Inhibition of mTOR affects protein stability of OGT

Autophagy regulates cellular homeostasis through degradation of aged or damaged subcellular organelles and components. Interestingly, autophagy-deficient beta cells, for example Atg7-mutant mice, exhibited hypoinsulinemia and hyperglycemia. Also, autophagy response is diminished in heart of diabetic mice. These results implied that autophagy and diabetes are closely connected and affect each other. Although protein O-GlcNAcylation is up-regulated in hyperglycemia and diabetes, and O-GlcNAcylated proteins play an important role in metabolism and nutrient sensing, little is known whether autophagy affects O-GlcNAc modification and vice versa. In this study, we suppressed the action of mTOR by treatment of mTOR catalytic inhibitors (PP242 and Torin1) to induce autophagic flux. Results showed a decrease in global O-GlcNAcylation, which is due to decreased OGT protein and increased OGA protein. Interestingly, knockdown of ATG genes or blocking of lysosomal degradation enhanced protein stability of OGT. In addition, when proteasomal inhibitor was treated together with mTOR inhibitor, protein level of OGT almost recovered to control level. These data suggest that mTOR inhibition is a more efficient way to reduce protein level of OGT rather than that of CHX treatment. We also showed that not only proteasomal degradation regulated OGT stability but autophagic degradation also affected OGT stability in part. We concluded that mTOR signaling regulates protein O-GlcNAc modification through adjustment of OGT stability.     

JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy

Starvation induces autophagy to preserve cellular homeostasis in virtually all eukaryotic organisms. However, the mechanisms by which starvation induces autophagy are not completely understood. In mammalian cells, the antiapoptotic protein, Bcl-2, binds to Beclin 1 during nonstarvation conditions and inhibits its autophagy function. Here we show that starvation induces phosphorylation of cellular Bcl-2 at residues T69, S70, and S87 of the nonstructured loop; Bcl-2 dissociation from Beclin 1; and autophagy activation. In contrast, viral Bcl-2, which lacks the phosphorylation site-containing nonstructured loop, fails to dissociate from Beclin 1 during starvation. Furthermore, the stress-activated signaling molecule, c-Jun N-terminal protein kinase 1 (JNK1), but not JNK2, mediates starvation-induced Bcl-2 phosphorylation, Bcl-2 dissociation from Beclin 1, and autophagy activation. Together, our findings demonstrate that JNK1-mediated multisite phosphorylation of Bcl-2 stimulates starvation-induced autophagy by disrupting the Bcl-2/Beclin 1 complex. These findings define a mechanism that cells use to regulate autophagic activity in response to nutrient status.     

Novel compounds for the modulation of mTOR and autophagy to treat neurodegenerative diseases

Most neurodegenerative diseases show a disruption of autophagic function and display abnormal accumulation of toxic protein aggregates that promotes cellular stress and death. Therefore, induction of autophagy has been proposed as a reasonable strategy to help neurons clear abnormal protein aggregates and survive. The kinase mammalian target of rapamycin (mTOR) is a major regulator of the autophagic process and is regulated by starvation, growth factors, and cellular stressors. The phosphoinositide 3-kinase (PI3K)/ protein kinase B (Akt) pathway, which promotes cellular survival, is the main modulator upstream of mTOR, and alterations in this pathway are common in neurodegenerative diseases, e.g. Alzheimer’s disease (AD) and Parkinson’s disease (PD). In the present work we revised mammalian target of rapamycin complex 1 (mTORC1) pathway and mTORC2 as a complementary an important element in mTORC1 signaling. In addition, we revised the extracellular signal regulated kinase (ERK) pathway, which has become relevant in the regulation of the autophagic process and cellular survival through mTORC2 signaling. Finally, we summarize novel compounds that promote autophagy and neuronal protection in the last five years.     

Hyperactivation of Mammalian Target of Rapamycin Complex 1 (mTORC1) Promotes Breast Cancer Progression through Enhancing Glucose Starvation-induced Autophagy and Akt Signaling

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation. Recent studies have suggested that constitutive activation of mTORC1 in normal cells could lead to malignant tumor development in several tissues. However, the mechanisms of mTORC1 hyperactivation to promote the growth and metastasis of breast or other cancers are still not well characterized. Here, using a new inducible deletion system, we show that deletion of Tsc1 in mouse primary mammary tumor cells, either before or after their transplantation, significantly increased their growth in vivo. The increase in tumor growth was completely rescued by rapamycin treatment, suggesting a major contribution from mTORC1 hyperactivation. Interestingly, glucose starvation-induced autophagy, but not amino acid starvation-induced autophagy, was increased significantly in Tsc1-null tumor cells. Further analysis of these cells also showed an increased Akt activation but no significant changes in Erk signaling. Together, these results provide insights into the mechanism by which hyperactivation of mTORC1 promotes breast cancer progression through increasing autophagy and Akt activation in vivo.     

Proteomics analysis of starved cells revealed Annexin A1 as an important regulator of autophagic degradation

Macroautophagy is involved in the bulk degradation of long-lived cytosolic proteins and subcellular organelles, which is important for the survival of cells during starvation. To identify potential players of the autophagy process, we subjected HCT116 cells cultured in complete medium and in Earle’s balanced salt solution to proteomics analysis. In approximately 1500 protein spots detected, we characterized 52 unique proteins, whose expression levels were significantly changed following starvation. Notably, we found that Annexin A1 was significantly upregulated following starvation at both mRNA and protein levels. Inhibition of Annexin A1 expression with specific siRNA did not alter starvation-induced autophagy as measured by the level of lipidated LC3, but significantly reversed autophagy degradation as measured by the level of p62/SQSTM 1. Thus Annexin A1 seemed to be positively upregulated during starvation to promote autophagic degradation. Overall, the data presented in this study established a expression profile of the proteome in starved cells, which allowed the identification of proteins with potential significance in starvation-induced autophagy.     

Withdrawal of Essential Amino Acids Increases Autophagy by a Pathway Involving Ca2+/Calmodulin-dependent Kinase Kinase-β (CaMKK-β)

Autophagy is the main lysosomal catabolic process that becomes activated under stress conditions, such as amino acid starvation and cytosolic Ca²⁺ upload. However, the molecular details on how both conditions control autophagy are still not fully understood. Here we link essential amino acid starvation and Ca²⁺ in a signaling pathway to activate autophagy. We show that withdrawal of essential amino acids leads to an increase in cytosolic Ca²⁺, arising from both extracellular medium and intracellular stores, which induces the activation of adenosine monophosphate-activated protein kinase (AMPK) via Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β). Furthermore, we show that autophagy induced by amino acid starvation requires AMPK, as this induction is attenuated in its absence. Subsequently, AMPK activates UNC-51-like kinase (ULK1), a mammalian autophagy-initiating kinase, through phosphorylation at Ser-555 in a process that requires CaMKK-β. Finally, the mammalian target of rapamycin complex C1 (mTORC1), a negative regulator of autophagy downstream of AMPK, is inhibited by amino acid starvation in a Ca²⁺-sensitive manner, and CaMKK-β appears to be important for mTORC1 inactivation, especially in the absence of extracellular Ca²⁺. All these results highlight that amino acid starvation regulates autophagy in part through an increase in cellular Ca²⁺ that activates a CaMKK-β-AMPK pathway and inhibits mTORC1, which results in ULK1 stimulation.     

Translation inhibitors induce formation of cholesterol ester-rich lipid droplets

Lipid droplets (LDs) in non-adipocytes contain triglycerides (TG) and cholesterol esters (CE) in variable ratios. TG-rich LDs are generated when unsaturated fatty acids are administered, but the conditions that induce CE-rich LD formation are less well characterized. In the present study, we found that protein translation inhibitors such as cycloheximide (CHX) induced generation of CE-rich LDs and that TIP47 (perilipin 3) was recruited to the LDs, although the expression of this protein was reduced drastically. Electron microscopy revealed that LDs formed in CHX-treated cells possess a distinct electron-dense rim that is not found in TG-rich LDs, whose formation is induced by oleic acid. CHX treatment caused upregulation of mTORC1, but the CHX-induced increase in CE-rich LDs occurred even when rapamycin or Torin1 was given along with CHX. Moreover, the increase in CE was seen in both wild-type and autophagy-deficient Atg5-null mouse embryonic fibroblasts, indicating that mTORC1 activation and suppression of autophagy are not necessary to induce the observed phenomenon. The results showed that translation inhibitors cause a significant change in the lipid ester composition of LDs by a mechanism independent of mTORC1 signaling and autophagy.     

CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation

mTORC1 (mammalian target of rapamycin complex 1) integrates information regarding availability of nutrients and energy to coordinate protein synthesis and autophagy. Using ribonucleic acid interference screens for autophagy-regulating phosphatases in human breast cancer cells, we identify CIP2A (cancerous inhibitor of PP2A [protein phosphatase 2A]) as a key modulator of mTORC1 and autophagy. CIP2A associates with mTORC1 and acts as an allosteric inhibitor of mTORC1-associated PP2A, thereby enhancing mTORC1-dependent growth signaling and inhibiting autophagy. This regulatory circuit is reversed by ubiquitination and p62/SQSTM1-dependent autophagic degradation of CIP2A and subsequent inhibition of mTORC1 activity. Consistent with CIP2A’s reported ability to protect c-Myc against proteasome-mediated degradation, autophagic degradation of CIP2A upon mTORC1 inhibition leads to destabilization of c-Myc. These data characterize CIP2A as a distinct regulator of mTORC1 and reveals mTORC1-dependent control of CIP2A degradation as a mechanism that links mTORC1 activity with c-Myc stability to coordinate cellular metabolism, growth, and proliferation.     

ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy

In response to nutrient stress, cells start an autophagy program that can lead to adaptation or death. The mechanisms underlying the signaling from starvation to the initiation of autophagy are not fully understood. In the current study we show that the absence or inactivation of PARP-1 strongly delays starvation-induced autophagy. We have found that DNA damage is an early event of starvation-induced autophagy as measured by γ-H2AX accumulation and comet assay, with PARP-1 knockout cells displaying a reduction in both parameters. During starvation, ROS-induced DNA damage activates PARP-1, leading to ATP depletion (an early event after nutrient deprivation). The absence of PARP-1 blunted AMPK activation and prevented the complete loss of mTOR activity, leading to a delay in autophagy. PARP-1 depletion favors apoptosis in starved cells, suggesting a pro-survival role of autophagy and PARP-1 activation after nutrient deprivation. In vivo results show that neonates of PARP-1 mutant mice subjected to acute starvation, also display deficient liver autophagy, implying a physiological role for PARP-1 in starvation-induced autophagy. Thus, the PARP signaling pathway is a key regulator of the initial steps of autophagy commitment following starvation.     

Role of Inositol Trisphosphate Receptors in Autophagy in DT40 Cells

Previous studies have shown that small interfering RNA knockdown and pharmacological inhibition of inositol 1,4,5-trisphosphate receptors (IP₃Rs) stimulate autophagy. We have investigated autophagy in chicken DT40 cell lines containing targeted deletions of all three IP₃R isoforms (triple knock-out (TKO) cells). Using gel shifts of microtubule-associated protein 1 light chain 3 as a marker of autophagy, we find that TKO cells have enhanced basal autophagic flux even under nutrient-replete conditions. Stable DT40 cell lines derived from TKO cells containing the functionally inactive D2550A IP₃R mutant did not suppress autophagy in the same manner as wild-type receptors. This suggests that the channel function of the receptor is important in its regulatory role in autophagy. There were no marked differences in the phosphorylation state of AMP-activated protein kinase, Akt, or mammalian target of rapamycin between wild-type and TKO cells. The amount of immunoprecipitated complexes of Bcl-2-Beclin-1 and Beclin-1-Vps34 were also not different between the two cell lines. The major difference noted was a substantially decreased mTORC1 kinase activity in TKO cells based on decreased phosphorylation of S6 kinase and 4E-BP1. The discharge of intracellular stores with thapsigargin stimulated mTORC1 activity (measured as S6 kinase phosphorylation) to a greater extent in wild-type than in TKO cells. We suggest that basal autophagic flux may be negatively regulated by IP₃R-dependent Ca²⁺ signals acting to maintain an elevated mTORC1 activity in wild-type cells and that Ca²⁺ regulation of this enzyme is defective in TKO cells. The protective effect of a higher autophagic flux in cells lacking IP₃Rs may play a role in the delayed apoptotic response observed in these cells.     

Nutrient-dependent mTORC1 Association with the ULK1–Atg13–FIP200 Complex Required for Autophagy

Autophagy is an intracellular degradation system, by which cytoplasmic contents are degraded in lysosomes. Autophagy is dynamically induced by nutrient depletion to provide necessary amino acids within cells, thus helping them adapt to starvation. Although it has been suggested that mTOR is a major negative regulator of autophagy, how it controls autophagy has not yet been determined. Here, we report a novel mammalian autophagy factor, Atg13, which forms a stable ~3-MDa protein complex with ULK1 and FIP200. Atg13 localizes on the autophagic isolation membrane and is essential for autophagosome formation. In contrast to yeast counterparts, formation of the ULK1–Atg13–FIP200 complex is not altered by nutrient conditions. Importantly, mTORC1 is incorporated into the ULK1–Atg13–FIP200 complex through ULK1 in a nutrient-dependent manner and mTOR phosphorylates ULK1 and Atg13. ULK1 is dephosphorylated by rapamycin treatment or starvation. These data suggest that mTORC1 suppresses autophagy through direct regulation of the ~3-MDa ULK1–Atg13–FIP200 complex.     

Role and regulation of starvation-induced autophagy in the Drosophila fat body

In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Here, we show that signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor.     

Induction of Autophagy by Amino Acid Starvation in Fish Cells

Autophagy is well established as a starvation-induced process in yeast and mammalian cells and tissues. To elucidate the cellular mechanisms induced by starvation in fish, we characterized the induction of autophagy in cultured zebrafish cells under starvation conditions. As an autophagic marker protein, the microtubule-associated protein 1-light chain 3B protein (MAP1-LC3B) was cloned from the fish cells, and its expression and localization were characterized. In zebrafish embryonic (ZE) cells, posttranslational modifications produced two distinct forms of MAP1-LC3B, i.e., a cytosolic form and a membrane-bound form (types I and II, respectively). Immunofluorescence microscopy revealed fluorescently labeled autophagosomes in cells stably transfected with a green fluorescent protein (GFP)–MAP1-LC3B fusion protein and showed that this protein accumulated in punctate dots in a time-dependent manner in response to amino acid starvation. Starvation also induced the degradation of long-lived proteins. Treatment with 3-methyladenine and wortmannin, two class-III inhibitors of phosphoinositide 3-kinase (PI3K), repressed autophagy under starvation conditions, indicating that the PI3K class-III pathway regulates starvation-induced autophagy in fish.     

Autophagy gets a brake: DAP1, a novel mTOR substrate,is activated to suppress the autophagic process

Autophagy, a highly regulated catabolic process, is controlled by the action of positive and negative regulators. While many of the positive mediators of autophagy have been identified, very little is known about negative regulators that might counterbalance the process. We recently identified death-associated protein 1 (DAP1) as a suppressor of autophagy and as a novel direct substrate of mammalian target of rapamycin (mTOR). We found that DAP1 is functionally silent in cells growing under rich nutrient supplies through mTOR-dependent inhibitory phosphorylation on two sites, which were mapped to Ser3 and Ser51. During amino acid starvation, mTOR activity is turned off resulting in a rapid reduction in the phosphorylation of DAP1. This caused the conversion of the protein into a suppressor of autophagy, thus providing a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under conditions of nutrient deprivation. Based on these studies we propose the “gas and brake” concept in which mTOR, the main sensor that regulates autophagy in response to amino acid deprivation, also controls the activity of a specific balancing brake to prevent the overactivation of autophagy.Key words: DAP1, mTOR, autophagy, amino acid starvation, phosphorylationIn recent years, many of the genes controlling and executing the autophagic process have been identified. Most of these genes act as positive mediators of the various steps of the process, including the ULK1 complex, which regulates the induction step, the Vps34-Beclin 1 complex that participates in the vesicle nucleation step and two ubiquitin-like pathways, the Atg12-Atg5 and the LC3-phosphatidylethanolamine (PE) conjugation steps, which play a central role in the vesicle elongation process. To date, only a few negative regulators of autophagy have been identified, including mTOR and the anti-apoptotic Bcl-2 family members. mTOR Ser/Thr kinase is a central suppressor of autophagy acting at the initiating regulatory steps of the process. Many signaling pathways act to inhibit mTOR activity, thus relieving its inhibitory effects on autophagy. The anti-apoptotic Bcl-2 and Bcl-X_L proteins, on the other hand, act at the nucleation step, by directly binding to Beclin 1''s BH3 domain, thus reducing the activation of Vps34 and subsequent autophagy. This inhibition can be relieved through dissociation of the complex, following either JNK-1 mediated phosphorylation of Bcl-2 or DAP kinase-mediated phosphorylation of the BH3 domain of Beclin 1.DAP1 is a small (∼15 kDa), ubiquitously expressed protein, rich in prolines and lacking known functional motifs. DAP1 was isolated more than a decade ago in our laboratory using a functional approach to gene cloning aimed at identifying novel mediators of IFNγ-induced cell death in mammalian cell cultures. Until recently, very little was known about the cellular and molecular functions of DAP1, mainly due to the lack of homology to other known proteins and the lack of functional motifs that could indicate a possible cellular function and studies in mammalian systems were missing.Recently, we discovered that DAP1 is another negative regulator of autophagy; yet, interestingly, its suppressive activity is selectively turned on during the autophagic process. Moreover, we found that DAP1 suppressive activity is tightly linked to the status of mTOR kinase activity. Under nutrient-rich culture conditions, DAP1 is phosphorylated by mTOR on two sites, Ser3 and Ser51, resulting in its inactivation. In response to nutrient deprivation, mTOR is inhibited and DAP1 undergoes rapid dephosphorylation. By knocking down the endogenous DAP1 and introducing either the phosphomimetic or the nonphosphorylatible DAP1 mutants, we found that the dephosphorylation leads to activation of the autophagic suppressive function of DAP1, whereas the phophorylated form is inactive. These results led to a “gas and brake” model, in which at the same time that autophagy is induced, some brakes such as DAP1 are also activated to provide a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under nutrient-deprivation conditions (Fig. 1). Notably, balancing autophagy is extremely important, since deregulated or excessive autophagy has been implicated in the pathogenesis of diverse diseases, such as certain types of neuronal degeneration and cancer and also in cellular aging.Open in a separate windowFigure 1“Gas and brake” model. During nutrient-rich conditions, active mTORC1 phosphorylates and inactivates the components of the ULK1 complex, ULK1 and Atg13, thus preventing the induction of autophagy. DAP1 is also inactivated simultaneously by mTORC1-mediated phosphorylation on Ser3 and Ser51. In addition, mTORC1 phosphorylates and activates p70S6K and 4E-BP1, which mediate the protein translation and cell growth activities of mTOR. Upon nutrient starvation, mTORC1 activity is attenuated, leading to dephosphorylation and activation of ULK1. ULK1, in turn, undergoes autophosphorylation and phosphorylates Atg13 and FIP200 resulting in ULK1 complex activation and induction of autophagy. On the other hand, activation of DAP1 by dephosphorylation, results in suppression of autophagy, thus inserting a brake into the process of autophagy. Note that the inactive proteins/complexes are faded out.The current challenge is to identify the molecular basis of the suppressive functions of DAP1 on autophagy. We have recently shown that DAP1 knockdown enhances LC3 lipidation and autophagosome accumulation both during amino acid starvation and rapamycin treatment. In addition, preliminary data indicate that the knockdown of DAP1 has no effect on mTOR complex 1 (mTORC1) activity in cells, at least during the first hours of starvation. Accordingly, DAP1 may function between the mTORC1 and the LC3 conjugation systems. The potential targets may fall into one of the multiprotein complexes functioning downstream of mTOR such as the ULK1 complex, the Vps34-Beclin 1 complex and more. Future studies will be performed to identify the molecular mechanism by which DAP1 suppresses autophagy. The lack of known functional motifs in the DAP1 protein sequence suggests that this small proline-rich protein may function as an adaptor blocking autophagy by binding to critical protein partners that still await identification.Although autophagy is primarily a protective process for the cell, it can also play a role in cell death. In response to prolonged starvation, autophagy can act either as a cell survival mechanism or be recruited as a cell death executer. In the future it would be interesting to examine whether the autophagy enhancement resulting from DAP1 knockdown contributes to increased cell death in our system or even may convert the survival properties of autophagy into death induction. This will fit the “gas and brake” model, in which autophagy, which is initially recruited as a cell survival mechanism, is converted into cell death machinery when a certain threshold is crossed due to the loss of the “brake” by the knockdown of DAP1.To date, very little is known about the putative mechanisms that restrict the intensity of the autophagic flux to maintain the continuous benefits of this process under stress. Therefore, the ability of DAP1 to counterbalance and buffer the process in a manner that is tightly linked to the status of a central player in autophagy (i.e., mTOR) is an important discovery in this field and provides a target for future drug design.     

Inhibition of autophagosome formation by the benzoporphyrin derivative verteporfin

Autophagy enables cells to degrade and recycle cytoplasmic materials both as a housekeeping mechanism and in response to extracellular stress such as nutrient deprivation. Recent studies indicate that autophagy also functions as a protective mechanism in response to several cancer therapy agents, making it a prospective therapeutic target. Few pharmacological inhibitors suitable for testing the therapeutic potential of autophagy inhibition in vivo are known. An automated microscopy assay was used to screen >3,500 drugs and pharmacological agents and identified one drug, verteporfin, as an inhibitor of autophagosome accumulation. Verteporfin is a benzoporphyrin derivative used in photodynamic therapy, but it inhibits autophagy without light activation. Verteporfin did not inhibit LC3/Atg8 processing or membrane recruitment in response to autophagic stimuli, but it inhibited drug- and starvation-induced autophagic degradation and the sequestration of cytoplasmic materials into autophagosomes. Transient exposure to verteporfin in starvation conditions reduced cell viability whereas cells in nutrient-rich medium were unaffected by drug treatment. Analysis of structural analogs indicated that the activity of verteporfin requires the presence of a substituted cyclohexadiene at ring A of the porphyrin core but that it can tolerate a number of large substituents at rings C and D. The existence of an autophagy inhibitor among FDA-approved drugs should facilitate the investigation of the therapeutic potential of autophagy inhibition in vivo.     

AMP-activated protein kinase: a universal regulator of autophagy?

Autophagy is a lysosomal pathway involved in the turnover of cellular macromolecules and organelles. Starvation and various other stresses increase autophagic activity above the low basal levels observed in unstressed cells, where it is kept down by mammalian target of rapamycin complex 1 (mTORC1). In starved cells, LKB1 activates AMP-activated protein kinase (AMPK) that inhibits mTORC1 activity via a pathway involving tuberous sclerosis complex 1 and 2 (TSC1/2) and its substrate Rheb. The present study suggests hat AMPK inhibits mTORC1 and autophagy also in nonstarved cells. Various Ca(2+) mobilizing agents (vitamin D compounds, thapsigargin, ATP and ionomycin) activate MPK via activation of Ca(2+)/calmodulin-dependent kinase kinase-beta (CaMKK-beta), and his pathway is required for Ca(2+)-induced autophagy. Thus, we propose that an increase in free cytosolic Ca(2+) ([Ca(2+)](c)) induces autophagy via the CaMKK/beta-AMPK-TSC1/2-Rheb-mTORC1 signaling pathway and that AMPK is a more general regulator of autophagy than previously expected.     

Autophagy induction by tetrahydrobiopterin deficiency

Tetrahydrobiopterin (BH₄) deficiency is a genetic disorder associated with a variety of metabolic syndromes such as phenylketonuria (PKU). In this article, the signaling pathway by which BH₄ deficiency inactivates mTORC1 leading to the activation of the autophagic pathway was studied utilizing BH₄-deficient Spr^-/- mice generated by the knockout of the gene encoding sepiapterin reductase (SR) catalyzing BH₄ synthesis. We found that mTORC1 signaling was inactivated and autophagic pathway was activated in tissues from Spr^-/- mice. This study demonstrates that tyrosine deficiency causes mTORC1 inactivation and subsequent activation of autophagic pathway in Spr^-/- mice. Therapeutic tyrosine diet completely rescued dwarfism and mTORC1 inhibition but inactivated autophagic pathway in Spr^-/- mice. Tyrosine-dependent inactivation of mTORC1 was further supported by mTORC1 inactivation in Pah^enu2 mouse model lacking phenylalanine hydroxylase (Pah). NIH3T3 cells grown under the condition of tyrosine restriction exhibited autophagy induction. However, mTORC1 activation by RhebQ64L, a positive regulator of mTORC1, inactivated autophagic pathway in NIH3T3 cells under tyrosine-deficient conditions. In addition, this study first documents mTORC1 inactivation and autophagy induction in PKU patients with BH₄ deficiency.Key words: tetrahydrobiopterin, autophagy, mTORC1, tyrosine, phenylalanine, phenylketonuria, Akt, AMPK