Phytochemical screening, antioxidant analyses, and in vitro and in vivo antimalarial activities of herbal medicinal plant - Rotheca serrata (L.) Steane & Mabb.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria is a major global health concern that is presently challenged by the emergence of Plasmodium falciparum (Pf) resistance to mainstay artemisinin-based combination therapies (ACTs). Hence, the discovery of novel and effective antimalarial drugs is pivotal to treating and controlling malaria. For many years, traditional plant-based herbal medicines have been employed in the treatment of various illnesses. Rotheca serrata (L.) Steane & Mabb. belongs to the Lamiaceae family that has been traditionally used to treat, cure, and prevent numerous diseases including malaria. AIM: The present investigation sought to assess the phytoconstituents, antioxidant, cytotoxicity, antimalarial activities of Rotheca serrata extract and its fractions. The in vitro antiplasmodial activity was assessed in chloroquine-sensitive Pf3D7 and artemisinin-resistant PfCam3.IR539T cultures, and the in vivo antimalarial activity was analyzed in Plasmodium berghei (Pb) ANKA strain-infected BALB/c mouse model. MATERIALS AND METHODS: The fresh leaves of Rotheca serrata were extracted in methanol (RsMeOH crude leaf extract). A portion of the extract was used to prepare successive solvent fractions using ethyl acetate (RsEA) and hexane (RsHex). The in vitro antiplasmodial activity was evaluated using [3H]-hypoxanthine incorporation assays against Pf3D7 and PfCam3.IR539T cultures. In vitro cytotoxicity study on HeLa, HEK-293T, and MCF-7 cell lines was carried out using MTT assay. The human red blood cells (RBCs) were used to perform the hemolysis assays. In vitro antioxidant studies and detailed phytochemical analysis were performed using GC-MS and FTIR. The four-day Rane's test was performed to evaluate the in vivo antimalarial activity against Pb ANKA strain-infected mice. RESULTS: Phytochemical quantification of Rotheca serrata extract (RsMeOH) and its fractions (RsEA and RsHex) revealed that RsMeOH crude extract and RsEA fraction had higher contents of total phenol and flavonoid than RsHex fraction. The RsEA fraction showed potent in vitro antiplasmodial activity against Pf3D7 and PfCam3.IR539T with IC50 values of 9.24 ± 0.52 µg/mL and 17.41 ± 0.43 µg/mL, respectively. The RsMeOH crude extract exhibited moderate antiplasmodial activity while the RsHex fraction showed the least antiplasmodial activity. The GC-MS and FTIR analysis of RsMeOH and RsEA revealed the presence of triterpenes, phenols, and hydrocarbons as major constituents. The RsMeOH crude extract was non-hemolytic and non-cytotoxic to HeLa, HEK-293T, and MCF-7 cell lines. The in vivo studies showed that a 1200 mg/kg dose of RsMeOH crude extract could significantly suppress parasitemia by ∼63% and prolong the survival of treated mice by ∼10 days. The in vivo antiplasmodial activity of RsMeOH was better than the RsEA fraction. CONCLUSION: The findings of this study demonstrated that traditionally used herbal medicinal plants like R. serrata provide a platform for the identification and isolation of potent bioactive phytochemicals that in turn can promote the antimalarial drug research. RsMeOH crude extract and RsEA fraction showed antiplasmodial, antimalarial and antioxidant activities. Chemical fingerprinting analysis suggested the presence of bioactive phytocompounds that are known for their antimalarial effects. Further detailed investigations on RsMeOH crude extract and RsEA fraction would be needed for the identification of the entire repertoire of the active antimalarial components with potent pharmaceutical and therapeutic values.

Antimalarial activity of Toona ciliata MJ Roem aqueous methanolic leaf extract and its antioxidant and phytochemical properties.

Background and aim: Malaria is a global health issue causing substantial morbidity and mortality. Screening of various traditionally important medicinal plants is a key source for the discovery of new antimalarials. We evaluated the antimalarial and antioxidant activities, and performed detailed phytochemical analyses of Toona ciliata MJ Roem aqueous methanolic leaf extract (TcMLE). Experimental procedures: In vitro antiplasmodial studies in Plasmodium falciparum (Pf) 3D7 and PfCam3.IR539T strains were performed by [3H]-hypoxanthine uptake assays. In vitro cytotoxicity in HeLa and HEK293T cell lines was evaluated using MTT assays. Hemolysis assay was performed using RBCs. Phytochemical analysis by GC-MS and in vitro antioxidant studies by DPPH and ABTS assays were performed. In vivo antimalarial studies in Pb-infected mice were carried out using Rane's test and Peters' 4-day test. Results and conclusions: TcMLE showed significant in vitro antioxidant activity and had phytochemicals reported for antimalarial activity. In vitro studies showed prominent antiplasmodial activity against Pf3D7 strain (IC50 ∼22 µg/ml) and PfCam3. IR539Tstrain (IC50 value ∼43 µg/ml). In vitro cytotoxicity studies, in vitro hemolytic assays, and in vivo acute toxicity studies further suggested that TcMLE is nontoxic. In vivo antimalarial studies using Rane's test showed a significant decrease in parasitemia by ∼70% at 1200 mg/kg doses and delayed the mortality of mice by ∼10-14 days. Peters' 4-day test also showed a similar pattern. The present study demonstrated the antimalarial potential of TcMLE. These findings deliver a platform for further studies to identify the active components of TcMLE and discover new antimalarials.

Antioxidant, Diabetic and Inflammatory Activities of Alpinia calcarata Roscoe Extract.

BACKGROUND: Alpinia calcarata (AC) Roscoe of Zingiberaceae popularly known as lesser galangal has a widespread occurrence in China, India, Sri-Lanka, Bangladesh, Malaysia, Indonesia and Thailand. Essential oil (Eoil) was obtained from leaves/rhizomes of AC via hydro-distillation process. METHODS: To identify chemical ingredients in oil from leaves/rhizomes of AC through GC/MS technique for volatile components and their anti-oxidant, inflammatory/diabetic activities. RESULTS: The 38 and 65 components were found to make up 99.9 and 99.6 %, respectively in total of Eoil composition of AC leaves/rhizomes. Key chemical constituents were eucalyptol (28.7 % in leaves; 25.4 % in rhizomes), camphor (12.8 % in leaves; 4.2 % in rhizomes), and carotol (9.8 % in leaves; 5.6 % in rhizomes) found in oil of AC leaves/rhizomes. Colorimetric assay showed anti-oxidant activities in leaves and rhizomes are IC50 =71.01±0.71 µg/mL and IC50 =73.83±0.49 µg/mL, respectively in the Eoils. Eoils had high anti-oxidant capabilities in IC50 -values of AC-L-Eoil=43.09±0.82&AC-Rh-Eoil=68.11±0.87 in reducing power in µg/mL was found. Albumin test of rhizome oil had IC50 -values of 15.19±0.25 µg/mL. Concentrations range of 7.81 µg/mL and 250 µg/mL in the Eoils of AC leaves and rhizome, respectively by α-glucosidase inhibition assay. CONCLUSION: Our findings demonstrated that leaf oil was slightly more promising results than rhizome oil of AC extract, which was ultimately showed medicinal potential of secondary metabolites with anti-oxidant, diabetic/inflammatory activities. Further, Eoils of AC have a wide range of pharmacological potential and promising anti-diabetic effects.

Signaling pathways and regulation of gene expression in hematopoietic cells.

Cellular functions are regulated by signal transduction pathway networks consisting of protein-modifying enzymes that control the activity of many downstream proteins. Protein kinases and phosphatases regulate gene expression by reversible phosphorylation of transcriptional factors, which are their direct substrates. Casein kinase II (CK2) is a serine/threonine kinase that phosphorylates a large number of proteins that have critical roles in cellular proliferation, metabolism and survival. Altered function of CK2 has been associated with malignant transformation, immunological disorders and other types of diseases. Protein phosphatase 1 (PP1) is a serine/threonine phosphatase, which regulates the phosphorylation status of many proteins that are essential for cellular functions. IKAROS is a DNA-binding protein, which functions as a regulator of gene transcription in hematopoietic cells. CK2 directly phosphorylates IKAROS at multiple phosphosites which determines IKAROS activity as a regulator of gene expression. PP1 binds to IKAROS via the PP1-consensus recognition site and dephosphorylates serine/threonine residues that are phosphorylated by CK2. Thus, the interplay between CK2 and PP1 signaling pathways have opposing effects on the phosphorylation status of their mutual substrate - IKAROS. This review summarizes the effects of CK2 and PP1 on IKAROS role in regulation of gene expression and its function as a tumor suppressor in leukemia.

Planispine A Sensitized Cancer Cells to Cisplatin by Inhibiting the Fanconi Anemia Pathway.

The use of cisplatin as a chemotherapeutic drug is impeded by the development of drug resistance. Combination therapies of a chemosensitizer for cisplatin have been studied, but with little success, and the search for an effective combination therapy is continuing. Our earlier reports have shown that Zanthoxylum armatum DC. extract enhances the apoptotic effect of cisplatin in cancer cell lines. In this study, we purified and identified the bioactive phytocompound through bio-assay-guided purification, using column chromatography and HPLC. Chemical characterization using NMR and mass spectrometry revealed the compound as planispine A, with molecular structure C25H30O6 and molecular weight, 426.16 g/mol. Planispine A was found to inhibit cancer cell proliferation in a dose-dependent manner and to sensitize the cancer cells to cisplatin-augmented apoptotic cell death, in a caspase-dependent manner. A combination of planispine A and cisplatin induced S-phase cell cycle arrest, and reduced the expression of survival proteins such as cyclin D1. Interestingly, planispine A inhibits the Fanconi anemia pathway, as shown by reduced FANCD2 foci formation and FANCD2 monoubiquitination, which revealed the molecular mechanism of chemo-sensitization of cancer cells to cisplatin. Evaluation of this combination therapy in cisplatin-resistant tumors may lead to more efficient cisplatin treatment.

Mass spectrometry analysis of affinity-purified cytoplasmic translation initiation complexes from human and fly cells.

eIF5-mimic protein (5MP) controls translation through binding to the ribosomal pre-initiation complex (PIC) and alters non-AUG translation rates for cancer oncogenes and repeat-expansions in neurodegenerative diseases. Here, we describe a semi-quantitative protocol for detecting 5MP-associated proteins in cultured human and fly cells. We detail one-step anti-FLAG affinity purification and whole-lane mass spectrometry analysis of samples resolved by SDS-PAGE. This protocol allows for quantitative evaluation of the effect of 5MP mutations on its molecular interactions, to elucidate translational control by 5MP. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).

Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein.

eIF5-mimic protein (5MP) controls translation through its interaction with eukaryotic translation initiation factor (eIF) 2 and eIF3 and alters non-AUG translation rates for oncogenes in cancer and repeat expansions in neurodegenerative disease. To precisely evaluate the effect of 5MP mutations on binding affinity against eIFs, here we describe two label-free protocols of affinity measurement for 5MP binding to eIF2 or eIF3 protein segments, termed isothermal titration calorimetry (ITC) and bio-layer interferometry (BLI), starting with how to purify proteins used. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).

Phytochemical screening, cytotoxicity assessment and evaluation of in vitro antiplasmodial and in vivo antimalarial activities of Mentha spicata L. methanolic leaf extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria causes extensive morbidity and mortality, and the decreasing efficacy of artemisinin and its partner drugs has posed a serious concern. Therefore, it is important to identify new antimalarials, and the natural compounds from plants provide a promising platform. Mentha spicata L. representing the Lamiaceae family has been used in traditional medicine for various diseases including malaria. AIM OF THE STUDY: This study was aimed at evaluating the antiplasmodial activity of M. spicata methanolic leaf extract using Plasmodium falciparum (Pf) cultures (Pf3D7 and artemisinin (ART)-resistant PfCam3.IR539T strains) and antimalarial activity using Plasmodium berghei (Pb)-infected mice. Dry leaf powder and methanolic leaf extract were examined for in vivo antimalarial activity and the efficacy of oral versus parenteral administration was compared. MATERIALS AND METHODS: Leaves of M. spicata were collected and extracted using 70% methanol in water (v/v). [3H]-hypoxanthine incorporation assays and Giemsa-stained smears were used to assess the in vitro antiplasmodial activity of M. spicata methanolic extract against Pf3D7 and ART-resistant PfCam3.IR539T strains. Cytotoxicity was evaluated in HeLa and HEK-293T cell lines using MTT assays. Hemolysis assays were performed using red blood cells (RBCs). In vivo antimalarial activities of M. spicata dry leaf powder and methanolic leaf extract were examined in P. berghei-infected mice by Rane's curative test and Peters' 4-day suppressive test. RESULTS: Phytochemical screening of M. spicata methanolic leaf extract indicated the presence of reducing sugars, phenolic compounds, flavonoids, glycosides, sterols, saponins, alkaloids, coumarins, tannins, carbohydrates, and proteins. In vitro studies carried out using Pf cultures showed that M. spicata methanolic leaf extract had significant antiplasmodial activity against Pf3D7 cultures with a 50% inhibitory concentration (IC50) of 57.99 ± 2.82 µg/ml. The extract was also effective against ART-resistant PfCam3.IR539T strain with an IC50 of 71.23 ± 3.85 µg/ml. The extract did not show significant in vitro cytotoxicity, hemolysis, and in vivo toxicity. In vivo studies performed using Pb-infected mice treated with M. spicata dry leaf powder and methanolic leaf extract showed ∼50% inhibition in parasite growth at 1500 mg/kg and 1000 mg/kg doses, respectively. There was also a significant delay in the mortality of treated mice. Parenteral administration was found to be appropriate for the in vivo treatment. CONCLUSIONS: Our in vitro and in vivo findings from Pf and Pb parasites suggested the therapeutic potential of M. spicata leaf extract as an antimalarial. M. spicata leaf extract could also inhibit the growth of ART-resistant Pf strain. Further studies on fractionation and active component analysis of M. spicata leaf extract would be required to identify the bioactive phytochemicals having pharmaceutical and therapeutic values. Such efforts would help us in developing new antimalarials to combat malaria.

Translational recoding by chemical modification of non-AUG start codon ribonucleotide bases.

In contrast to prokaryotes wherein GUG and UUG are permissive start codons, initiation frequencies from non-AUG codons are generally low in eukaryotes, with CUG being considered as strongest. Here, we report that combined 5-cytosine methylation (5mC) and pseudouridylation (Ψ) of near-cognate non-AUG start codons convert GUG and UUG initiation strongly favored over CUG initiation in eukaryotic translation under a certain context. This prokaryotic-like preference is attributed to enhanced NUG initiation by Ψ in the second base and reduced CUG initiation by 5mC in the first base. Molecular dynamics simulation analysis of tRNAiMet anticodon base pairing to the modified codons demonstrates that Ψ universally raises the affinity of codon:anticodon pairing within the ribosomal preinitiation complex through partially mitigating discrimination against non-AUG codons imposed by eukaryotic initiation factor 1. We propose that translational control by chemical modifications of start codon bases can offer a new layer of proteome diversity regulation and therapeutic mRNA technology.

Human oncoprotein 5MP suppresses general and repeat-associated non-AUG translation via eIF3 by a common mechanism.

eIF5-mimic protein (5MP) is a translational regulatory protein that binds the small ribosomal subunit and modulates its activity. 5MP is proposed to reprogram non-AUG translation rates for oncogenes in cancer, but its role in controlling non-AUG initiated synthesis of deleterious repeat-peptide products, such as FMRpolyG observed in fragile-X-associated tremor ataxia syndrome (FXTAS), is unknown. Here, we show that 5MP can suppress both general and repeat-associated non-AUG (RAN) translation by a common mechanism in a manner dependent on its interaction with eIF3. Essentially, 5MP displaces eIF5 through the eIF3c subunit within the preinitiation complex (PIC), thereby increasing the accuracy of initiation. In Drosophila, 5MP/Kra represses neuronal toxicity and enhances the lifespan in an FXTAS disease model. These results implicate 5MP in protecting cells from unwanted byproducts of non-AUG translation in neurodegeneration.

Design of Potential IKK-ß Inhibitors using Molecular Docking and Molecular Dynamics Techniques for their Anti-cancer Potential.

AIM AND OBJECTIVE: To evaluate a set of seventy phytochemicals for their potential ability to bind the inhibitor of nuclear factor kappaB kinase beta (IKK-ß) which is a prime target for cancer and inflammatory diseases. MATERIALS AND METHODS: Seventy phytochemicals were screened against IKK-ß enzyme using DFT-based molecular docking technique and the top docking hits were carried forward for molecular dynamics (MD) simulation protocols. The ADME-Toxicity analysis was also carried out for the top docking hits. RESULTS: Sesamin, matairesinol and resveratrol were found to be the top docking hits with a total score of -413 kJ/mol, -398.11 kJ/mol and 266.73 kJ/mol, respectively. Glu100 and Gly102 were found to be the most common interacting residues. The result from MD simulation observed a stable trajectory with a binding free energy of -107.62 kJ/mol for matairesinol, -120.37 kJ/mol for sesamin and -40.56 kJ/mol for resveratrol. The ADME-Toxicity prediction observed that these compounds fall within the permissible area of Boiled-Egg and it does not violate any rule for pharmacological criteria, drug-likeness etc. Conclusion: The study interprets that dietary phytochemicals are potent inhibitors of IKK-ß enzyme with favorable binding affinity and less toxic effects. In fact, there is a gradual rise in the use of plant-derived molecules because of its lesser side effects compared to chemotherapy. The study has also provided an insight by which the phytochemicals inhibited the IKK-ß enzyme. The investigation would also provide in understanding the inhibitory mode of certain dietary phytochemicals in treating cancer.

Gcn2 eIF2α kinase mediates combinatorial translational regulation through nucleotide motifs and uORFs in target mRNAs.

The protein kinase Gcn2 is a central transducer of nutritional stress signaling important for stress adaptation by normal cells and the survival of cancer cells. In response to nutrient deprivation, Gcn2 phosphorylates eIF2α, thereby repressing general translation while enhancing translation of specific mRNAs with upstream ORFs (uORFs) situated in their 5'-leader regions. Here we performed genome-wide measurements of mRNA translation during histidine starvation in fission yeast Schizosaccharomyces pombe. Polysome analyses were combined with microarray measurements to identify gene transcripts whose translation was up-regulated in response to the stress in a Gcn2-dependent manner. We determined that translation is reprogrammed to enhance RNA metabolism and chromatin regulation and repress ribosome synthesis. Interestingly, translation of intron-containing mRNAs was up-regulated. The products of the regulated genes include additional eIF2α kinase Hri2 amplifying the stress signaling and Gcn5 histone acetyl transferase and transcription factors, together altering genome-wide transcription. Unique dipeptide-coding uORFs and nucleotide motifs, such as '5'-UGA(C/G)GG-3', are found in 5' leader regions of regulated genes and shown to be responsible for translational control.

Rutaecarpine exhibits anti-diabetic potential in high fat diet-multiple low dose streptozotocin induced type 2 diabetic mice and in vitro by modulating hepatic glucose homeostasis.

Rutaecarpine, an indolopyridoquinazoline alkaloid, attracted attentions because of possessing various biological activities. The objective of this study was to investigate the effect of rutaecarpine on glucose and lipid metabolism in high fat diet-multiple low dose streptozotocin induced type 2 diabetic (HFD-db) mice and to understand the mechanism of action. HFD-db mice showed impaired glucose metabolism and lipid profile. Oral administration of rutaecarpine reduced the blood glucose levels, decreased blood hemoglobin A1c (HbA1c) levels, improved glucose tolerance and restored insulin sensitivity in HFD-db mice. Rutaecarpine also decreased body weight gain, water intake and visceral fat gain in HFD-db mice. Total cholesterol, triglycerides, very low density lipoprotein and low density lipoprotein were reduced and high density lipoprotein level was augmented in rutaecarpine treated HFD-db mice. Rutaecarpine also reduced the elevated levels of serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, urea and creatinine in HFD-db mice. Rutaecarpine significantly promoted the rate of glucose consumption, glucose uptake and glycolysis in C2C12 myotubes. Western blotting results showed that rutaecarpine augmented p-GSK-3ß and p-AMPK expression, and suppressed G6Pase expression in HepG2 cells. These results suggest that rutaecarpine might be having therapeutic importance to fight against type 2 diabetes mellitus associated with dyslipidemia.

The Interaction between the Ribosomal Stalk Proteins and Translation Initiation Factor 5B Promotes Translation Initiation.

Ribosomal stalk proteins recruit translation elongation GTPases to the factor-binding center of the ribosome. Initiation factor 5B (eIF5B in eukaryotes and aIF5B in archaea) is a universally conserved GTPase that promotes the joining of the large and small ribosomal subunits during translation initiation. Here we show that aIF5B binds to the C-terminal tail of the stalk protein. In the cocrystal structure, the interaction occurs between the hydrophobic amino acids of the stalk C-terminal tail and a small hydrophobic pocket on the surface of the GTP-binding domain (domain I) of aIF5B. A substitution mutation altering the hydrophobic pocket of yeast eIF5B resulted in a marked reduction in ribosome-dependent eIF5B GTPase activity in vitro In yeast cells, the eIF5B mutation affected growth and impaired GCN4 expression during amino acid starvation via a defect in start site selection for the first upstream open reading frame in GCN4 mRNA, as observed with the eIF5B deletion mutant. The deletion of two of the four stalk proteins diminished polyribosome levels (indicating defective translation initiation) and starvation-induced GCN4 expression, both of which were suppressible by eIF5B overexpression. Thus, the mutual interaction between a/eIF5B and the ribosomal stalk plays an important role in subunit joining during translation initiation in vivo.

Zerumbone reduces proliferation of HCT116 colon cancer cells by inhibition of TNF-alpha.

Zerumbone is a known anti-cancer herbal compound. However, the actual protein target is not fully understood or known. This investigation focus on the association of zerumbone in HCT116 colon cancer cell proliferation and its link with TNF-alpha. The study shows that with the increasing concentration of zerumbone, there was a reduction of HCT116 cells proliferation based on the cell line study and hence higher TNF-alpha inhibition based on the TNF-alpha assay. The study also emphasizes on the computational aspect by investigating the molecular docking analysis of zerumbone against TNF-alpha. The docked complex was further validated using molecular dynamics simulation studies. The docking analysis observed that alpha-beta unsaturated carbonyl scaffold is an important moiety for the anti-cancer activity of zerumbone. Furthermore, the DFT analysis also confirms the reactivity nature of zerumbone based on the frontier molecular orbital analysis.

Competition between translation initiation factor eIF5 and its mimic protein 5MP determines non-AUG initiation rate genome-wide.

In the human genome, translation initiation from non-AUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly understood. Here, we show that the non-AUG initiation rate is nearly consistent under a fixed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome profiling we identified a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUG-initiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP.

Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5.

During eukaryotic translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome and recruits the gate-keeper protein eIF1 and eIF5 to the decoding center. This is largely mediated by the N-terminal domain (NTD) of eIF3c, which can be divided into three parts: 3c0, 3c1, and 3c2. The N-terminal part, 3c0, binds eIF5 strongly but only weakly to the ribosome-binding surface of eIF1, whereas 3c1 and 3c2 form a stoichiometric complex with eIF1. 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 faces 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the 40S, whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association, facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon.

Overexpression of eIF5 or its protein mimic 5MP perturbs eIF2 function and induces ATF4 translation through delayed re-initiation.

ATF4 is a pro-oncogenic transcription factor whose translation is activated by eIF2 phosphorylation through delayed re-initiation involving two uORFs in the mRNA leader. However, in yeast, the effect of eIF2 phosphorylation can be mimicked by eIF5 overexpression, which turns eIF5 into translational inhibitor, thereby promoting translation of GCN4, the yeast ATF4 equivalent. Furthermore, regulatory protein termed eIF5-mimic protein (5MP) can bind eIF2 and inhibit general translation. Here, we show that 5MP1 overexpression in human cells leads to strong formation of 5MP1:eIF2 complex, nearly comparable to that of eIF5:eIF2 complex produced by eIF5 overexpression. Overexpression of eIF5, 5MP1 and 5MP2, the second human paralog, promotes ATF4 expression in certain types of human cells including fibrosarcoma. 5MP overexpression also induces ATF4 expression in Drosophila The knockdown of 5MP1 in fibrosarcoma attenuates ATF4 expression and its tumor formation on nude mice. Since 5MP2 is overproduced in salivary mucoepidermoid carcinoma, we propose that overexpression of eIF5 and 5MP induces translation of ATF4 and potentially other genes with uORFs in their mRNA leaders through delayed re-initiation, thereby enhancing the survival of normal and cancer cells under stress conditions.

Induction of apoptosis by zerumbone isolated from Zingiber zerumbet (L.) Smith in protozoan parasite Leishmania donovani due to oxidative stress

Abstract In the present context of emergence of resistance aligned with the conventional anti-leishmanial drugs and occasional treatment failure compelled us to continue the search for replaceable therapeutic leads against Leishmaniainfection. Various ginger spices of the Zingiberaceae family are widely used as spices, flavouring agents, and medicines in Southeast Asia because of their unique flavour as well as due to their medicinal properties. Zerumbone, a natural component of Zingiber zerumbet (L.) Smith, has been studied for its pharmacological potential as antiulcer, antioxidant, anticancer, and antimicrobial. In this study, we have shown that zerumbone could induce ROS mediated apoptosis in Leishmania donovani promastigotes and also found effective in reducing intracellular amastigotes in infected-macrophages. We emphasized the potential of zerumbone to be employed in the development of new therapeutic drugs against L. donovaniinfection and provided the basis for future research on the application of transitional medicinal plants.