A distinct protein posttranslational modifications-linked OsATL32-OsPPKL2-OsGSK2 loop modulates rice immunity against blast disease.

Protein posttranslational modifications play crucial roles in plant immunity through modulating a complicated signaling network mediated by different hormones. We previously demonstrated that OsATL32, an ATL-type E3 ligase, negatively contributes to rice immunity against Magnaporthe oryzae. Here, we show that OsATL32 forms a loop with OsPPKL2 and OsGSK2 through distinct protein posttranslational modifications to modulate rice immunity. OsATL32 ubiquitinates OsPPKL2, a protein phosphatase with Kelch-like repeat domains that exerts positive roles in regulating rice immunity against M. oryzae and chitin-triggered immune responses, for degradation. The glycogen synthase kinase 2 (OsGSK2), which acts as a negative regulator of rice immunity against M. oryzae and chitin-triggered immune responses, phosphorylates OsATL32 to elevate its protein stability and E3 ligase activity on OsPPKL2. Moreover, OsPPKL2 directly dephosphorylates OsGSK2, affecting its kinase activity on substrates including OsATL32 for phosphorylation. Like OsGSK2 as a BR signaling repressor, OsATL32 negatively regulates BR signaling; conversely, OsPPKL2 plays a positive role in BR signaling. These findings provide a molecular mechanism in which OsATL32 serves as a node connecting BR signaling and immunity by associating with OsPPKL2 and OsGSK2, assembling into a distinct protein posttranslational modifications-linked loop that functions in rice BR signaling and immunity.

Analyses of genes encoding the Glycogen Synthase Kinases in rice and Arabidopsis reveal mechanisms which regulate their expression during development and responses to abiotic stresses.

Plant Glycogen Synthase Kinases (GSKs) enable a crosstalk among the brassinosteroid signaling and phytohormonal- and stress-response pathways to regulate various physiological processes. Initial information about regulation of the GSK proteins' activity was obtained, however, mechanisms that modulate expression of the GSK genes during plant development and stress responses remain largely unknown. Taking into account the importance of the GSK proteins, combined with the lack of in-depth knowledge about modulation of their expression, research in this area may provide a significant insight into mechanisms regulating these aspects of plant biology. In the current study, a detailed analysis of the GSK promoters in rice and Arabidopsis was performed, including identification of the CpG/CpNpG islands, tandem repeats, cis-acting regulatory elements, conserved motifs, and transcription factor-binding sites. Moreover, characterization of expression profiles of the GSK genes in different tissues, organs and under various abiotic stress conditions was performed. Additionally, protein-protein interactions between products of the GSK genes were predicted. Results of this study provided intriguing information about these aspects and insight into various regulatory mechanisms that influence non-redundant and diverse functions of the GSK genes during development and stress responses. Therefore, they may constitute a reference for future research in other plant species.

Inhibition of Glycogen Synthase Kinase 3ß Enhances Functions of Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells in the Blood-Brain Barrier.

Brain microvascular endothelial cells (BMECs) are essential component of the blood-brain barrier (BBB). BMECs strictly regulate the entry of various molecules into the central nervous system from the peripheral circulation by forming tight junctions and expressing various influx/efflux transporters and receptors. In vitro BBB models have been widely reported with primary BMECs isolated from animals, although it is known that the expression patterns and levels of transporters and receptors in BMECs differ between humans and animals. Recently, several methods to differentiate BMECs from human induced pluripotent stem (hiPS) cell have been developed. However, the expression of P-glycoprotein (P-gp), which is a key efflux transporter, in hiPS cell-derived BMECs was detected at a relatively low level compared with primary human BMECs. In this study, we examined the involvement of the canonical Wnt signaling pathway, which contributes to the development of BBB formation, in the regulation of P-gp expression in hiPS cell-derived BMECs. We found that the barrier integrity was significantly enhanced in hiPS cell-derived BMECs treated with glycogen synthase kinase-3ß (GSK-3ß) inhibitors, which are known to positively regulate the canonical Wnt signaling pathway. In addition, our data also showed P-gp expression level was increased by treatment with GSK-3ß inhibitors. In conclusion, physiological barrier function and P-gp expression in BMECs can be enhanced by the canonical Wnt signaling pathway. Our results may be useful for promoting the development of drugs for central nervous system diseases using in vitro BBB model.

Mulberry polyphenols ameliorate atherogenic migration and proliferation by degradation of K-Ras and downregulation of its signals in vascular smooth muscle cell.

Extra-proliferation and increased migration of vascular smooth cells con-tribute to the formation of atherosclerosis. Ras small G proteins play a critical role in the prolif-eration and migration of a wide range of cells. Mulberry, an economic fruit in Asia, exhibits anti-inflammation, anti-migration, and anti-oxidant properties. The mechanisms of action of mulberry extracts on K-Ras small G protein-induced proliferation and migration of vascular smooth muscle cell have not been extensively investigated. In this study, we explored the effects of mulberry polyphenol extracts (MPE) on the proliferation and migration of K-Ras-overexpressing A7r5 smooth muscle cells. The overexpression of K-Ras enhanced the ex-pression and activity of matrix metalloproteinase (MMP)-2, promoted vascular endothelial growth factor (VEGF) production, and eventually triggered the migration of A7r5 cells. Treatment with MPE attenuated K-Ras-induced phenomenon. In addition, MPE blocked K-Ras-induced actin fibril stress. MPE dose-dependently diminished K-Ras-induced Rho A, Rac1, CDC42, and phosphorylated focal adhesion kinase (FAK) expression. MPE elevated Rho B ex-pression. Phosphorylated AKT and glycogen synthase kinase (GSK) induced by K-Ras were also repressed by MPE treatment. MPE enhanced the interaction of IκB with NFκB. MPE restored the G0/G1 population and p21 and p27 expressions, which were repressed by K-Ras. Finally, MPE triggered the degradation of K-Ras by ubiquitination. MPE inhibited the migration and proliferation of vascular smooth cell through K-Ras-induced pathways and eventually pre-vented atherosclerosis.

Glycogen synthase kinase homolog Rim11 regulates lipid synthesis through the phosphorylation of Pah1 phosphatidate phosphatase in yeast.

Pah1 phosphatidate (PA) phosphatase plays a major role in triacylglycerol synthesis in Saccharomyces cerevisiae by producing its precursor diacylglycerol and concurrently regulates de novo phospholipid synthesis by consuming its precursor PA. The function of Pah1 requires its membrane localization, which is controlled by its phosphorylation state. Pah1 is dephosphorylated by the Nem1-Spo7 protein phosphatase, whereas its phosphorylation occurs by multiple known and unknown protein kinases. In this work, we show that Rim11, a yeast homolog of mammalian glycogen synthase kinase-3ß, is a protein kinase that phosphorylates Pah1 on serine (Ser12, Ser602, and Ser818) and threonine (Thr163, Thr164, Thr522) residues. Enzymological characterization of Rim11 showed that its Km for Pah1 (0.4 µM) is similar to those of other Pah1-phosphorylating protein kinases, but its Km for ATP (30 µM) is significantly higher than those of these same kinases. Furthermore, we demonstrate Rim11 phosphorylation of Pah1 does not require substrate prephosphorylation but was increased ∼2-fold upon its prephosphorylation by the Pho85-Pho80 protein kinase. In addition, we show Rim11-phosphorylated Pah1 was a substrate for dephosphorylation by Nem1-Spo7. Finally, we demonstrate the Rim11 phosphorylation of Pah1 exerted an inhibitory effect on its PA phosphatase activity by reduction of its catalytic efficiency. Mutational analysis of the major phosphorylation sites (Thr163, Thr164, and Ser602) indicated that Rim11-mediated phosphorylation at these sites was required to ensure Nem1-Spo7-dependent localization of the enzyme to the membrane. Overall, these findings advance our understanding of the phosphorylation-mediated regulation of Pah1 function in lipid synthesis.

Brain insulin resistance and the therapeutic value of insulin and insulin-sensitizing drugs in Alzheimer's disease neuropathology.

The incidence of Alzheimer's disease (AD) is significantly higher in people with diabetes. Insulin and insulin receptor (IR) signaling intermediates are expressed in the brain. Insulin exerts multiple function in the brain. The role of compromised IR signaling in AD pathogenesis and the therapeutic value of insulin attract broad attention. This review summarizes the collective insulin action in the brain related to key factors of AD pathogenesis, updates the key features of insulin resistance in the AD brain and assesses the therapeutic potential of insulin and insulin-sensitizing drugs. Insulin stimulates neural growth and survival, suppresses amyloidogenic processing of the amyloid precursor protein (AßPP) and inhibits the Tau phosphorylation kinase, glycogen synthase kinase 3ß. Central nervous IR signaling regulates systemic metabolism and increases glucose availability to neurons. The expression of IR and its downstream effectors is reduced in AD brain tissues. Insulin and insulin-sensitizing drugs can improve cognitive function in AD patients and AD animal models. Systemic insulin delivery is less effective than intranasal insulin treatment. The penetrance of insulin-sensitizing drugs to the blood brain barrier is problematic and new brain-prone drugs need be developed. Insulin resistance manifested by the degradation and the altered phosphorylation of IR intermediates precedes overt AD syndrome. Type 3 diabetes as a pure form of brain insulin resistance without systemic insulin resistance is proposed as a causal factor in AD. Further research is needed for the identification of critical factors leading to impaired IR signaling and the development of new molecules to stimulate brain IR signaling.

NUMB as a Therapeutic Target for Melanoma.

The upregulation of the adaptor protein NUMB triggers melanocytic differentiation from multipotent skin stem cells, which share many properties with aggressive melanoma cells. Although NUMB acts as a tumor suppressor in various human cancer types, little is known about its role in melanoma. In this study, we investigated the role of NUMB in melanoma progression and its regulatory mechanism. Analysis of The Cancer Genome Atlas melanoma datasets revealed that high NUMB expression in melanoma tissues correlates with improved patient survival. Moreover, NUMB expression is downregulated in metastatic melanoma cells. NUMB knockdown significantly increased the invasion potential of melanoma cells in a three-dimensional collagen matrix in vitro and in the lungs of a mouse model in vivo; it also significantly upregulated the expression of the NOTCH target gene CCNE. Previous studies suggested that Wnt signaling increases NUMB expression. By mimicking Wnt stimulation through glycogen synthase kinase-3 inhibition, we increased NUMB expression in melanoma cells. Furthermore, a glycogen synthase kinase-3 inhibitor reduced the invasion of melanoma cells in a NUMB-dependent manner. Together, our results suggest that NUMB suppresses invasion and metastasis in melanoma, potentially through its regulation of the NOTCH‒CCNE axis and that the inhibitors that upregulate NUMB can exert therapeutic effects in melanoma.

WRKY53 integrates classic brassinosteroid signaling and the mitogen-activated protein kinase pathway to regulate rice architecture and seed size.

In rice (Oryza sativa) and other plants, plant architecture and seed size are closely related to yield. Brassinosteroid (BR) signaling and the mitogen-activated protein kinase (MAPK) pathway (MAPK kinase kinase 10 [MAPKKK10]-MAPK kinase 4 [MAPKK4]-MAPK6) are two major regulatory pathways that control rice architecture and seed size. However, their possible relationship and crosstalk remain elusive. Here, we show that WRKY53 mediated the crosstalk between BR signaling and the MAPK pathway. Biochemical and genetic assays demonstrated that glycogen synthase kinase-2 (GSK2) phosphorylates WRKY53 and lowers its stability, indicating that WRKY53 is a substrate of GSK2 in BR signaling. WRKY53 interacted with BRASSINAZOLE-RESISTANT 1(BZR1); they function synergistically to regulate BR-related developmental processes. We also provide genetic evidence showing that WRKY53 functions in a common pathway with the MAPKKK10-MAPKK4-MAPK6 cascade in leaf angle and seed size control, suggesting that WRKY53 is a direct substrate of this pathway. Moreover, GSK2 phosphorylated MAPKK4 to suppress MAPK6 activity, suggesting that GSK2-mediated BR signaling might also regulated MAPK pathway. Together, our results revealed a critical role for WRKY53 and uncovered sophisticated levels of interplay between BR signaling and the MAPK pathway in regulating rice architecture and seed size.

Wnt/ß­catenin signaling modulates piperine­mediated antitumor effects on human osteosarcoma cells.

The plant extract piperine is used as a traditional Chinese medicine due to its anti­inflammatory effects and efficacy against numerous types of cancer. The aim of the present study was to investigate the antitumor mechanism of piperine in human osteosarcoma U2OS and 143B cell lines. The effects of piperine on cell apoptosis and invasion of human osteosarcoma cells were assessed using flow cytometry and Transwell assays. Moreover, western blotting was used to measure the effects of piperine on the protein expression levels of the metastasis markers matrix metalloproteinase­2 (MMP­2) and vascular endothelial growth factor (VEGF). In addition, the involvement of the Wnt/ß­catenin signaling pathway in modulating the effects of piperine was examined via western blot analysis. The results of MTT and Transwell invasion assays indicated that piperine treatment dose­dependently reduced U2OS and 143B cell viability and invasion. Furthermore, a significant reduction was identified in MMP­2, VEGF, glycogen synthase kinase­3ß and ß­catenin protein expression levels, as well as the expression levels of their target proteins cyclooxygenase­2, cyclin D1 and c­myc, in U2OS cells after piperine treatment. In addition, similar results were observed in 143B cells. Therefore, the present study demonstrated the efficacy of piperine in osteosarcoma, and identified that the Wnt/ß­catenin signaling pathway may modulate the antitumor effects of piperine on human U2OS and 143B cells.

AMPKα Subunit Ssp2 and Glycogen Synthase Kinases Gsk3/Gsk31 are involved in regulation of sterol regulatory element-binding protein (SREBP) activity in fission yeast.

Sterol regulatory element-binding protein (SREBP), a highly conserved family of membrane-bound transcription factors, is an essential regulator for cellular cholesterol and lipid homeostasis in mammalian cells. Sre1, the homolog of SREBP in the fission yeast Schizosaccharomyces pombe (S. pombe), regulates genes involved in the transcriptional responses to low sterol as well as low oxygen. Previous study reported that casein kinase 1 family member Hhp2 phosphorylated the Sre1 N-terminal transcriptional factor domain (Sre1N) and accelerated Sre1N degradation, and other kinases might exist for regulating the Sre1 function. To gain insight into the mechanisms underlying the Sre1 activity and to identify additional kinases involved in regulation of Sre1 function, we developed a luciferase reporter system to monitor the Sre1 activity through its binding site called SRE2 in living yeast cells. Here we showed that both ergosterol biosynthesis inhibitors and hypoxia-mimic CoCl2 caused a dose-dependent increase in the Sre1 transcription activity, concurrently, these induced transcription activities were almost abolished in Δsre1 cells. Surprisingly, either AMPKα Subunit Ssp2 deletion or Glycogen Synthase Kinases Gsk3/Gsk31 double deletion significantly suppressed ergosterol biosynthesis inhibitors- or CoCl2-induced Sre1 activity. Notably, the Δssp2Δgsk3Δgsk31 mutant showed further decreased Sre1 activity when compared with their single or double deletion. Consistently, the Δssp2Δgsk3Δgsk31 mutant showed more marked temperature sensitivity than any of their single or double deletion. Moreover, the fluorescence of GFP-Sre1N localized at the nucleus in wild-type cells, but significantly weaker nuclear fluorescence of GFP-Sre1N was observed in Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells. On the other hand, the immunoblot showed a dramatic decrease in GST-Sre1N levels in the Δgsk3Δgsk31 or the Δssp2Δgsk3Δgsk31 cells but not in the Δssp2 cells. Altogether, our findings suggest that Gsk3/Gsk31 may regulate Sre1N degradation, while Ssp2 may regulate not only the degradation of Sre1N but also its translocation to the nucleus.

Effects of a Tripeptide on Mitogen-Activated Protein Kinase and Glycogen Synthase Kinase Activation in a Cell Line Derived from the Foetal Hippocampus of a Trisomy 16 Mouse: an Animal Model of Down Syndrome.

Down syndrome (DS) is a developmental disorder that results from the trisomy of chromosome 21. DS patients show several abnormalities including cognitive deficits. Here, we show enhanced activation of the extracellular signal-regulated kinase (ERK), a kinase that critically regulates synaptic plasticity and memory, in a hippocampal cell line derived from trisomy 16 mouse foetus. In addition, these cells show enhanced activation of p38 mitogen-activated protein kinase (p38 MAPK). The hyper-activation of ERK and p38 MAPK is significantly reduced by a small peptide, Gly-Pro-Glu (GPE), derived from insulin-like growth factor-1. In addition, the trisomic cells show reduced level of inhibitory phosphorylation of glycogen synthase kinase-3ß (GSK-3ß), which is enhanced by GPE. Furthermore, the trisomic cells do not show ERK activation in response to KCl depolarization or forskolin treatment. Importantly, ERK activation by these stimuli is observed after GPE treatment of the cells. These results suggest that GPE may help reduce aberrant signalling in the trisomic neurons by affecting MAPK and GSK-3ß activation.

Single Step Purification of Glycogen Synthase Kinase Isoforms from Small Scale Transient Expression in HEK293 Cells with a Calcium-Dependent Fragment Complementation System.

Purification of proteins for the biophysical analysis of protein interactions occurring in human cells can benefit from methods that facilitate the capture of small amounts of natively processed protein obtained using transient mammalian expression systems. We have used a novel calcium-dependent fragment complementation-based affinity method to effectively purify full length glycogen synthase kinase 3 (GSK3) α and ß isoforms to study their interaction with amyloid ß peptide (Aß42). Using these proteins, purified from 1 mg of total cell lysate, we measured an apparent KD of ≤100 pM between GSK3α/ß and immobilized Aß42 with surface plasmon resonance technology. This approach can be used to retrieve useful quantities of protein for biophysical experiments with small scale mammalian cell culture.

Phosphorylation of the Gα protein Gpa2 promotes protein kinase A signaling in yeast.

Heterotrimeric G proteins are important molecular switches that facilitate transmission of a variety of signals from the outside to the inside of cells. G proteins are highly conserved, enabling study of their regulatory mechanisms in model organisms such as the budding yeast Saccharomyces cerevisiae Gpa2 is a yeast Gα protein that functions in the nutrient signaling pathway. Using Phos-tag, a highly specific phosphate binding tag for separating phosphorylated proteins, we found that Gpa2 undergoes phosphorylation and that its level of phosphorylation is markedly increased upon nitrogen starvation. We also observed that phosphorylation of Gpa2 depends on glycogen synthase kinase (GSK). Disrupting GSK activity diminishes Gpa2 phosphorylation levels in vivo, and the purified GSK isoforms Mck1 and Ygk3 are capable of phosphorylating Gpa2 in vitro Functionally, phosphorylation enhanced plasma membrane localization of Gpa2 and promoted nitrogen starvation-induced activation of protein kinase A. Together, the findings of our study reveal a mechanism by which GSK- and nutrient-dependent phosphorylation regulates subcellular localization of Gpa2 and its ability to activate downstream signaling.

Identification of GSK3ß inhibitor kenpaullone as a temozolomide enhancer against glioblastoma.

Cancer stem cells are associated with chemoresistance and rapid recurrence of malignant tumors, including glioblastoma (GBM). Although temozolomide (TMZ) is the most effective drug treatment for GBM, GBM cells acquire resistance and become refractory to TMZ during treatment. Therefore, glioma stem cell (GSC)-targeted therapy and TMZ-enhancing therapy may be effective approaches to improve GBM prognosis. Many drugs that suppress the signaling pathways that maintain GSC or enhance the effects of TMZ have been reported. However, there are no established therapies beyond TMZ treatment currently in use. In this study, we screened drug libraries composed of 1,301 existing drugs using cell viability assays to evaluate effects on GSCs, which led to selection of kenpaullone, a kinase inhibitor, as a TMZ enhancer targeting GSCs. Kenpaullone efficiently suppressed activity of glycogen synthase kinase (GSK) 3ß. Combination therapy with kenpaullone and TMZ suppressed stem cell phenotype and viability of both GSCs and glioma cell lines. Combination therapy in mouse models significantly prolonged survival time compared with TMZ monotherapy. Taken together, kenpaullone is a promising drug for treatment of GBM by targeting GSCs and overcoming chemoresistance to TMZ.

Silencing of microRNA-708 promotes cell growth and epithelial-to-mesenchymal transition by activating the SPHK2/AKT/ß-catenin pathway in glioma.

Aberrant microRNA-708 (miR-708) expression is frequently reported in cancer studies; however, its role in glioma has not been examined in detail. We investigated miR-708 function in glioma and revealed that miR-708 expression was significantly down-regulated in glioma tissues and cell lines. Restoration of miR-708 inhibited glioma cell growth and invasion both in vitro and in vivo. The oncogene SPHK2 (sphingosine kinase 2) was identified as a downstream target of miR-708 using luciferase and western blot assays. miR-708 inhibited AKT/ß-catenin signaling, which is activated by SPHK2. In addition, we revealed that miR-708 was transcriptionally repressed by EZH2 (enhancer of zeste homolog 2)-induced histone H3 lysine 27 trimethylation and promoter methylation. In summary, our findings revealed that miR-708 is a glioma tumor suppressor and suggest that miR-708 is a potential therapeutic target for glioma patients.

Brassinosteroid Regulates Root Development with Highly Redundant Genes in Hexaploid Wheat.

Brassinosteroid (BR) plays an important role in plant development and biotic and abiotic stress tolerance, but its specific function remains largely unknown in wheat (Triticum aestivum L.), preventing its utilization in this important crop. In this study, the function of BR and its underlying cytological role in wheat root development were comprehensively investigated. Our findings demonstrated that BR has a conserved function in regulating root length in wheat, and novel roles in regulating lateral root emergence and root diameter were uncovered. Analyses of BR homologous gene composition and evolutionary divergence demonstrated that the genetic framework of the wheat BR pathway was close to that of rice, but contained highly redundant homologous copies of genes from the subgenome A, B and D. These homologous copies showed active expression and shared a conserved BR response. The expression of wheat DWF4 and glycogen synthase kinase (GSK) genes in Arabidopsis confirmed that multiple homologous copies maintained their conserved function in regulating root development, highlighting their redundant status and indicating that a special challenge exists in wheat gene modification to deal with this high redundancy. However, our results suggested that the hypermorphic effect of T. aestivum GSK (TaGSK) genes with point mutations may be an effective approach to overcome this redundancy in the manipulation of BR signaling in wheat. Our study provides fundamental data uncovering the function of BR in wheat root development, the underlying genetic basis and a possible strategy to manipulate BR signaling in hexaploid wheat.

Targeting kinases in Parkinson's disease: A mechanism shared by LRRK2, neurotrophins, exenatide, urate, nilotinib and lithium.

Several kinases have been implicated in the pathogenesis of Parkinson's disease (PD), most notably leucine-rich repeat kinase 2 (LRRK2), as LRRK2 mutations are the most common genetic cause of a late-onset parkinsonism that is clinically indistinguishable from sporadic PD. More recently, several other kinases have emerged as promising disease-modifying targets in PD based on both preclinical studies and clinical reports on exenatide, the urate precursor inosine, nilotinib and lithium use in PD patients. These kinases include protein kinase B (Akt), glycogen synthase kinases-3ß and -3α (GSK-3ß and GSK-3α), c-Abelson kinase (c-Abl) and cyclin-dependent kinase 5 (cdk5). Activities of each of these kinases are involved either directly or indirectly in phosphorylating tau or increasing α-synuclein levels, intracellular proteins whose toxic oligomeric forms are strongly implicated in the pathogenesis of PD. GSK-3ß, GSK-3α and cdk5 are the principle kinases involved in phosphorylating tau at sites critical for the formation of tau oligomers. Exenatide analogues, urate, nilotinib and lithium have been shown to affect one or more of the above kinases, actions that can decrease the formation and increase the clearance of intraneuronal phosphorylated tau and α-synuclein. Here we review the current preclinical and clinical evidence supporting kinase-targeting agents as potential disease-modifying therapies for PD patients enriched with these therapeutic targets and incorporate LRRK2 physiology into this novel model.

Histone acetyltransferase MoHat1 acetylates autophagy-related proteins MoAtg3 and MoAtg9 to orchestrate functional appressorium formation and pathogenicity in Magnaporthe oryzae.

Macroautophagy/autophagy is critical for normal appressorium formation and pathogenicity of the rice blast fungus Magnaporthe oryzae, but the molecular base of autophagy linked to pathogenicity remains elusive in this or other pathogenic fungi. We found that MoHat1, a histone acetyltransferase (HAT) homolog, had a role in the regulation of autophagy through the acetylation of autophagy related proteins MoAtg3 and MoAtg9. We also found that MoHat1 was subject to regulation by the protein kinase MoGsk1 that modulated the translocation of MoHat1 from the nucleus to the cytoplasm with the assistance of MoSsb1, a protein chaperone. The alternation of intracellular location affected MoHat1 in the modification of cytosolic autophagy proteins that maintained normal autophagy. Furthermore, we provided evidence linking acetylation of MoAtg3 and MoAtg9 by MoHat1 to functional appressorium development and pathogenicity. Together with the first report of MoAtg9 being subject to acetylation regulation by MoHat1, our studies depicted how MoHat1 regulated autophagy in conjunction with MoGsk1 and how normal autophagy was linked to appressorium formation and function and pathogenicity of M. oryzae. Abbreviations: A/Ala: alanine; AP: autophagosome; Atg genes/proteins: autophagy-related genes/proteins; BiFC: bimolecular fluorescence complementation; co-IP: co-immunoprecipitation; DAPI: 4', 6-diamidino-2-phenylindole; D/Asp: aspartic acid; GFP: green fluorescent protein; GSK3: glycogen synthase kinase 3; HAT: histone acetyltransferase; Hsp70: heat-shock protein 70; IH: invasive hyphae; K/Lys: lysine; MMS: methyl methanesulfonate; Mo: Magnaporthe oryzae; PAS: phagophore assembly site; PE: phosphatidylethanolamine; PtdIns3K: phosphatidylinositol 3-kinase; R/Arg: arginine; S/Ser: serine; T/Thr: threonine; TOR: target of rapamycin; WT: wild type; YFP: yellow fluorescent protein.

In Silico Computations of Selective Phytochemicals as Potential Inhibitors Against Major Biological Targets of Diabetes Mellitus.

BACKGROUND: In the past few years, several developments have been made to understand and control the complications and harmful side-effects associated with the disorder diabetes mellitus (DM). Many new steps have been taken in a better understanding of the pathophysiology of the disease. With the advancement in the field of medical sciences, various novel therapies have been developed to efficiently control the pathological effects of diabetes mellitus. Recently, phytochemicals possessing various medicinal properties have opened up a new vast range of opportunities to design novel therapeutic drugs against diabetes mellitus. OBJECTIVE: The present study aims to identify and screen phytochemicals as potent and novel inhibitors against diabetes mellitus. METHODS: Three major biological targets of diabetes mellitus named Cytochrome P450, glycogen synthase kinase and PPARγ are targeted using phytochemicals by performing pharmacological properties prediction, molecular docking and density functional theory studies. RESULTS: Out of 108 phytochemicals, 20, 12 and 3 phytochemicals showed higher binding affinity values as compared to chemically synthesized drugs against cytochrome P450, glycogen synthase kinase and PPARγ, respectively. CONCLUSION: The screened phytochemicals have strong inhibitory potential against diabetes mellitus and in future, these compounds, holding immense potential, can be considered as candidate drugs for treating diabetes mellitus.