metabolic-analysis-of-drosophila-melanogaster-larval-and-adult-brains

Metabolic Analysis of <em>Drosophila melanogaster</em> Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Providence College

The Salvador-Warts-Hippo (Hippo) pathway is a conserved regulator of organ size and is deregulated in human cancers. In epithelial tissues, the Hippo pathway is regulated by fundamental cell biological properties, such as polarity and adhesion, and coordinates these with tissue growth. Despite its importance in disease, development, and regeneration, the complete set of proteins that regulate Hippo signaling remain undefined. To address this, we used proteomics to identify proteins that bind to the Hippo (Hpo) kinase. Prominent among these were PAK-interacting exchange factor (known as Pix or RtGEF) and G-protein-coupled receptor kinase-interacting protein (Git). Pix is a conserved Rho-type guanine nucleotide exchange factor (Rho-GEF) homologous to Beta-PIX and Alpha-PIX in mammals. Git is the single Drosophila melanogaster homolog of the mammalian GIT1 and GIT2 proteins, which were originally identified in the search for molecules that interact with G-protein-coupled receptor kinases. Pix and Git form an oligomeric scaffold to facilitate sterile 20-like kinase activation and have also been linked to GTPase regulation. We show that Pix and Git regulate Hippo-pathway-dependent tissue growth in D. melanogaster and that they do this in parallel to the known upstream regulator Fat cadherin. Pix and Git influence activity of the Hpo kinase by acting as a scaffold complex, rather than enzymes, and promote Hpo dimerization and autophosphorylation of Hpo's activation loop. Therefore, we provide important new insights into an ancient signaling network that controls the growth of metazoan tissues.

The GTPase regulatory proteins Pix and Git control tissue growth via the Hippo pathway.

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that triggers adaptive responses upon low oxygen conditions and plays a crucial role in cancer metabolism and therapy resistance. Tetrathiomolybdate (TM), a therapy option for copper overload disorder, has also been shown to be capable of limiting tumor angiogenesis, although its underlying mechanism remains unclear. Using ovarian and endometrial cancer cell lines, we observed that TM downregulates HIF-1α protein levels and HIF-transcriptional targets involved in tumor angiogenesis and glycolysis, but did not affect HIF-1α protein synthesis. TM-mediated HIF-1α downregulation was suppressed when HIF-prolyl hydroxylase activity was pharmacologically inhibited using deferoxamine or dimethyloxaloylglycine, and also when the oxygen-dependent degradation domains of HIF-1α, which are responsible for the interaction with HIF-prolyl hydroxylase, were deleted. These findings suggest that TM causes HIF-1α downregulation in a HIF-prolyl hydroxylase-dependent manner. Our studies showed that TM inhibits the activity of the copper-dependent mitochondrial complex IV and reduces mitochondrial respiration, thereby possibly increasing oxygen availability, which is crucial for HIF-prolyl hydroxylase activity. Pimonidazole staining also showed that TM elevates oxygen tension in hypoxic cells. Our studies provide mechanistic evidence for TM-mediated HIF-1α regulation and suggest its therapeutic potential as a method of blocking angiogenesis in ovarian and endometrial tumors. 

Tetrathiomolybdate inhibits mitochondrial complex IV and mediates degradation of hypoxia-inducible factor-1α in cancer cells.

Many neuronal and glial diseases have been associated with changes in metabolism. Therefore, metabolic reprogramming has become an important area of research to better understand disease at the cellular level, as well as to identify targets for treatment. Model systems are ideal for interrogating metabolic questions in a tissue dependent context. However, while new tools have been developed to study metabolism in cultured cells there has been less progress towards studies in vivo and ex vivo.

Article	Year
The GTPase regulatory proteins Pix and Git control tissue growth via the Hippo pathway. Current biology : CB\| PubMed ID: 25484297	2015
Tetrathiomolybdate inhibits mitochondrial complex IV and mediates degradation of hypoxia-inducible factor-1α in cancer cells. Scientific reports\| PubMed ID: 26469226	2015
A novel ex vivo method for measuring whole brain metabolism in model systems. Journal of neuroscience methods\| PubMed ID: 29287743	2018

Marla Tipping

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