ABSTRACT
Hutchinson-Gilford progeria syndrome (HGPS) is a very rare fatal disease characterized for accelerated aging. Although the causal agent, a point mutation in LMNA gene, was identified more than a decade ago, the molecular mechanisms underlying HGPS are still not fully understood and, currently, there is no cure for the patients, which die at a mean age of thirteen. With the aim of unraveling non-previously altered molecular pathways in the premature aging process, human cell lines from HGPS patients and from healthy parental controls were studied in parallel using Next-Generation Sequencing (RNAseq) and High-Resolution Quantitative Proteomics (iTRAQ) techniques. After selection of significant proteins and transcripts and crosschecking of the results a small set of protein/transcript pairs were chosen for validation. One of those proteins, ribose-phosphate pyrophosphokinase 1 (PRPS1), is essential for nucleotide synthesis. PRPS1 loss-of-function mutants present lower levels of purine. PRPS1 protein and transcript levels are detected as significantly decreased in HGPS cell lines vs. healthy parental controls. This modulation was orthogonally confirmed by targeted techniques in cell lines and also in an animal model of Progeria, the ZMPSTE24 knock-out mouse. In addition, functional experiments through supplementation with S-adenosyl-methionine (SAMe), a metabolite that is an alternative source of purine, were done. Results indicate that SAMe has a positive effect in the proliferative capacity and reduces senescence-associated Beta-galactosidase staining of the HPGS cell lines. Altogether, our data suggests that nucleotide and, specifically, purine-metabolism, are altered in premature aging, opening a new window for the therapeutic treatment of the disease.
Subject(s)
Lamin Type A/genetics , Progeria/genetics , Purines/metabolism , RNA, Messenger/genetics , Ribose-Phosphate Pyrophosphokinase/genetics , Adult , Animals , Cell Line , Cell Proliferation , Child , Computational Biology/methods , Disease Models, Animal , Female , Founder Effect , Gene Expression Profiling , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Humans , Lamin Type A/deficiency , Membrane Proteins/deficiency , Membrane Proteins/genetics , Metalloendopeptidases/deficiency , Metalloendopeptidases/genetics , Mice , Mice, Knockout , Progeria/drug therapy , Progeria/metabolism , Progeria/pathology , RNA, Messenger/metabolism , Ribose-Phosphate Pyrophosphokinase/deficiency , S-Adenosylmethionine/pharmacology , beta-Galactosidase/genetics , beta-Galactosidase/metabolismABSTRACT
Imidazolidin-4-ones are commonly employed as skeletal modifications in bioactive oligopeptides, either as proline surrogates or for protection of the N-terminal amino acid against aminopeptidase- and endopeptidase-catalyzed hydrolysis. Imidazolidin-4-one synthesis usually involves the reaction of an alpha-aminoamide moiety with a ketone or an aldehyde to yield an imine, followed by intramolecular cyclization. We have unexpectedly found that imidazolidin-4-one formation is stereoselective when benzaldehydes containing o-carboxyl or o-methoxycarbonyl substituents are reacted with alpha-aminoamide derivatives of the antimalarial drug primaquine. A systematic computational and experimental study on the stereoselectivity of imidazolidin-4-one formation from primaquine alpha-aminoamides and various substituted benzaldehydes has been carried out, and they have allowed us to conclude that intramolecular hydrogen-bonds involving the C=O oxygen of the o-substituent play a crucial role.
Subject(s)
Amides/chemistry , Antimalarials/chemistry , Benzaldehydes/chemistry , Computational Biology , Primaquine/chemistry , Antimalarials/chemical synthesis , Catalysis , Magnetic Resonance Spectroscopy , Molecular Conformation , Molecular Structure , Primaquine/chemical synthesis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , StereoisomerismABSTRACT
The design of vaccines for RNA viral diseases is complicated by the high genetic variability of the viruses, which favors the selection of escape mutants. A case in point is foot-and-mouth disease virus (FMDV), for which only limited protection has been observed in vaccination with single peptides. We have explored the potential of immunogens of higher sequence diversity, covering a broad range of field or culture-induced mutations at the immunodominant site A of FMDV, serotype C. Four mixotope-type peptide libraries, containing ca. 3 x 10(3) or ca. 3 x 10(5) peptides each, in either linear or cyclic form, and combining most significant mutations found or induced at site A have been synthesized and used to immunize guinea-pigs. Substantial levels of serum conversion have been observed for all four mixotope libraries, as well as for single peptides, linear or cyclic, corresponding to the consensus site A sequence. The specificity and neutralizing ability of the anti-mixotope and -peptide antibodies have been evaluated by direct ELISA and by plaque reduction and micro-neutralization assays, respectively. Challenge experiments with an infectious, guinea-pig-adapted FMDV strain, have shown higher protection rates in animals immunized with the cyclic versions, either in single sequence or in combinatorial mixotope form.
