scholarly journals Mammalian cell polyamine homeostasis is altered by the radioprotector WR1065

1998 ◽  
Vol 335 (2) ◽  
pp. 329-334 ◽  
Author(s):  
John L. A. MITCHELL ◽  
Jennifer RUPERT ◽  
Aviva LEYSER ◽  
Gary G. JUDD
Keyword(s):  
Ornithine Decarboxylase ◽  
Mammalian Cells ◽  
Regulatory Protein ◽  
Polyamine Metabolism ◽  
Intact Cells ◽  
Polyamine Synthesis ◽  
Radiation Induced ◽  
Htc Cells ◽  
Low Levels ◽  
The Stability

Mammalian cells become more susceptible to radiation-induced death and mutagenesis when restricted in their production of the natural polyamines putrescine, spermidine and spermine. The effects of polyamine deprivation are reversed by N-(2-mercaptoethyl)-1,3-diaminopropane (WR1065), a simple aminothiol that has been extensively studied for its radioprotectant properties. Because this compound and its oxidized derivative WR33278 bear some resemblance to the polyamines, it was hypothesized that radioprotection by WR1065 or its metabolites is derived, at least in part, from their ability to supplement the natural polyamines. To evaluate the ability of these aminothiol compounds to emulate polyamine function in intact cells, rat liver hepatoma (HTC) cells were treated with radioprotective doses of WR1065; the ability of this compound to affect various aspects of normal polyamine metabolism was monitored. Although cellular WR1065 was maintained at levels exceeding those of the polyamines, this aminothiol did not have any polyamine-like effect on the initial polyamine biosynthetic enzyme, ornithine decarboxylase, or on polyamine degradative reactions. On the contrary, treatment with relatively low levels of WR1065 resulted in an unexpected increase in putrescine and spermidine synthesis. WR1065 treatment enhanced the stability, and consequently the activity, of ornithine decarboxylase. This stabilization seems to result from a WR1065-induced delay in the synthesis of antizyme, a critical regulatory protein required in the feedback modulation of polyamine synthesis and transport. The increase in cellular spermidine induced by WR1065 might explain its antimutagenic properties, but is probably not a factor in protection against cell killing by radiation. This is the first evidence that compounds can be designed to control polyamine levels by targeting the activity of the regulatory protein antizyme.

scholarly journals Proteasome pathway operates for the degradation of ornithine decarboxylase in intact cells

1996 ◽  
Vol 317 (1) ◽  
pp. 77-80 ◽  
Author(s):  
Yasuko MURAKAMI ◽  
Nobuyuki TANAHASHI ◽  
Keiji TANAKA ◽  
Satoshi ŌMURA ◽  
Shin-ichi HAYASHI
Keyword(s):  
Ornithine Decarboxylase ◽  
Proteasome Inhibitor ◽  
Mammalian Cells ◽  
Degradation Pathway ◽  
Dependent Manner ◽  
Intact Cells ◽  
Htc Cells ◽  
Proteasome Pathway ◽  
Hypertonic Shock

Ornithine decarboxylase (ODC) is degraded in an ATP-dependent manner in vitro by the 26 S proteasome in the presence of antizyme, an ODC destabilizing protein induced by polyamines. In the present study we examined whether the proteasome catalyses ODC degradation in living mammalian cells. Lactacystin, the most selective proteasome inhibitor, strongly inhibited the degradation of ODC that had been induced in hepatoma tissue-culture (HTC) cells by refeeding with fresh medium. Furthermore the inhibitor inhibited the rapid degradation of ODC that had been induced by hypotonic shock. Interestingly, hypertonic shock was found to increase the proportion of ODC present as a complex with antizyme (the ratio of ODC–antizyme complex to total ODC). Cycloheximide, which partly inhibits rapid ODC degradation caused by hypertonic shock, also partly inhibited the increase in the ratio of ODC–antizyme complex to total ODC. These results suggest that a common ODC degradation pathway, namely the antizyme-dependent and 26 S proteasome-catalysed ODC degradation pathway, is also operating in intact cells for osmoregulated ODC degradation.

scholarly journals Osmotic stress induces variation in cellular levels of ornithine decarboxylase-antizyme

1998 ◽  
Vol 329 (3) ◽  
pp. 453-459 ◽  
Author(s):  
L. A. John MITCHELL ◽  
G. Gary JUDD ◽  
Aviva LEYSER ◽  
Chung-youl CHOE
Keyword(s):  
Steady State ◽  
Ornithine Decarboxylase ◽  
Osmotic Shock ◽  
Regulatory Protein ◽  
Feedback Regulation ◽  
Integral Role ◽  
Htc Cells ◽  
Steady State Levels ◽  
The Common ◽  
Translational Start

The polyamines, and especially putrescine, play an integral role in the physiological response of cells to varying extracellular osmotic conditions. Ornithine decarboxylase (ODC) synthesis and stability, as well as the activity of the polyamine transporter, had all been reported to be very sensitive to media osmolarity in different cells and tissues, yet the mechanism of this complex, co-ordinated response was not known. In this study we have determined that all these aspects of osmotic-shock response may be mediated by the common regulatory protein, ODC-antizyme. HTC cells were induced for antizyme and then exposed to media of reduced osmotic strength. Both antizyme activity and protein decreased rapidly, under these conditions, to new steady-state levels that depended upon the degree of reduction in media tonicity. This antizyme reduction was found to be due to a rapid increase in antizyme degradation, with a half-life decrease from 75 min down to 45 min occurring immediately upon exchanging media. In complementary experiments, increased media tonicity induced elevated antizyme levels and stability. The sensitivity of antizyme turnover to osmotic conditions was also observed in DH23b cells, which contain elevated levels of more stable antizyme. Interestingly, the two main antizyme proteins, AZ-1 and AZ-2 (presumably products from the first and second translational start sites), differed in their responses to these changing osmotic conditions. Just as feedback regulation of antizyme synthesis provides an effective mechanism for maintaining stable polyamine levels, these studies suggest that alteration in the rate of antizyme degradation may be the mechanism whereby cells adjust steady-state polyamine levels in response to stimulation or stress.

scholarly journals Ornithine decarboxylase activity in embryos depends on temperature of development rather than on the stage of development. Molecular adaptation to temperature changes in poikilothermic animals

1983 ◽  
Vol 216 (3) ◽  
pp. 597-604 ◽  
Author(s):  
A A Neyfakh ◽  
K N Yarygin ◽  
S I Gorgolyuk
Keyword(s):  
Ornithine Decarboxylase ◽  
Mammalian Cells ◽  
Decarboxylase Activity ◽  
Polyamine Synthesis ◽  
Temperature Changes ◽  
Physiological Limits ◽  
Stage Of Development ◽  
Key Enzyme ◽  
Synthesis And Degradation ◽  
Misgurnus Fossilis

The activity of ornithine decarboxylase (ODC) (the key enzyme of polyamine synthesis) in different poikilothermic animals depends on the temperatures at which they were kept just before the enzyme assay. With an increase in temperature (within physiological limits) ODC activity rises 5-25-fold within several hours. With a decrease in temperature it falls at the same rate. This effect, studied on loach (Misgurnus fossilis) embryos in detail, was also shown for embryos, larvae and some adult tissues of many species. It is not, however, observed in homoiothermic animals (chick embryos and mammalian cells), nor in bacteria and plants. Changes in polyamine concentrations follow those in ODC activity, but more slowly and to a lesser extent. It is assumed that modulation of ODC activity changes as a result of its synthesis and degradation. We suggest that the temperature-dependence of ODC activity is a mechanism of adaptation which maintains the optimal cellular concentration of polyamines for each temperature.

scholarly journals Ornithine decarboxylase, the rate-limiting enzyme of polyamine synthesis, modifies brain pathology in a mouse model of tuberous sclerosis complex

2020 ◽  
Vol 29 (14) ◽  
pp. 2395-2407
Author(s):  
David Kapfhamer ◽  
James McKenna ◽  
Caroline J Yoon ◽  
Tracy Murray-Stewart ◽  
Robert A Casero ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tuberous Sclerosis ◽  
Ornithine Decarboxylase ◽  
Tuberous Sclerosis Complex ◽  
Neurodevelopmental Disorder ◽  
Polyamine Metabolism ◽  
Polyamine Synthesis ◽  
Radial Glial ◽  
Rate Limiting ◽  
And Oxidative Stress

Abstract Tuberous sclerosis complex (TSC) is a rare autosomal dominant neurodevelopmental disorder characterized by variable expressivity. TSC results from inactivating variants within the TSC1 or TSC2 genes, leading to constitutive activation of mechanistic target of rapamycin complex 1 signaling. Using a mouse model of TSC (Tsc2-RG) in which the Tsc2 gene is deleted in radial glial precursors and their neuronal and glial descendants, we observed increased ornithine decarboxylase (ODC) enzymatic activity and concentration of its product, putrescine. To test if increased ODC activity and dysregulated polyamine metabolism contribute to the neurodevelopmental defects of Tsc2-RG mice, we used pharmacologic and genetic approaches to reduce ODC activity in Tsc2-RG mice, followed by histologic assessment of brain development. We observed that decreasing ODC activity and putrescine levels in Tsc2-RG mice worsened many of the neurodevelopmental phenotypes, including brain growth and neuronal migration defects, astrogliosis and oxidative stress. These data suggest a protective effect of increased ODC activity and elevated putrescine that modify the phenotype in this developmental Tsc2-RG model.

scholarly journals A Phosphomimetic Mutation at Ser-138 Renders Iron Regulatory Protein 1 Sensitive to Iron-Dependent Degradation

2003 ◽  
Vol 23 (19) ◽  
pp. 6973-6981 ◽  
Author(s):  
Carine Fillebeen ◽  
Danielle Chahine ◽  
Annie Caltagirone ◽  
Phillip Segal ◽  
Kostas Pantopoulos
Keyword(s):  
Mammalian Cells ◽  
Regulatory Protein ◽  
Proteasome Inhibitors ◽  
Iron Chelator ◽  
Binding Activity ◽  
Iron Regulatory Protein ◽  
Cluster Assembly ◽  
The Stability ◽  
Iron Responsive Elements ◽  
Iron Regulatory Protein 1

ABSTRACT Iron regulatory protein 1 (IRP1) binds to mRNA iron-responsive elements (IREs) and thereby controls the expression of IRE-containing mRNAs. In iron-replete cells, assembly of a cubane [4Fe-4S] cluster inhibits IRE-binding activity and converts IRP1 to a cytosolic aconitase. Earlier experiments with Saccharomyces cerevisiae suggested that phosphomimetic mutations of Ser-138 negatively affect the stability of the cluster (N. M. Brown, S. A. Anderson, D. W. Steffen, T. B. Carpenter, M. C. Kennedy, W. E. Walden, and R. S. Eisenstein, Proc. Natl. Acad. Sci. USA 95:15235-15240, 1998). Along these lines, we show here that a highly purified preparation of recombinant human IRP1 bearing a phosphomimetic S138E substitution (IRP1S138E) lacks aconitase activity, which is a hallmark of [4Fe-4S] cluster integrity. Similarly, IRP1S138E expressed in mammalian cells fails to function as aconitase. Furthermore, we demonstrate that the impairment of [4Fe-4S] cluster assembly in mammalian cells sensitizes IRP1S138E to iron-dependent degradation. This effect can be completely blocked by the iron chelator desferrioxamine or by the proteasome inhibitors MG132 and lactacystin. As expected, the stability of wild-type or phosphorylation-deficient IRP1S138A is not affected by iron manipulations. Ser-138 and flanking sequences appear to be highly conserved in the IRP1s of vertebrates, whereas insect IRP1 orthologues and nonvertebrate IRP1-like molecules contain an S138A substitution. Our data suggest that phosphorylation of Ser-138 may provide a basis for an additional mechanism for the control of vertebrate IRP1 activity at the level of protein stability.

scholarly journals Effects of diamines on ornithine decarboxylase activity in control and virally transformed mouse fibroblasts

1979 ◽  
Vol 180 (1) ◽  
pp. 87-94 ◽  
Author(s):  
D R Bethell ◽  
A E Pegg
Keyword(s):  
Cell Line ◽  
Ornithine Decarboxylase ◽  
Mammalian Cells ◽  
Decarboxylase Activity ◽  
3T3 Cells ◽  
Polyamine Synthesis ◽  
Ornithine Decarboxylase Activity ◽  
3T3 Fibroblasts ◽  
Naturally Occurring ◽  
Alpha Omega

1. The induction of ornithine decarboxylase activity in mouse 3T3 fibroblasts or an SV-40 transformed 3T3 cell line by serum was prevented by addition of the naturally occurring polyamines putrescine (butane-1,4-diamine) and spermidine. Much higher concentrations of these amines were required to fully suppress ornithine decarboxylase activity in the transformed SV-3T3 cells than in the 3T3 fibroblasts. 2. Synthetic alpha omega-diamines with 3–12 carbon atoms also prevented the increase in ornithine decarboxylase activity induced by serum in these cells. The longer chain diamines were somewhat more potent than propane-1,3-diamine in this effect, but the synthetic diamines were less active than putrescine in the 3T3 cells. There was little difference between the responses of 3T3 and SV-3T3 cells to the synthetic diamines propane-1,3-diamine and heptane-1,7-diamine. 3. These results are discussed in relation to the control of polyamine synthesis in mammalian cells.

Effect of l,3-diaminopropan-2-o1, an inhibitor of ornithine decarboxylase, on the metabolic response of liver to insulin

1982 ◽  
Vol 60 (12) ◽  
pp. 1493-1498 ◽  
Author(s):  
Yu-Wan Hu ◽  
Douglas E. Hall ◽  
Margaret E. Brosnan
Keyword(s):  
Ornithine Decarboxylase ◽  
Glycogen Content ◽  
Metabolic Response ◽  
Diabetic Rats ◽  
Polyamine Metabolism ◽  
Polyamine Synthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Streptozotocin Diabetic Rats

The effect of diaminopropanol, an inhibitor of polyamine synthesis, on the metabolic response of liver to insulin was studied in streptozotocin-diabetic rats. Insulin elicited a prompt and very marked increase in ornithine and S-adenosylmethionine decarboxylase activities and in putrescine concentration. Pretreatment of rats with diaminopropanol prevented the increase in the decarboxylases and resulted in decreased spermidine and spermine content of liver. The insulin-induced increase in glycogen content was depressed by 50% and the increase in the rate of lipogenesis in vivo was completely prevented by prior injection of diaminopropanol. These studies implicate altered polyamine metabolism in the metabolic response of liver of streptozotocin-diabetic rats to insulin.

scholarly journals Purification of A1 adenosine receptor–G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling

1999 ◽  
Vol 338 (3) ◽  
pp. 729-736 ◽  
Author(s):  
Zhenhai GAO ◽  
Anna S. ROBEVA ◽  
Joel LINDEN
Keyword(s):  
G Protein ◽  
Protein Complexes ◽  
Regulatory Protein ◽  
Chinese Hamster ◽  
Down Regulation ◽  
Intact Cells ◽  
Low Level ◽  
Receptor Purification ◽  
Guanine Nucleotide Binding ◽  
The Stability

We examined the effects of exposing A1 adenosine receptors (A1ARs) to an agonist on the stability and phosphorylation state of receptor–guanine nucleotide-binding regulatory protein (R–G-protein) complexes. Non-denatured recombinant human A1ARs extended on the N-terminus with hexahistidine (His6) and the FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) epitope (H/F) were purified to near homogeneity from stably transfected Chinese-hamster ovary (CHO)-K1 cells. Purified receptors have pharmacological properties similar to receptors in membranes. G-proteins were co-purified with 15±2% of H/F-A1AR unless receptor–G-protein (R–G) complexes were uncoupled by pre-treating cell membranes with GTP. By silver staining, purified A1AR–G-protein complexes contain receptors, G-protein α and β subunits and an unidentified 97 kDa protein. Pretreating intact cells with N6-cyclopentyladenosine (CPA) for 24 h decreased both the total number of receptors measured in membranes and the number of purified A1ARs by about 50%. In contrast, pretreating cells with CPA decreased the number of R–G complexes measured in membranes (54±6%) significantly less than it decreased the number of purified R–G complexes (78±3%) as detected by 125I-N6-(4-aminobenzyl)adenosine binding or by Western blotting Giα2. The effect of CPA to decrease the fraction of receptors purified as R–G complexes was not associated with any change in low-level A1AR phosphorylation (found on serine), or low-level phosphorylation of G-protein α or β subunits or the 97 kDa protein. These experiments reveal a novel aspect of agonist-induced down-regulation, namely a diminished stability of receptor–G-protein complexes that is manifested as uncoupling during receptor purification.

scholarly journals Cathepsin S Cleaves BAX as a Novel and Therapeutically Important Regulatory Mechanism for Apoptosis

Pharmaceutics ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 339
Author(s):  
Surinder M. Soond ◽  
Lyudmila V. Savvateeva ◽  
Vladimir A. Makarov ◽  
Neonila V. Gorokhovets ◽  
Paul A. Townsend ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Regulatory Mechanism ◽  
Renal Clear Cell Carcinoma ◽  
Cathepsin S ◽  
Apoptotic Pathway ◽  
Intact Cells ◽  
Unique Role ◽  
Bax Protein ◽  
Anti Cancer

Certain lysosomal cathepsin proteins have come into focus as being good candidates for therapeutic targeting, based on them being over-expressed in a variety of cancers and based on their regulation of the apoptotic pathway. Here, we report novel findings that highlight the ability of cathepsin S expression to be up-regulated under Paclitaxel-stimulatory conditions in kidney cell lines and it being able to cleave the apoptotic p21 BAX protein in intact cells and in vitro. Consistent with this, we demonstrate that this effect can be abrogated in vitro and in mammalian cells under conditions that utilize dominant-inhibitory cathepsin S expression, cathepsin S expression-knockdown and through the activity of a novel peptide inhibitor, CS-PEP1. Moreover, we report a unique role for cathepsin S in that it can cleave a polyubiquitinated-BAX protein intermediate and is a step that may contribute to down-regulating post-translationally-modified levels of BAX protein. Finally, CS-PEP1 may possess promising activity as a potential anti-cancer therapeutic against chemotherapeutic-resistant Renal Clear Cell Carcinoma kidney cancer cells and for combined uses with therapeutics such as Paclitaxel.

scholarly journals Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1834
Author(s):  
Tomoko Okada ◽  
Toshihiko Ogura
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Related Gene ◽  
Autophagosome Formation ◽  
Intact Cells ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Difference ◽  
Related Proteins ◽  
Scanning Electron

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

Sign in / Sign up

Export Citation Format

Share Document