Binding Protein Complex Research Articles

Uphill energy transfer mechanism for photosynthesis in an Antarctic alga

Prasiola crispa, an aerial green alga, forms layered colonies under the severe terrestrial conditions of Antarctica. Since only far-red light is available at a deep layer of the colony, P. crispa has evolved a molecular system for photosystem II (PSII) excitation using far-red light with uphill energy transfer. However, the molecular basis underlying this system remains elusive. Here, we purified a light-harvesting chlorophyll (Chl)-binding protein complex from P. crispa (Pc-frLHC) that excites PSII with far-red light and revealed its ring-shaped structure with undecameric 11-fold symmetry at 3.13 Å resolution. The primary structure suggests that Pc-frLHC evolved from LHCI rather than LHCII. The circular arrangement of the Pc-frLHC subunits is unique among eukaryote LHCs and forms unprecedented Chl pentamers at every subunit‒subunit interface near the excitation energy exit sites. The Chl pentamers probably contribute to far-red light absorption. Pc-frLHC’s unique Chl arrangement likely promotes PSII excitation with entropy-driven uphill excitation energy transfer.

Critical Roles of METTL3 in Translation Regulation of Cancer.

Aberrant translation, a characteristic feature of cancer, is regulated by the complex and sophisticated RNA binding proteins (RBPs) in the canonical translation machinery. N6-methyladenosine (m6A) modifications are the most abundant internal modifications in mRNAs mediated by methyltransferase-like 3 (METTL3). METTL3 is commonly aberrantly expressed in different tumors and affects the mRNA translation of many oncogenes or dysregulated tumor suppressor genes in a variety of ways. In this review, we discuss the critical roles of METTL3 in translation regulation and how METTL3 and m6A reader proteins in collaboration with RBPs within the canonical translation machinery promote aberrant translation in tumorigenesis, providing an overview of recent efforts aiming to 'translate' these results to the clinic.

Synthesis, docking, and biological investigations of new coumarin-piperazine hybrids as potential antibacterial and anticancer agents

Structurally diversified coumarin analogs were found to display a remarkable array of affinity with the different molecular targets. 4-hydroxy-7-methylcoumarin was synthesized from m-cresol and malonic acid. In basic media, the 4-hydroxy group of coumarin is replaced further by the bromopropoxy group. Several potent piperazine-containing scaffolds are known for their versatility and medicinal significance. A series of novel piperazines possessing coumarin derivatives were synthesized from these 4-bromopropoxycoumarin derivatives. These analogs were evaluated for their antibacterial and anticancer activity. Compound 3 (MIC: 12.5 µg/ml, E. coli) and compound 6 (MIC: 12.5 µg/ml, E. coli, and 25 µg/ml, S. aureus) showed potent activity against bacterial pathogens. Compound 3 showed excellent antibacterial activity against S. aureus with a MIC of 6.25 µg/ml. Compound 6 showed marvelous activity against the MCF-7 cell line with IC50 of 0.003451 µM, which is far better than the reference Doxorubicin. This is also proclaimed by a docking interaction study of these compounds. In a 100-ns MD simulation, slight RMSD variations and a convergent pattern indicate that the compounds 3 and 6 bound protein complexes did not change structurally or conformationally throughout the simulation run. Furthermore, an overall drug-likeness parameter and insilico predicted ADME properties indicated that compounds could be potential leads for the development of drugs.

LncRNA ELF3-AS1 inhibits gastric cancer by forming a negative feedback loop with SNAI2 and regulates ELF3 mRNA stability via interacting with ILF2/ILF3 complex

BackgroundThe biological function of lncRNA ELF3-AS1 remains largely unknown in cancers. The cause of SNAI2 overexpression in tumor metastasis remains largely unclear. The molecular mechanisms underlying the high co-expression of antisense lncRNAs and adjacent protein-coding genes remains unclear.MethodsRNA-seq, CHIP and dual-luciferase reporter assay were performed to identify lncRNAs regulated by SNAI2. MicroRNA-seq and RNA-seq studies were conducted to reveal the biological function of ELF3-AS1 in GC. RNA pulldown and CHIRP assays were conducted to identify the protein that interacts with ELF3-AS1.ResultsA total of 123 lncRNAs were identified to be regulated by SNAI2 in GC by RNA sequencing. The ELF3 gene and antisense lncRNA ELF3-AS1 were both transcriptionally repressed by SNAI2 or SNAI1. Down-regulation of ELF3-AS1 and ELF3 predicted poor prognosis in GC. Nuclear localized lncRNA ELF3-AS1 negatively regulated GC cell cycle progression via suppressing G1/S transition and histone synthesis. ELF3-AS1 mainly inhibited GC metastasis by repressing SNAI2 signaling. Additionally, ELF3-AS1 modulated ELF3 mRNA stability by RNA-RNA interaction. The RNA duplexes formed by ELF3 mRNA and lncRNA ELF3-AS1 directly interacted with the double-stranded RNA (dsRNA) binding protein complex ILF2/ILF3 (NF45/NF90). In turn, the ILF2/ILF3 complex dynamically regulated the expression of ELF3-AS1 and ELF3 by affecting the dsRNA stability.ConclusionsThe SNAI2-ELF3-AS1 feedback loop regulates ELF3 expression at transcriptional and post-transcriptional levels and drives gastric cancer metastasis by maintaining SNAI2 overexpression. The ILF2/ILF3 complex plays a critical role in regulating dsRNA stability. In addition, our work provides a direct evidence that head-to-head antisense lncRNAs can share promoters with neighboring coding genes, which make their expression subject to similar transcriptional regulation, leading to high co-expression.

The Mycobacterium tuberculosis PE15/PPE20 complex transports calcium across the outer membrane.

The mechanisms by which nutrients traverse the Mycobacterium tuberculosis (Mtb) outer membrane remain mostly unknown and, in the absence of classical porins, likely involve specialized transport systems. Calcium ions (Ca2+) are an important nutrient and serve as a second messenger in eukaryotes, but whether bacteria have similar Ca2+ signaling systems is not well understood. To understand the basis for Ca2+ transport and signaling in Mtb, we determined Mtb's transcriptional response to Ca2+. Overall, only few genes changed expression, suggesting a limited role of Ca2+ as a transcriptional regulator. However, 2 of the most strongly down-regulated genes were the pe15 and ppe20 genes that code for members of a large family of proteins that localize to the outer membrane and comprise many intrinsically disordered proteins. PE15 and PPE20 formed a complex and PPE20 directly bound Ca2+. Ca2+-associated phenotypes such as increased ATP consumption and biofilm formation were reversed in a pe15/ppe20 knockout (KO) strain, suggesting a direct role in Ca2+ homeostasis. To test whether the PE15/PPE20 complex has a role in Ca2+ transport across the outer membrane, we created a fluorescence resonance energy transfer (FRET)-based Ca2+ reporter strain. A pe15/ppe20 KO in the FRET background showed a specific and selective loss of Ca2+ influx that was dependent on the presence of an intact outer cell wall. These data show that PE15/PPE20 form a Ca2+-binding protein complex that selectively imports Ca2+, show a distinct transport function for an intrinsically disordered protein, and support the emerging idea of a general family-wide role of PE/PPE proteins as idiosyncratic transporters across the outer membrane.

Letrozole treatment alters hippocampal gene expression in common marmosets (Callithrix jacchus)

Aromatase inhibitors (AIs) are a class of drugs commonly given to patients with estrogen receptor (ER)-dependent breast cancers to reduce estrogenic stimulation. However, AIs like Letrozole are associated with negative side effects such as cognitive deficits, sleep disturbances and hot flashes. We have previously shown that these negative effects can be recapitulated in common marmosets (Callithrix jacchus) treated with Letrozole (20 μg daily) for 4 weeks and that marmosets treated with Letrozole show increased levels of estradiol in the hippocampus (Gervais et al., 2019). In order to better understand the mechanisms through which AIs affect cognitive function and increase steroid levels in the hippocampus, we used bulk, paired-end RNA-sequencing to examine differentially expressed genes among Letrozole-treated (LET; n = 8) and vehicle-treated (VEH; n = 8) male and female animals. Gene ontology results show significant reduction across hundreds of categories, some of the most significant being inflammatory response, stress response, MHC Class II protein complex binding, T-cell activation, carbohydrate binding and signaling receptor binding in LET animals. GSEA results indicate that LET females, but not LET males, show enrichment for hormonal gene sets. Based on the transcriptional changes observed, we conclude that AIs may differentially affect the sexes in part due to processes mediated by the CYP-450 superfamily. Ongoing studies will further investigate the longitudinal effects of AIs on behavior and whether AIs increase the risk of stress-induced neurodegeneration.

TMIC-49. CHITINASE-3-LIKE 1 PROTEIN COMPLEXES REGULATE PD-1 SIGNALING-MEDIATED IMMUNOSUPPRESSION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM), the most common and lethal brain tumor with a median survival rate of only 15 months, remains largely incurable despite intensive multimodal treatment, including immunotherapeutic strategies being tested in clinical trials. GBM is highly immunosuppressive and resistant to immunotherapy because glioma cells escape from effective antitumor immunity through programing the tumor microenvironment (TME). Owing to the tremendous heterogeneity and plasticity of tumor cells and the surrounding TME, understanding the mechanisms of immune evasion by GBM remains elusive. We have recently discovered that the Chitinase-3-like-1 (CHI3L1)-Galectin-3 (Gal3) protein binding complex can selectively promote tumor-associated macrophage migration and infiltration with a protumor M2-like phenotype and T cell-mediated immunosuppression, which are governed by a transcriptional program of NF-κB/CEBPβ in the CHI3L1/Gal3-PI3K/AKT/mTOR axis. The immunoprecipitation coupled to liquid chromatography-mass spectrometry analysis revealed that galectin 3–binding protein (Gal3BP) competes with Gal3 to bind with CHI3L1 for negative regulation of the CHI3L1-Gal3 mediated processes. Interestingly, a newly-developed Gal3BP mimetic peptide can disrupt CHI3L1-Gal3 interaction, resulting in decreasing migration of M2-like bone marrow-derived macrophages (BMDMs), increasing CD8+ T cell infiltration, reversing immunosuppression, and inhibiting tumor progression in vitro and in vivo. Analyzing PD-1 signaling activation, we found that the Gal3BP mimetic peptide significantly decreased PD-L1 expression in tumor cells. Correlation analysis showed that CHI31L and Gal3 (encoded by LGALS3 gene) are significantly associated with both PD-L1 and PD-L2 in GBM patient samples. Furthermore, overexpression of CHI3L1 increased expression levels of PD-L1 and PD-L2, and CHI3L1 deletion decreased their expression in GBM patient-derived neurosphere lines. The treatment with recombinant CHI3L1 protein significantly increased PD-L1 and PD-L2 expression in M2-like BMDMs (with high levels of endogenous Gal3). Collectively, these data suggest that CHI3L1 protein complexes control the GBM immunosuppressive microenvironment by PD-1/PD-L1/PD-L2 signaling, providing new immunotherapeutic strategies for this brain cancer.

Interdependence of fucoxanthin biosynthesis and fucoxanthin-chlorophyll a/c binding proteins in Phaeodactylum tricornutum under different light intensities

Fucoxanthin (FX), a primary carotenoid, is associated with the fucoxanthin-chlorophyll a/c binding protein (FCP) complex integrated into the thylakoid membrane (TM) which functions as a light-harvesting complex in the diatom Phaeodactylum tricornutum. Here, we aimed to elucidate the FX production regulated by different light intensities via the correlation of FX biosynthesis and apoproteins composing of FCP complex. High light (HL) accelerated P. tricornutum growth more than low light (LL). The maximum values of FX content and productivity obtained under LL (1.7 mg g−1 and 2.12 mg L−1 day−1, respectively) were substantially higher than those obtained under HL (0.54 mg g−1 and 0.79 mg L−1 day−1, respectively). Notably, proteome and photosynthetic pigment analyses revealed the enrichment of FCP antennae in the LL culture TM fractions but not the HL culture. Semi-quantification of FCP antenna protein using LC–MS/MS and RNA transcriptome analyses revealed that PtLhcf5 and PtLhcf8 played crucial roles in FCP biosynthesis under LL. P. tricornutum cultured under light transition exhibited FCP formation only in the early growth stage to meet the increased photosynthetic activity requirements under LL. Meanwhile, FCP degradation could be triggered by HL throughout the cultivation period. Therefore, FX production was highly correlated with FCP formation, and LL conditions in the early growth stage were critical for higher FX productivity.

MPAD: A Database for Binding Affinity of Membrane Protein–protein Complexes and their Mutants

Membrane protein complexes are crucial for a large variety of biological functions which are mainly dictated by their binding affinity. Due to the intricate nature of their structure, however, the binding affinity of membrane proteins is less explored compared to globular proteins. Mutations in these complexes affect their binding affinity, as well as impair critical functions, and may lead to diseases. Although experimental binding affinity data have expanded in the literature, they are dispersed and it is necessary to compile them into a reliable and comprehensive database. Hence, we developed MPAD (Membrane Protein complex binding Affinity Database), which contains experimental binding affinities of membrane protein–protein complexes and their mutants along with sequence, structure, and functional information, membrane-specific features, experimental conditions, as well as literature information. MPAD has an easy-to-use interface and options to build search queries, display, sort, download, and upload the data are among the other features available to users. This database can be used to understand the factors influencing the binding affinity in membrane proteins when compared to globular proteins as well as the impact of mutations on binding affinity, which may have potential applications to structure-based drug design. MPAD can be freely accessed at https://web.iitm.ac.in/bioinfo2/mpad.

Electrostatic Contributions to the Binding Free Energy of Nicotine to the Acetylcholine Binding Protein.

Biomolecular binding relies on specific attractive interactions between two partner molecules, including electrostatics, dispersion, hydrophobicity, and solvation. Assessing the contributions of electrostatic interactions to binding is key to the understanding of ligand binding mechanisms and the design of improved biomolecular binders. For example, nicotine is a well-known agonist of nicotinic acetylcholine receptors (nAChRs), but the molecular mechanisms for the differential action of nicotine on brain and muscle nAChRs remain elusive. In this work, we have chosen the acetylcholine binding protein (AChBP) in complex with nicotine as a model system to interrogate the electrostatic contributions to nicotine binding. Our absolute binding free energy simulations confirm that nicotine binds AChBP predominantly in its protonated (charged) form. By comparing energetic contributions from decomposed interactions for either neutral or charged nicotine, our calculations shed light on the nature of the binding of nicotine to the AChBP. The preferred binding of charged nicotine over neutral nicotine originates from its stronger electrostatic interactions with AChBP, a cation-π interaction to a tryptophan residue and a hydrogen bond between nicotine and the backbone carbonyl of the tryptophan, whereas the major force driving the binding process appears to be van der Waals interactions. The various nonelectrostatic terms can also indirectly modulate the electrostatic interactions through fine-tuning the binding pose of the ligand in the binding site, providing an explanation of why the binding specificity of nicotine to the brain versus muscle nAChRs is driven by electrostatic interaction, given that the immediate binding site residues, including the key tryptophan residue, are identical in the two receptors.

Transcriptomic analysis reveals distinct mechanisms of adaptation of a polar picophytoplankter under ocean acidification conditions

Human emissions of carbon dioxide are causing irreversible changes in our oceans and impacting marine phytoplankton, including a group of small green algae known as picochlorophytes. Picochlorophytes grown in natural phytoplankton communities under future predicted levels of carbon dioxide have been demonstrated to thrive, along with redistribution of the cellular metabolome that enhances growth rate and photosynthesis. Here, using next-generation sequencing technology, we measured levels of transcripts in a picochlorophyte Chlorella, isolated from the sub-Antarctic and acclimated under high and current ambient CO2 levels, to better understand the molecular mechanisms involved with its ability to acclimate to elevated CO2. Compared to other phytoplankton taxa that induce broad transcriptomic responses involving multiple parts of their cellular metabolism, the changes observed in Chlorella focused on activating gene regulation involved in different sets of pathways such as light harvesting complex binding proteins, amino acid synthesis and RNA modification, while carbon metabolism was largely unaffected. Triggering a specific set of genes could be a unique strategy of small green phytoplankton under high CO2 in polar oceans.

Inhibition of the CtBP complex and FBXO11 enhances MHC class II expression and anti-cancer immune responses.

There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ Tcell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.

PR-DUB preserves Polycomb repression by preventing excessive accumulation of H2Aub1, an antagonist of chromatin compaction.

The Polycomb repressive complexes PRC1, PRC2, and PR-DUB repress target genes by modifying their chromatin. In Drosophila, PRC1 compacts chromatin and monoubiquitinates histone H2A at lysine 118 (H2Aub1), whereas PR-DUB is a major H2Aub1 deubiquitinase, but how H2Aub1 levels must be balanced for Polycomb repression remains unclear. We show that in early embryos, H2Aub1 is enriched at Polycomb target genes, where it facilitates H3K27me3 deposition by PRC2 to mark genes for repression. During subsequent stages of development, H2Aub1 becomes depleted from these genes and is no longer enriched when Polycomb maintains them repressed. Accordingly, Polycomb targets remain repressed in H2Aub1-deficient animals. In PR-DUB catalytic mutants, high levels of H2Aub1 accumulate at Polycomb target genes, and Polycomb repression breaks down. These high H2Aub1 levels do not diminish Polycomb protein complex binding or H3K27 trimethylation but increase DNA accessibility. We show that H2Aub1 interferes with nucleosome stacking and chromatin fiber folding in vitro. Consistent with this, Polycomb repression defects in PR-DUB mutants are exacerbated by reducing PRC1 chromatin compaction activity, but Polycomb repression is restored if PRC1 E3 ligase activity is removed. PR-DUB therefore acts as a rheostat that removes excessive H2Aub1 that, although deposited by PRC1, antagonizes PRC1-mediated chromatin compaction.

Proteomic profiling of aspergillus flavus endophthalmitis derived extracellular vesicles in an in-vivo murine model.

EVs play an important role in cell communication. In our study proteomic profiling of EVs isolated from A. flavus infected mice provided new insights into the understanding of the pathobiology of A. flavus endophthalmitis and validation of these proteins can serve as biomarkers.

Integration of protein context improves protein-based COVID-19 patient stratification

BackgroundClassification of disease severity is crucial for the management of COVID-19. Several studies have shown that individual proteins can be used to classify the severity of COVID-19. Here, we aimed to investigate whether integrating four types of protein context data, namely, protein complexes, stoichiometric ratios, pathways and network degrees will improve the severity classification of COVID-19.MethodsWe performed machine learning based on three previously published datasets. The first was a SWATH (sequential window acquisition of all theoretical fragment ion spectra) MS (mass spectrometry) based proteomic dataset. The second was a TMTpro 16plex labeled shotgun proteomics dataset. The third was a SWATH dataset of an independent patient cohort.ResultsBesides twelve proteins, machine learning also prioritized two complexes, one stoichiometric ratio, five pathways, and five network degrees, resulting a 25-feature panel. As a result, a model based on the 25 features led to effective classification of severe cases with an AUC of 0.965, outperforming the models with proteins only. Complement component C9, transthyretin (TTR) and TTR-RBP (transthyretin-retinol binding protein) complex, the stoichiometric ratio of SAA2 (serum amyloid A proteins 2)/YLPM1 (YLP Motif Containing 1), and the network degree of SIRT7 (Sirtuin 7) and A2M (alpha-2-macroglobulin) were highlighted as potential markers by this classifier. This classifier was further validated with a TMT-based proteomic data set from the same cohort (test dataset 1) and an independent SWATH-based proteomic data set from Germany (test dataset 2), reaching an AUC of 0.900 and 0.908, respectively. Machine learning models integrating protein context information achieved higher AUCs than models with only one feature type.ConclusionOur results show that the integration of protein context including protein complexes, stoichiometric ratios, pathways, network degrees, and proteins improves phenotype prediction.

Proteomics analysis of lysine crotonylation and 2-hydroxyisobutyrylation reveals significant features of systemic lupus erythematosus

Introduction/objectivesTo seek significant features of systemic lupus erythematosus (SLE) by utilizing bioinformatics analysis.MethodLiquid chromatography-tandem mass spectrometry (LC–MS/MS) was used to quantify lysine crotonylation (Kcr) and lysine 2-hydroxyisobutyrylation (Khib) in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus (SLE) patients and normal controls.ResultsSeventy-six differentially modified proteins (DMPs) dually modified by Kcr and Khib were identified between SLE patients and healthy people. GO enrichment analysis prompted significant enrichment of seventy-six DMPs in MHC class II protein complex binding and leukocyte migration. KEGG pathways were enriched in antigen processing and presentation pathway and leukocyte transendothelial migration pathway. Six DMPs (CLTC, HSPA1B, HSPA8, HSP90AB1, HSPD1, and PDIA3) were identified in antigen processing and presentation pathway, of which HSPA8 was the core protein. Significant changes of Kcr and Khib in HSPA8 may increase ATP hydrolysis and promote antigen binding to MHC II molecule. In leukocyte transendothelial migration pathway, 7 DMPs (ACTN1, ACTN4, EZR, MSN, RAC1, RHOA, and VCL) were identified. MSN was the protein with the most modification sites in this pathway. In amino terminal ferm region of MSN, Kcr and Khib expression change may lead to the adhesion between leukocytes and endothelial cells, which was an important step of leukocyte migration.ConclusionKcr and Khib may promote the antigen presentation and jointly regulate the tissue damage mediated by leukocyte migration in SLE patients, which may play key roles in the pathogenesis of SLE probably.Key Points• Antigen processing and presentation and leukocyte transendothelial migration may play key roles in the pathogenesis of SLE.

Macrophages-Related Genes Biomarkers in the Deterioration of Atherosclerosis.

BackgroundThe macrophages are involved in all stages of cardiovascular diseases, demonstrating the correlation between inflammation, atherosclerosis, and myocardial infarction (MI). Here, we aim to investigate macrophages-related genes in the deterioration of atherosclerosis.MethodsGSE41571 was downloaded and the abundance of immune cells was estimated by utilizing the xCell. By utilizing the limma test and correlation analysis, differentially expressed macrophages-related genes (DEMRGs) were documented. The functional pathways and the protein–protein interaction (PPI) network were analyzed and the hub DEMRGs were obtained. The hub DEMRGs and their interactions were analyzed using NetworkAnalyst 3.0 and for validation, the expressions of hub DEMRGs were analyzed using the GSE135055 and GSE116250 datasets as well as atherosclerosis and MI mice model.ResultsA total of 509 differentially expressed genes (DEGs) were correlated with the abundance of macrophages and were identified as DEMRGs (Pearson correlation coefficients (PCC) > 0.6), which were mainly enriched in extracellular structure organization, lysosomal membrane, MHC protein complex binding, and so on. After screening out, 28 hub DEMRGs were obtained with degrees ≥20, including GNAI1 (degree = 113), MRPS2 (degree = 56), HCK (degree = 45), SOCS3 (degree = 40), NET1 (degree = 28), and so on. After validating using Gene Expression Omnibus (GEO) datasets and the atherosclerosis and MI mice model, eight proteins were validated using ApoE-/- and C57 mice. The expression levels of proteins, including SYNJ2, NET1, FZD7, LCP2, HCK, GNB2, and PPP4C were positively correlated to left ventricular ejection fraction (LVEF), while that of EIF4EBP1 was negatively correlated to LVEF.ConclusionThe screened hub DEMRGs, SYNJ2, NET1, FZD7, LCP2, HCK, GNB2, EIF4EBP1, and PPP4C, may be therapeutic targets for treatment and prediction in the patients with plaque progression and MI recurrent events. The kit of the eight hub DEMRGs may test plaque progression and MI recurrent events and help in the diagnosis and treatment of MI-induced heart failure (HF), thus decreasing mortality and morbidity.

A screening method for binding synthetic metallo-complexes to haem proteins

The introduction of a second coordination sphere, in the form of a protein scaffold, to synthetic catalysts can be beneficial for their reactivity and substrate selectivity. Here we present semi-native polyacrylamide gel electrophoresis (semi-native PAGE) as a rapid screening method for studying metal complex-protein interactions. Such a screening is generally performed using electron spray ionization mass spectrometry (ESI-MS) and/or UV–Vis spectroscopy. Semi-native PAGE analysis has the advantage that it does not rely on spectral changes of the metal complex upon protein interaction and can be applied for high-throughput screening and optimization of complex binding. In semi-native PAGE non-denatured protein samples are loaded on a gel containing sodium dodecyl sulphate (SDS), leading to separation based on differences in structural stability. Semi-native PAGE gel runs of catalyst-protein mixtures were compared to gel runs obtained with native and denaturing PAGE. ESI-MS was additionally realised to confirm protein-complex binding. The general applicability of semi-native PAGE was investigated by screening the binding of various cobalt- and ruthenium-based compounds to three types of haem proteins.

Nonlinear molecular dynamics of quercetin in Gynocardia odorata and Diospyros malabarica fruits: Its mechanistic role in hepatoprotection.

Liver performs number of critical physiological functions in human system. Intoxication of liver leads to accumulation of free radicals that eventually cause damage, fibrosis, cirrhosis and cancer. Carbon tetrachloride (CCl4) belongs to hepatotoxin is converted to a highly reactive free radical by cytochrome P450 enzymes that causes liver damage. Plant extracts derived quercetin has substantial role in hepatoprotection. This study highlights the possible mechanism by which quercetin plays significant role in hepatoprotection. HPLC analysis revealed the abundance of quercetin in the fruit extracts of Gynocardia odorata and Diospyros malabarica, were isolated, purified and subjected to liver function analysis on Wistar rats. Post quercetin treatment improved liver function parameters in the hepatotoxic Wistar rats by augmenting bilirubin content, SGOT and SGPT activity. Gene expression profile of quercetin treated rats revealed down regulation of HGF, TIMP1 and MMP2 expressed during CCl4 induction. In silico molecular mechanism prediction suggested that quercetin has a high affinity for cell signaling pathway proteins BCL-2, JAK2 and Cytochrome P450 Cyp2E1, which all play a significant role in CCl4 induced hepatotoxicity. In silico molecular docking and molecular dynamics simulation have shown that quercetin has a plausible affinity for major signaling proteins in liver. MMGBSA studies have revealed high binding of quercetin (ΔG) -41.48±11.02, -43.53±6.55 and -39.89±5.78 kcal/mol, with BCL-2, JAK2 and Cyp2E1, respectively which led to better stability of the quercetin bound protein complexes. Therefore, quercetin can act as potent inhibitor against CCl4 induced hepatic injury by regulating BCL-2, JAK2 and Cyp2E1.

GABAA receptor participation in anxiolytic and anticonvulsant effects of (E)-3-(furan-2-yl)-1-(2hydroxy-3,4,6-trimethoxyphenyl)prop-2-en-1-one in adult zebrafish

Anxiety is a mental disorder that affects 25% of patients with epilepsy, and treatments for anxiety and seizures involve the use of benzodiazepines, a class of drugs that have many adverse effects such as decreased motor coordination, drowsiness, and sedation. Thus, new types of drugs with minimal side effects are of immediate requirement. Chalcones comprise a class of compounds with important therapeutic potential and have recently been investigated for their potential as anxiolytic and anticonvulsant agents. Therefore, this study aimed to evaluate the anxiolytic and anticonvulsant effects of the synthetic chalcone (E)-3-(furan-2-yl)-1-(2hydroxy-3,4,6-trimethoxyphenyl)prop-2-en-1-one (FURCHAL) using adult zebrafish as an animal model. Anxiolytic potential was assessed using the light/dark test and the anticonvulsant effect in 3-stage pentylenetetrazol (PTZ)-induced seizure tests. The mechanisms of the anxiolytic effect were analyzed using γ-aminobutyric acid (GABA) and the serotoninergic system. The anxiolytic effect of FURCHAL was verified by a reduction in fish locomotion, similar to diazepam (DZP), which may involve the GABAA receptor, as there was no reversal in the anxiolytic behavior of animals treated with FURCHAL by serotonergic antagonists. In addition, pretreatment with flumazenil blocked the anticonvulsant effect of FURCHAL and DZP at all three stages, indicating that FURCHAL also has anticonvulsant effects and that the presence of the α,β unsaturated aromatic system and heterocyclic moiety in FURCHAL provided greater affinity for the GABAA receptors. Molecular docking revealed that the interactions involved in the formation of the protein-binding complex FURCHAL-GABAA are formed by three H-bonds involving the oxygen atoms of FURCHAL, and notably, complexes operated in the same region of the DZP site. Thus, this study adds new evidence and highlights that FURCHAL can potentially be used to develop compounds with anxiolytic and anticonvulsant properties.