Molecular dynamics simulation studies of GSK-3β ATP competitive inhibitors: understanding the factors contributing to selectivity

Glycogen synthase kinase-3 is a constitutively acting, multifunctional serine threonine kinase, the role of which has been implicated in several physiological pathways and has emerged as a promising target for the treatment of type-II diabetes and Alzheimer’s disease. In order to provide a detailed understanding of the origin of selectivity determinants of ATP competitive inhibitors, molecular dynamics simulations in combination with MM-PBSA binding energy calculations were performed using crystal structures of GSK-3β and CDK-2 in complex with 12 ATP competitive inhibitors. Analysis of energy contributions indicate that electrostatic interaction energy dictates the selectivity of ATP competitive inhibitors against CDK-2. Key interactions as well as residues that potentially make a major contribution to the binding free energy were identified at the ATP binding site. This analysis stresses the need for the inhibitors to interact with Lys85, Thr138, and Arg141 in the binding site of GSK-3β to show selectivity. The residue-wise energy decomposition analysis further suggested the additional role of Gln185 in determining the selectivity of maleimides. The results obtained in this study can be utilized to design new selective GSK-3 ATP competitive inhibitors.     

Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2 alpha kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization

Heme-regulated eIF2alpha kinase (HRI) is an important enzyme that modulates protein synthesis during cellular emergency/stress conditions, such as heme deficiency in red cells. It is essential to identify the heme axial ligand(s) and/or binding sites to establish the heme regulation mechanism of HRI. Previous reports suggest that a His residue in the N-terminal region and a Cys residue in the C-terminal region trans to the His are axial ligands of the heme. Moreover, mutational analyses indicate that a residue located in the kinase insertion (KI) domain between Kinase I and Kinase II domains in the C-terminal region is an axial ligand. In the present study, we isolate the KI domain of mouse HRI and employ site-directed mutagenesis to identify the heme axial ligand. The optical absorption spectrum of the Fe(III) hemin-bound wild-type KI displays a broad Soret band at around 373nm, while that of the Fe(II) heme-bound protein contains a band at 422nm. Spectral titration studies conducted for both the Fe(III) hemin and Fe(II) heme complexes with KI support a 1:1 stoichiometry of heme iron to protein. Resonance Raman spectra of Fe(III) hemin-bound KI suggest that thiol is the axial ligand in a 5-coordinate high-spin heme complex as a major form. Electron spin resonance (ESR) spectra of Fe(III) hemin-bound KI indicate that the axial ligands are OH(-) and Cys. Since Cys385 is the only cysteine in KI, the residue was mutated to Ser, and its spectral characteristics were analyzed. The Soret band position, heme spectral titration behavior and ESR parameters of the Cys385Ser mutant were markedly different from those of wild-type KI. Based on these spectroscopic findings, we conclude that Cys385 is an axial ligand of isolated KI.     

Rewired Metabolism in Drug-resistant Leukemia Cells: A METABOLIC SWITCH HALLMARKED BY REDUCED DEPENDENCE ON EXOGENOUS GLUTAMINE*

Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid β-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.     

Physico-mechanical properties of chemically treated palm and coir fiber reinforced polypropylene composites

In this work, palm and coir fiber reinforced polypropylene bio-composites were manufactured using a single extruder and injection molding machine. Raw palm and coir were chemically treated with benzene diazonium salt to increase their compatibility with the polypropylene matrix. Both raw and treated palm and coir fiber at five level of fiber loading (15, 20, 25, 30 and 35 wt.%) was utilized during composite manufacturing. Microstructural analysis and mechanical tests were conducted. Comparison has been made between the properties of the palm and coir fiber composites. Treated fiber reinforced specimens yielded better mechanical properties compared to the raw composites, while coir fiber composites had better mechanical properties than palm fiber ones. Based on fiber loading, 30% fiber reinforced composites had the optimum set of mechanical properties.     

Adaptation of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> to Thermal Stress Yields a Thermotolerant Variant Which Also Exhibits Improved Survival at pH 2

Loss in probiotic viability upon exposure to stressful storage and transport conditions has plagued the probiotic market worldwide. Lactobacillus acidophilus is an important probiotic that is added to various functional foods. It is known to be fairly labile and susceptible to temperature variations that it encounters during processing and storage which increases production cost. It has been repeatedly demonstrated that pre-exposure to sub-lethal doses of stress, particularly, temperature and pH, leads to improved survival of various probiotics when they subsequently encounter the same stress of a much greater magnitude. Attempts to adapt L. acidophilus to temperatures as high as 65 °C to arrive at a thermotolerant variant have not been reported previously. To improve viability at elevated temperatures, we gradually adapted the L. acidophilus NCFM strain to survival at 65 °C for 40 min. Following adaptation, the variant showed a 2-log greater survival compared to wild-type at 65 °C. Interestingly, this thermotolerant variant also demonstrated a 2-log greater stability compared to wild-type at pH 2.0. The improved pH and temperature stress tolerance exhibited by this variant remained unaltered even when the strain was lyophilized. Moreover, the thermotolerant variant demonstrated improved stability compared to wild-type when stored for up to a week at 37 and 42 °C. Probiotic properties of the variant such as adherence to epithelial cells and antibacterial activity remained unaltered. This strain can potentially help address the issue of significant loss in viable cell counts of L. acidophilus which is typically encountered during probiotic manufacture and storage.     

Evaluation of Neurotransmitter Alterations in Four Distinct Brain Regions After Rapid Eye Movement Sleep Deprivation (REMSD) Induced Mania-Like Behaviour in Swiss Albino Mice

A number of neurotransmitter systems have been implicated in contributing to the pathology of mood disorders, including those of dopamine (DA), serotonin (5-HT), norepinephrine (NE) and γ-aminobutyric acid (GABA). Rapid eye movement sleep deprivation (REMSD) alters most of the neurotransmitters, which may have adverse behavioural changes and other health consequences like mania and other psychiatric disorders. The exact role of REMSD altered neurotransmitter levels and the manner in which emerging consequences lead to mania-like behaviour is poorly understood. Thus, we sought to verify the levels of neurotransmitter changes after 48, 72 and 96 h of REMSD induced mania-like behaviour in mice. We performed modified multiple platform (MMP) method of depriving the REM sleep and one group maintained as a control. To measure the hyperactivity through locomotion, exploration and behavioural despair, we performed the Open Field Test (OFT) and the Forced Swim Test (FST). Quantitative determinations of DA, 5-HT, NE and GABA concentrations in four distinct brain regions (cerebral cortex, hippocampus, midbrain, and pons) were determined by the spectrofluorimetric method. These experiments showed higher locomotion and increased swimming, struggling/climbing and decreased mobility among REMSD animals as well as disrupted concentrations of the majority of the studied neurotransmitters during REMSD. Our study indicated that REMSD results in mania-like behaviour in mice and associated disruption to neurotransmitter levels, although the exact mechanisms by which these take place remain to be determined.     

Prognostic role of EGR1 in breast cancer: a systematic review

EGR1 (early growth response 1) is dysregulated in many cancers and exhibits both tumor suppressor and promoter activities, making it an appealing target for cancer therapy. Here, we used a systematic multiomics analysis to review the expression of EGR1 and its role in regulating clinical outcomes in breast cancer (BC). EGR1 expression, its promoter methylation, and protein expression pattern were assessed using various publicly available tools. COSMIC-based somatic mutations and cBioPortal-based copy number alterations were analyzed, and the prognostic roles of EGR1 in BC were determined using Prognoscan and Kaplan-Meier Plotter. We also used bc-GenEx-Miner to investigate the EGR1 co-expression profile. EGR1 was more often downregulated in BC tissues than in normal breast tissue, and its knockdown was positively correlated with poor survival. Low EGR1 expression levels were also associated with increased risk of ER+, PR+, and HER2-BCs. High positive correlations were observed among EGR1, DUSP1, FOS, FOSB, CYR61, and JUN mRNA expression in BC tissue. This systematic review suggested that EGR1 expression may serve as a prognostic marker for BC patients and that clinicopathological parameters influence its prognostic utility. In addition to EGR1, DUSP1, FOS, FOSB, CYR61, and JUN can jointly be considered prognostic indicators for BC.     

Plasma proteome analysis of cervical intraepithelial neoplasia and cervical squamous cell carcinoma

Although cervical cancer is preventable with early detection, it remains the second most common malignancy among women. An understanding of how proteins change in their expression during a particular diseased state such as cervical cancer will contribute to an understanding of how the disease develops and progresses. Potentially, it may also lead to the ability to predict the occurrence of the disease. With this in mind, we aimed to identify differentially expressed proteins in the plasma of cervical cancer patients. Plasma from control, cervical intraepithelial neoplasia (CIN) grade 3 and squamous cell carcinoma (SCC) stage IV subjects was resolved by two-dimensional gel electrophoresis and the resulting proteome profiles compared. Differentially expressed protein spots were then identified by mass spectrometry. Eighteen proteins were found to be differentially expressed in the plasma of CIN 3 and SCC stage IV samples when compared with that of controls. Competitive ELISA further validated the expression of cytokeratin 19 and tetranectin. Functional analyses of these differentially expressed proteins will provide further insight into their potential role(s) in cervical cancer-specific monitoring and therapeutics.     

Analysis of p53 and NF-κB signaling in modulating the cardiomyocyte fate during hypertrophy

Cardiac hypertrophy leading to eventual heart failure is the most common cause of mortality throughout the world. The triggering mechanisms for cardiac hypertrophy are not clear but both apoptosis and cell proliferation have been reported in sections of failing hearts. In this study, we utilized both angiotensin II (AngII) treatment of cardiomyocytes and aortic ligation in rats (Rattus norvegicus, Wistar strain) for induction of hypertrophy to understand the cellular factors responsible for activation of apoptotic or anti-apoptotic pathway. Hypertrophy markers (ANF, β-MHC), apoptotic proteins (Bax, Bad, Fas, p53, caspase-3, PARP), and anti-apoptotic or cell proliferation marker proteins (Bcl2, NF-κB, Ki-67) were induced significantly during hypertrophy, both in vitro as well as in vivo. Co-localization of both active caspase-3 and Ki-67 was observed in hypertrophied myocytes. p53 and NF-κBp65 binding to co-activator p300 was also increased in AngII treated myocytes. Inhibition of p53 resulted in downregulation of apoptosis, NF-κB activation, and NF-κB-p300 binding; however, NF-κB inhibition did not inhibit apoptosis or p53-p300 binding. Blocking of either p53 or NF-κB by specific inhibitors resulted in decrease in cell proliferation and hypertrophy markers, suggesting that p53 initially binds to p300 and then this complex recruits NF-κB. Thus, these results indicate the crucial role of p53 in regulating both apoptotic and cell proliferation during hypertrophy.