Faculty Opinions recommendation of RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.

Cysteine levels are carefully regulated in mammals to balance metabolic needs against the potential for cytotoxicity. It has been postulated that one of the major regulators of intracellular cysteine levels in mammals is cysteine dioxygenase (CDO). Hepatic expression of this catabolic enzyme increases dramatically in response to increased cysteine availability and may therefore be part of a homeostatic response to shunt excess toxic cysteine to more benign metabolites such as sulfate or taurine. Direct experimental evidence, however, is lacking to support the hypothesis that CDO is capable of altering steady-state intracellular cysteine levels. In this study, we expressed either the wild-type (WT) or a catalytically inactivated mutant (H86A) isoform of CDO in HepG2/C3A cells (which do not express endogenous CDO protein) and cultured them in different concentrations of extracellular cysteine. WT CDO, but not H86A CDO, was capable of reducing intracellular cysteine levels in cells incubated in physiologically relevant concentrations of cysteine. WT CDO also decreased the glutathione pool and potentiated the toxicity of CdCl2. These results demonstrate that CDO is capable of altering intracellular cysteine levels as well as glutathione levels.

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DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

The Journal of Cell Biology ◽

10.1083/jcb.125.3.625 ◽

1994 ◽

Vol 125 (3) ◽

pp. 625-638 ◽

Cited By ~ 176

Author(s):

J Lukas ◽

H Müller ◽

J Bartkova ◽

D Spitkovsky ◽

A A Kjerulff ◽

...

Keyword(s):

Cell Cycle ◽

Complex Formation ◽

Cyclin D1 ◽

Cell Lines ◽

T Antigen ◽

Regulatory Function ◽

Retinoblastoma Gene ◽

Checkpoint Control ◽

Wild Type ◽

In Cells

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.

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Seipin traps triacylglycerols to facilitate their nanoscale clustering in the ER membrane

10.1101/2020.10.26.355065 ◽

2020 ◽

Author(s):

Xavier Prasanna ◽

Veijo T. Salo ◽

Shiqian Li ◽

Katharina Ven ◽

Helena Vihinen ◽

...

Keyword(s):

Lipid Droplet ◽

In Silico ◽

Lipid Membrane ◽

Wild Type ◽

Binding Interface ◽

Biomolecular Simulations ◽

Hydrophobic Helices ◽

Tag Clustering ◽

Er Membrane ◽

In Cells

AbstractSeipin is a disk-like oligomeric ER protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the α3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While seipin-S166D mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.

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The zebrafish T-box genesno tailandspadetailare required for development of trunk and tail mesoderm and medial floor plate

Development ◽

10.1242/dev.129.14.3311 ◽

2002 ◽

Vol 129 (14) ◽

pp. 3311-3323 ◽

Cited By ~ 6

Author(s):

Sharon L. Amacher ◽

Bruce W. Draper ◽

Brian R. Summers ◽

Charles B. Kimmel

Keyword(s):

Embryonic Development ◽

Neural Tube ◽

Transcriptional Regulators ◽

Floor Plate ◽

Wild Type ◽

T Box ◽

No Tail ◽

Ventral Neural Tube ◽

Plate Formation ◽

In Cells

T-box genes encode transcriptional regulators that control many aspects of embryonic development. Here, we demonstrate that the mesodermally expressed zebrafish spadetail (spt)/VegT and no tail (ntl)/Brachyury T-box genes are semi-redundantly and cell-autonomously required for formation of all trunk and tail mesoderm. Despite the lack of posterior mesoderm in spt–;ntl– embryos, dorsal-ventral neural tube patterning is relatively normal, with the notable exception that posterior medial floor plate is completely absent. This contrasts sharply with observations in single mutants, as mutations singly in ntl or spt enhance posterior medial floor plate development. We find that ntl function is required to repress medial floor plate and promote notochord fate in cells of the wild-type notochord domain and that spt and ntl together are required non cell-autonomously for medial floor plate formation, suggesting that an inducing signal present in wild-type mesoderm is lacking in spt–;ntl– embryos.

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Abstract 357: Manganese Superoxide Dismutase: a Novel Mediator of Heart Failure Development and Progression

Circulation Research ◽

10.1161/res.117.suppl_1.357 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Sumitra Miriyala ◽

Mini Chandra ◽

Benjamin Maxey ◽

Daret K St. Clair ◽

Manikandan Panchatcharam

Keyword(s):

Superoxide Dismutase ◽

Oxygen Consumption ◽

Manganese Superoxide Dismutase ◽

Consumption Rate ◽

Primary Source ◽

Wild Type ◽

Membrane Pore ◽

Manganese Superoxide ◽

Rate Of Oxygen Consumption ◽

In Cells

Manganese Superoxide Dismutase (MnSOD), an antioxidant enzyme that catalyzes the conversion of superoxide radicals (O 2 •-) in mitochondria. Constitutive activation mitochondrial reactive oxygen species (ROS) has been implicated in both the pathogenesis and the progression of cardiovascular disease. Absence of SOD2 (gene that encodes MnSOD) is found to be embryonic lethal in animal models due to impairment of mitochondrial function, most noticeably in the heart. In our earlier investigation, we have shown that the MnSOD mimetic, MnTnBuOE-2-PyP 5+ distributes 3-fold more in mitochondria than in cytosol. The exceptional ability of MnTnBuOE-2-PyP 5+ to dismute O 2 •- parallels its ability to reduce ONOO– and CO3–. Based on our earlier reports, we have generated mice that specifically lack MnSOD in cardiomyocytes (Mhy6-SOD2 Δ ). These mice showed early mortality ~4 months due to cardiac mitochondrial dysfunction. Oxidative phosphorylation (OXPHOS) in mitochondria is the predominant mode for O 2 consumption in cells, and the mitochondria are the primary source of ROS in cells due to leaked electrons. FACS analyses using Mito-Tracker Green indicated that the mass of mitochondria per cell was slightly decreased in the Mhy6-SOD2 Δ to the wild type. We then examined OXPHOS levels in Mhy6-SOD2 Δ v.s. wild type using a Seahorse XF analyzer. The rate of oxygen consumption per cells was signi[[Unable to Display Character: ﬁ]]cantly lower in Mhy6-SOD2 Δ cardiomyocytes than that in wild type. The most noticeable difference in the O 2 consumption was found in the presence of FCCP (H+ ionophore / uncoupler). FCCP is an inner membrane pore opener which resets the proton gradient between the mitochondrial matrix and the interspace, resulting in continuous transport of protons and consuming O 2 at the maximum potential. Remarkably, while the FCCP treatment increased O 2 consumption in wild type, the treatment showed no effect on the O 2 consumption in the Mhy6-SOD2 Δ cardiomyocytes. The result indicated that the low basal OXPHOS activity in Mhy6-SOD2 Δ was due to unusually low OXPHOS potential. We examined glycolysis in these cells by measuring extracellular acidi[[Unable to Display Character: ﬁ]]cation (ECAR) and the pattern exactly opposite to that of oxygen consumption rate (OCR) was observed for glycolysis rates between Mhy6-SOD2 Δ and wild type.

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The lethality of p60v-src in Saccharomyces cerevisiae and the activation of p34CDC28 kinase are dependent on the integrity of the SH2 domain

Journal of Cell Science ◽

10.1242/jcs.105.2.519 ◽

1993 ◽

Vol 105 (2) ◽

pp. 519-528

Author(s):

F. Boschelli ◽

S.M. Uptain ◽

J.J. Lightbody

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Cell Cycle Control ◽

Sh2 Domain ◽

Wild Type ◽

Type V ◽

Cdc28 Kinase ◽

In Cells

The lethal effects of the expression of the oncogenic protein tyrosine kinase p60v-src in Saccharomyces cerevisiae are associated with a loss of cell cycle control at the G1/S and G2/M checkpoints. Results described here indicate that the ability of v-Src to kill yeast is dependent on the integrity of the SH2 domain, a region of the Src protein involved in recognition of proteins phosphorylated on tyrosine. Catalytically active v-Src proteins with deletions in the SH2 domain have little effect on yeast growth, unlike wild-type v-Src protein, which causes accumulation of large-budded cells, perturbation of spindle microtubules and increased DNA content when expressed. The proteins phosphorylated on tyrosine in cells expressing v-Src differ from those in cells expressing a Src protein with a deletion in the SH2 domain. Also, unlike the wild-type v-Src protein, which drastically increases histone H1-associated Cdc28 kinase activity, c-Src and an altered v-Src protein have no effect on Cdc28 kinase activity. These results indicate that the SH2 domain is functionally important in the disruption of the yeast cell cycle by v-Src.

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STAT5A-Deficient Mice Demonstrate a Defect in Granulocyte-Macrophage Colony-Stimulating Factor–Induced Proliferation and Gene Expression

Blood ◽

10.1182/blood.v90.5.1768 ◽

1997 ◽

Vol 90 (5) ◽

pp. 1768-1776 ◽

Cited By ~ 131

Author(s):

Gerald M. Feldman ◽

Louis A. Rosenthal ◽

Xiuwen Liu ◽

Mark P. Hayes ◽

Anthony Wynshaw-Boris ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding Proteins ◽

Colony Stimulating Factor ◽

Wild Type ◽

Stat Proteins ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor ◽

In Cells

Abstract Responses of cells to cytokines typically involve the activation of a family of latent DNA binding proteins, referred to as signal transducers and activators of transcription (STAT) proteins, which are critical for the expression of early response genes. Of the seven known STAT proteins, STAT5 (originally called mammary gland factor) has been shown to be activated by several cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5, which are known to play important roles in growth and differentiation of hematopoietic precursors. In this report we have used mice that are deficient in STAT5A (one of two homologues of STAT5) to study the role of STAT5A in GM-CSF stimulation of cells. When bone marrow–derived macrophages were generated by differentiation with macrophage-CSF (M-CSF), exposure of cells from wild-type mice to GM-CSF resulted in a typical pattern of assembly of DNA binding proteins specific for the gamma activation sequence (GAS) element within the β-casein promoter. However, in cells from the STAT5A null mouse one of the shifted bands was absent. Immunoblotting analysis in the null mice showed that lack of STAT5A protein resulted in no alteration in activation of STAT5B by tyrosine phosphorylation. Proliferation experiments revealed that, when exposed to increasing concentrations of GM-CSF, cells derived from the null mice grew considerably more slowly than cells derived from the wild-type mice. Moreover, expression of GM-CSF–dependent genes, CIS and A1, was markedly inhibited in cells derived from null mice as compared with those of wild-type mice. The decreased expression observed with A1, a bcl-2 like gene, may account in part for the suppression of growth in cells from the null mice. These data suggest that the presence of STAT5A during the GM-CSF–induced assembly of STAT5 dimers is critical for the formation of competent transcription factors that are required for both gene expression and cell proliferation.

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Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells

AJP Cell Physiology ◽

10.1152/ajpcell.00415.2001 ◽

2002 ◽

Vol 282 (5) ◽

pp. C973-C979 ◽

Cited By ~ 10

Author(s):

Lukas Schwake ◽

Andreas W. Henkel ◽

Hans D. Riedel ◽

Thorsten Schlenker ◽

Matthias Both ◽

...

Keyword(s):

Hela Cells ◽

Single Cells ◽

Hereditary Hemochromatosis ◽

Inhibitory Effect ◽

Membrane Capacitance ◽

Wild Type ◽

Time Resolved ◽

Cell Membrane Capacitance ◽

Endocytic Vesicles ◽

In Cells

The hereditary hemochromatosis protein HFE is known to complex with the transferrin receptor; however, its function regarding endocytosis of transferrin is unclear. We performed patch-clamp capacitance measurements in transfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNA under the control of a tetracycline-sensitive promoter. Whole cell experiments in cells with suppressed expression of wild-type HFE revealed a decrease in membrane capacitance, reflecting predominance of endocytosis in the presence of transferrin. Cells overexpressing C282Y-mutant HFE displayed less intense capacitance decreases, whereas no significant decrease was observed in cells overexpressing wild-type HFE. The formation of single endocytic vesicles in cells with suppressed expression of wild-type HFE was greatly increased in the presence of transferrin as revealed by cell-attached recordings. According to their calculated diameters, many of these vesicles corresponded to clathrin-coated vesicles. These results suggest that wild-type HFE negatively modulates the endocytic uptake of transferrin. This inhibitory effect is attenuated in cells expressing C282Y-mutant HFE. Time-resolved measurements of cell membrane capacitance provide a powerful tool to study transferrin-induced endocytosis in single cells.

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