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The Journal of Pharmacology and... Oct 2012Modeling the binding sites for spermine and ifenprodil on the regulatory (R) domains of the N-methyl-D-aspartate receptor GluN1 and GluN2B subunits was carried out after...
Modeling the binding sites for spermine and ifenprodil on the regulatory (R) domains of the N-methyl-D-aspartate receptor GluN1 and GluN2B subunits was carried out after measuring spermine stimulation and ifenprodil inhibition at receptors containing GluN1 and GluN2B R domain mutants. Models were constructed based on the published crystal structure of the GluN1 and GluN2B R domains, which form a heterodimer (Nature 475:249-253, 2011). The experimental results and modeling suggest that a binding site for spermine was formed by the residues near the cleft between the R1 and R2 lobes of the GluN1 R domain (GluN1R) together with residues on the surface of the R2 (C-terminal side) lobe of the GluN2B R domain (GluN2BR). The ifenprodil binding site included residues on the surface of the R1 lobe (N-terminal side) of GluN1R together with residues near the cleft between the R1 and R2 lobes of GluN2BR. It was confirmed using a Western blot analysis that GluN1R and GluN2BR formed a heterodimer. Models of spermine and ifenprodil binding to the heterodimer were constructed. The modeling suggests that an open space between the two R1 lobes of GluN1R and GluN2BR is promoted through spermine binding and that the R1 lobes of GluN1R and GluN2BR approach each other through ifenprodil binding--an effect opposite to that seen with the binding of spermine.
Topics: Amino Acid Sequence; Animals; Female; Molecular Sequence Data; Piperidines; Protein Binding; Protein Multimerization; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits; Rats; Receptors, N-Methyl-D-Aspartate; Spermine; Xenopus laevis
PubMed: 22743575
DOI: 10.1124/jpet.112.192286 -
BMC Cancer Oct 2010Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates...
BACKGROUND
Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme.
METHODS
BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined.
RESULTS
Both the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors.
CONCLUSIONS
This study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials.
Topics: Aged; Aged, 80 and over; Animals; Antineoplastic Agents; Breast Neoplasms; Female; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Mice; Middle Aged; Oxidoreductases Acting on CH-NH Group Donors; Polyamines; Recombinant Proteins; Spermine; Polyamine Oxidase
PubMed: 20946629
DOI: 10.1186/1471-2407-10-555 -
Nature Communications Sep 2023Native mass spectrometry (MS) is a powerful technique for interrogating membrane protein complexes and their interactions with other molecules. A key aspect of the...
Native mass spectrometry (MS) is a powerful technique for interrogating membrane protein complexes and their interactions with other molecules. A key aspect of the technique is the ability to preserve native-like structures and noncovalent interactions, which can be challenging depending on the choice of detergent. Different strategies have been employed to reduce charge on protein complexes to minimize activation and preserve non-covalent interactions. Here, we report the synthesis of a class of polyamine detergents tailored for native MS studies of membrane proteins. These detergents, a series of spermine covalently attached to various alkyl tails, are exceptional charge-reducing molecules, exhibiting a ten-fold enhanced potency over spermine. Addition of polyamine detergents to proteins solubilized in maltoside detergents results in improved, charge-reduced native mass spectra and reduced dissociation of subunits. Polyamine detergents open new opportunities to investigate membrane proteins in different detergent environments that have thwarted previous native MS studies.
Topics: Polyamines; Membrane Proteins; Detergents; Spermine; Mass Spectrometry
PubMed: 37709761
DOI: 10.1038/s41467-023-41429-w -
Molecular Microbiology Jun 2017Growth of Pseudomonas aeruginosa on spermine requires a functional γ-glutamylpolyamine synthetase PauA2. Not only subjected to growth inhibition by spermine, the pauA2...
Growth of Pseudomonas aeruginosa on spermine requires a functional γ-glutamylpolyamine synthetase PauA2. Not only subjected to growth inhibition by spermine, the pauA2 mutant became more sensitive to β-lactam antibiotics in human serum. To explore PauA2 as a potential target of drug development, suppressors of the pauA2 mutant, which alleviated toxicity, were isolated from selection plates containing spermine. These suppressors share common phenotypic changes including delayed growth rate, retarded swarming motility, and pyocyanin overproduction. Genome resequencing of a representative suppressor revealed a unique C T mutation at the phoU gene that results in Ser Leu substitution and a constitutive expression of the Pho regulon. Identical phenotypes were also observed in a ΔpauA2ΔphoU double knockout mutant and complemented by the wild-type phoU gene. Accumulation of polyphosphate granules and spermine resistance in the suppressor were reversed concomitantly when expressing exopolyphosphatase PPX from a recombinant plasmid, or by the introduction of deletion alleles in pstS pstC for phosphate uptake, phoB for Pho regulation, and ppk for polyphosphate synthesis. In conclusion, this study identifies polyphosphate accumulation due to an activated Pho regulon and phosphate uptake by the phoU mutation as a potential protection mechanism against spermine toxicity.
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Membrane Transport Proteins; Mutation; Phosphates; Polyphosphates; Promoter Regions, Genetic; Pseudomonas aeruginosa; Regulon; Spermine; Transcription Factors
PubMed: 28370665
DOI: 10.1111/mmi.13678 -
Scientific Reports Oct 2019According to previous research, natural polyamines exert a role in regulating cell committment and differentiation from stemness during skeletal development. In order to...
According to previous research, natural polyamines exert a role in regulating cell committment and differentiation from stemness during skeletal development. In order to assess whether distinct polyamine patterns are associated with different skeletal cell types, primary cultures of stem cells, chondrocytes or osteoblasts were dedicated for HPLC analysis of intracellular polyamines. Spermine (SPM) and Spermidine (SPD) levels were higher in adipose derived stem cells (ASC) compared to mature skeletal cells, i.e. chondrocytes and osteoblasts, confirming the connection of polyamine content with stemness. To establish whether polyamines can protect ASC against oxidative DNA damage in a 3-D differentiation model, the level of γH2AX was measured by western blot, and found to correlate with age and BMI of patients. Addition of either polyamine to ASC was able to hinder DNA damage in the low micromolecular range, with marked reduction of γH2AX level at 10 µM SPM and 5 µM SPD. Molecular analysis of the mechanisms that might underlie the protective effect of polyamine supplementation evidences a possible involvement of autophagy. Altogether, these results support the idea that polyamines are able to manage both stem cell differentiation and cell oxidative damage, and therefore represent appealing tools for regenerative and cell based applications.
Topics: Adult; Aged; Cells, Cultured; DNA Damage; Histones; Humans; Mesenchymal Stem Cells; Middle Aged; Spermidine; Spermine
PubMed: 31582764
DOI: 10.1038/s41598-019-50543-z -
International Journal of Molecular... May 2017Zeta potential and nanoparticle size were determined on film forming solutions of native and heat-denatured proteins of bitter vetch as a function of pH and of different...
Zeta potential and nanoparticle size were determined on film forming solutions of native and heat-denatured proteins of bitter vetch as a function of pH and of different concentrations of the polyamines spermidine and spermine, both in the absence and presence of the plasticizer glycerol. Our results showed that both polyamines decreased the negative zeta potential of all samples under pH 8.0 as a consequence of their ionic interaction with proteins. At the same time, they enhanced the dimension of nanoparticles under pH 8.0 as a result of macromolecular aggregations. By using native protein solutions, handleable films were obtained only from samples containing either a minimum of 33 mM glycerol or 4 mM spermidine, or both compounds together at lower glycerol concentrations. However, 2 mM spermidine was sufficient to obtain handleable film by using heat-treated samples without glycerol. Conversely, brittle materials were obtained by spermine alone, thus indicating that only spermidine was able to act as an ionic plasticizer. Lastly, both polyamines, mainly spermine, were found able to act as "glycerol-like" plasticizers at concentrations higher than 5 mM under experimental conditions at which their amino groups are undissociated. Our findings open new perspectives in obtaining protein-based films by using aliphatic polycations as components.
Topics: Nanoparticles; Plant Proteins; Plastics; Polymerization; Seeds; Spermine; Vicia
PubMed: 28489025
DOI: 10.3390/ijms18051026 -
The Journal of Physiology May 19961. A cloned inwardly rectifying K+ channel, IRK2, was expressed in a human cell line, human embryonic kidney (HEK) 293T. Its electrophysiological properties were...
1. A cloned inwardly rectifying K+ channel, IRK2, was expressed in a human cell line, human embryonic kidney (HEK) 293T. Its electrophysiological properties were examined using the patch clamp technique in the whole-cell, cell-attached and inside-out patch configurations. 2. The cells transfected with IRK2 cDNA exhibited a K+ current which showed classical properties of inwardly rectifying K+ channels at both whole-cell and single-channel levels. 3. In the inside-out patch configuration, intracellular Mg2+ (Mg2+i blocked the outward currents in a voltage-dependent and virtually time-independent manner. Mg2+i (1-100 microM) caused a decrease in the unitary current amplitude of the IRK2 channel by inducing subconducting levels. 4. In the absence of Mg2+i, intracellular spermine blocked the outwardly flowing IRK2 currents in a voltage- and time-dependent manner. Spermine (1-100 nM) did not affect the unitary channel current amplitude but reduced the channel open probability. The spermine block showed a slower time and steeper voltage dependence than the Mg2+i++ block. 5. When both these blockers were present, Mg2+i apparently attenuated the inhibitory effect of spermine on the outwardly flowing IRK2 currents. This interaction was voltage and time dependent, and could be well explained by a model in which Mg2+i and spermine competitively bind to the channel with their individual first-order kinetics. This competition would induce time-dependent transits of the channel between the Mg2+i -and spermine-blocked states via a single open state, thereby preserving a certain size of persistent outward currents at depolarized potentials.
Topics: Barium; Binding, Competitive; Cell Line; Cesium; Cloning, Molecular; Electrophysiology; Humans; Kidney; Kinetics; Magnesium; Patch-Clamp Techniques; Potassium; Potassium Channels; Potassium Channels, Inwardly Rectifying; Spermine
PubMed: 8735700
DOI: 10.1113/jphysiol.1996.sp021370 -
Journal of Natural Products Jun 2021An extract of a sp. soft coral showed inhibitory activity against the E3-ubiquitin ligase casitas B-lineage lymphoma proto-oncogene B (Cbl-b). Subsequent...
Sinularamides A-G, Terpenoid-Derived Spermidine and Spermine Conjugates with Casitas B-Lineage Lymphoma Proto-Oncogene B (Cbl-b) Inhibitory Activities from a sp. Soft Coral.
An extract of a sp. soft coral showed inhibitory activity against the E3-ubiquitin ligase casitas B-lineage lymphoma proto-oncogene B (Cbl-b). Subsequent bioassay-guided separation of the extract provided a series of terpenoid-derived spermidine and spermine amides that were named sinularamides A-G (-). Compounds - represent new natural products; however, sinularamide A () was previously reported as a synthetic end product. The structures of sinularamides A-G (-) were elucidated by analysis of spectroscopic and spectrometric data from NMR, IR, and HRESIMS experiments and by comparison with literature data. All of the isolated compounds showed Cbl-b inhibitory activities with IC values that ranged from approximately 6.5 to 33 μM.
Topics: Adaptor Proteins, Signal Transducing; Animals; Anthozoa; Molecular Structure; Palau; Proto-Oncogene Proteins c-cbl; Spermidine; Spermine; Terpenes
PubMed: 34038132
DOI: 10.1021/acs.jnatprod.1c00367 -
International Journal of Molecular... Feb 2024The work presents the synthesis of a series of linear polyamidoamines by polycondensation of sebacoyl dichloride with endogenous polyamines: putrescine, spermidine,...
The work presents the synthesis of a series of linear polyamidoamines by polycondensation of sebacoyl dichloride with endogenous polyamines: putrescine, spermidine, spermine, and norspermidine-a biogenic polyamine not found in the human body. During the synthesis carried out via interfacial reaction, hydrophilic, semi-crystalline polymers with an average viscosity molecular weight of approximately 20,000 g/mol and a melting point of approx. 130 °C were obtained. The structure and composition of the synthesized polymers were confirmed based on NMR and FTIR studies. The cytotoxicity tests performed on human fibroblasts and keratinocytes showed that the polymers obtained with spermine and norspermidine were strongly cytotoxic, but only in high concentrations. All the other examined polymers did not show cytotoxicity even at concentrations of 2000 µg/mL. Simultaneously, the antibacterial activity of the obtained polyamides was confirmed. These polymers are particularly active against , and virtually all the polymers obtained demonstrated a strong inhibitory effect on the growth of cells of this strain. Antimicrobial activity of the tested polymer was found against strains like , , and . The broadest spectrum of bactericidal action was demonstrated by polyamidoamines obtained from spermine, which contains two amino groups in the repeating unit of the chain. The obtained polymers can be used as a material for forming drug carriers and other biologically active compounds in the form of micro- and nanoparticles, especially as a component of bactericidal creams and ointments used in dermatology or cosmetology.
Topics: Humans; Spermine; Escherichia coli; Polyamines; Anti-Bacterial Agents; Polymers; Spermidine
PubMed: 38473823
DOI: 10.3390/ijms25052576 -
The Journal of Biological Chemistry Jul 1989Exposure of human colon tumor (HT 29 cells) to N1,N12-bis(ethyl)spermine and analogs produced a rapid loss of intracellular polyamines. This loss was brought about...
Exposure of human colon tumor (HT 29 cells) to N1,N12-bis(ethyl)spermine and analogs produced a rapid loss of intracellular polyamines. This loss was brought about predominantly by an increased excretion of spermidine. N1,N11-Bis(ethyl)norspermine and N1,N12-Bis(ethyl)spermine were potent inducers of spermidine/spermine N1-acetyltransferase, and this induction facilitated the efflux of polyamines by enhancing the conversion of spermine into spermidine. N1,N14-Bis(ethyl)homospermine, which did not induce spermidine/spermine N1-acetyltransferase, also caused the loss of spermidine from the cell but was less effective in bringing about the decline in intracellular spermine. These results indicate that cellular polyamine levels can be regulated by excretion of spermidine and that the bis(ethyl)spermine derivatives deplete intracellular polyamine content by interference with this process.
Topics: Acetylation; Acetyltransferases; Colonic Neoplasms; Humans; Polyamines; Radiometry; Spermine; Tumor Cells, Cultured
PubMed: 2745415
DOI: No ID Found