The first evidence of biological activity for free Hypusine, an enigmatic amino acid discovered in the '70s.

Hypusine amino acid [Nε-(4-amino-2-hydroxybutyl)-lysine] was first isolated in 1971 from bovine brain extracts. Hypusine originates from a post-translational modification at the eukaryotic translation initiation factor 5A (eIF5A), a protein produced by archaebacteria and eukaryotes. The eIF5A protein is the only one described containing the hypusine residue, which is essential for its activity. Hypusine as a free amino acid is a consequence of proteolytic degradation of eIF5A. Herein, we showed, for the first time, evidence of biological activity for the free hypusine. C6 rat glioma cells were treated with hypusine, and different cellular parameters were evaluated. Hypusine treatment significantly reduced C6 cell proliferation and potently suppressed their clonogenic capacity without leading to apoptosis. Hypusine also decreased the Eif5A transcript content and the global protein synthesis profile that may occur due to negative feedback in response to high hypusine concentration, controlling the content of newly synthesized eIF5A, which can affect the translation process. Besides, hypusine treatment also altered cellular metabolism by changing the pathways for energy production, reducing cellular respiration coupled with oxidative phosphorylation, and increasing the anaerobic metabolism. These observed results and the relationship between eIF5A and tumor processes led us to test the combination of hypusine with the chemotherapeutic drug temozolomide. Combining temozolomide with hypusine reduced the MTT conversion to the same levels as those observed using double temozolomide dosage alone, demonstrating a synergetic action between the compounds. Thus, since 1971, this is the first study showing evidence of biological activity for hypusine not associated with being an essential component of the eiF5A protein. Finding out the molecular targets of hypusine are the following efforts to completely characterize its biological activity.

Insulin action on protein synthesis and its association with eIF5A expression and hypusination.

The hormone insulin plays a central role in the metabolism of carbohydrates, lipids, and proteins. In relation to protein metabolism, insulin stimulates amino acid uptake and activates protein synthesis in responsive cells by modulation of signal transduction pathways, such as associated to Akt/PkB, mTOR, S6Ks, 4E-BP1, and several translation initiation/elongation factors. In this context, there is no information on direct cellular treatment with insulin and effects on eukaryotic translation initiation factor 5A (eIF5A) regulation. The eIF5A protein contains an exclusive amino acid residue denominated hypusine, which is essential for its activity and synthesized by posttranslational modification of a specific lysine residue using spermidine as substrate. The eIF5A protein is involved in cellular proliferation and differentiation processes, as observed for satellite cells derived from rat muscles, revealing that eIF5A has an important role in muscle regeneration. The aim of this study was to determine whether eIF5A expression and hypusination are influenced by direct treatment of insulin on L6 myoblast cells. We observed that insulin increased the content of eIF5A transcripts. This effect occurred in cells treated or depleted of fetal bovine serum, revealing a positive insulin effect independent of other serum components. In addition, it was observed that hypusination follows the maintenance of eIF5A protein content in the serum depleted cells and treated with insulin. These results demonstrate that eIF5A is modulated by insulin, contributing the protein synthesis machinery control, as observed by puromycin incorporation in nascent proteins.

Alternative Start Codon Connects eIF5A to Mitochondria.

Eukaryotic translation initiation factor 5A (eIF5A), a protein containing the amino acid residue hypusine required for its activity, is involved in a number of physiological and pathological cellular processes. In humans, several EIF5A1 transcript variants encode the canonical eIF5A1 isoform B, whereas the hitherto uncharacterized variant A is expected to code for a hypothetical eIF5A1 isoform, referred to as isoform A, which has an additional N-terminal extension. Herein, we validate the existence of eIF5A1 isoform A and its production from transcript variant A. In fact, variant A was shown to encode both eIF5A1 isoforms A and B. Mutagenic assays revealed different efficiencies in the start codons present in variant A, contributing to the production of isoform B at higher levels than isoform A. Immunoblotting and mass spectrometric analyses showed that isoform A can undergo hypusination and acetylation at specific lysine residues, as observed for isoform B. Examination of the N-terminal extension suggested that it might confer mitochondrial targeting. Correspondingly, we found that isoform A, but not isoform B, co-purified with mitochondria when the proteins were overproduced. These findings suggest that eIF5A1 isoform A has a role in mitochondrial function. J. Cell. Physiol. 231: 2682-2689, 2016. © 2016 Wiley Periodicals, Inc.

Titanium-protein nanocomposites as new biomaterials produced by high-pressure torsion.

The development of new biomaterials with outstanding mechanical properties and high biocompatibility has been a significant challenge in the last decades. Nanocrystalline metals have provided new opportunities in producing high-strength biomaterials, but the biocompatibility of these nanometals needs to be improved. In this study, we introduce metal-protein nanocomposites as high-strength biomaterials with superior biocompatibility. Small proportions of bovine serum albumin (2 and 5 vol%), an abundant protein in the mammalian body, are added to titanium, and two nanocomposites are synthesized using a severe plastic deformation process of high-pressure torsion. These new biomaterials show not only a high hardness similar to nanocrystalline pure titanium but also exhibit better biocompatibility (including cellular metabolic activity, cell cycle parameters and DNA fragmentation profile) compared to nano-titanium. These results introduce a pathway to design new biocompatible composites by employing compounds from the human body.

Glucoregulatory and Anti-Inflammatory Activities of Peptide Fractions Separated by Electrodialysis with Ultrafiltration Membranes from Salmon Protein Hydrolysate and Identification of Four Novel Glucoregulatory Peptides.

Natural bioactive peptides are suitable candidates for preventing the development of Type 2 diabetes (T2D), by reducing the various risk factors. The aim of this study was to concentrate glucoregulatory and anti-inflammatory peptides, from salmon by-products, by electrodialysis with ultrafiltration membrane (EDUF), and to identify peptides responsible for these bioactivities. Two EDUF configurations (1 and 2) were used to concentrate anionic and cationic peptides, respectively. After EDUF separation, two fractions demonstrated interesting properties: the initial fraction of the EDUF configuration 1 and the final fraction of the EDUF configuration 2 both showed biological activities to (1) increase glucose uptake in L6 muscle cells in insulin condition at 1 ng/mL (by 12% and 21%, respectively), (2) decrease hepatic glucose production in hepatic cells at 1 ng/mL in basal (17% and 16%, respectively), and insulin (25% and 34%, respectively) conditions, and (3) decrease LPS-induced inflammation in macrophages at 1 g/mL (45% and 30%, respectively). More impressive, the initial fraction of the EDUF configuration 1 (45% reduction) showed the same effect as the phenformin at 10 µM (40%), a drug used to treat T2D. Thirteen peptides were identified, chemically synthesized, and tested in-vitro for these three bioactivities. Thus, four new bioactive peptides were identified: IPVE increased glucose uptake by muscle cells, IVDI and IEGTL decreased hepatic glucose production (HGP) of insulin, whereas VAPEEHPTL decreased HGP under both basal condition and in the presence of insulin. To the best of our knowledge, this is the first time that (1) bioactive peptide fractions generated after separation by EDUF were demonstrated to be bioactive on three different criteria; all involved in the T2D, and (2) potential sequences involved in the improvement of glucose uptake and/or in the regulation of HGP were identified from a salmon protein hydrolysate.

Ultrahigh hardness and biocompatibility of high-entropy alloy TiAlFeCoNi processed by high-pressure torsion.

Despite significant studies on mechanical properties of high-entropy alloys (HEAs), there have been limited attempts to examine the biocompatibility of these alloys. In this study, a lattice-softened high-entropy alloy TiAlFeCoNi with ultrahigh hardness (examined by Vickers method), low elastic modulus (examined by nanoindentation) and superior activity for cell proliferation/viability/cytotoxicity (examined by MTT assay) was developed by employing imperial data and thermodynamic calculations. The designated alloy after casting was processed further by high-pressure torsion (HPT) to improve its hardness via the introduction of nanograins, dislocations and order-disorder transformation. The TiAlFeCoNi alloy with the L21-BCC crystal structure exhibited 170-580% higher hardness and 260-1020% better cellular metabolic activity compared to titanium and Ti-6Al-7Nb biomaterials, suggesting the high potential of HEAs for future biomedical applications.

Transcriptome analysis in Parhyale hawaiensis reveal sex-specific responses to AgNP and AgCl exposure.

Analysis of the transcriptome of organisms exposed to toxicants offers new insights for ecotoxicology, but further research is needed to enhance interpretation of results and effectively incorporate them into useful environmental risk assessments. Factors that must be clarified to improve use of transcriptomics include assessment of the effect of organism sex within the context of toxicant exposure. Amphipods are well recognized as model organisms for toxicity evaluation because of their sensitivity and amenability to laboratory conditions. To investigate whether response to metals in crustaceans differs according to sex we analyzed the amphipod Parhyale hawaiensis after exposure to AgCl and Ag nanoparticles (AgNP) via contaminated food. Gene specific analysis and whole genome transcriptional profile of male and female organisms were performed by both RT-qPCR and RNA-seq. We observed that expression of transcripts of genes glutathione transferase (GST) did not differ among AgCl and AgNP treatments. Significant differences between males and females were observed after exposure to AgCl and AgNP. Males presented twice the number of differentially expressed genes in comparison to females, and more differentially expressed were observed after exposure to AgNP than AgCl treatments in both sexes. The genes that had the greatest change in expression relative to control were those genes related to peptidase and catalytic activity and chitin and carbohydrate metabolic processes. Our study is the first to demonstrate sex specific differences in the transcriptomes of amphipods upon exposure to toxicants and emphasizes the importance of considering gender in ecotoxicology.