Whitehead Institute for Biomedical Research, MIT & HHMI, Cambridge, MA, USA.
The mechanisms that regulate organismal growth (mass accumulation) and coordinate it with the availability of nutrients were unknown until two decades ago. We now appreciate that one pathway – the mTOR pathway – is the major nutrient-sensitive regulator of growth in animals and plays a central role in physiology, metabolism, the aging process, and common diseases (reviewed in (Saxton and Sabatini 2017)). The mTOR protein kinase is the target of the drug rapamycin and the catalytic subunit of two multi-protein complexes, mTORC1 and mTORC2, that control distinct branches of the pathway. Rapamycin is a very interesting small molecule that was first isolated from bacteria collected on Easter Island, an island in the South Pacific which is also known as RapaNui.
We now appreciate that mTOR Complex 1 (mTORC1) pathway is one of the central signaling systems of mammals and is a major regulator of growth at the cell, organ, and whole body levels. It balances the activities of anabolic and catabolic systems, such as protein, lipid, and nucleotide synthesis versus autophagy. In addition, it is deregulated in many common human diseases, such as cancer and neurological disorders like epilepsy and also plays a key role in the aging process (Saxton and Sabatini 2017). Because of the potential of mTORC1 inhibitors to ameliorate aging-related diseases, there is great interest in developing small molecule inhibitors that are truly specific for mTORC1 and do not also inhibit mTORC2, which the best-known mTORC1 inhibitor (rapamycin) can also target (Sarbassov, Ali et al. 2006, Lamming, Ye et al. 2012).
A fascinating aspect of the mTORC1 pathway is that it senses many diverse stimuli, including growth factors, nutrients, and various forms of stress. A major focus of our laboratory has been to elucidate the mechanisms through which mTORC1 senses nutrients, amino acids in particular. In my presentation at the Pontifical Academy of Sciences I focused on our work on nutrient sensing.
Nutrient Sensing Pathway Components
We now appreciate that nutrients, including amino acids, activate mTORC1 by promoting its translocation to the lysosomal surface where Rheb, its activator, is located (see summary diagram in Figure 1). Rheb is a small GTP binding protein that directly interacts with mTORC1 and strongly stimulates its kinase activity. The translocation process depends on a second set of GTP binding proteins, the heterodimeric Rag GTPases. The Rag GTPases in turn are regulated by a large set of lysosomally-localized protein complexes, including GATOR1, GATOR2, Ragulator, KICSTOR, FLCN-FNIP. GATOR1, a negative regulator of the cytosolic branch of the nutrient-sensing pathway, has three subunits: Depdc5, Nprl2, and Nprl3, and is a GTPase Activating Protein (GAP) for RagA. We used site-directed mutagenesis, GTP hydrolysis assays, coimmunoprecipitation experiments, and structural analyses and identified Arg-78 on Nprl2 as the arginine finger that carries out the GATOR1 GAP function. Substitutions of this arginine render mTORC1 signaling insensitive to nutrient starvation and are found in cancers such as glioblastoma. None of the GATOR1 components have sequence homology to other proteins and so in a long-standing collaboration with Dr. Zhiheng Yu (HHMI Janelia Farms) we used Cryo-EM to solve two structures: GATOR1 and GATOR1 bound to the Rag GTPases. GATOR1 adopts an extended architecture with a cavity in the middle. Nprl2 is as a link between Depdc5 and Nprl3, and Depdc5. Biochemical analyses revealed that two binding modes must exist between the Rag GTPases and GATOR1.
KICSTOR is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and is required for nutrient deficiency to inhibit mTORC1 in human cells. KICSTOR localizes to lysosomes, binds and recruits GATOR1 to the lysosomal surface, and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases. In mice lacking SZT2, mTORC1 signaling is increased in several tissues, including in the brain. Just like for GATOR1 components, several KICSTOR components are mutated in neurological diseases like epilepsy.
We recently found that Ragulator and SLC38A9 are each unique guanine exchange factors (GEFs) that collectively push the Rag GTPases toward the active state. Ragulator triggers GTP release from RagC, thus resolving the locked inactivated state of the Rag GTPases. Upon arginine binding, SLC38A9 converts RagA from the GDP- to the GTP-loaded state, and therefore activates the Rag GTPase heterodimer.
Because mTORC1 senses lysosomal amino acids in addition to cytosolic ones, we became interested in developing methods to measure metabolite levels in organelles. We first developed the MitoIP method for the mitochondrial matrix and more recently an analogous method (LysoIP) for lysosomes. We are using these methods to understand how electron transport chain inhibition affects matrix metabolites, how mTORC1 regulates lysosomal amino acid efflux, and to de-orphan the function of lysosomal storage disease genes. In unpublished work we generated mice expressing the transgenes necessary to implement the methods and we are beginning to validate conclusions in vivo that we first made in cultured cells.
We used the LysoIP approach to measure the metabolite content of lysosomes from cells lacking SLC38A9 and uncovered an unexpectedly central role for it in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through cytosolic Sestrin2. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the lysosomal release of the amino acids needed for cell growth.
Lastly, in work that is under review, we used Cryo-EM to solve the structure of mTORC1 bound to the complex of the Rag GTPases with Ragulator, which comprise the docking site for mTORC1 on the lysosomal surface. Using it, we generated a model for how mTORC1 docks on the lysosome, revealing what we believe to be its active state. Using structure-guided mutagenesis, we defined the key residues mediating the interaction between Rag-Ragulator and Raptor, the subunit of mTORC1 that directly binds to them. Mutants that disrupt the interaction inhibit mTORC1 and prevent its lysosomal localization. It is very gratifying for us to see the structure of a complex that we have worked on for so many years.
The GATOR1, GATOR2, and Ragulator complexes interact with a number of direct sensors of individual amino acids and metabolites in the cytosol. The Sestrin family of proteins (Sestrin1, Sestrin2, and Sestrin3) directly sense leucine in the cytosol while the CASTOR family of proteins (CASTOR1, CASTOR2) directly sense cytosolic arginine. SAMTOR senses methionine but does so indirectly as it binds to S-adenosyl-methionine, a product of methionine catabolism that is used in hundreds of methylation reactions. Lastly, SLC38A9 senses arginine in the lysosome ((Sancak, Peterson et al. 2008, Sancak, Bar-Peled et al. 2010, Zoncu, Bar-Peled et al. 2011, Bar-Peled, Schweitzer et al. 2012, Bar-Peled, Chantranupong et al. 2013, Petit, Roczniak-Ferguson et al. 2013, Tsun, Bar-Peled et al. 2013, Chantranupong, Wolfson et al. 2014, Jung, Genau et al. 2015, Rebsamen, Pochini et al. 2015, Wang, Tsun et al. 2015, Chantranupong, Scaria et al. 2016, Saxton, Chantranupong et al. 2016, Saxton, Knockenhauer et al. 2016, Wolfson, Chantranupong et al. 2016, Castellano, Thelen et al. 2017, Gu, Orozco et al. 2017, Peng, Yin et al. 2017, Wolfson, Chantranupong et al. 2017, Wyant, Abu-Remaileh et al. 2017). Our recent works suggest that SLC38A9 senses lysosomal arginine as an indication that ribosomes, which are very high in arginine content, are being degraded in lysosomes and can serve as a source of nucleotides under periods of starvation. These nucleotides are released from the lysosome and recycled into RNA in the cytosol.
Our identification of nutrient-sensors upstream of mTORC1 revealed a novel group of targets amenable to therapeutic intervention (Chantranupong, Wolfson et al. 2014, Wang, Tsun et al. 2015, Chantranupong, Scaria et al. 2016, Wolfson, Chantranupong et al. 2016, Gu, Orozco et al. 2017, Wyant, Abu-Remaileh et al. 2017). We solved the structures of two of these and defined their nutrient-binding pockets, which enables a rational approach to developing a novel class of mTORC1 pathway inhibitors (Saxton, Chantranupong et al. 2016, Saxton, Knockenhauer et al. 2016, Saxton, Knockenhauer et al. 2016). Gratifyingly, a small molecule activator of mTORC1 (NVP-5138) that acts by mimicking the action of leucine on Sestrin1/2 (Kato, Pothula et al. 2019, Sengupta, Giaime et al. 2019) recently entered clinical testing (clinicaltrials.gov) for treatment-resistant depression, in which there is substantial evidence that depressed neuronal mTORC1 signaling plays a pathogenic role (Li, Lee et al. 2010, Jernigan, Goswami et al. 2011, Koike, Iijima et al. 2011, Li, Liu et al. 2011, Dwyer, Lepack et al. 2012, Chandran, Iyo et al. 2013, Voleti, Navarria et al. 2013, Yang, Hu et al. 2013, Yu, Zhang et al. 2013, Ota, Liu et al. 2014, Zhou, Wang et al. 2014, Dwyer, Maldonado-Aviles et al. 2015).
We have also made some progress in understanding what happens to animals when we deregulate its capacity to sense the absence of nutrients in vivo. We have generated mice in which mTORC1 is no longer inhibited when we starve the animals for nutrients but under the fed condition is not hyperactive. As far as we can tell, these animals are fine as long as they are in nutrient replete conditions. Upon starvation their fitness drops very quickly, most likely because they cannot induce autophagy to liberate nutrients from their internal stores. We can rescue their fitness defect by inhibiting mTORC1 with rapamycin or simply injecting glucose into the animals as it appears to be the nutrient that becomes limiting first. In fact, the mice perish when glucose in the blood becomes undetectable using a clinical-grade glucometer. We find equivalent results when we perform these experiments when the animals are neonates or young adults. In addition, the adult animals appear to suffer seizures, which are very similar to those caused by mutations in GATOR1, the Rag negative regulator, in people.
While we have made progress in understanding how mTORC1 senses nutrients, several major questions remain unanswered, many of which revolve around the GATOR2 complex. This complex has emerged as the central integrator of cytosolic nutrient signals to mTORC1 and acts upstream of GATOR1, which negatively regulates RagA/B by acting as a GTPase Activating Protein (GAP) for them. Despite its importance, GATOR2 remains very mysterious. First, we do not understand its biochemical activity, how it signals to other pathway components, nor its structure. Second, we know little about how its regulation by nutrient sensors like Sestrin2 impacts organ physiology in vivo. Third, while our previous work has helped elucidate how amino acids signal to mTORC1, we still have almost no mechanistic insight into how glucose regulates the GATOR1-GATOR2 axis to control mTORC1 activity. In unpublished work, we find that mTORC1 does not directly sense glucose but rather an intermediate in glycolysis. We are currently trying to identify the sensor for this intermediate.
In addition, we are currently generating mice in which we are eliminating each nutrient sensor one by one or introducing mutations into them that prevent their capacity to bind a particular amino acid. In preliminary data we find that these animals have a fitness defect when placed on diets lacking the cognate nutrient for the sensor. Fortunately, the mTORC1 pathway still contains many mysteries for us to solve.
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