scholarly journals Characteristics of Human OAS1 Isoform Proteins

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 152 ◽  
Author(s):  
Han Di ◽  
Husni Elbahesh ◽  
Margo A. Brinton
Keyword(s):  
Mammalian Cells ◽  
Cellular Localization ◽  
Hek293 Cells ◽  
Poly I:C ◽  
Synthetase Activity ◽  
Binding Partner ◽  
Rrna Cleavage ◽  
Functional Capacities ◽  
Two Hybrid

The human OAS1 (hOAS1) gene produces multiple possible isoforms due to alternative splicing events and sequence variation among individuals, some of which affect splicing. The unique C-terminal sequences of the hOAS1 isoforms could differentially affect synthetase activity, protein stability, protein partner interactions and/or cellular localization. Recombinant p41, p42, p44, p46, p48, p49 and p52 hOAS1 isoform proteins expressed in bacteria were each able to synthesize trimer and higher order 2′-5′ linked oligoadenylates in vitro in response to poly(I:C). The p42, p44, p46, p48 and p52 isoform proteins were each able to induce RNase-mediated rRNA cleavage in response to poly(I:C) when overexpressed in HEK293 cells. The expressed levels of the p42 and p46 isoform proteins were higher than those of the other isoforms, suggesting increased stability in mammalian cells. In a yeast two-hybrid screen, Fibrillin1 (FBN1) was identified as a binding partner for hOAS1 p42 isoform, and Supervillin (SVIL) as a binding partner for the p44 isoform. The p44-SVIL interaction was supported by co-immunoprecipitation data from mammalian cells. The data suggest that the unique C-terminal regions of hOAS1 isoforms may mediate the recruitment of different partners, alternative functional capacities and/or different cellular localization.

scholarly journals Complex II subunit SDHD is critical for cell growth and metabolism, which can be partially restored with a synthetic ubiquinone analog

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Aloka B. Bandara ◽  
Joshua C. Drake ◽  
David A. Brown
Keyword(s):  
Mammalian Cells ◽  
Krebs Cycle ◽  
Atp Synthesis ◽  
Hek293 Cells ◽  
Knockout Mutant ◽  
Complex Ii ◽  
Growth Defects ◽  
Transport Chain ◽  
Mutant Cells

Abstract Background Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist. Results Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells. Conclusions Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.

Enhanced xenobiotic transporter expression in normal teleost hepatocytes: response to environmental and chemotherapeutic toxins

2001 ◽  
Vol 204 (2) ◽  
pp. 217-227
Author(s):  
J.A. Albertus ◽  
R.O. Laine
Keyword(s):  
Mammalian Cells ◽  
Cellular Localization ◽  
Fundulus Heteroclitus ◽  
Environmental Pollutants ◽  
Human Tumor ◽  
Aquatic Organisms ◽  
In Vivo Studies ◽  
Multixenobiotic Resistance

Many aquatic organisms are resistant to environmental pollutants, probably because their inherent multi-drug-resistant protein extrusion pump (pgp) can be co-opted to handle man-made pollutants. This mechanism of multixenobiotic resistance is similar to the mechanism of multidrug resistance exhibited in chemotherapy-resistant human tumor cells. In the present study, a variety of techniques were used to characterize this toxin defense system in killifish (Fundulus heteroclitus) hepatocytes. The cellular localization and activity of the putative drug efflux system were evaluated. In addition, in vitro and in vivo studies were used to examine the range of expression of this putative drug transporter in the presence of environmental and chemotherapeutic toxins. The broad range of pgp expression generally observed in transformed mammalian cells was found in normal cells of our teleost model. Our findings suggest that the expression of the pgp gene in the killifish could be an excellent indicator of toxin levels or stressors in the environment.

scholarly journals Mammalian Septins Regulate Microtubule Stability through Interaction with the Microtubule-binding Protein MAP4

2005 ◽  
Vol 16 (10) ◽  
pp. 4648-4659 ◽  
Author(s):  
Brandon E. Kremer ◽  
Timothy Haystead ◽  
Ian G. Macara
Keyword(s):  
Mammalian Cells ◽  
Spectroscopic Analysis ◽  
Molecular Function ◽  
Rich Region ◽  
Intact Cells ◽  
Pronounced Increase ◽  
Binding Partner ◽  
Microtubule Stability ◽  
Proline Rich Region

Mammalian septins constitute a family of at least 12 GTP-binding proteins that can form hetero-oligomers and that are sometimes found in association with actin or microtubule filaments. However, their functions are not understood. Using RNA interference, we found that suppression of septin expression in HeLa cells caused a pronounced increase in microtubule stability. Mass spectroscopic analysis of proteins coprecipitating with Sept6 identified the microtubule-associated protein MAP4 as a septin binding partner. A small, proline-rich region in the C-terminal half of MAP4 bound directly to a Sept 2:6:7 heterotrimer, and to the Sept2 monomer. The trimer blocked the ability of this MAP4 fragment to bind and bundle microtubules in vitro. In intact cells, MAP4 was required for the stabilization of microtubules induced by septin depletion. Moreover, septin depletion increased the number of cells with abnormal nuclei, and this effect was blocked by gene silencing of MAP4. These data identify a novel molecular function for septins in mammalian cells: the modulation of microtubule dynamics through interaction with MAP4.

scholarly journals A Mammalian Ortholog of Saccharomyces cerevisiae Vac14 That Associates with and Up-Regulates PIKfyve Phosphoinositide 5-Kinase Activity

2004 ◽  
Vol 24 (23) ◽  
pp. 10437-10447 ◽  
Author(s):  
Diego Sbrissa ◽  
Ognian C. Ikonomov ◽  
Jana Strakova ◽  
Rajeswari Dondapati ◽  
Krzysztof Mlak ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Ectopic Expression ◽  
Multivesicular Body ◽  
Hek293 Cells ◽  
Lipid Kinase ◽  
Morphological Alterations ◽  
Protein Levels ◽  
Interfering Rna

ABSTRACT Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P2, produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P2 production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P2 conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P2 synthesis and endomembrane homeostasis in mammalian cells.

scholarly journals The NS3 protein of rice hoja blanca virus suppresses RNA silencing in mammalian cells

2008 ◽  
Vol 89 (1) ◽  
pp. 336-340 ◽  
Author(s):  
Esther Schnettler ◽  
Hans Hemmes ◽  
Rob Goldbach ◽  
Marcel Prins
Keyword(s):  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Hek293 Cells ◽  
Rnai Pathway ◽  
Luciferase Expression ◽  
Hairpin Rna ◽  
Rnai Suppressor ◽  
Ns3 Protein ◽  
Rnai Response

The NS3 protein of the tenuivirus rice hoja blanca virus (RHBV) has previously been shown to represent the viral RNA interference (RNAi) suppressor and is active in both plant and insect cells by binding short interfering RNAs (siRNAs) in vitro. Using a firefly luciferase-based silencing assay it is described here that NS3 is also active in mammalian cells. This activity is independent of the inducer molecule used. Using either synthetic siRNAs or a short hairpin RNA construct, NS3 was able to significantly suppress the RNAi-mediated silencing of luciferase expression in both monkey (Vero) and human (HEK293) cells. These results support the proposed mode of action of NS3 to act by sequestering siRNAs, the key molecules of the RNAi pathway conserved in all eukaryotes. The possible applications of this protein in modulating RNAi and investigating the proposed antiviral RNAi response in mammalian cell systems are discussed.

scholarly journals Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Amal Alex ◽  
Valentina Piano ◽  
Soumitra Polley ◽  
Marchel Stuiver ◽  
Stephanie Voss ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Mammalian Cells ◽  
Cellular Localization ◽  
Mitotic Checkpoint ◽  
Transferase Activity ◽  
Checkpoint Signaling ◽  
Chemically Modified ◽  
Promising Tool

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.

scholarly journals Complex II Subunit SDHD is Critical for Cell Growth and Metabolism, Which can be Partially Restored with A Synthetic Ubiquinone Analog

2020 ◽  
Author(s):  
Aloka B Bandara ◽  
David A Brown ◽  
Joshua Drake
Keyword(s):  
Mammalian Cells ◽  
Krebs Cycle ◽  
Atp Synthesis ◽  
Hek293 Cells ◽  
Knockout Mutant ◽  
Complex Ii ◽  
Growth Defects ◽  
Transport Chain ◽  
Mutant Cells

Abstract Background: Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist. Results: Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells. Conclusions: Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.

scholarly journals Native adiponectin in serum binds to mammalian cells expressing T-cadherin, but not AdipoRs or calreticulin

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Shunbun Kita ◽  
Shiro Fukuda ◽  
Norikazu Maeda ◽  
Iichiro Shimomura
Keyword(s):  
Cell Surface ◽  
Human Serum ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Hek293 Cells ◽  
C2c12 Myotubes ◽  
Binding Partner ◽  
Surface Localization ◽  
Significant Attenuation ◽  
Cell Surface Localization

Adiponectin is an adipocyte-derived atypically abundant circulating factor that protects various organs and tissues through its receptors, AdipoRs, calreticulin, and T-cadherin. To identify the major binding partner of circulating native adiponectin, we expressed these receptors on the surface of HEK293 cells. Adiponectin, either that in mouse or human serum, purified from serum, or produced by mammalian cells, bound to cells expressing T-cadherin, but not to those expressing AdipoR1 or calreticulin. The stable introduction of T-cadherin and AdipoR1 into CHO cells resulted in the cell surface localization of these receptors. Native adiponectin in serum bound to cells expressing T-cadherin, not to those expressing AdipoR1. The knockdown of T-cadherin, but not AdipoRs resulted in the significant attenuation of native adiponectin binding to C2C12 myotubes. Therefore, native adiponectin binding depended on the amount of T-cadherin expressed in HEK293 cells, CHO cells, and C2C12 myotubes. Collectively, our mammalian cell-based studies suggest that T-cadherin is the major binding partner of native adiponectin in serum.

scholarly journals Anti-Inflammatory Functions of Alverine via Targeting Src in the NF-κB Pathway

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 611
Author(s):  
Chae Young Lee ◽  
Han Gyung Kim ◽  
Sang Hee Park ◽  
Seok Gu Jang ◽  
Kyung Ja Park ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Inflammatory Responses ◽  
Contractile Activity ◽  
Hek293 Cells ◽  
Poly I:C ◽  
Raw264.7 Cells ◽  
Gastric Ulcers ◽  
Anti Inflammatory

Alverine, a smooth muscle relaxant, is used to relieve cramps or spasms of the stomach and intestine. Although the effects of alverine on spontaneous and induced contractile activity are well known, its anti-inflammatory activity has not been fully evaluated. In this study, we investigated the anti-inflammatory effects of alverine in vitro and in vivo. The production of nitric oxide (NO) in RAW264.7 cells activated by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly (I:C)) was reduced by alverine. The mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α) was also dose-dependently inhibited by treatment with alverine. In reporter gene assays, alverine clearly decreased luciferase activity, mediated by the transcription factor nuclear factor κB (NF-κB) in TIR-domain-containing adapter-inducing interferon-β (TRIF)- or MyD88-overexpressing HEK293 cells. Additionally, phosphorylation of NF-κB subunits and upstream signaling molecules, including p65, p50, AKT, IκBα, and Src was downregulated by 200 μM of alverine in LPS-treated RAW264.7 cells. Using immunoblotting and cellular thermal shift assays (CETSAs), Src was identified as the target of alverine in its anti-inflammatory response. In addition, HCl/EtOH-stimulated gastric ulcers in mice were ameliorated by alverine at doses of 100 and 200 mg/kg. In conclusion, alverine reduced inflammatory responses by targeting Src in the NF-κB pathway, and these findings provide new insights into the development of anti-inflammatory drugs.

scholarly journals The Second Catalytic Domain of Protein Tyrosine Phosphatase δ (PTPδ) Binds to and Inhibits the First Catalytic Domain of PTPς

1998 ◽  
Vol 18 (5) ◽  
pp. 2608-2616 ◽  
Author(s):  
Megan J. Wallace ◽  
Christopher Fladd ◽  
Jane Batt ◽  
Daniela Rotin
Keyword(s):  
Mammalian Cells ◽  
Catalytic Domain ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Regulatory Function ◽  
Yeast Two Hybrid ◽  
Protein Tyrosine ◽  
Catalytic Domains ◽  
Two Hybrid

ABSTRACT The LAR family protein tyrosine phosphatases (PTPs), including LAR, PTPδ, and PTPς, are transmembrane proteins composed of a cell adhesion molecule-like ectodomain and two cytoplasmic catalytic domains: active D1 and inactive D2. We performed a yeast two-hybrid screen with the first catalytic domain of PTPς (PTPς-D1) as bait to identify interacting regulatory proteins. Using this screen, we identified the second catalytic domain of PTPδ (PTPδ-D2) as an interactor of PTPς-D1. Both yeast two-hybrid binding assays and coprecipitation from mammalian cells revealed strong binding between PTPς-D1 and PTPδ-D2, an association which required the presence of the wedge sequence in PTPς-D1, a sequence recently shown to mediate D1-D1 homodimerization in the phosphatase RPTPα. This interaction was not reciprocal, as PTPδ-D1 did not bind PTPς-D2. Addition of a glutathione S-transferase (GST)–PTPδ-D2 fusion protein (but not GST alone) to GST–PTPς-D1 led to ∼50% inhibition of the catalytic activity of PTPς-D1, as determined by an in vitro phosphatase assay againstp-nitrophenylphosphate. A similar inhibition of PTPς-D1 activity was obtained with coimmunoprecipitated PTPδ-D2. Interestingly, the second catalytic domains of LAR (LAR-D2) and PTPς (PTPς-D2), very similar in sequence to PTPδ-D2, bound poorly to PTPς-D1. PTPδ-D1 and LAR-D1 were also able to bind PTPδ-D2, but more weakly than PTPς-D1, with a binding hierarchy of PTPς-D1>>PTPδ-D1>LAR-D1. These results suggest that association between PTPς-D1 and PTPδ-D2, possibly via receptor heterodimerization, provides a negative regulatory function and that the second catalytic domains of this and likely other receptor PTPs, which are often inactive, may function instead to regulate the activity of the first catalytic domains.

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