Ennio Cocca

c9,t11-Conjugated linoleic acid ameliorates steatosis by modulating mitochondrial uncoupling and Nrf2 pathway

Oxidative stress, hepatic steatosis, and mitochondrial dysfunction are key pathophysiological features of nonalcoholic fatty liver disease. A conjugated linoleic acid (CLA) mixture of cis9,trans11 (9,11-CLA) and trans10,cis12 (10,12-CLA) isomers enhanced the antioxidant/detoxifying mechanism via the activation of nuclear factor E2-related factor-2 (Nrf2) and improved mitochondrial function, but less is known about the actions of specific isomers. The differential ability of individual CLA isomers to modulate these pathways was explored in Wistar rats fed for 4 weeks with a lard-based high-fat diet (L) or with control diet (CD), and, within each dietary treatment, two subgroups were daily administered with 9,11-CLA or 10,12-CLA (30 mg/day). The 9,11-CLA, but not 10,12-CLA, supplementation to CD rats improves the GSH/GSSG ratio in the liver, mitochondrial functions, and Nrf2 activity. Histological examination reveals a reduction of steatosis in L-fed rats supplemented with both CLA isomers, but 9,11-CLA downregulated plasma concentrations of proinflammatory markers, mitochondrial dysfunction, and oxidative stress markers in liver more efficiently than in 10,12-CLA treatment. The present study demonstrates the higher protective effect of 9,11-CLA against diet-induced pro-oxidant and proinflammatory signs and suggests that these effects are determined, at least in part, by its ability to activate the Nrf2 pathway and to improve the mitochondrial functioning and biogenesis.

Conjugated linoleic acid protects against gliadin-induced depletion of intestinal defenses.

SCOPE The involvement of oxidative stress in gluten-induced toxicity has been evidenced in vitro and in clinical studies but has never been examined in vivo. We recently demonstrated the protective activity of conjugated linoleic acid (CLA), which functions by the activation of nuclear factor erythroid 2-related factor2 (Nrf2), a key transcription factor for the synthesis of antioxidant and detoxifying enzymes (phase 2). Here, we evaluate the involvement of nuclear factor erythroid 2-related factor2 in gliadin-mediated toxicity in human Caco-2 intestinal cells and in gliadin-sensitive human leukocyte antigen-DQ8 transgenic mice (DQ8) and the protective activity of CLA. METHODS AND RESULTS Gliadin effects in differentiated Caco-2 cells and in DQ8 mice, fed with a gliadin-containing diet with or without CLA supplementation, were evaluated by combining enzymatic, immunochemical, immunohistochemical, and quantitative real-time PCR (qRT-PCR) assays. Gliadin toxicity was accompanied by downregulation of phase 2 and elevates proteasome-acylpeptide hydrolase activities in vitro and in vivo. Notably, gliadin was unable to generate severe oxidative stress extent or pathological consequences in DQ8 mice intestine comparable to those found in celiac patients and the alterations produced were hampered by CLA. CONCLUSION The beneficial effects of CLA against the depletion of crucial intestinal cytoprotective defenses indicates a novel nutritional approach for the treatment of intestinal disease associated with altered redox homeostasis.

Mapping of alpha- and beta-globin genes on Antarctic fish chromosomes by fluorescence in-situ hybridization.

The pathways and mechanisms of genomic change that have led to the peculiar haemoglobinless phenotype of the white-blooded Antarctic icefishes (16 species in the family Channichthyidae) constitute an important model for understanding the rapid diversification of the Antarctic notothenioid fish flock. To provide complementary structural information on genomic change at globin-gene loci in Antarctic fish species, cytogenetic studies and in-situ chromosomal mapping have been undertaken. Using a DNA probe containing one α- and one β-globin gene from the embryonic/juvenile globin gene cluster of the red-blooded species Notothenia coriiceps, we mapped the cluster on the chromosomes of Antarctic teleosts by fluorescence in-situ hybridization. As anticipated on the basis of its molecular organization, the cluster was located on a single chromosome pair in all of the red-blooded fish species probed (N. coriiceps, N. angustata, Trematomus hansoni, T. pennellii). In contrast, the α/β-globin probe did not recognize complementary sequences on the chromosomes of the white-blooded species Chionodraco hamatus and Channichthys rhinoceratus. These results represent the first example of chromosomal mapping of embryonic/juvenile globin genes in teleostean fishes. Beyond its relevance to the evolutionary history of Antarctic notothenioids, this work contributes to our understanding of the evolution of the chromosomal loci of globin genes in fishes and other vertebrates.

Structure, function and molecular adaptations of haemoglobins of the polar cartilaginous fish Bathyraja eatonii and Raja hyperborea

Cartilaginous fish are very ancient organisms. In the Antarctic sea, the modern chondrichthyan genera are poorly represented, with only three species of sharks and eight species of skates; the paucity of chondrichthyans is probably an ecological consequence of unusual trophic or habitat conditions in the Southern Ocean. In the Arctic, there are 26 species belonging to the class Chondrichthyes. Fish in the two polar regions have been subjected to different regional histories that have influenced the development of diversity: Antarctic marine organisms are highly stenothermal, in response to stable water temperatures, whereas the Arctic communities are exposed to seasonal temperature variations. The structure and function of the oxygen-transport haem protein from the Antarctic skate Bathyraja eatonii and from the Arctic skate Raja hyperborea (both of the subclass Elasmobranchii, order Rajiformes, family Rajidae) is reported in the present paper. These species have a single major haemoglobin (Hb 1; over 80% of the total). The Bohr-proton and the organophosphate-binding sites are absent. Thus the haemoglobins of northern and southern polar skates appear functionally similar, whereas differences were observed with several temperate elasmobranchs. Such evidence suggests that, in temperate and polar habitats, physiological adaptations have evolved along distinct pathways, whereas, in this case, the effect of the differences characterizing the two polar environments is negligible.

Identification of a novel helitron transposon in the genome of Antarctic fish

Rolling-circle (RC) eukaryotic transposons, known as helitrons, are found in a wide range of organisms, from protist to mammals. Autonomous helitrons have a distinctive open reading frame (ORF) encoding a polypeptide that contains typical domains for RC replication (RCR): the Rep (RCR initiator) and the DNA helicase domains. These elements are believed to have an important role in the host genome evolution, owing to their frequent capture of host genes, some of which can evolve into novel genes or become essential for helitron transposition. We conducted a molecular analysis of the suborder Notothenioidei, a group of Perciformes that currently dominate the Antarctic waters by virtue of their remarkable cold-adaptation ability. A novel helitron from the genome of the icefish species Chionodraco hamatus, belonging to the Channichthyidae, the most derived Notothenioids family, was isolated, characterized and designated as HeliNoto (8.9 kb). Its ORF was compared to homologous sequences from different species in a comprehensive phylogenetic analysis. For the first time the putative functional domains of a helitron were subjected to a well accurate structural analysis including chromosomal localization. Finally, the distribution of HeliNoto among Notothenioids was investigated.

Evolutionary Studies on Teleost Hemoglobin Sequences

During cold adaptation, the blood of Antarctic fish has acquired some features which clearly differentiate these fish from fish of temperate and tropical climates. A much lower erythrocyte and hemoglobin content counteracts the temperature-induced viscosity increase in the blood and, consequently, cardiac work is greatly facilitated. At the extreme end of such evolution, the blood of the Notothenioid family Channichthyidae (a unique case among vertebrates) is characterized by the total lack of hemoglobin.

A novel class of bifunctional acylpeptide hydrolases – potential role in the antioxidant defense systems of the Antarctic fish Trematomus bernacchii

Oxidative challenge is an important factor affecting the adaptive strategies of Antarctic fish, but data on antioxidant defenses in these organisms remain scarce. In this context, a key role could be played by acylpeptide hydrolase (APEH), which was recently hypothesized to participate in the degradation of oxidized and cytotoxic proteins, although its physiological function is still not fully clarified. This study represents the first report on piscine members of this enzyme family, specifically from the Antarctic teleost Trematomus bernacchii. The cDNAs corresponding to two apeh genes were isolated, and the respective proteins were functionally and structurally characterized with the aim of understanding the biological significance of these proteases in Antarctic fish. Both APEH isoforms (APEH‐1Tb and APEH‐2Tb) showed distinct temperature‐kinetic behavior, with significant differences in the Km values. Moreover, beside the typical acylpeptide hydrolase activity, APEH‐2Tb showed remarkable oxidized protein endohydrolase activity towards oxidized BSA, suggesting that this isoform could play a homeostatic role in removing oxidatively damaged proteins, sustaining the antioxidant defense systems. The 3D structures of both APEHs were predicted, and a possible relationship was found between the substrate specificity/affinity and the marked changes in the number of charged residues and hydrophobicity properties surrounding their catalytic sites. Our results demonstrated the occurrence of two APEH isoforms in T. bernacchii, belonging to different phylogenetic clusters, identified for the first time, and showing distinct molecular and temperature–kinetic behaviors. In addition, we suggest that the members of the new cluster ‘APEH‐2’ could participate in reactive oxygen species detoxification as phase 3 antioxidant enzymes, enhancing the protein degradation machinery.

RedOx Status, Proteasome and APEH: Insights into Anticancer Mechanisms of t10,c12-Conjugated Linoleic Acid Isomer on A375 Melanoma Cells

This study describes the investigation of the efficiency of conjugated linoleic acid (CLA) isomers in reducing cancer cells viability exploring the role of the oxidative stress and acylpeptide hydrolase (APEH)/proteasome mediated pathways on pro-apoptotic activity of the isomer trans10,cis12 (t10,c12)-CLA. The basal activity/expression levels of APEH and proteasome (β-5 subunit) were preliminarily measured in eight cancer cell lines and the functional relationship between these enzymes was clearly demonstrated through their strong positive correlation. t10,c12-CLA efficiently inhibited the activity of APEH and proteasome isoforms in cell-free assays and the negative correlation between cell viability and caspase 3 activity confirmed the pro-apoptotic role of this isomer. Finally, modulatory effects of t10,c12-CLA on cellular redox status (intracellular glutathione, mRNA levels of antioxidant/detoxifying enzymes activated through NF-E2-related factor 2, Nrf2, pathway) and on APEH/β-5 activity/expression levels, were investigated in A375 melanoma cells. Dose- and time-dependent variations of the considered parameters were established and the resulting pro-apoptotic effects were shown to be associated with an alteration of the redox status and a down-regulation of APEH/proteasome pathway. Therefore, our results support the idea that these events are involved in ROS-dependent apoptosis of t10,c12-CLA-treated A375 cells. The combined inhibition, triggered by t10,c12-CLA, via the modulation of APEH/proteasome and Nrf2 pathway for treating melanoma, is suggested as a subject for further in vivo studies.

Identification and Characterisation of a Novel Acylpeptide Hydrolase from Sulfolobus Solfataricus: Structural and Functional Insights

A novel acylpeptide hydrolase, named APEH-3Ss, was isolated from the hypertermophilic archaeon Sulfolobus solfataricus. APEH is a member of the prolyl oligopeptidase family which catalyzes the removal of acetylated amino acid residues from the N terminus of oligopeptides. The purified enzyme shows a homotrimeric structure, unique among the associate partners of the APEH cluster and, in contrast to the archaeal APEHs which show both exo/endo peptidase activities, it appears to be a “true” aminopeptidase as exemplified by its mammalian counterparts, with which it shares a similar substrate specificity. Furthermore, a comparative study on the regulation of apeh gene expression, revealed a significant but divergent alteration in the expression pattern of apeh-3Ss and apehSs (the gene encoding the previously identified APEHSs from S. solfataricus), which is induced in response to various stressful growth conditions. Hence, both APEH enzymes can be defined as stress-regulated proteins which play a complementary role in enabling the survival of S. solfataricus cells under different conditions. These results provide new structural and functional insights into S. solfataricus APEH, offering a possible explanation for the multiplicity of this enzyme in Archaea.

A New APEH Cluster with Antioxidant Functions in the Antarctic Hemoglobinless Icefish Chionodraco hamatus.

Acylpeptide hydrolase (APEH) is a ubiquitous cytosolic protease that plays an important role in the detoxification of oxidised proteins. In this work, to further explore the physiological role of this enzyme, two apeh cDNAs were isolated from the Chionodraco hamatus icefish, which lives in the highly oxygenated Antarctic marine environment. The encoded proteins (APEH-1Ch and APEH-2Ch) were characterised in comparison with the uniquely expressed isoform from the temperate fish Dicentrarchus labrax (APEH-1Dl) and the two APEHs from the red-blooded Antarctic fish Trematomus bernacchii (APEH-1Tb and APEH-2Tb). Homology modelling and kinetic characterisation of the APEH isoforms provided new insights into their structure/function properties. APEH-2 isoforms were the only ones capable of hydrolysing oxidised proteins, with APEH-2Ch being more efficient than APEH-2Tb at this specific function. Therefore, this ability of APEH-2 isoforms is the result of an evolutionary adaptation due to the pressure of a richly oxygenated environment. The lack of expression of APEH-2 in the tissues of the temperate fish used as the controls further supported this hypothesis. In addition, analysis of gene expression showed a significant discrepancy between the levels of transcripts and those of proteins, especially for apeh-2 genes, which suggests the presence of post-transcriptional regulation mechanisms, triggered by cold-induced oxidative stress, that produce high basal levels of APEH-2 mRNA as a reserve that is ready to use in case of the accumulation of oxidised proteins.