scholarly journals Development of the First Fluorescence Screening Assay for the SLC39A2 Zinc Transporter

2014 ◽  
Vol 19 (6) ◽  
pp. 909-916 ◽  
Author(s):  
Marie-Christine Franz ◽  
Alexandre Simonin ◽  
Stefanie Graeter ◽  
Matthias A. Hediger ◽  
Gergely Kovacs
Keyword(s):  
Intracellular Signaling ◽  
Hek293 Cells ◽  
Zinc Transporters ◽  
Cellular Functions ◽  
Screening Assay ◽  
Prostate Epithelial Cells ◽  
Transporter Family ◽  
Prostate Cancer Development ◽  
Compound Screening ◽  
Dna And Protein Synthesis

Zinc is an essential micronutrient that is crucial for many vital cellular functions such as DNA and protein synthesis, metabolism, and intracellular signaling. Therefore, the intracellular zinc concentration is tightly regulated by zinc transporters and zinc-binding proteins. The members of the SCL39 transporter family transport zinc into the cytosol. The SLC39A2 (hZIP2) protein is highly expressed in prostate epithelial cells and was found to be involved in prostate cancer development. Thus far, there is no specific modulator available for the SLC39 transporters. The aim of this study was to develop a screening assay for compound screening targeting hZIP2. Employing the pIRES2-DsRed Express 2 bicistronic vector, we detected human ZIP2 expression at the plasma membrane in transiently transfected HEK293 cells. Using the FLIPR Tetra fluorescence plate reader, we demonstrated that ZIP2 transports Cd2+ with an apparent Km value of 53.96 nM at an extracellular pH of 6.5. The cadmium influx via hZIP2 was inhibited by zinc in a competitive manner. We found that hZIP2 activity can be measured using cadmium in the range of 0.1 to 10 µM with our assay. In summary, for the first time we developed an assay for human ZIP2 that can be adapted to other zinc transporters.

scholarly journals Stress-Based High-Throughput Screening Assays to Identify Inhibitors of Cell Envelope Biogenesis

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 808
Author(s):  
Maurice Steenhuis ◽  
Corinne M. ten Hagen-Jongman ◽  
Peter van Ulsen ◽  
Joen Luirink
Keyword(s):  
Outer Membrane ◽  
High Throughput ◽  
Stress Responses ◽  
High Throughput Screening ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Compound Screening ◽  
Outer Membrane Biogenesis

The structural integrity of the Gram-negative cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addition of antibacterial compounds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compound screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.

Angiotensin II early regulated genes in H295R human adrenocortical cells

2004 ◽  
Vol 19 (1) ◽  
pp. 106-116 ◽  
Author(s):  
Damian G. Romero ◽  
Maria Plonczynski ◽  
Gaston R. Vergara ◽  
Elise P. Gomez-Sanchez ◽  
Celso E. Gomez-Sanchez
Keyword(s):  
Gene Expression ◽  
Angiotensin Ii ◽  
Intracellular Signaling ◽  
Zona Glomerulosa ◽  
Ang Ii ◽  
Cellular Functions ◽  
Cellular Mechanisms ◽  
Mediated Effects ◽  
Aldosterone Production ◽  
H295r Cells

Evidence for the dysregulation of aldosterone synthesis in cardiovascular pathophysiology has renewed interest in the control of its production. Cellular mechanisms by which angiotensin II (ANG II) stimulates aldosterone synthesis in the adrenal zona glomerulosa are incompletely understood. To elucidate the mechanism of intracellular signaling by ANG II stimulation in the adrenal, we have studied immediate-early regulated genes in human adrenal H295R cells using cDNA microarrays. H295R cells were stimulated with ANG II for 3 h. Gene expression was analyzed by microarray technology and validated by real-time RT-PCR. Eleven genes were found to be upregulated by ANG II. These encode the proteins for ferredoxin, Nor1, Nurr1, c6orf37, CAT-1, A20, MBLL, M-Ras, RhoB, GADD45α, and a novel protein designated FLJ45273 . Maximum expression levels for all genes occurred 3–6 h after ANG II stimulation. This increase was dose dependent and preceded maximal aldosterone production. Other aldosterone secretagogues, K+and endothelin-1 (ET-1), also induced the expression of these genes with variable efficiency depending on the gene and with lower potency than ANG II. ACTH had negligible effect on gene expression except for the CAT-1 and Nurr1 genes. These ANG II-stimulated genes are involved in several cellular functions and are good candidate effectors and regulators of ANG II-mediated effects in adrenal zona glomerulosa.

Cartilage Oligomeric Matrix Protein affects prostate cancer development by altering main cellular functions

Immunobiology ◽  
2016 ◽  
Vol 221 (10) ◽  
pp. 1156
Author(s):  
Konstantinos Papadakos ◽  
Emelie Englund ◽  
Giacomo Canesin ◽  
Emma Persson ◽  
Neelanjan Vishnu ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cartilage Oligomeric Matrix Protein ◽  
Matrix Protein ◽  
Cancer Development ◽  
Cellular Functions ◽  
Prostate Cancer Development

scholarly journals Integration of Rap1 and Calcium Signaling

2020 ◽  
Vol 21 (5) ◽  
pp. 1616 ◽  
Author(s):  
Ramoji Kosuru ◽  
Magdalena Chrzanowska
Keyword(s):  
Intracellular Signaling ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Nucleotide Exchange ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins ◽  
Cardiac Excitation

Ca2+ is a universal intracellular signal. The modulation of cytoplasmic Ca2+ concentration regulates a plethora of cellular processes, such as: synaptic plasticity, neuronal survival, chemotaxis of immune cells, platelet aggregation, vasodilation, and cardiac excitation–contraction coupling. Rap1 GTPases are ubiquitously expressed binary switches that alternate between active and inactive states and are regulated by diverse families of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Active Rap1 couples extracellular stimulation with intracellular signaling through secondary messengers—cyclic adenosine monophosphate (cAMP), Ca2+, and diacylglycerol (DAG). Much evidence indicates that Rap1 signaling intersects with Ca2+ signaling pathways to control the important cellular functions of platelet activation or neuronal plasticity. Rap1 acts as an effector of Ca2+ signaling when activated by mechanisms involving Ca2+ and DAG-activated (CalDAG-) GEFs. Conversely, activated by other GEFs, such as cAMP-dependent GEF Epac, Rap1 controls cytoplasmic Ca2+ levels. It does so by regulating the activity of Ca2+ signaling proteins such as sarcoendoplasmic reticulum Ca2+-ATPase (SERCA). In this review, we focus on the physiological significance of the links between Rap1 and Ca2+ signaling and emphasize the molecular interactions that may offer new targets for the therapy of Alzheimer’s disease, hypertension, and atherosclerosis, among other diseases.

scholarly journals Armc8 is an evolutionarily conserved armadillo protein involved in cell–cell adhesion complexes through multiple molecular interactions

2019 ◽  
Vol 39 (8) ◽  
Author(s):  
Ismail Sahin Gul ◽  
Paco Hulpiau ◽  
Ellen Sanders ◽  
Frans van Roy ◽  
Jolanda van Hengel
Keyword(s):  
Intracellular Signaling ◽  
Protein Structures ◽  
Hybrid Approach ◽  
Evolutionary Relationship ◽  
Similar Process ◽  
Cellular Functions ◽  
Human Ortholog ◽  
Armadillo Repeat ◽  
Relationship Of ◽  
Cell Cell

Abstract Armadillo-repeat-containing protein 8 (Armc8) belongs to the family of armadillo-repeat containing proteins, which have been found to be involved in diverse cellular functions including cell–cell contacts and intracellular signaling. By comparative analyses of armadillo repeat protein structures and genomes from various premetazoan and metazoan species, we identified orthologs of human Armc8 and analyzed in detail the evolutionary relationship of Armc8 genes and their encoded proteins. Armc8 is a highly ancestral armadillo protein although not present in yeast. Consequently, Armc8 is not the human ortholog of yeast Gid5/Vid28. Further, we performed a candidate approach to characterize new protein interactors of Armc8. Interactions between Armc8 and specific δ-catenins (plakophilins-1, -2, -3 and p0071) were observed by the yeast two-hybrid approach and confirmed by co-immunoprecipitation and co-localization. We also showed that Armc8 interacts specifically with αE-catenin but neither with αN-catenin nor with αT-catenin. Degradation of αE-catenin has been reported to be important in cancer and to be regulated by Armc8. A similar process may occur with respect to plakophilins in desmosomes. Deregulation of desmosomal proteins has been considered to contribute to tumorigenesis.

scholarly journals The Spectrinome: The Interactome of a Scaffold Protein Creating Nuclear and Cytoplasmic Connectivity and Function

2019 ◽  
Vol 244 (15) ◽  
pp. 1273-1302 ◽  
Author(s):  
Steven R. Goodman ◽  
Daniel Johnson ◽  
Steven L. Youngentob ◽  
David Kakhniashvili
Keyword(s):  
Cell Surface ◽  
Intracellular Signaling ◽  
Cellular Protein ◽  
Human Diseases ◽  
Scaffolding Protein ◽  
Eukaryotic Cells ◽  
Cellular Functions ◽  
Cellular Compartment ◽  
Atlas Data ◽  
And Function

We provide a review of Spectrin isoform function in the cytoplasm, the nucleus, the cell surface, and in intracellular signaling. We then discuss the importance of Spectrin’s E2/E3 chimeric ubiquitin conjugating and ligating activity in maintaining cellular homeostasis. Finally we present spectrin isoform subunit specific human diseases. We have created the Spectrinome, from the Human Proteome, Human Reactome and Human Atlas data and demonstrated how it can be a useful tool in visualizing and understanding spectrins myriad of cellular functions. Impact statement Spectrin was for the first 12 years after its discovery thought to be found only in erythrocytes. In 1981, Goodman and colleagues 1 found that spectrin-like molecules were ubiquitously found in non-erythroid cells leading to a great multitude of publications over the next thirty eight years. The discovery of multiple spectrin isoforms found associated with every cellular compartment, and representing 2-3% of cellular protein, has brought us to today’s understanding that spectrin is a scaffolding protein, with its own E2/E3 chimeric ubiquitin conjugating ligating activity that is involved in virtually every cellular function. We cover the history, localized functions of spectrin isoforms, human diseases caused by mutations, and provide the spectrinome: a useful tool for understanding the myriad of functions for one of the most important proteins in all eukaryotic cells.

scholarly journals CD81 Associates with 14-3-3 in a Redox-regulated Palmitoylation-dependent Manner

2004 ◽  
Vol 279 (19) ◽  
pp. 19401-19406 ◽  
Author(s):  
Krista L. Clark ◽  
Alisha Oelke ◽  
Megan E. Johnson ◽  
Kenneth D. Eilert ◽  
Patrick C. Simpson ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Cellular Proliferation ◽  
Vital Role ◽  
Adapter Proteins ◽  
Dependent Manner ◽  
Cellular Functions ◽  
Intracellular Loop ◽  
Cellular Redox ◽  
Cytoplasmic Tails ◽  
Molecular Mechanism Of Action

As a member of the tetraspanin superfamily of proteins, CD81 has been linked to a number of biologic functions including cellular proliferation, differentiation, activation, and degranulation. As a co-receptor for hepatitis C virus, and a requirement for hepatocytes for infectivity of humanPlasmodium falciparumand rodentP. yoeliisporozoite infectivity, CD81 may also play a vital role in pathology. Despite the importance of CD81 in multiple cellular functions, the molecular mechanism of action of CD81 in these processes has remained elusive. Here we report an association between CD81 and the epsilon isoform of 14-3-3, a serine/threonine-binding intracellular signaling protein. Furthermore, we provide evidence that in human, this association is influenced by the palmitoylation state of the CD81 cytoplasmic tails. We have generated a series of CD81 cysteine mutants to identify palmitoylated intracellular motifs of CD81, and reveal palmitoylation on the N- and C-terminal tails as well as the intracellular loop between transmembrane domains 2 and 3. One of these mutants lacks all five of its intracellular cysteines and therefore cannot be palmitoylated. This unpalmitoylated version of CD81 shows constitutive association with 14-3-3. Interestingly, we find that under oxidative conditions, CD81 palmitoylation is inhibited and that condition correlates with the association of CD81 and 14-3-3. These finding suggest that CD81 signaling events could be mediated by 14-3-3 adapter proteins, and these signals may be dependent on cellular redox.

scholarly journals Multifaceted Roles of Mitochondrial Components and Metabolites in Metabolic Diseases and Cancer

2020 ◽  
Vol 21 (12) ◽  
pp. 4405 ◽  
Author(s):  
Jean Nakhle ◽  
Anne-Marie Rodriguez ◽  
Marie-Luce Vignais
Keyword(s):  
Intracellular Signaling ◽  
Metabolic Diseases ◽  
Tca Cycle ◽  
Target Cells ◽  
Intercellular Signaling ◽  
Cellular Functions ◽  
Mitochondrial Dysfunctions ◽  
Metabolic Functions ◽  
Transport Chain ◽  
Cellular Components

Mitochondria are essential cellular components that ensure physiological metabolic functions. They provide energy in the form of adenosine triphosphate (ATP) through the electron transport chain (ETC). They also constitute a metabolic hub in which metabolites are used and processed, notably through the tricarboxylic acid (TCA) cycle. These newly generated metabolites have the capacity to feed other cellular metabolic pathways; modify cellular functions; and, ultimately, generate specific phenotypes. Mitochondria also provide intracellular signaling cues through reactive oxygen species (ROS) production. As expected with such a central cellular role, mitochondrial dysfunctions have been linked to many different diseases. The origins of some of these diseases could be pinpointed to specific mutations in both mitochondrial- and nuclear-encoded genes. In addition to their impressive intracellular tasks, mitochondria also provide intercellular signaling as they can be exchanged between cells, with resulting effects ranging from repair of damaged cells to strengthened progression and chemo-resistance of cancer cells. Several therapeutic options can now be envisioned to rescue mitochondria-defective cells. They include gene therapy for both mitochondrial and nuclear defective genes. Transferring exogenous mitochondria to target cells is also a whole new area of investigation. Finally, supplementing targeted metabolites, possibly through microbiota transplantation, appears as another therapeutic approach full of promises.

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