Final Report Summary - INNATEDNASENSOR (A proteomic screen to identify cytoplasmic DNA sensor proteins mediating type I Interferon production)
Cells need to sense their external and intracellular environment and been able to integrate these different inputs to control fundamental process such as growth, proliferation and survival. Nutrient availability is a crucial factor in the cellular decision to growth and proliferate or to engage recycling processes. A central role in this context is played by the mechanistic target of rapamycin complex 1 (mTORC1) that act as a central regulator of cellular metabolism by integrating the presence of growth factors, energy levels, glucose and amino acids to modulate cellular responses, such as protein, nucleotide and lipid synthesis. The mechanisms controlling mTORC1 activation have been at the center of intense research as its deregulation is critically involved in a variety of diseases including cancer and metabolic disorders. Past studies have demonstrated that amino acids availability is a crucial factor for mTORC1 as in their absence it cannot be activated even in presence of growth factors. The lysosomal Ragulator/RAG GTPase complex has been shown to be required for amino acid-dependent regulation of mTORC1. Despite this, the molecular mechanisms by which amino acid levels are sensed are still poorly understood. Our work succeeded in identifying the previously uncharacterized putative amino acid transporter member 9 of the solute carrier family 38 (SLC38A9) as a key component of the machinery that control amino acid-mediated mTOR activation. Our data show that SLC38A9 localized in the lysosomal compartment and, through an extensive proteomic analysis, we could demonstrate that it is an integral component of the lysosomal amino acid sensing machinery that controls mTORC1 activity in dependency of amino acid availability. SLC38A9 interacts with the Ragulator/RAG GTPase complex through its cytoplasmic N-terminal region in an amino acid regulated manner. In vitro transporter assay demonstrated that SLC38A9 is indeed able to binds and transport amino acids. Gain- and loss-of-function experiments support a positive role for SLC38A9 in regulating mTORC1: silencing of its expression impaired amino acid-induced activation whereas its overexpression resulted in sustained mTORC1 activity upon amino acid starvation. Altogether our work identifies SLC38A9 as a physical and functional component of the lysosomal amino acid-sensing machinery regulating mTORC1. SLC38A9 is the first component of this complex shown to physically engage amino acids, supporting a role at the core of the amino acid sensing mechanism. We proposed that SLC38A9 act a transceptor (transporter-receptor) in which amino acid engagement is used for allosteric signal transduction rather than mere transport. The finding may lead to new opportunities to interfere with the mTOR pathway by targeting SLC38A9 in situation where aberrant mTORC1 activation is thought to promote pathological conditions such as cancer and metabolic disorders. Tightly interconnected with the ability to sense the environmental and cellular metabolic status and stress conditions is the decision between the engagement of survival pathways or regulated cell death programs. Similarly to the well-recognized regulated nature of apoptosis, some forms of necrotic cell death have been shown to be regulated and to depend from a genetically encoded program. Necroptosis has emerged in the recent years as a programmed form of necrosis that can be triggered by different immune receptors such as the cytoplasmic DNA sensor DAI and other cellular stress conditions. While providing a beneficial, protective effect during infection, necroptosis has been proposed to contribute to the pathogenesis of many diseases associated with sterile inflammation. Considering the potential benefit in blocking this cell demise pathway in selected pathophysiological conditions, we applied different screening approaches in to order to identify inhibitors of necroptotic cell death and potential molecular targets. By performing a chemical screen on TNF-induced necroptosis, we identified two FDA-approved anticancer agents, ponatinib and pazopanib, as potent inhibitors of this pathway. Extensive molecular and functional characterization of these drugs demonstrated that they target key components of the necroptotic signaling pathway: ponatinib blocking RIPK1 and RIPK3 activity and pazopanib targeting RIPK1. Using a proteomic approach to identify interactors of the crucial mediator of necroptosis MLKL, we could demonstrate that it is a novel HSP90 client and that HSP90 inhibition results in MLKL destabilization and block of necroptosis. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis as well as of HSP90 as a candidate target highlights potential strategies for the treatment of pathologies caused or aggravated by necroptotic cell death.