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mTOR

History/Discovery

mTOR is considered to be the mammalian target of rapamycin. Rapamycin was discovered in a soil sample from Easter Island in the 1970s. Researchers studied this sample and found that the bacterium Streptomyces hygroscopicus made an antifungal, which they named rapamycin after the island's name Rapa Nui, which it was called by the locals meaning "navel of the world." Studies on rapamycin revealed that it was a powerful antifungal agent that could arrest fungal activity at the G1 phase. It was then tested in rats as a potential antifungal drug in humans, and was found to also greatly suppress their immune system by blocking the G1 to S phase transition in T-lymphocytes. This has led to its clinical use as an immunosupressant following organ transplantation.

In 1991, a genetic screen was performed on Saccharomyces cerevisiae to elucidate what rapamycin was specifically targeting to initiate this response. It was found that knockout of three genes allowed for the fungus' resistance to rapamycin. Two of the genes were called targets of rapamycin, or TOR, while the third gene was already characterized to be Fpr1, which is now known to be a binding protein in the TOR complexes. In 1994, the mammalian target of rapamycin (mTOR) was identified at the rapamycin target in mammals.

mTOR Structure

The mammalian target of rapamycin (mTOR) is considered a 'master switch' of cellular processes, regulating transcription of proteins required for cell growth and proliferation by sensing energy and nutrient levels. As a result, it plays an important role in various human diseases, including cancer and diabetes. mTOR consists of two structures, mTOR complex 1 (mTORc1) and mTOR complex 2 (mTORc2). mTORc1, named for its putative sensitivity to rapamycin, is the better-studied component of the mTOR protein and is thought to play the principal role in the initial mTOR protein; it is believed to regulate cell growth, proliferation, and survival by integrating hormones, growth factors, nutrients, stressors, and energy signals. mTORc2 is thought to regulate cytoskeleton and cell survival in response to insulin, although the upstream signaling pathways have yet to be fully elucidated.

mTORc1 and 2 share the same core protein complex of 2549 amino acids, which contains seven conserved domains, written from the N- to the C- terminus as HEAT, FAT, FRB, kinase, NR, and FATC domains. The largest domain is called the HEAT repeat domain, named for the Huntingtin, Ef2, A subunit of PP2A, TOR1 amino acid repeats that it contains. The HEAT domain is implicated in protein-protein interactions. The FAT domain I need to research more about. The FRB domain is specifically targeted by rapamycin for inhibition for the mTOR complexes. It is important to note, that only the mTORc1 complex has been shown to be inhibited by rapamycin. The kinase domain is the catalytic domain responsible for phosphorylating serine and threonine residues on target proteins. Attached to the kinase domain is the mLST8 domain, the mammalian ortholog of the LST8 protein first discovered in yeast. mTORc2 has been shown to require this domain for function, and it has been suggested that it is also required in mTORc1. The NR (negative regulatory) domain is the putative negative regulatory region of the mTOR complexes. The FATC domain, or FAT domain on the C-terminus, has been shown to be necessary for the kinase function of mTOR, as a single amino acid deletion from this sequence prevents such activity.

There are two complexes that contain the mTOR core protein: mTOR complex 1 and mTOR complex 2 named mTORC1 and mTORC2 respectively.

The mTOR core protein is 2549 amino acids long and contains a HEAT repeats domain, FAT domain, FRB domain, kinase domain, NR domain, and FATC domain. The HEAT domain spans the first half of the core protein and consists of many tandem HEAT repeats which stands for the amino acid sequences for the domains of for Huntingtin, EF3, A subunit of PP2A, and TOR1. These HEAT repeats are considered to be for protein-protein interactions with either Raptor or Rictor for mTORC1 or mTORC2, respectively. FAT lies downstream of the HEAT region and will interact with the FAT C terminal domain and this interaction is thought to modulate the kinase activity of mTOR. This FATC domain is so conserved that changing one amino acid in this sequence has been shown to disrupt mTOR activity. The FRB (FKB12-rapamycin binding) domain is the stretch of amino acids that rapamycin binds to in order to inhibit mTOR activity. The NR domain is located just before the FATC domain on the C terminus and is a putative negative regulatory region, where substrates can bind to inhibit mTOR activity.

The mTORC1 and mTORC2 complexes differ due to the associated proteins that make up their complexes.

mTORc1 and mTORc2 both have specific regulatory proteins, known as Raptor and Rictor, respectively. I will go on to talk about Raptor and Rictor next time.

mTORC1

Upstream of mTORC1

The role of mTOR is to activate translation of proteins. In order for cells to grow and proliferate by manufacturing more proteins, the cells must ensure that they have the resources available for protein production. Thus, for protein production, and therefore mTORC1 activation, cells must have adequate energy resources, nutrient availability, oxygen abundance, and proper growth factors in order for protein translation to begin.

All of these variables for protein synthesis affect mTORC1 activation by interacting with the TSC1/TSC2 complex. TSC2 is a GTP-ase activating protein (GAP). Its GAP activity interacts with Rheb by hydrolyzing the GTP of the active Rheb-GTP complex, converting it to the inactive Rheb-GDP complex. The active Rheb-GTP activates mTORC1 through unelucidated pathways. Thus, many of the pathways that influence mTORC1 activation do so through the activation or inactivation of the TSC1/TSC2 heterodimer. This control is usually performed through phosphorylation of the complex, which can cause the dimer to dissociate losing its GAP activity, or the phosphorylation can cause the heterodimer to have more active GAP activity, depending on the kinase phosphorylating the dimer.

Growth Factors

Growth factors like insulin can activate mTORC1 through the receptor tyrosine kinase (RTK) pathway. Ultimately Akt phosphorylates TSC2 on serine residue 939, serine residue 981, and threonine residue 1462. These phosphorylated sites will recruit the cytosolic anchoring protein 14-3-3 to TSC2, disrupting the TSC1/TSC2 dimer. When TSC2 is not associated with TSC1, TSC2 loses its GAP activity and can no longer hydrolyze Rheb-GTP. This results in continued activation of mTORC1, allowing for protein synthesis via insulin signaling.

Akt will also phosphorylate PRAS40, causing it to fall off of Raptor on mTORC1. PRAS40 prevents Raptor from recruiting mTORC1's substrates 4E-BP1 and S6K-1. Thus when PRAS40 falls off of Raptor, the two substrates are recruited to mTORC1 and thereby activated in this way.

Because insulin is a factor that is secreted by pancreatic beta cells upon glucose elevation in the blood, this signaling ensures that there is energy for protein synthesis to take place. In a negative feedback loop, S6K-1 is able to phosphorylate the insulin receptor, and inhibit its sensitivity to insulin. This has great significance in diabetes mellitus, which is due to insulin resistance.

Mitogens

Mitogens like insulin like growth factor 1 (IGF1) can activate the Ras-ERK pathway, which can control the TSC1/TSC2 complex as well as directly have the same downstream role that mTORC1 has. In this pathway, the G protein Ras is tethered to the plasma membrane via a farnesyl group and is its inactive GDP state. Upon growth factor binding to the adjacent receptor tyrosine kinase, the adaptor protein GRB2 gets binds with its SH2 domains. This recruits the GEF called Sos, which activates the Ras G protein. Ras activates Raf (MAPKKK), which activates Mek (MAPKK), which activates Erk (MAPK). Erk can go on to activate RSK. Erk will phosphorylate the serine residue 644 on TSC2, while RSK will phosphorylate serine residue 1798 on TSC2. These phosphorylations will cause the heterodimer to fall apart, and not be able to deactivate Rheb. This, thus keeps mTORC1 active.

RSK has also been shown to phosphorylate Raptor, which helps it overcome the inhibitory effects of PRAS40.

Cytokines

Cytokines like tumor necrosis factor alpha (TFNalpha), can induce mTOR activity through IKK beta. IKK beta can phosphorylate TSC1 at serine residue 487 and TSC1 at serine residue 511. This causes the heterodimer TSC complex to fall apart, keeping Rheb in its active GTP bound state.

Energy Status

In order for translation to take place, abundant sources of energy, particularly in the form of ATP, need to be present. If these levels of ATP are not present, due to its hydrolysis into other forms like AMP, and the ratio of AMP to ATP molecules gets too high, AMPK will become activated. AMPK will go on to inhibit energy consuming pathways such as protein synthesis.

AMPK can phosphorylate TSC2 on serine residue 1387, which will now activate the GAP activity of this complex, causing Rheb-GTP to be hydrolyzed into Rheb-GDP. This inactivates mTORC1, and no protein synthesis occurs through this pathway.

AMPK can also phosphorylate Raptor on two serine residues. This phosphorylated Raptor now recruits 14-3-3, to bind to it, preventing Raptor from being part of the mTORC1 complex. Since mTORC1 cannot recruit its substrates without Raptor, no protein synthesis via mTORC1 occurs.

LBK1 is a known tumor suppressor that can activate AMPK. More studies on this aspect of mTORC1 may help shed light on its strong link to cancer.

Hypoxic Stress

When oxygen levels in the cell are low, it will limit its energy expenditure through the inhibition of protein synthesis. Under hypoxic conditions, hypoxia inducible factor one alpha (HIF-1 alpha) will stabilize and activate transcription of REDD1. After translation, this REDD1 protein will bind to TSC2, which prevents 14-3-3 from inhibiting the TSC complex. Thus, TSC retains its GAP activity towards Rheb, causing Rheb to remain bound to GDP, and mTORC1 inactive.

Due to the lack of synthesis of ATP in the mitochondria under hypoxic stress, AMPK will also become active and thus inhibit mTORC1 through its processes.

Wnt Pathway

The Wnt pathway is responsible for cellular growth and proliferation during organismal development. Thus it could be reasoned that activation of this pathway also activates mTORC1. Activation of the Wnt pathway inhibits glycogen synthase kinase 3 beta (GSK3 beta). When the Wnt pathway is not active, GSK3 beta is able to phosphorylate TSC2 on two serine residues of 1341 and 1337, in conjunction with AMPK phosphorylating serine residue 1345. It has been studied that the AMPK is required to first phosphorylate residue 1345 before GSK3 beta can phosphorylate its target serine residues. This phosphorylation of TSC2 would inactivate this complex, if GSK3 beta were active. Since the Wnt pathway inhibits GSK3 signaling, when the Wnt pathway is active, so also is the mTORC1 pathway. Now, mTORC1 can activate protein synthesis for the developing organism.

Amino Acids

Even if a cell has the proper energy for protein synthesis, if it does not have the amino acid building blocks for proteins, no protein synthesis will occur. Consequentially, mTORC1 signaling is sensitive to amino acid levels in the cell. Studies have shown that depriving amino acid levels inhibits mTORC1 signaling to the point where both energy abundance and amino acids are necessary for mTORC1 to function. When amino acids are introduced to a deprived cell, the presence of amino acids causes Rag GTPases heterodimers to switch to its active conformation. Active Rag heterodimers interact with RAPTOR, localizing mTORC1 to the surface of late endosomes and lysosome where the Rag GTPases are located. This allows mTORC1 to physically interact with RHEB, which is activated by growth factors such as insulin. The interaction between the Rags and mTORC1 brings mTORC1 to the surface of endosomes and lysosomes where Rheb is located. This is where Rheb-GTP activates mTORC1.

Downstream of mTORC1

mTOC1 activates transcription and translation through its interactions with 4E-BP1 and S6K.

4E-BP1

Activated mTORC1 will phosphorylate transcription inhibitor 4E-BP1, releasing it from eukaryotic translation initiation factor 4E (eIF4E). eIF4E is now free to join the eukaryotic translation initiation factor 4G (eIF4G) and the eukaryotic translation initiation factor 4A (eIF4A). This complex then binds to the 5' cap of mRNA and will recruit the helicase eukaryotic translation initiation factor A (eIF4A) and its cofactor eukaryotic translation initiation factor 4B (eIF4B). The helicase is required to remove hairpin loops that arise in the 5' untranslated regions of mRNA, that prevent premature translation of proteins. Once the initiation complex is assembled at the 5' cap of mRNA, it will recruit the 40S small ribosomal subunit that is now capable of scanning for the AUG codon start site, because the hairpin loop has been eradicated by the eIF4A helicase.

S6K

mTORC1 will also activate S6K, which is responsible for the recruitment of eIF4B to the initiation complex by phosphorylating its serine residue 422.

S6K also can phosphorylate programmed cell death 4 (PDCD4), which marks it for degradation by ubiquidin ligase Beta-TrCP. PDCD4 is a tumor suppressor that binds to eIF4A and prevents it from being incorporated into the initiation complex.

Active S6K can bind to the SKAR scaffold protein that can get recruited to exon junction complexes. Exon junction complexes span the mRNA region where two exons come together after an intron has been spliced out. Once S6K binds to this complex, increased translation on these mRNA regions occurs.

Hypophosphorylated S6K is located on the eIR3 scaffold complex. Active mTORC1 gets recruited to the scaffold, and once there, will phosphorylate S6K to make it active.

mTORC1 Role in Human Diseases and Aging
mTOR was found to be related to aging in 2001 when the ortholog of S6K, SCH9, was deleted in S. cerevisiae, doubling its lifespan. As a result, mTORC1 signaling was focused on and techniques used to inhibit its activity in C. elegans, fruitflies, and mice significantly increased their lifespans relative to the control organisms for the respective species.

Based on upstream signaling of mTORC1, a clear relationship between food consumption and mTORC1 activity has been observed. Most specifically, carbohydrate consumption activates mTORC1 through the insulin growth factor pathway. In addition, amino acid consumption will stimulate mTORC1 through the branched chain amino acid/Rag pathway. Thus dietary restriction inhibits mTORC1 signaling through both upstream pathways of mTORC that converge on the lysosome.

Dietary restriction has been shown to significantly increase lifespan in the human model of Rhesus monkeys as well as protect against their age related decline. More specifically, Rhesus monkeys on a calorie restricted diet had significantly less chance of developing cardiovascular disease, diabetes, cancer, and age related cognitive decline than those monkeys who were not placed on the calorie restricted diet.

Stem Cells
Conservation of stem cells in the body has been shown to help prevent against premature aging. mTORC1 activity plays a critical role in the growth and proliferation of stem cells. Knocking out mTORC1 results in embryonic lethality due to lack of trophoblast development. Treating stem cells with rapamycin will also slow their proliferation, conserving the stem cells in their undifferentiated condition.

mTORC1 plays a role in the differentiation and proliferation of hematopoietic stem cells. Its upregulation has been shown to cause premature aging in hematopoietic stem cells. Conversely, inhibiting mTOR restores and regenerates the hematopoietic stem cell line. Rapamycin is used clinically as an immunosupressant and prevents the proliferation of T cells and B cells. Paradoxically, even though rapamycin is a federally approved immunosuppressant, its inhibition of mTORC1 results in better quantity and quality of functional memory T cells. mTORC1 inhibition with rapamycin improves the ability of naïve T cells to become memory precursor cells during the expansion phase of T cell development. This inhibition also allows for an increase in quality of these memory T cells that become mature T cells during the contraction phase of their development. mTORC1 inhibition with rapamycin has also been linked to a dramatic increase of B cells in old mice, enhancing their immune systems. This paradox of rapamycin inhibiting the immune system response has been linked to several reasons, including its interaction with T-regulatory cells. The mechanisms mTORC1's inhibition on proliferation and differentiation of hematopoietic stem cells has yet to be fully elucidated.

Autophagy
Autophagy is the major degradation pathway in eukaryotic cells and is essential for the removal of damaged organelles via macroautophagy or proteins and smaller cellular debris via microautophagy from the cytoplasm. Thus, autophagy is a way for the cell to recycle old and damaged materials by breaking them down into their smaller components, allowing for the resynthesis of newer and healthier cellular structures. Autophagy can thus remove aggregates of proteins and damaged organelles, that can lead to cellular dysfunction.

Upon activation, mTORC1 will phosphorylate Atg 13, preventing it from entering the ULK1 kinase complex, which consists of Atg1-Atg17-Atg101. This prevents the structure from being recruited to the preautophagosomal structure at the plasma membrane, inhibiting autophagy. .

mTORC1's ability to inhibit autophagy while at the same time stimulate protein synthesis and cell growth can result in accumulations of damaged proteins and organelles, contributing to damage at the cellular level. Because autophagy appears to decline with age, activation of autophagy may help promote longevity in humans. Problems in proper autophagy processes have been linked to diabetes, cardiovascular disease, neurodegenerative diseases, and cancer.

Reactive Oxygen Species
Reactive oxygen species can damage the DNA and proteins in cells. A majority of them arise in the mitochondria.

Deletion of the TOR1 gene in yeast increases mitochondrial respiration by enhancing the translation of mitochondrial DNA that encodes for the complexes involved in the electron transport chain. When this electron transport chain is not as efficient, the unreduced oxygen molecules in the mitochondrial cortex may accumulate and begin to produce reactive oxygen species. It is important to note that both cancer cells as well as those cells with greater levels of mTORC1 both rely more on glycolysis in the cytosol for ATP production rather than through oxidative phoshphorylation in the inner membrane of the mitochondria.

Inhibition of mTORC1 has also been shown to increase transcription of the NRF2 gene, which is a transcription factor that is able to regulate the expression of electrophilic response elements as well as antioxidants in response to increased levels of reactive oxygen species.

mTORC1 Inhibition
Rapamycin was the first known inhibitor of mTORC1, considering that mTORC1 was discovered as being the target of rapamycin. Rapamycin will bind to cytosolic FKBP12 and act as a scaffold molecule, allowing this protein to dock on the FBP regulatory region on mTORC1. The binding of the FKBP12-rapamycin complex to the FBP regulatory region inhibits mTORC1 through processes not yet known.

Rapamycin itself is not very water soluble and is not very stable, so scientists developed rapamycin analogs, called rapalogs, to overcome these two problems with rapamycin. These drugs are considered the first generation inhibitors of mTOR.

Siroliumus, which is the drug name for rapamycin, was approved by the FDA in 1999 to prevent against host rejection in patients undergoing kidney transplantation. In 2003, it was approved as a stent covering for people who want to widen their arteries to prevent against heart attacks and stuff. In 2007, they began being approved for treatments against cancers such as renal cell carcinoma. In 2008 they were approved for mantle cell lymphoma. mTORC1 inhibitors have recently been approved for treatment of pancreatic cancer. In 2010 they were approved for treatment of tuberous sclerosis.

The second generation of inhibitors were created to overcome problems with upstream signaling upon the introduction of first generation inhibitors to the treated cells. One problem with the first generation inhibitors of mTORC1 is that there is a negative feedback loop from phosphorylated S6K, that can inhibit the insulin RTK via phosphorylation. When this negative feedback loop is no longer there, the upstream regulators of mTORC1 become more active than they would otherwise would have been under normal mTORC1 activity. Another problem is that since mTORC2 is resistant to rapamycin, and it too acts upstream of mTORC1 by activating Akt. Thus signaling upstream of mTORC1 still remains very active upon its inhibition via rapamycin and the rapalogs.

Second generation inhibitors are able to bind to the ATP binding site on the kinase domain of the mTOR core protein itself and abolish activity of both mTOR complexes. In addition, since the mTOR and the PI3K proteins are both in the same PIKK family of kinases, some second generation inhibitors have dual inhibition towards the mTOR complexes as well as PI3K, which acts upstream of mTORC1. As of 2011, these second generation inhibitors were in phase II of testing.

There have been several dietary compounds that have been suggested to inhibit mTOR signaling including EGCG, resveratrol, curcumin, caffeine, and alcohol.

There are currently more than 1,300 clinical trials underway for the mTOR complex inhibitors.