress, indicating its involvement in potato defense against high-temperature stress, while the expression levels of StGPXL4 and StGPXL5 were significantly downregulated. The expression of StGPXL1, StGPXL2, StGPXL3, and StGPXL6 was significantly upregulated under drought stress, indicating their involvement in potato defense against drought stress. After MeJA hormone treatment, the expression level of StGPXL6 was significantly upregulated, indicating its involvement in the chemical defense mechanism of potatoes. The expression of all StGPXL genes is inhibited under biotic stress, which indicates that GPXL is a multifunctional gene family, which may endow plants with resistance to various stresses. This study will help deepen the understanding of the function of the potato GPXL gene family, provide comprehensive information for the further analysis of the molecular function of the potato GPXL gene family as well as a theoretical basis for potato molecular breeding.

Novel Arginine End-Tagging Antimicrobial Peptides to Combat Multidrug-Resistant Bacteria.

The emergence of multidrug-resistant microorganisms has been termed one of the most common global health threats, emphasizing the discovery of new antibacterial agents. To address this issue, we engineered peptides harboring "RWWWR" as a central motif plus arginine (R) end-tagging and then tested them in vitro and in vivo. Our results demonstrate that Pep 6, one of the engineered peptides, shows great potential in combating Escherichia coli bacteremia and the Staphylococcus aureus skin burn infection model, which induces a 62-90% reduction in bacterial burden. Remarkably, after long serial passages of S. aureus and E. coli for 30 days, Pep 6 is still highly efficient in killing pathogens, compared with 64- and 128-fold increase in minimal inhibitory concentrations (MICs) for vancomycin and polymyxin B, respectively. We also found that Pep 6 exhibited robust biofilm-inhibiting activity and eliminated 61.33% of the mature methicillin-resistant Staphylococcus aureus (MRSA) biofilm with concentration in the MIC level. These results suggest that the RWWWR motif and binding of arginine end-tagging could be harnessed as a new agent for combating multidrug-resistant bacteria.

Screening of Severe Acute Respiratory Syndrome Coronavirus 2 RNA-Dependent RNA Polymerase Inhibitors Using Computational Approach.

The detrimental effect of coronavirus disease 2019 (COVID-19) pandemic has manifested itself as a global crisis. Currently, no specific treatment options are available for COVID-19, so therapeutic interventions to tackle the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection must be urgently established. Therefore, cohesive and multidimensional efforts are required to identify new therapies or investigate the efficacy of small molecules and existing drugs against SARS-CoV-2. Since the RNA-dependent RNA Polymerase (RdRP) of SARS-CoV-2 is a promising therapeutic target, this study addresses the identification of antiviral molecules that can specifically target SARS-CoV-2 RdRP. The computational approach of drug development was used to screen the antiviral molecules from two antiviral libraries (Life Chemicals [LC] and ASINEX) against RdRP. Here, we report six antiviral molecules (F3407-4105, F6523-2250, F6559-0746 from LC and BDG 33693278, BDG 33693315, LAS 34156196 from ASINEX), which show substantial interactions with key amino acid residues of the active site of SARS-CoV-2 RdRP and exhibit higher binding affinity (>7.5 kcalmol-1) than Galidesivir, an Food and Drug Administration-approved inhibitor of the same. Further, molecular dynamics simulation and Molecular Mechanics Poisson-Boltzmann Surface Area results confirmed that identified molecules with RdRP formed higher stable RdRP-inhibitor(s) complex than RdRP-Galidesvir complex. Our findings suggest that these molecules could be potential inhibitors of SARS-CoV-2 RdRP. However, further in vitro and preclinical experiments would be required to validate these potential inhibitors of SARS-CoV-2 protein.

Genome-wide analysis of the NF-Y gene family and their roles in relation to fruit development in Tartary buckwheat (Fagopyrum tataricum).

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor playing crucial roles in various biological process in plant. However, thorough research on NF-Y gene family of Tartary buckwheat (Fagopyrum tataricum) is little. In this study, 38 FtNF-Y genes (12 FtNF-YAs, 17 FtNF-YBs, and 9 FtNF-YCs) were identified and renamed on the basis of their subfamily and chromosomal location. Their gene structure, genomic mapping, motif composition, conserved domain, phylogenetic relationships, cis-acting elements and gene expression were investigated. Illustration of gene structures and conserved domains of FtNF-Ys revealed their functional conservation and specificity. Construction of phylogenetic trees of NF-Ys in Tartary buckwheat, Arabidopsis, tomato, rice and banana, allowed us to predict functional similarities among NF-Ys from different species. Gene expression analysis displayed that twenty-four FtNF-Ys were expressed in all the tissues and the transcript levels of them were different, suggesting their function varieties. Moreover, expression profiles of twenty FtNF-Ys along five different fruit development stages acquired by real-time quantitative PCR (RT-qPCR) demonstrated distinct abundance diversity at different stages, providing some clues of potential fruit development regulators. Our study could provide helpful reference information for further function characterization of FtNF-Ys and for the fruit quality enhancement of Tartary buckwheat.

Targeted mutagenesis of CYP76AK2 and CYP76AK3 in Salvia miltiorrhiza reveals their roles in tanshinones biosynthetic pathway.

Salvia miltiorrhiza Bunge, belonging to Lamiaceae family, is one of the most important Chinese medicinal herbs. The dried roots, also called Danshen in Chinese, are usually used in the formula of Chinese traditional medicine due to the bioactive constituents known as phenolic acids and tanshinones, which are a group of abietane nor-diterpenoid quinone natural products. Cytochrome P450 enzymes (CYPs) usually play crucial roles in terpenoids synthesis, especially in hydroxylation processes. Up to now, several important P450 enzymes, such as CYP76AH1, CYP76AH3, CYP76AK1, CYP71D373, and CYP71D375, have been functionally characterized in the tanshinones biosynthetic pathway. Nevertheless, the tanshinones biosynthesis is a so complex network that more P450 enzymes should be identified and characterized. Here, we report two novel P450 enzymes CYP76AK2 and CYP76AK3 that are involved in tanshinones biosynthetic pathway. These two P450 enzymes were highly homologous to previously reported CYP76AK1 and showed the same expression profile as CYP76AK1. Also, CYP76AK2 and CYP76AK3 could be stimulated by MeJA and SA, resulting in increased expression. We used a triple-target CRISPR/Cas9 system to generate targeted mutagenesis of CYP76AK2 and CYP76AK3 in S. miltiorrhiza. The content of five major tanshinones was significantly reduced in both cyp76ak2 and cyp76ak3 mutants, indicating that the two enzymes might be involved in the biosynthesis of tanshinones. This study would provide a foundation for the catalytic function identification of CYP76AK2 and CYP76AK3, and further enrich the understanding of the network of tanshinones secondary metabolism synthesis as well.

Virus and helper component interactions favour the transmission of recombinant potato virus Y strains.

In recent years, several recombinant strains of potato virus Y, notably PVYNTN and PVYN:O have displaced the ordinary strain, PVYO, and emerged as the predominant strains affecting the USA potato crop. Previously we reported that recombinant strains were transmitted more efficiently than PVYO when they were acquired sequentially, regardless of acquisition order. In another recent study, we showed that PVYNTN binds preferentially to the aphid stylet over PVYO when aphids feed on a mixture of PVYO and PVYNTN. To understand the mechanism of this transmission bias as well as preferential virus binding, we separated virus and active helper component proteins (HC), mixed them in homologous and heterologous combinations, and then fed them to aphids using Parafilm sachets. Mixtures of PVYO HC with either PVYN:O or PVYNTN resulted in efficient transmission. PVYN:O HC also facilitated the transmission of PVYO and PVYNTN, albeit with reduced efficiency. PVYNTN HC failed to facilitate transmission of either PVYO or PVYN:O. When PVYO HC or PVYN:O HC was mixed with equal amounts of the two viruses, both viruses in all combinations were transmitted at high efficiencies. In contrast, no transmission occurred when combinations of viruses were mixed with PVYNTN HC. Further study evaluated transmission using serial dilutions of purified virus mixed with HCs. While PVYNTN HC only facilitated the transmission of the homologous virus, the HCs of PVYO and PVYN:O facilitated the transmission of all strains tested. This phenomenon has likely contributed to the increase in the recombinant strains affecting the USA potato crop.

Discovery of Hyperstable Noncanonical Plant-Derived Epidermal Growth Factor Receptor Agonist and Analogs.

Here, we report the discovery of the first plant-derived and noncanonical epidermal growth factor receptor (EGFR) agonist, the 36-residue bleogen pB1 from Pereskia bleo of the Cactaceae family. We show that bleogen pB1 is a low-affinity EGFR agonist using a suite of chemical, biochemical, cellular, and animal experiments which include incisor eruption and wound-healing mouse models. A focused positional scanning pB1 library of Ala- and d-amino acid scans yielded a high-affinity pB1 analog, [K29k]pB1, with a 60-fold-improved EGFR affinity and mitogenicity. We show that the potency of [K29k]pB1 and the epidermal growth factor (EGF) is comparable in a diabetic mouse wound-healing model. We also show that both bleogen pB1 and [K29k]pB1 are hyperstable, being >100-fold more stable than EGF against proteolytic degradation. Overall, our discovery of a noncanonical proteolytic-resistant EGFR agonist scaffold could open new avenues for developing wound healing and skin regeneration therapeutics and biomaterials.

Ralstonia solanacearum type III effector RipV2 encoding a novel E3 ubiquitin ligase (NEL) is required for full virulence by suppressing plant PAMP-triggered immunity.

Ralstonia solanacearum causes bacterial wilt disease in a broad range of plants, primarily through type Ⅲ secreted effectors. However, the R. solanacearum effectors promoting susceptibility in host plants remain limited. In this study, we determined that the R. solanacearum effector RipV2 functions as a novel E3 ubiquitin ligase (NEL). RipV2 was observed to be locali in the plasma membrane after translocatio into plant cells. Transient expression of RipV2 in Nicotiana benthamiana could induce cell death and suppress the flg22-induced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, mediating such effects as attenuation of the expression of several PTI-related genes and ROS bursts. Furthermore, we demonstrated that the conserved catalytic residue is highly important for RipV2. Transient expression of the E3 ubiquitin ligase catalytic mutant RipV2 C403A alleviated the PTI suppression ability and cell death induction, indicating that RipV2 requires its E3 ubiquitin ligase activity for its role in plant-microbe interactions. More importantly, mutation of RipV2 in R. solanacearum reduces the virulence of R. solanacearum on potato. In conclusion, we identified a NEL effector that is required for full virulence of R. solanacearum by suppressing plant PTI.

Identification and expression analysis of the WRKY gene family in Isatis indigotica.

The WRKY proteins, which represent one of the largest families of transcriptional regulators in plants, play pivotal roles in regulating multiple processes of growth and development, particularly in diverse stress responses. Isatis indigotica is widely used in Traditional Chinese Medicine and is famous for its use as a dye for the color indigo. However, reports of the WRKY gene family in I. indigotica are limited. In this study, 64 IiWRKY genes encoding proteins with the complete WRKY domain were identified from genome of I. indigotica. Based on their structure and phylogenetic relationships of this gene family in I. indigotica, the IiWRKY genes were classified into three groups: Group I (n = 13), Group II (n = 35) and Group III (n = 16). Sequence alignment revealed that IiWRKY proteins harbored two variants, WRKYRQK and WRKYGKK, of the highly conserved WRKYGQK motif. The number of exons in IiWRKY genes varied from two to 14, with most of IiWRKY genes containing three exons. Investigation of gene duplication demonstrated that 10 and 14 IiWRKY genes were incorporated in tandem and segmental duplication events, respectively. Finally, the expression profiles derived from transcriptome data and quantitative real-time PCR analysis showed distinct expression patterns of these IiWRKY gene in five different organs or in response to four abiotic stresses. Taken together, our results will contribute to functional analysis of IiWRKY genes, and also provide a basis for further clarification of the molecular mechanism of stress responses in this important herb.

Ush regulates hemocyte-specific gene expression, fatty acid metabolism and cell cycle progression and cooperates with dNuRD to orchestrate hematopoiesis.

The generation of lineage-specific gene expression programmes that alter proliferation capacity, metabolic profile and cell type-specific functions during differentiation from multipotent stem cells to specialised cell types is crucial for development. During differentiation gene expression programmes are dynamically modulated by a complex interplay between sequence-specific transcription factors, associated cofactors and epigenetic regulators. Here, we study U-shaped (Ush), a multi-zinc finger protein that maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches we reveal that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. We employ complementary biochemical approaches to characterise the molecular mechanisms of Ush-mediated gene regulation. We uncover distinct Ush isoforms one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, we use genetic assays to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. Our findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.

Truncated YY1 interacts with BASP1 through a 339KLK341 motif in YY1 and suppresses vascular smooth muscle cell growth and intimal hyperplasia after vascular injury.

AIMS: In-stent restenosis and late stent thrombosis are complications associated with the use of metallic and drug-coated stents. Strategies that inhibit vascular smooth muscle cell (SMC) proliferation without affecting endothelial cell (EC) growth would be helpful in reducing complications arising from percutaneous interventions. SMC hyperplasia is also a pathologic feature of graft stenosis and fistula failure. Our group previously showed that forced expression of the injury-inducible zinc finger (ZNF) transcription factor, yin yang-1 (YY1), comprising 414 residues inhibits neointima formation in carotid arteries of rabbits and rats. YY1 inhibits SMC proliferation without affecting EC growth in vitro. Identifying a shorter version of YY1 retaining cell-selective inhibition would make it more amenable for potential use as a gene therapeutic agent. METHODS AND RESULTS: We dissected YY1 into a range of shorter fragments (YY1A-D, YY1Δ) and found that the first two ZNFs in YY1 (construct YY1B, spanning 52 residues) repressed SMC proliferation. Receptor binding domain analysis predicts a three-residue (339KLK341) interaction domain. Mutation of 339KLK341 to 339AAA341 in YY1B (called YY1Bm) abrogated YY1B's ability to inhibit SMC but not EC proliferation and migration. Incubation of recombinant GST-YY1B and GST-YY1Bm with SMC lysates followed by precipitation with glutathione-agarose beads and mass spectrometric analysis identified a novel interaction between YY1B and BASP1. Overexpression of BASP1, like YY1, inhibited SMC but not EC proliferation and migration. BASP1 siRNA partially rescued SMC from growth inhibition by YY1B. In the rat carotid balloon injury model, adenoviral overexpression of YY1B, like full-length YY1, reduced neointima formation, whereas YY1Bm had no such effect. CD31+ immunostaining suggested YY1B could increase re-endothelialization in a 339KLK341-dependent manner. CONCLUSION: These studies identify a truncated form of YY1 (YY1B) that can interact with BASP1 and inhibit SMC proliferation, migration, and intimal hyperplasia after balloon injury of rat carotid arteries as effectively as full length YY1. We demonstrate the therapeutic potential of YY1B in vascular proliferative disease.

Buckwheat FeNramp5 Mediates High Manganese Uptake in Roots.

Manganese (Mn) is an essential element for plant growth and development, but transporters required for Mn uptake have only been identified in a few plant species. Here, we functionally characterized a member of the natural resistance-associated macrophage proteins (Nramps) family, FeNramp5 in buckwheat (Fagopyrum esculentum Moench), which is known as a species well adapted to acidic soils. FeNramp5 was mainly expressed in the roots, and its expression was upregulated by the deficiency of Mn and Fe. Furthermore, spatial and tissue-specific expression analysis showed that FeNramp5 was expressed in all tissues of the basal root regions. FeNramp5-GFP protein was localized to the plasma membrane when transiently expressed in buckwheat leaf protoplast. FeNramp5 showed the transport activity for Mn2+ and Cd2+ but not for Fe2+ when expressed in yeast. Furthermore, the transport activity for Mn2+ was higher in yeast expressing FeNramp5 than in yeast expressing AtNramp1. FeNramp5 was also able to complement the phenotype of Arabidopsis atnramp1 mutant in terms of the growth and accumulation of Mn and Cd. The absolute expression level of AtNramp1 was comparable to that of FeNramp5 in the roots, but buckwheat accumulated higher Mn than Arabidopsis when grown under the same condition. Further analysis showed that at least motif B in FeNramp5 seems important for its high transport activity for Mn. These results indicate that FeNramp5 is a transporter for the uptake of Mn and Cd and its higher transport activity for Mn is probably associated with higher Mn accumulation in buckwheat.

Functional analysis of whether the glycine residue of the GMN motif of the Arabidopsis MRS2/MGT/CorA-type Mg2+ channel protein AtMRS2-11 is critical for Mg2+ transport activity.

Magnesium (Mg2+) plays a critical role in many physiological processes. The AtMRS2/MGT family, which contains nine Arabidopsis genes (and two pseudogenes), belongs to a eukaryotic subset of the CorA superfamily of divalent cation transporters. AtMRS2-11/MGT10 possesses the signature GlyMetAsn sequence (the GMN motif) conserved in the CorA superfamily; however, little is known about the role of the GMN motif in AtMRS2. Direct measurement using the fluorescent dye mag-fura-2 revealed that reconstituted AtMRS2-11 mediated rapid Mg2+ uptake into proteoliposomes at extraliposomal Mg2+ concentrations of 10 and 20 mM. Mutations in the GMN motif, G417 to A, S or V, did not show a significant change in Mg2+ uptake relative to the wild-type protein. The G417W mutant exhibited a significant increase in Mg2+ uptake. The functional complementation assay in Escherichia coli strain TM2 showed that E. coli cells expressing AtMRS2-11 with mutations in G of the GMN motif did not grow in LB medium without Mg2+ supplementation, while growth was observed in LB medium supplemented with 0.5 mM Mg2+; no difference was observed between the growth of TM2 cells expressing the AtMRS2-11 G417W mutant and that of cells expressing wild-type AtMRS2-11. These results suggested that the Mg2+ transport activity of the AtMRS2-11 GMN-motif mutants was low at low physiological Mg2+ concentrations; thus, the Gly residue is critical for Mg2+ transport, and the Mg2+ transport activity of the GMN-motif mutants was increased at high Mg2+ concentrations.

MiR-CLIP reveals iso-miR selective regulation in the miR-124 targetome.

Many microRNAs regulate gene expression via atypical mechanisms, which are difficult to discern using native cross-linking methods. To ascertain the scope of non-canonical miRNA targeting, methods are needed that identify all targets of a given miRNA. We designed a new class of miR-CLIP probe, whereby psoralen is conjugated to the 3p arm of a pre-microRNA to capture targetomes of miR-124 and miR-132 in HEK293T cells. Processing of pre-miR-124 yields miR-124 and a 5'-extended isoform, iso-miR-124. Using miR-CLIP, we identified overlapping targetomes from both isoforms. From a set of 16 targets, 13 were differently inhibited at mRNA/protein levels by the isoforms. Moreover, delivery of pre-miR-124 into cells repressed these targets more strongly than individual treatments with miR-124 and iso-miR-124, suggesting that isomirs from one pre-miRNA may function synergistically. By mining the miR-CLIP targetome, we identified nine G-bulged target-sites that are regulated at the protein level by miR-124 but not isomiR-124. Using structural data, we propose a model involving AGO2 helix-7 that suggests why only miR-124 can engage these sites. In summary, access to the miR-124 targetome via miR-CLIP revealed for the first time how heterogeneous processing of miRNAs combined with non-canonical targeting mechanisms expand the regulatory range of a miRNA.

Inhibition of potato leafroll virus multiplication and systemic translocation by siRNA constructs against putative ATPase fold of movement protein.

Viruses cause many severe plant diseases, resulting in immense losses of crop yield worldwide. Therefore, developing novel approaches to control plant viruses is crucial to meet the demands of a growing world population. Recently, RNA interference (RNAi) has been widely used to develop virus-resistant plants. Once genome replication and assembly of virion particles is completed inside the host plant, mature virions or sometimes naked viral genomes spread cell-to-cell through plasmodesmata by interacting with the virus-encoded movement protein (MP). We used the RNAi approach to suppress MP gene expression, which in turn prevented potato leafroll virus (PLRV) systemic infection in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with MP siRNA constructs exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of MP gene expression is an efficient method for generating PLRV-resistant potato plants. Further, we identified novel ATPase motifs in MP that may be involved in DNA binding and translocation through plasmodesmata. We also showed that the ATPase activity of MP was stimulated in the presence of DNA/RNA. Overall, our findings provide a robust technology to generate PLRV-resistant potato plants, which can be extended to other species. Moreover, this approach also contributes to the study of genome translocation mechanisms of plant viruses.

Cytochrome P450 family: Genome-wide identification provides insights into the rutin synthesis pathway in Tartary buckwheat and the improvement of agricultural product quality.

Flavonoids can not only help plants resist ultraviolet and pathogen attacks, but also show a wide range of therapeutic prospects for human health, including antioxidant, anti-inflammatory and anti-hypertension. Tartary buckwheat, as medicinal and food homologous crop, is rich in flavonoids, among which rutin may prevent liver damage. The one of the major objectives of Tartary buckwheat breeding is to cultivate varieties that have large fruits, high flavonoids and nutrient contents. Members of the cytochrome P450 monooxygenase (CYP) superfamily play a vital role in the synthesis of flavonoids, plant growth and development. Whole-genome analyses of the CYP family have been performed in several plants, but the CYP family has not been characterized in Tartary buckwheat. In this study, 285 FtCYPs were identified from the genome to improve the rutin content and quality of Tartary buckwheat. By exploring the structure, motif composition, tandem and segmental duplication events of FtCYPs, as well as evolutionary relationships with CYPs in other plants, we preliminarily screened potential FtCYPs regulating rutin synthesis, growth and development. The expression levels of the FtCYPs in different organs and fruits at various periods were measured. This study provides a solid foundation for verifying the function of FtCYPs, cultivating high rutin Tartary buckwheat varieties.

Mechanism of Selenium Nanoparticles Inhibiting Advanced Glycation End Products.

Selenium nanoparticles (SeNPs) have been applied in fields of nanobiosensors, environment, nanomedicine, etc. as a result of their excellent characteristics. Early studies had shown that SeNPs have certain inhibition ability against glycation, but the inhibition mechanism, especially for the influence of SeNPs on the reaction activity of glycation sites, remains unclear. The aim of the presented research was to reveal the effects of SeNPs on the ß-lactoglobulin (ß-Lg)/d-ribose glycation system at the molecular level and explore the possible inhibitory mechanism of SeNPs on the formation of advanced glycation end products (AGEs) by analyzing the glycation sites via high-performance liquid chromatography (HPLC)-Orbitrap-tandem mass spectrometry (MS/MS). Changes in contents of AGE formation and free amino acid contents had indicated that SeNPs could significantly slow the glycation process, thus attenuating the formation of AGEs. HPLC-Orbitrap-MS/MS analysis revealed that, at 6, 12, and 24 h, the number of glycation sites of glycated ß-Lg decreased from 7, 7, and 9 to 5, 5, and 6 after the intervention of SeNPs, respectively. The glycation extent of each glycation site was controlled, and the dual-glycation ability of K8, K14, K47, K91, and K101 was changed. All of these results confirmed that SeNPs could indeed slow the process of protein glycation at the molecular level. This may be the reason for SeNPs reducing the formation of AGEs during glycation. Therefore, this study shed light on the insight of how SeNPs reduce the formation of AGEs.

A mitochondrial prolyl aminopeptidase PAP2 releases N-terminal proline and regulates proline homeostasis during stress response.

Most mitochondrial proteins are synthesised in the cytosol and targeted into the organelle via N-terminal targeting peptides that are cleaved upon import. The free targeting peptide is subsequently processed in a stepwise manner, with single amino acids released as final products. Here, we have characterised a proline-cleaving aminopeptidase in Arabidopsis thaliana, prolyl aminopeptidase-2 (PAP2, At3g61540). Activity assays show that PAP2 has a preferred activity to hydrolyse N-terminal proline. Protein localisation studies revealed that PAP2 is exclusively targeted to mitochondria. Characterisation of pap2 mutants show defective pollen, enhanced dark-induced senescence and increased susceptibility to abiotic stresses, which are likely attributed to a reduced level of accumulated free proline. Taken together, these results demonstrate the role of PAP2 in proline cleavage from mitochondrial peptides and proline homeostasis, which is required for the development of male gametophyte, tolerance to abiotic stresses, and leaf senescence.

A De novo Peptide from a High Throughput Peptide Library Blocks Myosin A -MTIP Complex Formation in Plasmodium falciparum.

Apicomplexan parasites, through their motor machinery, produce the required propulsive force critical for host cell-entry. The conserved components of this so-called glideosome machinery are myosin A and myosin A Tail Interacting Protein (MTIP). MTIP tethers myosin A to the inner membrane complex of the parasite through 20 amino acid-long C-terminal end of myosin A that makes direct contacts with MTIP, allowing the invasion of Plasmodium falciparum in erythrocytes. Here, we discovered through screening a peptide library, a de-novo peptide ZA1 that binds the myosin A tail domain. We demonstrated that ZA1 bound strongly to myosin A tail and was able to disrupt the native myosin A tail MTIP complex both in vitro and in vivo. We then showed that a shortened peptide derived from ZA1, named ZA1S, was able to bind myosin A and block parasite invasion. Overall, our study identified a novel anti-malarial peptide that could be used in combination with other antimalarials for blocking the invasion of Plasmodium falciparum.

Affinity Crystallography Reveals Binding of Pomegranate Juice Anthocyanins at the Inhibitor Site of Glycogen Phosphorylase: The Contribution of a Sugar Moiety to Potency and Its Implications to the Binding Mode.

Anthocyanins (ACNs) are dietary phytochemicals with an acknowledged therapeutic significance. Pomegranate juice (PJ) is a rich source of ACNs with potential applications in nutraceutical development. Glycogen phosphorylase (GP) catalyzes the first step of glycogenolysis and is a molecular target for the development of antihyperglycemics. The inhibitory potential of the ACN fraction of PJ is assessed through a combination of in vitro assays, ex vivo investigation in hepatic cells, and X-ray crystallography studies. The ACN extract potently inhibits muscle and liver isoforms of GP. Affinity crystallography reveals the structural basis of inhibition through the binding of pelargonidin-3-O-glucoside at the GP inhibitor site. The glucopyranose moiety is revealed as a major determinant of potency as it promotes a structural binding mode different from that observed for other flavonoids. This inhibitory effect of the ACN scaffold and its binding mode at the GP inhibitor binding site may have significant implications for future structure-based drug design endeavors.