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      The apo-structure of the leucine sensor Sestrin2 is still elusive

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          Abstract

          Sestrin2 is a GATOR2 interacting protein that directly binds leucine and is required for the inhibition of mTORC1 under leucine deprivation, indicating that it is a leucine sensor for the mTORC1 pathway. We recently reported the structure of Sestrin2 in complex with leucine (PDB ID: 5DJ4), and demonstrated that mutations in the leucine-binding pocket alter the affinity of Sestrin2 for leucine and result in a corresponding change in the leucine sensitivity of mTORC1 in cells. A lower resolution structure of human Sestrin2 (PDB ID: 5CUF), which was crystallized in the absence of exogenous leucine, showed Sestrin2 to be in a nearly identical conformation as the leucine-bound structure. Based on this observation, it has been argued that leucine binding does not affect the conformation of Sestrin2 and thus that Sestrin2 may not be a sensor for leucine. Here, we show that simple analysis of the reported “apo”-Sestrin2 structure reveals the clear presence of prominent, unmodeled electron density in the leucine-binding pocket that exactly accommodates the leucine observed in the higher resolution structure. Refining the reported “apo”-structure with leucine eliminates the large F O-F C difference density at this position and improves the working and free R-factors of the model. Consistent with this, our own structure of Sestrin2 crystallized in the absence of exogenous leucine also contains electron density that is best explained by leucine. Thus, the structure of apo-Sestrin2 remains elusive.

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          Sestrin2 is a leucine sensor for the mTORC1 pathway.

          Leucine is a proteogenic amino acid that also regulates many aspects of mammalian physiology, in large part by activating the mTOR complex 1 (mTORC1) protein kinase, a master growth controller. Amino acids signal to mTORC1 through the Rag guanosine triphosphatases (GTPases). Several factors regulate the Rags, including GATOR1, aGTPase-activating protein; GATOR2, a positive regulator of unknown function; and Sestrin2, a GATOR2-interacting protein that inhibits mTORC1 signaling. We find that leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction by binding to Sestrin2 with a dissociation constant of 20 micromolar, which is the leucine concentration that half-maximally activates mTORC1. The leucine-binding capacity of Sestrin2 is required for leucine to activate mTORC1 in cells. These results indicate that Sestrin2 is a leucine sensor for the mTORC1 pathway.
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            Signal integration by mTORC1 coordinates nutrient input with biosynthetic output.

            Flux through metabolic pathways is inherently sensitive to the levels of specific substrates and products, but cellular metabolism is also managed by integrated control mechanisms that sense the nutrient and energy status of a cell or organism. The mechanistic target of rapamycin complex 1 (mTORC1), a protein kinase complex ubiquitous to eukaryotic cells, has emerged as a critical signalling node that links nutrient sensing to the coordinated regulation of cellular metabolism. Here, we discuss the role of mTORC1 as a conduit between cellular growth conditions and the anabolic processes that promote cell growth. The emerging network of signalling pathways through which mTORC1 integrates systemic signals (secreted growth factors) with local signals (cellular nutrients - amino acids, glucose and oxygen - and energy, ATP) is detailed. Our expanding understanding of the regulatory network upstream of mTORC1 provides molecular insights into the integrated sensing mechanisms by which diverse cellular signals converge to control cell physiology.
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              Janus-faced Sestrin2 controls ROS and mTOR signalling through two separate functional domains

              Sestrins are stress-inducible metabolic regulators with two seemingly unrelated but physiologically important functions: reduction of reactive oxygen species (ROS) and inhibition of the mechanistic target of rapamycin complex 1 (mTORC1). How Sestrins fulfil this dual role has remained elusive so far. Here we report the crystal structure of human Sestrin2 (hSesn2), and show that hSesn2 is twofold pseudo-symmetric with two globular subdomains, which are structurally similar but functionally distinct from each other. While the N-terminal domain (Sesn-A) reduces alkylhydroperoxide radicals through its helix–turn–helix oxidoreductase motif, the C-terminal domain (Sesn-C) modified this motif to accommodate physical interaction with GATOR2 and subsequent inhibition of mTORC1. These findings clarify the molecular mechanism of how Sestrins can attenuate degenerative processes such as aging and diabetes by acting as a simultaneous inhibitor of ROS accumulation and mTORC1 activation.
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                Author and article information

                Journal
                101465400
                34171
                Sci Signal
                Sci Signal
                Science signaling
                1945-0877
                1937-9145
                28 September 2016
                20 September 2016
                20 September 2016
                31 October 2016
                : 9
                : 446
                : ra92
                Affiliations
                [1 ]Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA
                [2 ]Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
                [3 ]Howard Hughes Medical Institute, Cambridge, MA 02139, USA
                [4 ]Koch Institute for Integrative Cancer Research, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
                [5 ]Broad Institute of Harvard and Massachusetts Institute of Technology, 415 Main Street, Cambridge MA 02142, USA
                Author notes
                [* ] Corresponding author. sabatini@ 123456wi.mit.edu (D.M.S), tus@ 123456mit.edu (T.U.S)
                [6]

                Current Address: Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA

                Article
                PMC5087270 PMC5087270 5087270 nihpa819546
                10.1126/scisignal.aah4497
                5087270
                27649739
                d990994d-cf44-490f-aa0f-724160d5977b
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