Identification of FAH domain-containing protein 1 (FAHD1) as oxaloacetate decarboxylase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins occur in both prokaryotes and eukaryotes, where they carry out diverse enzymatic reactions, probably related to structural differences in their respective FAH domains; however, the precise relationship between structure of the FAH domain and the associated enzyme function remains elusive. In mammals, three FAH domain-containing proteins, FAHD1, FAHD2A, and FAHD2B, are known; however, their enzymatic function, if any, remains to be demonstrated. In bacteria, oxaloacetate is subject to enzymatic decarboxylation; however, oxaloacetate decarboxylases (ODx) were so far not identified in eukaryotes. Based on molecular modeling and subsequent biochemical investigations, we identified FAHD1 as a eukaryotic ODx enzyme. The results presented here indicate that dedicated oxaloacetate decarboxylases exist in eukaryotes.

Characterisation of Nox4 inhibitors from edible plants.

NADPH oxidases transport electrons from cytosolic NADPH through biological membranes to generate reactive oxygen species. NADPH oxidase 4, broadly expressed in humans, is an interesting pharmacological target, since its activity is deregulated in several diseases, including pulmonary fibrosis, diabetic nephropathy, and cardiac hypertrophy. Whereas several candidate NADPH oxidase 4 inhibitors were recently described, most of these compounds are either unspecific or toxic. Here we set out to identify new NADPH oxidase 4 inhibitors from edible plants, in an attempt to decrease the number of hits with toxic side effects. We screened a compound library prepared from edible plants for new bioactives with the ability to inhibit the activity of NADPH oxidase 4. Using both cell-based and cell-free assays, we identified several compounds with significant inhibitory activity towards NADPH oxidase 4. For selected compounds, the activity profile towards NADPH oxidase 2 and NADPH oxidase 5 was established, and controls were carried out to exclude general reactive oxygen species scavengers. A number of promising NADPH oxidase 4 inhibitors from edible plants was identified and characterised. Several new chemical entities are disclosed which act as NADPH oxidase 4 inhibitors, and the efficacies of our best hits, in particular several diarylheptanoids and lignans, are comparable to the best available pharmacological NADPH oxidase 4 inhibitors. These findings will provide valuable tools to study mechanisms of NADPH oxidase inhibition.

Insulin-like growth factor binding protein-6 delays replicative senescence of human fibroblasts.

Cellular senescence can be induced by a variety of mechanisms, and recent data suggest a key role for cytokine networks to maintain the senescent state. Here, we have used a proteomic LC-MS/MS approach to identify new extracellular regulators of senescence in human fibroblasts. We identified 26 extracellular proteins with significantly different abundance in conditioned media from young and senescent fibroblasts. Among these was insulin-like growth factor binding protein-6 (IGFBP-6), which was chosen for further analysis. When IGFBP-6 gene expression was downregulated, cell proliferation was inhibited and apoptotic cell death was increased. Furthermore, downregulation of IGFBP-6 led to premature entry into cellular senescence. Since IGFBP-6 overexpression increased cellular lifespan, the data suggest that IGFBP-6, in contrast to other IGF binding proteins, is a negative regulator of cellular senescence in human fibroblasts.

Role of endonuclease G in senescence-associated cell death of human endothelial cells.

Mitotic cells in culture show a limited replicative potential and after extended subculturing undergo a terminal growth arrest termed cellular senescence. When cells reach the senescent phenotype, this is accompanied by a significant change in the cellular phenotype and massive changes in gene expression, including the upregulation of secreted factors. In human fibroblasts, senescent cells also acquire resistance to apoptosis. In contrary, in human endothelial cells, both replicative and stress-induced premature senescence is accompanied by increased cell death; however mechanisms of cell death are poorly explored. In this communication, we addressed the role of endonuclease G (EndoG), a mitochondrial mediator of caspase-independent cell death, in senescence-associated cell death of human endothelial cells. Using immunofluorescence microscopy, we found, that EndoG is localized in the mitochondria in young cells, but relocalizes to the nucleus upon senescence. When EndoG gene expression was downregulated by lentiviral shRNA vectors, we found a significant reduction in the replicative life span and a corresponding increase in cell death. We also observed a slight shift in the cell death phenotype from necrosis to apoptosis. Together these observations suggest an important role of EndoG in the senescence program of human endothelial cells.