Microarray data of differentiated human keratinocytes treated with and without AMD1 inhibitor EGBG and rescued with spermidine/ spermine.
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ABSTRACT: Maintaining tissue homeostasis depends on a balance of cell proliferation, differentiation and apoptosis. Polyamine regulator, AMD1, is a crucial regulator of keratinocyte differentiation and AMD1 protein is upregulated on differentiation and highly expressed in the suprabasal layers of the human epidermis. During keratinocyte differentiation, elevated AMD1 promotes decreased putrescine and increased spermine levels. Inhibition of AMD1 results in reduced spermine levels and inhibition of keratinocyte differentiation. Supplementing AMD1 inhibited keratinocytes with exogenous spermidine/spermine rescued aberrant differentiation. Undifferentiated and differentiated keratinocytes that had been treated with and without AMD1 inhibitor EGBG in the presence or absence of spermidine/ spermine supplementation were subjected to microarray analysis. These data show that AMD1 up regulation is required for keratinocyte differentiation and that inhibition of AMD1 can be rescued by supplementation with the polyamines spermidine and spermine.
Project description:Microarray data of differentiated human keratinocytes treated with and without AMD1 inhibitor EGBG and rescued with spermidine/ spermine.
Project description:The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well established in vivo. In this work we have performed a microarray experiment in a polyamine mutant (delta-spe3 delta-fms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine and spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10-5 M) or spermine (10-5 M) compared to a control culture containing 10-8 M spermidine. 247 genes were up-regulated >2-fold, and 11 genes were up-regulated more than 10-fold after spermidine addition. Functional categorization of the genes showed induction of transport related genes, and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were down-regulated more than 2-fold, and 6 genes were down-regulated more than 8-fold after spermidine addition. A majority of the down-regulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine.
Project description:The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well established in vivo. In this work we have performed a microarray experiment in a polyamine mutant (delta-spe3 delta-fms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine and spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10-5 M) or spermine (10-5 M) compared to a control culture containing 10-8 M spermidine. 247 genes were up-regulated >2-fold, and 11 genes were up-regulated more than 10-fold after spermidine addition. Functional categorization of the genes showed induction of transport related genes, and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were down-regulated more than 2-fold, and 6 genes were down-regulated more than 8-fold after spermidine addition. A majority of the down-regulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine. Experiment Overall Design: 5 control replicates vs. 3 spermine (SP)-treated or 5 spermidine (SPD)-treated samples.
Project description:Bacterial and mammalian cells are rich in putrescine, spermidine and spermine. Polyamines can be incorporated into proteins in vitro. Very few naturally occurring polyaminated proteins have been identified. Bovine albumin and the recombinant universal stress protein from Francisella tularensis were used as models for mass spectrometry analysis of polyaminated proteins. The proteins were covalently bound to putrescine, spermidine, or spermine by the action of carbodiimide or microbial transglutaminase. Tryptic peptides were subjected to liquid chromatography tandem mass spectrometry. Adducts were identified by Protein Prospector software. We describe the search parameters for identifying polyaminated peptides in Protein Prospector and show convincing MMS spectra for adducts with putrescine, spermidine, and spermine. Manual evaluation led us to recognize signature ions for polyamine adducts on Asp, Glu, and Gln. Manual evaluation recognized neutral loss from putrescine, spermidine and spermine during the fragmentation process. Mechanisms for formation of signature ions and neutral loss are presented. Manual evaluation recognized a false positive adduct which had been formed during trypsinolysis by rearrangement of a peptide sequence. Another false positive was a 71 Da added mass on cysteine, which was initially assumed to be putrescine, but was actually a propionamide adduct for a sample extracted from a polyacrylamide gel. The type of information presented in this report serves as a model for identifying naturally occurring polyaminated proteins.
Project description:<p>Transcription factor p63 is a key regulator of epidermal keratinocyte proliferation and differentiation. Mutations in the p63 DNA-binding domain are associated with Ectrodactyly Ectodermal Dysplasia Cleft Lip/Palate (EEC) syndrome. Underlying molecular mechanism of these mutations however remain unclear. Here we characterized the transcriptome and epigenome of p63 mutant keratinocytes derived from EEC patients. The transcriptome of p63 mutant keratinocytes deviated from the normal epidermal cell identity. Epigenomic analyses showed an altered enhancer landscape in p63 mutant keratinocytes contributed by loss of p63-bound active enhancers and by unexpected gain of enhancers. The gained enhancers were frequently bound by deregulated transcription factors such as RUNX1. Reversing RUNX1 overexpression partially rescued deregulated gene expression and the altered enhancer landscape. Our findings identify an unreported disease mechanism whereby mutant p63 rewires the enhancer landscape and affects epidermal cell identity, consolidating the pivotal role of p63 in controlling the enhancer landscape of epidermal keratinocytes.</p>
Project description:Type 2 diabetes is a complex disorder due to defects in insulin secretion and insulin action. Therefore, we investigated mice overexpressing spermidine/spermine N1-acetyltransferase (SSAT) have reduced amount of white adipose tissue, high basal metabolic rate, improved glucose tolerance, high insulin sensitivity, and a low accumulation of triglycerides in liver and skeletal muscle. Our aim was to elucidate the molecular mechanisms leading to the phenotype of SSAT mice.
Project description:Cells counteract oxidative stress by altering metabolism, cell cycle and gene expression. However, the mechanisms that coordinate these adaptations are only marginally understood. Here we provide evidence that timing of these responses in yeast requires export of the polyamines spermidine and spermine. We show that during hydrogen peroxide (H2O2) exposure, the polyamine transporter Tpo1 controls spermidine and spermine concentrations and mediates induction of antioxidant proteins, including Hsp70, Hsp90, Hsp104 and Sod1. Moreover, Tpo1 determines a cell cycle delay during adaptation to increased oxidant levels, and affects H2O2 tolerance. Thus, central components of the stress response are timed through Tpo1‐controlled polyamine export.