Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP–HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β₄ can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI–MS (electrospray ionization–mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β₄ did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β₄ as well as of other proteins and peptides.     

Polyamines as cancer markers: applicable separation methods

Spermine, spermidine, putrescine and cadaverine are aliphatic amines widely spread in the human body. Their concentrations together with their acetyl conjugates increase significantly in the biological fluids and the affected tissues of cancer patients. Their concentrations decrease with the improvement in the patient’s condition on multiple therapy. Various chromatographic techniques are frequently used in monitoring concentrations of di- and polyamines in cancer. Among these techniques, thin-layer chromatography and liquid chromatography using pre- or postcolumn derivatization, separating on a reversed-phase or an ion-exchange column are the most commonly used. Besides, high-resolution capillary column gas chromatography (GC) is increasingly used over packed column GC, and in recent years, capillary zone electrophoresis has also gained some importance in polyamine determinations. The review examines the prospects and the limitations of polyamines as cancer markers using chromatographic and electrophoretic techniques.     

Ethanol perturbs polyamine metabolism in the phytopathogenic fungus Pyrenophora avenae

Biomass production by the plant pathogenic fungus Pyrenophora avenae was reduced following growth in 1, 3 and 6% ethanol. Although cadaverine concentration was not affected by growth in ethanol, putrescine and spermine concentrations were increased following growth in 3% ethanol and concentrations of spermidine and spermine were substantially increased following exposure to 6% ethanol. These changes were accompanied by significant increases in the activities of the polyamine biosynthetic enzymes ornithine decarboxylase and S-adenosylmethionine decarboxylase and in the flux of label from ornithine into the polyamines. Formation of the cadaverine derivatives aminopropylcadaverine and N,N-bis(3-aminopropyl)cadaverine was greatly increased in P. avenae exposed to 6% ethanol, probably via the action of lysine decarboxylase, S-adenosylmethionine decarboxylase and the aminopropyltransferases. There was also a doubling of polyamine oxidase activity following fungal growth in 6% ethanol.     

Formation of cadaverine derivatives in Saccharomyces cerevisiae

Abstract The higher homologues of cadaverine, aminopropylcadaverine (APC) and N , N - bis (3-aminopropyl)cadaverine (3APC) were formed by a wild-type strain of Saccharomyces cerevisiae , and by two mutant strains, spe 3-1 and spe 4-1, exhibiting point mutations in the genes for spermidine synthase and spermine synthase, respectively. This, together with the incomplete inhibition of APC and 3 APC formation in the presence of inhibitors of 5-adenosylmethionine decarboxylase and spermidine synthase, suggests that the cadaverine derivatives are formed partly by the operation of a different route. However, the yeast strains were unable to utilise [¹⁴C]aspartate and lysine to form APC and 3APC. Since the ornithine decarboxylase inhibitor adifluoromethylomithine (DFMO) greatly reduced the formation of APC and 3APC, it is suggested that these compounds are formed preferentially in these yeast strains from cadaverine formed by ODC. APC and 3APC formation in the yeast strains was increased substantially following exposure to 37 °C for 2 h.     

Biosynthesis of 5-Aminopentanoic Acid and 2-Piperidone from Cadaverine and 1-Piperideine in Mouse

1-Piperideine, 5-aminopentanoic acid, and its lactam, 2-piperidone, were identified as metabolites of cadaverine in 10,000 g mouse liver supernatants to which diamine oxidase had been added. Both metabolites were also found when the cadaverine metabolite 1-piperideine was incubated with the preparation which suggested that 1-piperideine is an intermediate in the formation of 5-aminopentanoic acid and 2-piperidone. Identification of the metabolites was based on gas chromatography-mass spectrometric analysis in comparison to authentic standards. Mouse brain homogenates converted 1-piperideine to 5-aminopentanoic acid. The results suggest that the metabolic fate of cadaverine may provide precursors of pharmacologically active analogues of GABA.     

Arabidopsis mutant plants with diverse defects in polyamine metabolism show unequal sensitivity to exogenous cadaverine probably based on their spermine content

Arabidopsis plants do not synthesize the polyamine cadaverine, a five carbon-chain diamine and structural analog of putrescine. Mutants defective in polyamine metabolic genes were exposed to exogenous cadaverine. Spermine-deficient spms mutant grew well while a T-DNA insertion mutant (pao4-1) of polyamine oxidase (PAO) 4 was severely inhibited in root growth compared to wild type (WT) or other pao loss-of-function mutants. To understand the molecular basis of this phenomenon, polyamine contents of WT, spms and pao4-1 plants treated with cadaverine were analyzed. Putrescine contents increased in all the three plants, and spermidine contents decreased in WT and pao4-1 but not in spms. Spermine contents increased in WT and pao4-1. As there were good correlations between putrescine (or spermine) contents and the degree of root growth inhibition, effects of exogenously added putrescine and spermine were examined. Spermine mimicked the original phenomenon, whereas high levels of putrescine evenly inhibited root growth, suggesting that cadaverine-induced spermine accumulation may explain the phenomenon. We also tested growth response of cadaverine-treated WT and pao4-1 plants to NaCl and found that spermine-accumulated pao4-1 plant was not NaCl tolerant. Based on the results, the effect of cadaverine on Arabidopsis growth and the role of PAO during NaCl stress are discussed.     

From zero to hero – Production of bio-based nylon from renewable resources using engineered Corynebacterium glutamicum

Polyamides are important industrial polymers. Currently, they are produced exclusively from petrochemical monomers. Herein, we report the production of a novel bio-nylon, PA5.10 through an integration of biological and chemical approaches. First, systems metabolic engineering of Corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. In this way, a hyper-producer, with a high diaminopentane yield of 41% in shake flask culture, was generated. Subsequent fed-batch production of C. glutamicum DAP-16 allowed a molar yield of 50%, a productivity of 2.2 g L⁻¹ h⁻¹, and a final titer of 88 g L⁻¹. The streamlined producer accumulated diaminopentane without generating any by-products. Solvent extraction from alkalized broth and two-step distillation provided highly pure diaminopentane (99.8%), which was then directly accessible for poly-condensation. Chemical polymerization with sebacic acid, a ten-carbon dicarboxylic acid derived from castor plant oil, yielded the bio-nylon, PA5.10. In pure form and reinforced with glass fibers, the novel 100% bio-polyamide achieved an excellent melting temperature and the mechanical strength of the well-established petrochemical polymers, PA6 and PA6.6. It even outperformed the oil-based products in terms of having a 6% lower density. It thus holds high promise for applications in energy-friendly transportation. The demonstration of a novel route for generation of bio-based nylon from renewable sources opens the way to production of sustainable bio-polymers with enhanced material properties and represents a milestone in industrial production.