Mutations in either the TSC1 or TSC2 tumor suppressor gene are responsible for Tuberous Sclerosis Complex. The gene products of TSC1 and TSC2 form a functional complex and inhibit the phosphorylation of S6K and 4EBP1, two key regulators of translation. Here, we describe that TSC2 is regulated by cellular energy levels and plays an essential role in the cellular energy response pathway. Under energy starvation conditions, the AMP-activated protein kinase (AMPK) phosphorylates TSC2 and enhances its activity. Phosphorylation of TSC2 by AMPK is required for translation regulation and cell size control in response to energy deprivation. Furthermore, TSC2 and its phosphorylation by AMPK protect cells from energy deprivation-induced apoptosis. These observations demonstrate a model where TSC2 functions as a key player in regulation of the common mTOR pathway of protein synthesis, cell growth, and viability in response to cellular energy levels.

Mutation in the TSC2 tumor suppressor causes tuberous sclerosis complex, a disease characterized by hamartoma formation in multiple tissues. TSC2 inhibits cell growth by acting as a GTPase-activating protein toward Rheb, thereby inhibiting mTOR, a central controller of cell growth. Here, we show that Wnt activates mTOR via inhibiting GSK3 without involving beta-catenin-dependent transcription. GSK3 inhibits the mTOR pathway by phosphorylating TSC2 in a manner dependent on AMPK-priming phosphorylation. Inhibition of mTOR by rapamycin blocks Wnt-induced cell growth and tumor development, suggesting a potential therapeutic value of rapamycin for cancers with activated Wnt signaling. Our results show that, in addition to transcriptional activation, Wnt stimulates translation and cell growth by activating the TSC-mTOR pathway. Furthermore, the sequential phosphorylation of TSC2 by AMPK and GSK3 reveals a molecular mechanism of signal integration in cell growth regulation.

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs. Two genes responsible for the disease, TSC1 and TSC2, have been identified. The TSC1 and TSC2 proteins, also called hamartin and tuberin, respectively, have been shown to regulate cell growth through inhibition of the mammalian target of rapamycin pathway. TSC1 is known to stabilize TSC2 by forming a complex with TSC2, which is a GTPase-activating protein for the Rheb small GTPase. We have identified HERC1 as a TSC2-interacting protein. HERC1 is a 532-kDa protein with an E3 ubiquitin ligase homology to E6AP carboxyl terminus (HECT) domain. We observed that the interaction of TSC1 with TSC2 appears to exclude TSC2 from interacting with HERC1. Disease mutations in TSC2, which result in its destabilization, allow binding to HERC1 in the presence of TSC1. Our study reveals a potential molecular mechanism of how TSC1 stabilizes TSC2 by excluding the HERC1 ubiquitin ligase from the TSC2 complex. Furthermore, these data reveal a possible biochemical basis of how certain disease mutations inactivate TSC2. Tuberous sclerosis complex (TSC)5 is an autosomal dominant genetic disorder affecting 1 in 6,000 -10,000 births (1). Mutations in either of the two genes, TSC1 (also called hamartin) or TSC2 (tuberin), cause the disorder characterized by benign tumor formation (hamartomas) in various organs and tissues. Complications of hamartomas in critical organs include renal failure, seizures, mental retardation, and autism (1). One of the hallmarks of TSC hamartomas is an increase in cell size, implicating TSC1 and TSC2 as negative regulators of cell growth (2-4).Recent studies have revealed the molecular mechanism for the tumor suppressor function of TSC1 and TSC2, which form a physical and functional complex (5). The TSC1⅐TSC2 complex suppresses cell growth by inhibiting the mammalian target of rapamycin, mTOR, which is a central controller of cell growth. TSC1/ TSC2 has GTPase-activating protein (GAP) activity toward the Rheb small GTPase (6, 7). Rheb acts upstream of and stimulates mTOR. TSC2 is the catalytic GAP subunit, while TSC1 enhances TSC2 function by stabilizing TSC2. The majority of disease-associated TSC1 mutations identified result in no TSC1 protein being expressed; therefore, the free TSC2 protein in TSC1 mutant cells is unstable (1). Similarly, many disease-derived TSC2 mutants are unstable due to weakened interaction with TSC1 (8, 9). However, the precise mechanism how TSC1 stabilizes TSC2 is largely unclear.In this report, we identified HERC1 as a TSC2-interacting protein. The COOH-terminal region of HERC1 has a HECT E3 ubiquitin ligase domain (10). Interestingly, HERC1 does not associate with TSC1. Moreover, TSC1 efficiently competes with HERC1 for TSC2 binding. Our study provides a potential biochemical mechanism of TSC1 in TSC2 stabilization by inhibiting the interaction between TSC2 and the E3 ubiquitin ligase HERC1. EXPERIMENTAL PROCEDURESPlasmids and Antibodies-HA-TSC2 and Myc-TSC1 we...

Aims The artery contains numerous cell types which contribute to multiple vascular diseases. However, the heterogeneity and cellular responses of these vascular cells during abdominal aortic aneurysm (AAA) progression have not been well characterized. Methods and results Single-cell RNA sequencing was performed on the infrarenal abdominal aortas (IAAs) from C57BL/6J mice at Days 7 and 14 post-sham or peri-adventitial elastase-induced AAA. Unbiased clustering analysis of the transcriptional profiles from >4500 aortic cells identified 17 clusters representing nine-cell lineages, encompassing vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells, immune cells (macrophages, T cells, B cells, and dendritic cells), and two types of rare cells, including neural cells and erythrocyte cells. Seurat clustering analysis identified four smooth muscle cell (SMC) subpopulations and five monocyte/macrophage subpopulations, with distinct transcriptional profiles. During AAA progression, three major SMC subpopulations were proportionally decreased, whereas the small subpopulation was increased, accompanied with down-regulation of SMC contractile markers and up-regulation of pro-inflammatory genes. Another AAA-associated cellular response is immune cell expansion, particularly monocytes/macrophages. Elastase exposure induced significant expansion and activation of aortic resident macrophages, blood-derived monocytes and inflammatory macrophages. We also identified increased blood-derived reparative macrophages expressing anti-inflammatory cytokines suggesting that resolution of inflammation and vascular repair also persist during AAA progression. Conclusion Our data identify AAA disease-relevant transcriptional signatures of vascular cells in the IAA. Furthermore, we characterize the heterogeneity and cellular responses of VSMCs and monocytes/macrophages during AAA progression, which provide insights into their function and the regulation of AAA onset and progression.

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Rationale: Nitro-oleic acid (OA-NO 2 ) is a bioactive, nitric-oxide derived fatty acid with physiologically relevant vasculoprotective properties in vivo. OA-NO 2 exerts cell signaling actions as a result of its strong electrophilic nature and mediates pleiotropic cell responses in the vasculature. Objective: The present study sought to investigate the protective role of OA-NO 2 in angiotensin (Ang) II–induced hypertension. Methods and Results: We show that systemic administration of OA-NO 2 results in a sustained reduction of Ang II–induced hypertension in mice and exerts a significant blood pressure lowering effect on preexisting hypertension established by Ang II infusion. OA-NO 2 significantly inhibits Ang II contractile response as compared to oleic acid (OA) in mesenteric vessels. The improved vasoconstriction is specific for the Ang II type 1 receptor (AT 1 R)-mediated signaling because vascular contraction by other G-protein–coupled receptors is not altered in response to OA-NO 2 treatment. From the mechanistic viewpoint, OA-NO 2 lowers Ang II–induced hypertension independently of peroxisome proliferation-activated receptor (PPAR)γ activation. Rather, OA-NO 2 , but not OA, specifically binds to the AT 1 R, reduces heterotrimeric G-protein coupling, and inhibits IP 3 (inositol-1,4,5-trisphosphate) and calcium mobilization, without inhibiting Ang II binding to the receptor. Conclusions: These results demonstrate that OA-NO 2 diminishes the pressor response to Ang II and inhibits AT 1 R-dependent vasoconstriction, revealing OA-NO 2 as a novel antagonist of Ang II–induced hypertension.