Parkinson's disease protein PARK7 prevents metabolite and protein damage caused by a glycolytic metabolite.

Cells are continuously exposed to potentially dangerous compounds. Progressive accumulation of damage is suspected to contribute to neurodegenerative diseases and aging, but the molecular identity of the damage remains largely unknown. Here we report that PARK7, an enzyme mutated in hereditary Parkinson's disease, prevents damage of proteins and metabolites caused by a metabolite of glycolysis. We found that the glycolytic metabolite 1,3-bisphosphoglycerate (1,3-BPG) spontaneously forms a novel reactive intermediate that avidly reacts with amino groups. PARK7 acts by destroying this intermediate, thereby preventing the formation of proteins and metabolites with glycerate and phosphoglycerate modifications on amino groups. As a consequence, inactivation of PARK7 (or its orthologs) in human cell lines, mouse brain, and Drosophila melanogaster leads to the accumulation of these damaged compounds, most of which have not been described before. Our work demonstrates that PARK7 function represents a highly conserved strategy to prevent damage in cells that metabolize carbohydrates. This represents a fundamental link between metabolism and a type of cellular damage that might contribute to the development of Parkinson's disease.

Oxygenation and adenosine deaminase support growth and proliferation of ex vivo cultured Drosophila wing imaginal discs.

The Drosophila wing imaginal disc has been an important model system over past decades for discovering novel biology related to development, signaling and epithelial morphogenesis. Novel experimental approaches have been enabled using a culture setup that allows ex vivo cultures of wing discs. Current setups, however, are not able to sustain both growth and cell-cycle progression of wing discs ex vivo We discover here a setup that requires both oxygenation of the tissue and adenosine deaminase activity in the medium, and supports both growth and proliferation of wing discs for 9 h. Nonetheless, further work will be required to extend the duration of the culturing and to enable live imaging of the cultured discs in the future.

Ecdysone regulates Drosophila wing disc size via a TORC1 dependent mechanism.

Most cells in a developing organ stop proliferating when the organ reaches a correct, final size. The underlying molecular mechanisms are not understood. We find that in Drosophila the hormone ecdysone controls wing disc size. To study how ecdysone affects wing size, we inhibit endogenous ecdysone synthesis and feed larvae exogenous ecdysone in a dose-controlled manner. For any given ecdysone dose, discs stop proliferating at a particular size, with higher doses enabling discs to reach larger sizes. Termination of proliferation coincides with a drop in TORC1, but not Dpp or Yki signaling. Reactivating TORC1 bypasses the termination of proliferation, indicating that TORC1 is a main downstream effector causing proliferation termination at the maximal ecdysone-dependent size. Experimental manipulation of Dpp or Yki signaling can bypass proliferation termination in hinge and notum regions, but not the pouch, suggesting that the mechanisms regulating proliferation termination may be distinct in different disc regions.

Crebl2 regulates cell metabolism in muscle and liver cells.

We previously identified Drosophila REPTOR and REPTOR-BP as transcription factors downstream of mTORC1 that play an important role in regulating organismal metabolism. We study here the mammalian ortholog of REPTOR-BP, Crebl2. We find that Crebl2 mediates part of the transcriptional induction caused by mTORC1 inhibition. In C2C12 myoblasts, Crebl2 knockdown leads to elevated glucose uptake, elevated glycolysis as observed by lactate secretion, and elevated triglyceride biosynthesis. In Hepa1-6 hepatoma cells, Crebl2 knockdown also leads to elevated triglyceride levels. In sum, this works identifies Crebl2 as a regulator of cellular metabolism that can link nutrient sensing via mTORC1 to the metabolic response of cells.

Phenotypic characterization of SETD3 knockout Drosophila.

Lysine methylation is a reversible post-translational modification that affects protein function. Lysine methylation is involved in regulating the function of both histone and non-histone proteins, thereby influencing both cellular transcription and the activation of signaling pathways. To date, only a few lysine methyltransferases have been studied in depth. Here, we study the Drosophila homolog of the human lysine methyltransferase SETD3, CG32732/dSETD3. Since mammalian SETD3 is involved in cell proliferation, we tested the effect of dSETD3 on proliferation and growth of Drosophila S2 cells and whole flies. Knockdown of dSETD3 did not alter mTORC1 activity nor proliferation rate of S2 cells. Complete knock-out of dSETD3 in Drosophila flies did not affect their weight, growth rate or fertility. dSETD3 KO flies showed normal responses to starvation and hypoxia. In sum, we could not identify any clear phenotypes for SETD3 knockout animals, indicating that additional work will be required to elucidate the molecular and physiological function of this highly conserved enzyme.

REPTOR and REPTOR-BP Regulate Organismal Metabolism and Transcription Downstream of TORC1.

TORC1 regulates growth and metabolism, in part, by influencing transcriptional programs. Here, we identify REPTOR and REPTOR-BP as transcription factors downstream of TORC1 that are required for ∼ 90% of the transcriptional induction that occurs upon TORC1 inhibition in Drosophila. Thus, REPTOR and REPTOR-BP are major effectors of the transcriptional stress response induced upon TORC1 inhibition, analogous to the role of FOXO downstream of Akt. We find that, when TORC1 is active, it phosphorylates REPTOR on Ser527 and Ser530, leading to REPTOR cytoplasmic retention. Upon TORC1 inhibition, REPTOR becomes dephosphorylated in a PP2A-dependent manner, shuttles into the nucleus, joins its partner REPTOR-BP to bind target genes, and activates their transcription. In vivo functional analysis using knockout flies reveals that REPTOR and REPTOR-BP play critical roles in maintaining energy homeostasis and promoting animal survival upon nutrient restriction.