l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria

As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M.     

Conjugated Linoleic Acid Is a Preferential Substrate for Fatty Acid Nitration

The oxidation and nitration of unsaturated fatty acids by oxides of nitrogen yield electrophilic derivatives that can modulate protein function via post-translational protein modifications. The biological mechanisms accounting for fatty acid nitration and the specific structural characteristics of products remain to be defined. Herein, conjugated linoleic acid (CLA) is identified as the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 10⁵ greater extent of nitration products as compared with bis-allylic linoleic acid. Multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification. Nitroalkene derivatives of CLA and their metabolites are detected in the plasma of healthy humans and are increased in tissues undergoing episodes of ischemia reperfusion. Dietary CLA and nitrite supplementation in rodents elevates NO₂-CLA levels in plasma, urine, and tissues, which in turn induces heme oxygenase-1 (HO-1) expression in the colonic epithelium. These results affirm that metabolic and inflammatory reactions yield electrophilic products that can modulate adaptive cell signaling mechanisms.     

Coexistence of mitochondrial and nuclear DNA mutations in a woman with mitochondrial encephalomyopathy and double cortex

We describe a 16-year-old girl with mental retardation, myoclonic epilepsy, ataxia, mitochondrial myopathy, sensorineural hearing loss, lactic acidosis, and MRI evidence of diffuse subcortical laminar heterotopia and agyria/pachygyria. Restriction fragment length polymorphism (RFLP) and DNA sequence analyses revealed two pathogenic mutations: a heteroplasmic m.3243A > G in muscle and blood, and a new heterozygous insertion at nt697 in the doublecortin gene (DCX), resulting in a frameshift after amino acid residue 232, with a premature stop codon at amino acid residue 244. This is yet another example of genetic “double trouble” resulting in a complex phenotype.     

Gliadin Peptide P31-43 Localises to Endocytic Vesicles and Interferes with Their Maturation

Background

Celiac Disease (CD) is both a frequent disease (1∶100) and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called “toxic” A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR)-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles.

Methods/Principal Findings

Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy.

Conclusions

P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs-mediated maturation to late endosomes in cells and intestinal biopsies. Consequently, in P31-43-treated cells, Receptor Tyrosin Kinase (RTK) activation is extended. This finding may explain the role played by gliadin peptides in inducing proliferation and other effects in enterocytes from CD biopsies.     

Farnesoid X Receptor, through the Binding with Steroidogenic Factor 1-responsive Element, Inhibits Aromatase Expression in Tumor Leydig Cells

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that regulates bile acid homeostasis. It is expressed in the liver and the gastrointestinal tract, but also in several non-enterohepatic tissues including testis. Recently, FXR was identified as a negative modulator of the androgen-estrogen-converting aromatase enzyme in human breast cancer cells. In the present study we detected the expression of FXR in Leydig normal and tumor cell lines and in rat testes tissue. We found, in rat Leydig tumor cells, R2C, that FXR activation by the primary bile acid chenodeoxycholic acid (CDCA) or a synthetic agonist GW4064, through a SHP-independent mechanism, down-regulates aromatase expression in terms of mRNA, protein levels, and its enzymatic activity. Transient transfection experiments, using vector containing rat aromatase promoter PII, evidenced that CDCA reduces basal aromatase promoter activity. Mutagenesis studies, electrophoretic mobility shift, and chromatin immunoprecipitation analysis reveal that FXR is able to compete with steroidogenic factor 1 in binding to a common sequence present in the aromatase promoter region interfering negatively with its activity. Finally, the FXR-mediated anti-proliferative effects exerted by CDCA on tumor Leydig cells are at least in part due to an inhibition of estrogen-dependent cell growth. In conclusion our findings identify for the first time the activators of FXR as negative modulators of the aromatase enzyme in Leydig tumor cell lines.     

Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology

We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a zinc-finger nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two ∼750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5–10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5′ overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5′ overhangs on the donor DNA. Finally, we extend this non-homologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.     

Critical role of transglutaminase and other stress proteins during neurodegenerative processes

Proteolytic stress, resulting from the intracellular accumulation of misfolded or aggregated proteins, which exceed the capacity of the ubiquitin–proteasome system to degrade them, plays a relevant role in neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s chorea. Most of toxic protein aggregates are characterised by the presence of isopeptide bonds (cross-links) catalysed by transglutaminase activity; further, several disease-specific proteins—tau, amyloid-beta, alpha-synuclein, huntingtin—are in vitro and/or in vivo substrates of transglutaminase 2. These findings suggest an important role for transglutaminase 2-mediated cross-linking reactions in neurodegeneration. Therefore, the use of transglutaminase activity inhibitors could ameliorate neuronal cell death. New therapeutic perspectives also arise from the possibility to prevent or reduce protein aggregation by enhancing the activation of heat shock proteins, which have been shown to be potent suppressors of neurodegeneration in cell cultures/animal models. Interestingly, some heat shock proteins have been shown to be in vitro or in vivo cross-linked by transglutaminase 2. These observations seem to suggest that transglutaminase activity could be involved in the stabilization of intracellular protein aggregates by interfering with proteasomal degradation of misfolded proteins. Further studies are needed to validate leading hypotheses and to open new prospects for developing therapeutic tools.