Depletion of Rictor, an essential protein component of mTORC2, decreases male lifespan

Rapamycin, an inhibitor of the mechanistic target of rapamycin (mTOR), robustly extends the lifespan of model organisms including mice. We recently found that chronic treatment with rapamycin not only inhibits mTOR complex 1 (mTORC1), the canonical target of rapamycin, but also inhibits mTOR complex 2 (mTORC2) in vivo. While genetic evidence strongly suggests that inhibition of mTORC1 is sufficient to promote longevity, the impact of mTORC2 inhibition on mammalian longevity has not been assessed. RICTOR is a protein component of mTORC2 that is essential for its activity. We examined three different mouse models of Rictor loss: mice heterozygous for Rictor, mice lacking hepatic Rictor, and mice in which Rictor was inducibly deleted throughout the body in adult animals. Surprisingly, we find that depletion of RICTOR significantly decreases male, but not female, lifespan. While the mechanism by which RICTOR loss impairs male survival remains obscure, we find that the effect of RICTOR depletion on lifespan is independent of the role of hepatic mTORC2 in promoting glucose tolerance. Our results suggest that inhibition of mTORC2 signaling is detrimental to males, which may explain in part why interventions that decrease mTOR signaling show greater efficacy in females

MCT1-mediated transport of a toxic molecule is an effective strategy for targeting glycolytic tumors

There is increasing evidence that oncogenic transformation modifies the metabolic program of cells. A common alteration is the upregulation of glycolysis, and efforts to target glycolytic enzymes for anticancer therapy are under way. Here, we performed a genome-wide haploid genetic screen to identify resistance mechanisms to 3-bromopyruvate (3-BrPA), a drug candidate that inhibits glycolysis in a poorly understood fashion. We identified the SLC16A1 gene product, MCT1, as the main determinant of 3-BrPA sensitivity. MCT1 is necessary and sufficient for 3-BrPA uptake by cancer cells. Additionally, SLC16A1 mRNA levels are the best predictor of 3-BrPA sensitivity and are most elevated in glycolytic cancer cells. Furthermore, forced MCT1 expression in 3-BrPA–resistant cancer cells sensitizes tumor xenografts to 3-BrPA treatment in vivo. Our results identify a potential biomarker for 3-BrPA sensitivity and provide proof of concept that the selectivity of cancer-expressed transporters can be exploited for delivering toxic molecules to tumors.National Institutes of Health (U.S.) (NIH CA103866)Jane Coffin Childs Memorial Fund for Medical Research (Fellowship)National Science Foundation (U.S.) (Fellowship)Howard Hughes Medical Institute (Investigator

Regulation of Protein Degradation by ADP-Ribosylation

Protein quality control is essential for cellular homeostasis. The Ubiquitin-Proteasome System (UPS) is responsible for the regulated breakdown of intracellular proteins. All proteins are not degraded at the same rate in the cell. For instance, global protein turnover rates in mammals change with an average time between 1-2 days. On the other hand, a handful of proteins such as myelin exhibits limited turnover for months or even years. The UPS recycles most of the short-lived proteins in a time range from minutes to days depending on their localization and post-translational modifications. The post-translational modification, poly-ADPribosylation has an estimated half-life of only 1-6 min. My thesis research aimed to reveal how the substrates of Tankyrase are degraded rapidly. Using Drosophila melanogaster, I described the role of Iduna E3 ubiquitin ligase in the regulation of Axin and Tankyrase proteolysis. I found that Iduna controls the proliferation of stem cells in the Drosophila midgut. Using a MS-based approach, I identified lysine 598 as an ADP-ribose acceptor site in Drosophila Tankyrase and showed that TnksK598A adult flies live significantly shorter than control flies. TnksK598A adult flies also reduce their flight, climbing abilities, global protein poly-ADP-ribosylation, and the activation of JNK signaling with age. Furthermore, I demonstrated that the ubcD1 ubiquitin-conjugating enzyme enhances the ubiquitin ligase activity of Iduna. Finally, I proposed a model by which poly-ADP-ribosylation brings together Tankyrase, target proteins, E2 ubiquitin-conjugating enzymes, E3 ubiquitin ligases and 26S proteasomes to accelerate the breakdown of target proteins. My work addresses the general question of how proteins can be rapidly turned over, focusing on the role of Tankyrase-mediated poly-ADP-ribosylation. This work provides novel mechanistic insights into the regulation of protein quality control. Ultimately, these results may be useful to guide the development of new therapies

Novel NLRP3/cryopyrin mutations and pro-inflammatory cytokine profiles in Behets syndrome patients

The role of mutations in NLRP3 in inflammatory features of Behets syndrome.Behets syndrome (BS) is a systemic inflammatory disorder with unknown etiology. Features of both innate and adaptive immunity have been claimed in the pathogenesis of BS. To test the possible dysregulation of the NLRP3/cryopyrin (Nod-like receptor with a pyrin domain 3) inflammasome, as a result of mutation(s), we performed single-strand conformation polymorphism analyses and/or sequencing of all the coding regions and intronexon boundaries of NLRP3/cryopyrin and ASC (apoptosis-associated speck-like protein containing CARD) genes from Turkish BS patients and healthy controls. At the same time, we determined pro-inflammatory cytokine secretion profiles of peripheral blood cells in response to LPS treatment using ELISA. BS patients with vascular involvement showed significantly increased levels of TNF- release at 2-, 4- and 8-h post-treatment and significantly increased IL-1 levels were detected at 2h (P 0.005) and 4h (P 0.025) (n 10). We identified four mutations in the NLRP3/cryopyrin gene, V200M (n 3/104) and T195M (n 1/104), in BS patients but none in control samples. No mutations were detected in the ASC gene. The effect of these NLRP3/cryopyrin mutants on ASC speck assembly and IL-1 secretion was tested and the V200M mutant was shown to induce IL-1 secretion. Thus, it is likely that certain mutations in NLRP3/cryopyrin in combination with yet unknown other factors may contribute to the pro-inflammatory cytokine profiles in BS patients

Slow TCA flux and ATP production in primary solid tumours but not metastases

Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP