Aminobutyl Group Research Articles

EIF5A hypusination, boosted by dietary spermidine, protects from premature brain aging and mitochondrial dysfunction.

Mitochondrial function declines during brain aging and is suspected to play a key role in age-induced cognitive decline and neurodegeneration. Supplementing levels of spermidine, a body-endogenous metabolite, has been shown to promote mitochondrial respiration and delay aspects of brain aging. Spermidine serves as the amino-butyl group donor for the synthesis of hypusine (Nε-[4-amino-2-hydroxybutyl]-lysine) at a specific lysine residue of the eukaryotic translation initiation factor 5A (eIF5A). Here, we show that in the Drosophila brain, hypusinated eIF5A levels decline with age but can be boosted by dietary spermidine. Several genetic regimes of attenuating eIF5A hypusination all similarly affect brain mitochondrial respiration resembling age-typical mitochondrial decay and also provoke a premature aging of locomotion and memory formation in adult Drosophilae. eIF5A hypusination, conserved through all eukaryotes as an obviously critical effector of spermidine, might thus be an important diagnostic and therapeutic avenue in aspects of brain aging provoked by mitochondrial decline.

Evaluation of a novel poly(amidoamine) with pendant aminobutyl group on the cellular properties of transfected bone marrow mesenchymal stem cells.

Stem cell-based gene therapy has been considered in the treatment of many degenerative diseases. Gene-modified stem cells should maintain its reproductive activity without losing stem cell properties, including genetic phenotype and differentiation potential. In the study, a novel poly (amidoamine) with pendant aminobutyl group (PAA-BA) designed by our group was used in the transfection of bone marrow mesenchymal stromal cells (BMSCs) and the cellular properties post-transfection were evaluated, including DNA content, colony forming capacity, genetic phenotype, and multi-directional differentiation. Two classical non-viral gene delivery vectors, polyethylenimine (PEI) and Lipofectamine 2000 (LP2000) were also used. Compared to non-transfected group, PAA-BA showed minor decreased DNA content but maintained BMSCs' phenotype, reproductive activity and multi-differentiation potential (osteogenic, chondrogenic, adipogenic, and neurogenic differentiation). Both PAA-BA and PEI transfected BMSCs demonstrated improved osteogenic differentiation ability at late stage but suppressed adipogenic as well as mature neural differentiation in vitro. LP2000 and PEI transfected BMSCs displayed significantly lower DNA content and reproductive activity. These findings suggest that PAA-BA is one of safe gene delivery vectors in BMSCs transfection and plays a role in stem cell's osteogenic and neurogenic differentiation. This study proposes the potential application of PAA-BA in BMSCs based gene therapy, in particular bone and nerve relative diseases. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 686-697, 2018.

The Essential Role of Spermidine in Growth of Agrobacterium tumefaciens Is Determined by the 1,3-Diaminopropane Moiety.

The ubiquitous polyamine spermidine is indispensable for eukaryotic growth and cell proliferation. A conserved vital function of spermidine across eukaryotes is conferred by its aminobutyl group that is transferred to a single lysine in translation factor eIF5A to form the essential hypusine post-translational modification required for cellular translation. In direct contrast, although spermidine is absolutely essential for growth of α-proteobacterial plant pathogen Agrobacterium tumefaciens, we have found, by employing a suite of natural polyamines and synthetic methylated spermidine analogues together with spermidine biosynthetic mutants, that it is solely the 1,3-diaminopropane moiety of spermidine that is required for growth. Indeed, any polyamine containing an intact terminal 1,3-diaminopropane moiety can replace spermidine for growth, including the simple diamine 1,3-diaminopropane itself, a paradigm shift in understanding polyamine function in bacteria. We have identified for the first time a spermidine retroconversion activity in bacteria, producing diamine putrescine from triamine spermidine; however, exogenously supplied tetraamine spermine is resistant to retroconversion. When spermidine levels are pharmacologically decreased, synthesis of spermine from spermidine is induced via the same biosynthetic enzymes, carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase that produce spermidine from putrescine, the first identification of a spermine biosynthetic pathway in bacteria. This also suggests that spermidine represses spermine biosynthesis, but when spermidine levels decrease, it is then converted by carboxyspermidine dehydrogenase and decarboxylase enzymes to spermine, which is resistant to retroconversion and constitutes a sequestered pool of protected 1,3-diaminopropane modules required for growth. We also identify an efficient N-acetylspermidine deacetylase activity, indicative of a sophisticated bacterial polyamine homeostasis system.

Cytotoxicity and in vivo tissue compatibility of poly(amidoamine) with pendant aminobutyl group as a gene delivery vector

To design successful polymeric gene delivery vehicles with good biocompatibility and highly efficient gene transfer ability is one of the great scientific challenges in modern gene therapy. Poly(amidoamine) with pendant aminobutyl group (PAA-BA) has been proved to exhibit high transfection efficiency against bone marrow stromal cells (BMSCs) in vitro. In this work, based on previous research, PAA-BA's biocompatibility including in vitro cytotoxicity determined by effect on BMSCs' morphology, viability, membrane damage and apoptosis/necrosis, and in vivo tissue compatibility determined by muscular and hepatic tissue response were further investigated in comparison to branched polyethylenimine (PEI) 25 kDa. The results demonstrated that PAA-BA possess much better cytocompatibility than PEI, yielding slight cell morphological change, high cell viability and mild effect on cell membrane damage as well as inducing less apoptotic/necrotic cells at optimal N/P ratio. PAA-BA also exhibited better tissue compatibility, reflected by no or less inflammatory response in the site of muscle injection at the same (0.03% w/v) or higher concentration (0.1% w/v) and no hepatic tissue morphological change with normal hepatocytes. We concluded that PAA-BA was promising and safe candidate for in vitro BMSCs gene delivery and had potential for in future in vivo gene therapy.

Mono‐ and Dinuclear CuII and ZnII Complexes of Cyclen‐Based Bis(macrocycles) Containing Two Aminoalkyl Pendant Arms of Different Lengths

AbstractThe basicity and coordination properties towards CuII and ZnII of the bis(macrocycles) L1, L2 and L3 have been investigated by means of potentiometric, 1H NMR and UV/Vis spectroscopic titrations in aqueous solutions. The synthesis of L1 and L3 is also described. The three ligands are composed of two [12]aneN4 units separated by a p‐phenylene spacer and differ in the length of the aminoalkyl pendant arms linked to each macrocyclic unit. L1–L3 form mono‐ and dinuclear complexes in aqueous solutions; in the dinuclear species each metal ion is coordinated by one of the two identical [12]aneN4 ligand moieties, as shown by the crystal structures of the complexes [Cu2L1]Cl4·8H2O, [Zn2L2](ClO4)4 and [Zn2L3](ClO4)4·H2O. In all structures the metal ion is pentacoordinate, and is bound to the four nitrogen donors of the cyclic unit and to the amine group of the side arm. The stability of both the [ML]2+ and [M2L]4+ complexes in aqueous solution decreases in the order L1 > L2 > L3. At the same time, both the [Cu2L]4+ and [Zn2L]4+ complexes show a different ability in proton binding among the three ligands, with the [M2L1]4+ complexes displaying the highest basicity. These results are explained in terms of the decreasing number of nitrogen donors involved in CuII or ZnII binding on passing from L1 to L3; in other words, while in the L1 dinuclear complexes each metal ion is coordinated to the four amine groups of a [12]aneN4 moiety and to the amine group of the side arm, in the L3 ones the metal cations are bound only to the four donor atoms of a cyclic moiety, the aminobutyl group not being coordinated. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Membrane binding and transport of N-aminoethyl-1,2-diamino ethane (dien) and N-aminopropyl-1,3-diamino propane (propen) by rat liver mitochondria and their effects on membrane permeability transition.

This investigation is aimed at defining the structural requirements for aliphatic polyamines to interact with mitochondrial binding sites, which are relevant for the regulation of the permeability transition and for mitochondrial polyamine uptake. The triamines N-aminoethyl-1,2-diaminoethane (dien) and N-aminopropyl-1,3-diaminopropane (propen), both symmetric polyamines, are accumulated to differing extents by an energy-dependent mechanism in liver mitochondria. Propen is also able completely to inhibit the permeability transition of mitochondria, induced by Ca2+ plus phosphate, with the same efficacy as the asymmetric ubiquitary triamine spermidine, whereas dien fails to exhibit this effect. The competitive inhibition of both triamines on spermidine transport demonstrates that they bind to the same site(s) of this polyamine and exploit its transport system. The binding of dien and propen to mitochondrial membrane was studied by applying a thermodynamic model of ligand-receptor interactions developed both for equilibrium and far-from-equilibrium binding processes. Results show the presence of two mono-coordinated binding sites, S1 and S2, for propen, and one monocoordinated binding site for dien, all exhibiting high capacity and low affinity. Comparisons of the binding parameters of these polyamines with those of other natural polyamines reveal that, besides flexibility and hydrophilicity, as previously suggested, protonation of the imino group and the symmetry of the molecules for S1, and the presence of an aminobutyl group for S2, also contribute to the polyamine interactions observed in the two sites.

Biosynthesis of spermidine, a direct precursor of pyrrolizidine alkaloids in root cultures of Senecio vulgaris L.

The polyamine spermidine is an essential biosynthetic precursor of pyrrolizidine alkaloids. It provides its aminobutyl group which is transferred to putrescine yielding homospermidine, the specific building block of the necine base moiety of pyrrolizidine alkaloids. The enzymatic formation of spermidine was studied in relation to the unique role of this polyamine as an alkaloid precursor. S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) and spermidine synthase (SPDS, EC 2.5.1.16) from root cultures of Senecio vulgaris were partially purified and characterized. The SAMDC-catalyzed reaction showed a pH optimum of 7.5, that of SPDS an optimum of 7.7. The Km value of SAMDC for its substrate S-adenosylmethionine (SAM) was 15 microM, while the apparent Km values of SPDS for its substrates decarboxylated SAM (dSAM) and putrescine were 4 microM and 21 microM, respectively. The relative molecular masses of the two enzymes, determined by gel filtration, were 29000 (SAMDC) and 37000 (SPDS). Studies with various potential inhibitors revealed, for most inhibitors, profiles that were similar to those established with the respective enzymes from other plant sources. However, putrescine which is not known to be an inhibitor of plant SAMDC, strongly inhibited the enzyme from S. vulgaris roots. Spermidine synthase was sensitive to inhibition by its product spermidine. In the presence of the stationary tissue concentrations of the two polyamines (ca. 0.1 mM each) the activities of SAMDC and SPDS would be inhibited by >80%. The results are discussed in relation to the role of spermidine in primary and secondary metabolism of alkaloid-producing S. vulgaris root cultures.

JSJ-1, an anti-spermidine monoclonal antibody with potential clinical applications.

Polyamines have been implicated in a wide variety of functions including nucleic acid synthesis and protein synthesis. Their levels have been shown to increase in response to cell growth and differentiation. Use of polyamines as prognostic indicators of proliferative disease conditions has been hindered by the lack of suitable rapid and sensitive assays. We report the characterization of an anti-spermidine antibody, JSJ-1, with novel putrescine cross reactivity. JSJ-1 cross-reacts more strongly with putrescine (11%) than with spermine (6%). This suggests that the aminobutyl group common to both putrescine and spermidine is an important element in the antibody-antigen interaction. We have demonstrated that antibody-spermidine binding is effected by increased ionic strength. This finding is consistent with the antibody-antigen interaction being ionic. The JSJ-1 antibody has been successfully used to detect increased polyamine levels in clinical serum samples and identify those with increased polyamine levels.

Biosynthetic incorporation of the aminobutyl group of spermidine into pyrrolizidine alkaloids

In short-term tracer experiments [ 3H]putrescine and [ 14C]spermidine were fed simultaneously to root cultures of Senecio vulgaris. The specific incorporation of the two tracers into putrescine, spermidine and pyrrolizidine alkaloids was followed over a period of 30 to 480 min. The results show that the aminobutyl moiety of spermidine is directly incorporated into the necine base of pyrrolizidine alkaloids. The same experiment carried out in the presence of the diamine oxidase inhibitor β-hydroxyethylhydrazine, which blocks the alkaloid biosynthesis after homospermidine, revealed the same specific incorporation into accumulating homospermidine. The results are in accordance with the substrate specificity of plant homospermidine synthase (EC 2.5.1.44) which uses both putrescine and spermidine as donor for the amino butyl group. Under physiological conditions more than half of the aminobutyl moiety of homospermidine comes directly from spermidine. This is the first report for the role of spermidine as aminobutyl donor in alkaloid biosynthesis. The importance of the linkage between spermidine and pyrrolizidine alkaloid biosynthesis is discussed. © 1997 Elsevier Science Ltd. All rights reserved

PCR-based cloning of the full-length Neurospora eukaryotic initiation factor 5A cDNA: polyhistidine-tagging and overexpression for protein affinity binding.

Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain a hypusine residue that is formed by transferring the aminobutyl moiety from spermidine to a specific lysine residue, followed by hydroxylation at the aminobutyl group. A simple PCR-based strategy was developed to obtain a full-length cDNA of Neurospora crassa eIF-5A. The strategy consists of (i) the design of a pair of key primers (21-mer) based on the highly conserved eIF-5A cDNA domains known in other species, (ii) PCR amplification of Neurospora cDNA using the two key primers to obtain the core sequence for the design of core primers, and (iii) combined use of the key primers, core primers and the universal primers, T3 and T7, to amplify the target sequence in a Neurospora cDNA library. The longest cDNA obtained was cloned into pBlueScript phagemid, and sequence analysis indicated that it encodes a polypeptide of 163 amino acid residues with a codon usage preference characteristic of abundant Neurospora genes. The Neurospora polypeptide showed 59% and 67% identity with human and yeast eIF-5A precursor protein respectively. We subcloned the Neurospora eIF-5A cDNA into pQE-30, which introduces six adjacent histidine residues to the N-terminus of the recombinant protein. The resulting plasmid, pQTy21, was overexpressed in Escherichia coli, and the soluble polyhistidine-tagged protein was purified by metal chelation chromatography. We obtained about 60 mg of purified eIF-5A precursor from 1 litre of culture in a single step using a Ni(II)-nitrilotriacetic acid (NTA)-agarose column. The histidine-tagged eIF-5A precursor protein could be recognized by anti-Neurospora crassa 21 kDa protein serum raised against wild-type eIF-5A precursor and could serve as the substrate protein for deoxyhypusine synthase. Using the histidine-tagged recombinant protein and the Ni(II)-NTA-agarose column, we constructed a protein affinity column and demonstrated an affinity binding between eIF-5A precursor and deoxyhypusine synthase in the presence of NAD+. One-step eIF-5A precursor affinity-column chromatography could lead to a 30-fold purification of deoxyhypusine synthase.

Biosynthetic labeling of hypusine in mammalian cells. Carbon-hydrogen bond fissions revealed by dual labeling.

Using a dual-label technique in which 3H- and 14C-labeled forms of putrescine and of spermidine were employed as biosynthetic precursors of hypusine, two -C-H bond cleavages were detected during production of this unique amino acid in Chinese hamster ovary cells. One of these cleavages occurs at C-1 of the 4-aminobutyl group during its transfer from the secondary amine nitrogen of spermidine to the nitrogen at the epsilon-position of a specific lysine residue in the polypeptide precursor of eukaryotic initiation factor 4D. Breakage of the other -C-H bond takes place at C-2 in this aminobutyl segment after it has been coupled to lysine to form the intermediate deoxyhypusine residue. Hydroxylation at this carbon atom, which constitutes the last step in hypusine biosynthesis, is the cause of bond cleavage. The data obtained are consistent with a notion that no additional -C-H bond fissions occur during hypusine biosynthesis. Our findings permit suggestion of a mechanism for enzymic aminobutyl group transfer in which 4-aminobutyraldehyde produced by oxidative cleavage of spermidine is coupled with the epsilon-amino group of a specific lysine residue to form an enzyme-bound imine intermediate.

Action of decarboxylated S-adenosylmethionine analogs in the spermidine-synthesizing system from rat prostate

Synthetic decarboxylated S-adenosylmethionine and its nine analogs, as well as four putrescine analogs, were examined in a polyamine-synthesizing system based on rat prostate spermidine synthase. In addition to the natural substrate, decarboxylated S-adenosylmethionine, S-5′-deoxyadenosyl-(5′)-3-ethylthiopropylamine, S-5′-deoxyadenosyl-(5′)-3-propylthiopropylamine, and S-5′-deoxyadenosyl-(5′)-3-butylthiopropylamine were all found to work as the aminopropyl donor to putrescine. Experiments with other analogs of decarboxylated S-adenosylmethionine indicated that the enzyme will transfer only the aminopropyl group, not an aminoethyl or aminobutyl group. The mammalian spermidine synthase could use only one of the two diastereoisomers of decarboxylated S-adenosylmethionine as the aminopropyl donor. The synthase could use 1,4-diamino-2-butene in addition to putrescine as the acceptor of the aminopropyl group from decarboxylated S-adenosylmethionine, but could not use 1,3-diaminopropane or cadaverine. Seven decarboxylated S-adenosylmethionine analogs were inhibitory to spermidine synthesis to varying degrees. Of these analogs, S-5′-deoxyadenosyl-(5′)-2-methylthioethylamine was the most potent inhibitor. Kinetic studies with some of the above compounds were also carried out.