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The Journal of Biological Chemistry Dec 1998The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its...
The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422-461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422-461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422-461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFkappaB binding sequences and monitoring tumor necrosis factor alpha-mediated NFkappaB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFkappaB translocation to the nucleus.
Topics: Amino Acid Sequence; Animals; Biological Transport; Genes, Reporter; Green Fluorescent Proteins; Half-Life; Luminescent Proteins; Mice; Microscopy, Fluorescence; Molecular Sequence Data; Mutagenesis; NF-kappa B; Ornithine Decarboxylase; Recombinant Fusion Proteins; Transcription, Genetic; Tumor Necrosis Factor-alpha
PubMed: 9857028
DOI: 10.1074/jbc.273.52.34970 -
Biochimica Et Biophysica Acta Jan 2014Most proteasome substrates are marked for degradation by ubiquitin conjugation, but some are targeted by other means. The properties of these exceptional cases provide... (Review)
Review
Most proteasome substrates are marked for degradation by ubiquitin conjugation, but some are targeted by other means. The properties of these exceptional cases provide insights into the general requirements for proteasomal degradation. Here the focus is on three ubiquitin-independent substrates that have been the subject of detailed study. These are Rpn4, a transcriptional regulator of proteasome homeostasis, thymidylate synthase, an enzyme required for production of DNA precursors and ornithine decarboxylase, the initial enzyme committed to polyamine biosynthesis. It can be inferred from these cases that proteasome association and the presence of an unstructured region are the sole prerequisites for degradation. Based on that inference, artificial substrates have been designed to test the proteasome's capacity for substrate processing and its limitations. Ubiquitin-independent substrates may in some cases be a remnant of the pre-ubiquitome world, but in other cases could provide optimized regulatory solutions. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.
Topics: Animals; DNA-Binding Proteins; Humans; Ornithine Decarboxylase; Proteasome Endopeptidase Complex; Protein Structure, Tertiary; Protein Unfolding; Proteolysis; Saccharomyces cerevisiae Proteins; Thymidylate Synthase; Transcription Factors; Ubiquitin
PubMed: 23684952
DOI: 10.1016/j.bbamcr.2013.05.008 -
The Journal of Biological Chemistry Nov 2018The polyamines spermidine, spermine, and their precursor putrescine are organic polycations involved in various cellular processes and are absolutely essential for... (Review)
Review
The polyamines spermidine, spermine, and their precursor putrescine are organic polycations involved in various cellular processes and are absolutely essential for cellular proliferation. Because of their crucial function in the cell, their intracellular concentration must be maintained at optimal levels. To a large extent, this regulation is achieved through the activity of an autoregulatory loop that involves two proteins, antizyme (Az) and antizyme inhibitor (AzI), that regulate the first enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC), and polyamine uptake activity in response to intracellular polyamine levels. In this Minireview, I will discuss what has been learned about the mechanism of Az expression and its physical interaction with both ODC and AzI in the regulation of polyamines.
Topics: Animals; Gene Expression Regulation, Enzymologic; Humans; Multigene Family; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Polyamines
PubMed: 30355739
DOI: 10.1074/jbc.TM118.003339 -
Medical Sciences (Basel, Switzerland) Apr 2022Parasites of the genus cause a variety of devastating and often fatal diseases in humans and domestic animals worldwide. The need for new therapeutic strategies is... (Review)
Review
Parasites of the genus cause a variety of devastating and often fatal diseases in humans and domestic animals worldwide. The need for new therapeutic strategies is urgent because no vaccine is available, and treatment options are limited due to a lack of specificity and the emergence of drug resistance. Polyamines are metabolites that play a central role in rapidly proliferating cells, and recent studies have highlighted their critical nature in . Numerous studies using a variety of inhibitors as well as gene deletion mutants have elucidated the pathway and routes of transport, revealing unique aspects of polyamine metabolism in parasites. These studies have also shed light on the significance of polyamines for parasite proliferation, infectivity, and host-parasite interactions. This comprehensive review article focuses on the main polyamine biosynthetic enzymes: ornithine decarboxylase, -adenosylmethionine decarboxylase, and spermidine synthase, and it emphasizes recent discoveries that advance these enzymes as potential therapeutic targets against parasites.
Topics: Animals; Leishmania; Ornithine Decarboxylase; Parasites; Polyamines; Spermidine Synthase
PubMed: 35645240
DOI: 10.3390/medsci10020024 -
Journal of Cell Science Jun 2023Polyamines promote cellular proliferation. Their levels are controlled by ornithine decarboxylase antizyme 1 (Az1, encoded by OAZ1), through the proteasome-mediated,...
Polyamines promote cellular proliferation. Their levels are controlled by ornithine decarboxylase antizyme 1 (Az1, encoded by OAZ1), through the proteasome-mediated, ubiquitin-independent degradation of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis. Az1-mediated degradation of other substrates such as cyclin D1 (CCND1), DNp73 (TP73) or Mps1 regulates cell growth and centrosome amplification, and the currently known six Az1 substrates are all linked with tumorigenesis. To understand whether Az1-mediated protein degradation might play a role in regulating other cellular processes associated with tumorigenesis, we employed quantitative proteomics to identify novel Az1 substrates. Here, we describe the identification of LIM domain and actin-binding protein 1 (LIMA1), also known as epithelial protein lost in neoplasm (EPLIN), as a new Az1 target. Interestingly, between the two EPLIN isoforms (α and β), only EPLIN-β is a substrate of Az1. The interaction between EPLIN-β and Az1 appears to be indirect, and EPLIN-β is degraded by Az1 in a ubiquitination-independent manner. Az1 absence leads to elevated EPLIN-β levels, causing enhanced cellular migration. Consistently, higher LIMA1 levels correlate with poorer overall survival of colorectal cancer patients. Overall, this study identifies EPLIN-β as a novel Az1 substrate regulating cellular migration.
Topics: Humans; Ornithine Decarboxylase; Ubiquitin; Protein Isoforms; Carcinogenesis; Cytoskeletal Proteins
PubMed: 37325974
DOI: 10.1242/jcs.260427 -
FASEB Journal : Official Publication of... May 2022Ornithine decarboxylase (ODC; EC 4.1.1.17) catalyzes the conversion of ornithine to putrescine, the rate-limiting first step for de novo polyamine biosynthesis....
Ornithine decarboxylase (ODC; EC 4.1.1.17) catalyzes the conversion of ornithine to putrescine, the rate-limiting first step for de novo polyamine biosynthesis. Previously, we reported that genetic knockdown of xanthine dehydrogenase 1 (XDH1)-a gene encoding the enzyme involved in the last two steps of uric acid synthesis-causes an increase in ODC transcript levels in fat body of blood-fed Aedes aegypti mosquitoes, suggesting a crosstalk at molecular level between XDH1 and ODC during nitrogen disposal. To further investigate the role of ODC in nitrogen metabolism, we conducted several biochemical and genetic analyses in sugar- and blood-fed A. aegypti females. Distinct ODC gene and protein expression patterns were observed in mosquito tissues dissected during the first gonotrophic cycle. Both pharmacological and RNA interference-mediated knockdown of ODC negatively impacted mosquito survival, disrupted nitrogen waste disposal, delayed oviposition onset, and decreased fecundity in vitellogenic blood-fed females. A lag in the expression of two major digestive serine proteases, a reduction of blood meal digestion in the midgut, and a decrease in vitellogenin yolk protein uptake in ovarian follicles were observed by western blots in ODC-deficient females. Moreover, genetic silencing of ODC showed a broad transcriptional modulation of genes encoding proteins involved in multiple metabolic pathways in mosquito fat body, midgut, and Malpighian tubules prior to and after blood feeding. All together, these data demonstrate that ODC plays an essential role in mosquito metabolism, and that ODC crosstalks with multiple genes and proteins to prevent deadly nitrogen perturbations in A. aegypti females.
Topics: Aedes; Animals; Female; Nitrogen; Ornithine; Ornithine Decarboxylase; Oviposition
PubMed: 35344219
DOI: 10.1096/fj.202200008R -
The Journal of Biological Chemistry May 2006Ornithine decarboxylase (ODC) initiates the polyamine biosynthetic pathway. The amount of ODC is altered in response to many growth factors, oncogenes, and tumor... (Review)
Review
Ornithine decarboxylase (ODC) initiates the polyamine biosynthetic pathway. The amount of ODC is altered in response to many growth factors, oncogenes, and tumor promoters and to changes in polyamine levels. Susceptibility to tumor development is increased in transgenic mice expressing high levels of ODC and is decreased in mice with reduced ODC due to loss of one ODC allele or elevated expression of antizyme, a protein that stimulates ODC degradation. This review describes key factors that contribute to the regulation of ODC levels, which can occur at the levels of transcription, translation, and protein turnover.
Topics: Alleles; Animals; Gene Expression Regulation, Enzymologic; Humans; Mice; Models, Biological; Ornithine Decarboxylase; Polyamines; Promoter Regions, Genetic; Protein Biosynthesis; RNA, Messenger; Transcription, Genetic
PubMed: 16459331
DOI: 10.1074/jbc.R500031200 -
Structural basis of binding and inhibition of ornithine decarboxylase by 1-amino-oxy-3-aminopropane.The Biochemical Journal Dec 2021Ornithine decarboxylase (ODC) is the rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are oncometabolites that are required for proliferation, and...
Ornithine decarboxylase (ODC) is the rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are oncometabolites that are required for proliferation, and pharmaceutical ODC inhibition is pursued for the treatment of hyperproliferative diseases, including cancer and infectious diseases. The most potent ODC inhibitor is 1-amino-oxy-3-aminopropane (APA). A previous crystal structure of an ODC-APA complex indicated that APA non-covalently binds ODC and its cofactor pyridoxal 5-phosphate (PLP) and functions by competing with the ODC substrate ornithine for binding to the catalytic site. We have revisited the mechanism of APA binding and ODC inhibition through a new crystal structure of APA-bound ODC, which we solved at 2.49 Å resolution. The structure unambiguously shows the presence of a covalent oxime between APA and PLP in the catalytic site, which we confirmed in solution by mass spectrometry. The stable oxime makes extensive interactions with ODC but cannot be catabolized, explaining APA's high potency in ODC inhibition. In addition, we solved an ODC/PLP complex structure with citrate bound at the substrate-binding pocket. These two structures provide new structural scaffolds for developing more efficient pharmaceutical ODC inhibitors.
Topics: Humans; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Propylamines; Protein Binding; Protein Domains
PubMed: 34796899
DOI: 10.1042/BCJ20210647 -
Plant Biotechnology Journal Oct 2022The polyamine putrescine (1,4-diaminobutane) contributes to cellular fitness in most organisms, where it is derived from the amino acids ornithine or arginine. In the...
The polyamine putrescine (1,4-diaminobutane) contributes to cellular fitness in most organisms, where it is derived from the amino acids ornithine or arginine. In the chemical industry, putrescine serves as a versatile building block for polyamide synthesis. The green microalga Chlamydomonas reinhardtii accumulates relatively high putrescine amounts, which, together with recent advances in genetic engineering, enables the generation of a powerful green cell factory to promote sustainable biotechnology for base chemical production. Here, we report a systematic investigation of the native putrescine metabolism in C. reinhardtii, leading to the first CO -based bio-production of putrescine, by employing modern synthetic biology and metabolic engineering strategies. A CRISPR/Cas9-based knockout of key enzymes of the polyamine biosynthesis pathway identified ornithine decarboxylase 1 (ODC1) as a gatekeeper for putrescine accumulation and demonstrated that the arginine decarboxylase (ADC) route is likely inactive and that amine oxidase 2 (AMX2) is mainly responsible for putrescine degradation in C. reinhardtii. A 4.5-fold increase in cellular putrescine levels was achieved by engineered overexpression of potent candidate ornithine decarboxylases (ODCs). We identified unexpected substrate promiscuity in two bacterial ODCs, which exhibited co-production of cadaverine and 4-aminobutanol. Final pathway engineering included overexpression of recombinant arginases for improved substrate availability as well as functional knockout of putrescine degradation, which resulted in a 10-fold increase in cellular putrescine titres and yielded 200 mg/L in phototrophic high cell density cultivations after 10 days.
Topics: Amino Acids; Arginine; Cadaverine; Carbon Dioxide; Carboxy-Lyases; Nylons; Ornithine; Ornithine Decarboxylase; Oxidoreductases; Polyamines; Putrescine
PubMed: 35748533
DOI: 10.1111/pbi.13879 -
Cell Reports. Medicine Nov 2023In preclinical models, α-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, delays the onset of type 1 diabetes (T1D) by reducing β cell... (Randomized Controlled Trial)
Randomized Controlled Trial
In preclinical models, α-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, delays the onset of type 1 diabetes (T1D) by reducing β cell stress. However, the mechanism of DFMO action and its human tolerability remain unclear. In this study, we show that mice with β cell ODC deletion are protected against toxin-induced diabetes, suggesting a cell-autonomous role of ODC during β cell stress. In a randomized controlled trial (ClinicalTrials.gov: NCT02384889) involving 41 recent-onset T1D subjects (3:1 drug:placebo) over a 3-month treatment period with a 3-month follow-up, DFMO (125-1,000 mg/m) is shown to meet its primary outcome of safety and tolerability. DFMO dose-dependently reduces urinary putrescine levels and, at higher doses, preserves C-peptide area under the curve without apparent immunomodulation. Transcriptomics and proteomics of DFMO-treated human islets exposed to cytokine stress reveal alterations in mRNA translation, nascent protein transport, and protein secretion. These findings suggest that DFMO may preserve β cell function in T1D through islet cell-autonomous effects.
Topics: Humans; Mice; Animals; Diabetes Mellitus, Type 1; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Eflornithine; Putrescine
PubMed: 37918404
DOI: 10.1016/j.xcrm.2023.101261