ARTICLE | doi:10.20944/preprints202106.0050.v1
Subject: Keywords: Montivipera bornmuelleri snake venom; F1F0-ATPase; Staphylococcus epidermidis; Escherichia coli; antibacterial activity; HCT116 cells; anticancer activity
Online: 2 June 2021 (07:21:59 CEST)
In this work, we pursued the biological characterization of the venom of Montivipera born-muelleri, a viper from the Lebanese mountains. In relativity to its antibacterial potential, the in-hibitory effect of this venom on the F1F0-ATPase enzymes of Gram-positive Staphylocoocus epider-midis and Gram-negative Escherichia coli bacteria was examined. In order to determine the de-gree of cytotoxicity of the venom on the HCT116 human colon cancer cell lines, the biological MTT proliferation and cell viability test were implemented. After validation of the enzymatic F1F0-ATPase model by the spectrophotometric method, using quercetin as the reference ligand, re-sults revealed that M. bornmuelleri venom is able to inhibit the activity of the enzyme of these two bacteria with a concentration of the order of 100-150 µg/mL. In addition, a venom concentration of 10 µg/mL was sufficient to kill the totality of HCT116 cell lines cultivated in vitro. These data show that M. bornmuelleri venom is a mixture of diverse molecules presenting activities of interest and is a potential source to explore in order to discover new drug candidates.
REVIEW | doi:10.20944/preprints202012.0320.v1
Subject: Medicine & Pharmacology, Allergology Keywords: Preeclampsia; Na/K-ATPase; marinobufagenin; Fli1
Online: 14 December 2020 (10:05:57 CET)
Despite prophylaxis and attempts to select a therapy, the frequency of preeclampsia does not decrease, and it still takes the leading position in the structure of maternal mortality and morbidity worldwide. In this review, we present a new theory of the etiology and pathogenesis of preeclampsia which is based on the interaction of Na/K-ATPase and its endogenous ligands including marinobufagenin. The signaling pathway of marinobufagenin involves an inhibition of transcriptional factor Fli1, a negative regulator of collagen synthesis, followed by deposition of collagen in the vascular tissues and altered vascular functions. Moreover, in vitro and in vivo neutralization of marinobufagenin is associated with restoration of Fli1. The inverse relationship between marinobufagenin and Fli1 opens new possibilities in the treatment of cancer: since Fli1 is a proto-oncogene, a hypothesis on suppression of Fli1 by cardiotonic steroids as potential anti-tumor therapeutic strategy is discussed as well. We propose a novel therapy of preeclampsia which is based on immunoneutralization of the marinobufagenin by monoclonal antibodies, which is capable to impair marinobufagenin-Na/K-ATPase interactions.
REVIEW | doi:10.20944/preprints202107.0244.v1
Subject: Life Sciences, Biochemistry Keywords: P4-ATPase; ABC transporter; phospholipid; cholesterol; membranes; cellular signaling
Online: 12 July 2021 (11:24:18 CEST)
Lipid composition in the cellular membranes plays an important role in maintaining the struc-tural integrity of cells and in regulating cellular signaling that controls functions of both mem-brane-anchored and cytoplasmic proteins. ATP-dependent ABC and P4-ATPase lipid transport-ers, two integral membrane proteins, are known to contribute to lipid translocation across the li-pid bilayers on the cellular membranes. In this review, we will highlight current knowledge about the role of cholesterol and phospholipids of cellular membrane in regulating cell signaling and how lipid transporters participate this process.
ARTICLE | doi:10.20944/preprints202009.0014.v1
Subject: Materials Science, Nanotechnology Keywords: mitochondria, F0F1-ATPase, carbon nanotubes, computational nanotoxicology.; QSAR; NanoQSAR
Online: 1 September 2020 (11:49:20 CEST)
Herein, we present a combination of experimental and computational study on the mitochondrial F0F1-ATPase nanotoxicity inhibition induced by single-walled carbon nanotubes (SWCNT-pristine, SWCNT-COOH). To this end, the in vitro inhibition responses in submitochondrial particles (SMP) as F0F1-ATPase enzyme were strongly dependent on the concentration assay (from 3 to 5 µg/ml) for both types of carbon nanotubes. Besides, both SWCNTs show an interaction inhibition pattern like the oligomycin A (the specific mitochondria F0F1-ATPase inhibitor). Furthermore, the best crystallography binding pose obtained for the docking complexes based on the free energy of binding (FEB), fit well with the previous in vitro evidences from the thermodynamics point of view. Following an affinity order as: FEB (oligomycin A/F0-ATPase complex) = -9.8 kcal/mol > FEB (SWCNT-COOH/F0-ATPase complex) = - 6.8 kcal/mol ~ FEB (SWCNT-pristine complex) = -5.9 kcal/mol. With predominance of van der Waals hydrophobic nanointeractions with key F0-ATPase binding site residues (Phe 55 and Phe 64). By the other hand, results on elastic network models, and fractal-surface analysis suggest that SWCNTs induce significant perturbations by triggering abnormal allosteric responses and signals propagation in the inter-residue network which could affect the substrate recognition ligand geometrical specificity of the F0F1-ATPase enzyme in order (SWCNT-pristine > SWCNT-COOH). Besides, the performed Nano-QSTR models for both SWCNTs show that this method may be used for the prediction of the nanotoxicity induced by SWCNT. Overall, the obtained results may open new avenues toward to the better understanding and prediction of new nanotoxicity mechanisms, rational drug-design based nanotechnology, and potential biomedical application in precision nanomedicine.
CONCEPT PAPER | doi:10.20944/preprints201806.0255.v1
Subject: Biology, Plant Sciences Keywords: morphogenesis, cell wall protein, Hechtian oscillator, calcium signalling, H+ ATPase
Online: 15 June 2018 (14:15:29 CEST)
Morphogenesis remains a riddle, wrapped in a mystery, inside an enigma. It remains a formidable problem viewed from many different perspectives of morphology, genetics, and computational modelling. We propose a biochemical reductionist approach that shows how both internal and external physical forces contribute to plant morphogenesis via mechanical stress-strain transduction from the primary cell wall tethered to the plasma membrane by a specific arabinogalactan protein (AGP). The resulting stress vector with direction defined by Hechtian adhesion sites, has a magnitude of a few picoNewtons amplified by a hypothetical Hechtian growth oscillator. This paradigm shift involves stress activated plasma membrane Ca2+channels and auxin-activated H+-ATPase. The proton pump dissociates periplasmic AGP-glycomodules that bind Ca2+. Thus, as the immediate source of cytosolic Ca2+ an AGP-Ca2+ capacitor directs vectorial exocytosis of cell wall precursors and auxin efflux (PIN) proteins. In toto these components comprise the Hechtian Oscillator and also the gravisensor. Thus interdependent auxin and Ca2+ morphogen gradients account for the predominance of AGPs. The size and location of a cell surface AGP-Ca2+ capacitor is essential to differentiation and explains AGP correlation with all stages of morphogenetic patterning from embryogenesis to root and shoot. Finally, evolutionary origins of the Hechtian Oscillator in the unicellular Chlorophycean algae reflect the ubiquitous role of chemiosmotic proton pumps that preceded DNA at the dawn of life.
REVIEW | doi:10.20944/preprints202112.0208.v1
Subject: Medicine & Pharmacology, General Medical Research Keywords: Spiral steroids; Ionotropin; digoxin-like materials; NaK-ATPase; potassium sparing diuretics
Online: 13 December 2021 (13:35:50 CET)
In addition to the classical steroids, which have cholesterol as a precursor, there are steroids with 7-dehydrocholesterol as a precursor. This review describes the identification of these steroids, their biosynthesis and some aspects of their function. There are three classes of these compounds, distinguished by the number of their carbon atoms, 23, 24 and 25. Each class has a spiral steroid and is a phosphodiester. Up until these investigations, no spiral steroids or steroid phosphodiesters were known. There are at least 13 compounds of which six have been purified to near homogeneity. Each one has been characterized by its mass and proposed composition. They function by regulating the NaK-ATPase. Based on the tissues in which they have been detected, each class of compound seems to regulate a different isoform of the NaK-ATPase. This is an important site of endocrine regulation.
ARTICLE | doi:10.20944/preprints202204.0230.v1
Subject: Life Sciences, Biochemistry Keywords: membrane pump theory; membrane potential; ion channel; Na/K ATPase; thermodynamics; elecrtromagnetism
Online: 26 April 2022 (06:11:34 CEST)
The membrane potential or resting potential of the neuron has been the subject of many studies. Although this theory explains the generation and maintenance of the membrane potential by direct or even facilitated diffusion, there are too many contradictions to doubt that these forces are sufficient or even at work in a process whose initial conditions are of rare complexity. The aim of this article is to show that already in the past, a competing theory has been developed whose hypothesis seems more scientifically sound. To confirm this last theory, Hirohisa Tamagawa carried out an experiment of great simplicity which makes it possible to invalidate the current theory and to question the teaching and the knowledge in Biology and Biophysics.
ARTICLE | doi:10.20944/preprints202204.0182.v1
Subject: Life Sciences, Biochemistry Keywords: membrane pump theory; membrane potential; ion channel; NA/K ATPase; Biophysics; Biology
Online: 20 April 2022 (03:42:28 CEST)
The generation and maintenance of membrane potential is a fundamental part of Membrane Pump Theory. One of the key points of this hypothesis is based on a natural or facilitated molecular diffusion through several types of ion channels and pumps like the Na/K ATPase. Following the principles of chemistry, electrostatics and geometry, it becomes clear that ion channels cannot function in this way. The ions channels cannot by their location have both a filter function and be ion concentrators, and the Na/K pump by its position in the membrane and by the proposed assumptions is not able to perform its regulatory function. The current model must absolutely be revised according to the current state of our knowledge and allow an advance in the understanding of the phenomena opening new research perspectives.
ARTICLE | doi:10.20944/preprints202011.0395.v1
Subject: Medicine & Pharmacology, Oncology & Oncogenics Keywords: TNBC; Ca2+-ATPase; SICE; HDAC inhibitors; EMT; WNT signaling; vorinostat; romidepsin; vimentin
Online: 13 November 2020 (20:50:48 CET)
The secretory pathway Ca2+-ATPase SPCA2 is a tumor suppressor in triple receptor negative breast cancer (TNBC), a highly aggressive molecular subtype that lacks tailored treatment options. Low expression of SPCA2 in TNBC confers poor survival prognosis in patients. Previous work has established that re-introducing SPCA2 to TNBC cells restores basal Ca2+ signaling, represses mesenchymal gene expression, mitigates tumor migration in vitro and metastasis in vivo. In this study, we examined the effect of histone deacetylase inhibitors (HDACi) in TNBC cell lines. We show that the pan-HDACi vorinostat and the class I HDACi romidepsin induce dose-dependent upregulation of SPCA2 transcript with concurrent downregulation of mesenchymal markers and tumor cell migration characteristic of epithelial phenotype. Silencing SPCA2 abolished the ability of HDACi to reverse epithelial to mesenchymal transition (EMT). Independent of ATPase activity, SPCA2 elevated resting Ca2+ levels to activate downstream components of non-canonical Wnt/Ca2+ signaling. HDACi treatment led to SPCA2-dependent phosphorylation of CAMKII and -catenin, turning Wnt signaling off. We conclude that SPCA2 mediates the efficacy of HDACi in reversing EMT in TNBC by a novel mode of non-canonical Wnt/Ca2+ signaling. Our findings provide incentive for screening epigenetic modulators that exploit Ca2+ signaling pathways to reverse EMT in breast tumors.
ARTICLE | doi:10.20944/preprints202110.0137.v1
Subject: Medicine & Pharmacology, Pathology & Pathobiology Keywords: Wilson disease; ATPase copper transporting beta; autophagosome-lysosome fusion; HepG2; LC3 interaction region
Online: 8 October 2021 (11:06:51 CEST)
Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which the copper metabolism and autophagy pathways interact mechanistically is vastly unexplored. Dysfunctional ATP7B, a copper-transporting ATPase, is involved in the development of monogenic Wilson disease, a disorder characterized by disturbed copper transport. Using in silico prediction, we found that ATP7B contains a number of potential binding sites for LC3, a central protein in autophagy pathway, so-called LC3 interaction regions (LIRs). The conserved LIR3, located at the C-terminal end of ATP7B, was found to directly interact with LC3B in vitro. Replacing the two conserved hydrophobic residues W1452 and L1455 of LIR3 significantly reduced interaction. Furthermore, autophagy was induced in normal human hepatocellular carcinoma cells (HepG2) leading to enhanced colocalization of ATP7B and LC3B on the autophagosome membranes. By contrast, HepG2 cells deficient of ATP7B (HepG2 ATP7B-/-) showed autophagy deficiency at elevated copper condition. This phenotype was complemented by heterologous ATP7B expression. These findings suggest a cooperative role of ATP7B and LC3B in autophagy-mediated copper clearance.
ARTICLE | doi:10.20944/preprints201807.0208.v1
Subject: Medicine & Pharmacology, General Medical Research Keywords: Na/K – ATPase; Markov chain; master equation; Src; oxidant stress; reactive oxygen species; aging
Online: 12 July 2018 (05:26:21 CEST)
Although the signaling function of Na/K ATPase is been studied for decades, the chasm between the pumping function and the signaling function of Na/K – ATPase is still an open issue. This article explores the relationship between ion pumping and signaling with attention to the amplification of oxidants through this signaling function. Starting with some experimental observations published by our laboratories and others, we develop some predictions regarding cellular oxidant stress.
ARTICLE | doi:10.20944/preprints202109.0023.v2
Subject: Medicine & Pharmacology, Gastroenterology Keywords: pSrc; Glutathione; Na/K-ATPase; metabolic prints; metabolomics; NAFLD; NASH; high-fat diet; fibrosis; inflammation
Online: 25 May 2022 (08:37:22 CEST)
BACKGROUND. Two sequelae of non-alcoholic steatohepatitis (NASH), ESLD and HCC, have become the leading causes for liver transplantation in the Western. The present study aims to approach the cellular metabolic disturbances involved in NASH progression that are associated with microbiota community changes. METHODS. Metabolic effects and microbiota community changes were explored in the murine with NASH progression by blocking the Na/K-ATPase/Src/reactive oxygen amplification loop using the synthetic targeting peptide pNaKtide. DNA from the terminal ileum microbiota habitat was obtained and amplified by PCR to develop DNA bacterial phylogenic sequence analysis of wild type and treated animals at 12, 24 and 48 weeks. Induced changes by pSrc normalization at 24 weeks were correlated with liver morphological changes, intestinal CD4+/CD8+ ratio, and liver macrophage CD14+ expression. Differences among groups were evaluated by ANOVA/t-test and Principal Component Analysis (PCA). RESULTS. Microbiota communities varied significantly at all time points (12, 24 and 48 weeks), with an increase of Verrucomicrobia and a decrease of Bacteroidetes and Firmicutes in the HFD group. Microbiota community changes regressed to their wild-type state at 24 weeks on treated animals, and those changes were associated with a decrease in liver inflammation and senescence, lower ileum CD4+/CD8+ T cells and higher liver CD14+ cells (p<0.05). Concomitantly, the metabolic disturbances in our diet-induced NASH model were normalized by NKA/Src signaling blockage and exercise with a paucity of apoptotic activity, mitigation of cell senescence, and regression of liver fibrosis (p<0.01). CONCLUSIONS. pSrc inhibition at caveolar α1-Na/K-ATPase rescinded NASH-related metabolic disturbances establishing resident physiological microbiota communities with concomitant paucity on apoptotic activity and regression of liver fibrosis; effects that were associated with both gut and liver T-lymphocyte responses.
ARTICLE | doi:10.20944/preprints201703.0232.v1
Subject: Life Sciences, Virology Keywords: Filamentous phage; M13; gp1; Zonula occludens toxin (Zot); phage assembly; assembly 27 complex; ATPase; membrane protein; molecular hinge; secretion; Walker motifs
Online: 31 March 2017 (11:13:40 CEST)
In contrast to lytic phages, filamentous phages are assembled in the inner membrane and secreted across the bacterial envelope without killing the host. For assembly and extrusion of the phage across the host cell wall, filamentous phages code for membrane-embedded morphogenesis proteins. In the outer membrane of E. coli, the protein gp4 forms a pore-like complex, while gp1 and gp11 form a complex in the inner membrane of the host. By comparing sequences with other filamentous phages, we identified putative Walker A and B motifs in gp1 with a conserved lysine in the Walker A motif (K14), and a glutamic and aspartic acid in the Walker B motif (D88, E89). In this work we demonstrate that both, Walker A and Walker B, are essential for phage production. The crucial role of these key residues suggest that gp1 is likely to be a molecular motor driving phage assembly. We further identified essential residues for the function of the assembly complex. Mutations in three out of six cysteine residues abolish phage production. Similarly, two out of six conserved glycine residues are crucial for gp1 function. We hypothesise that the residues represent molecular hinges allowing domain movement for nucleotide binding and phage assembly.