GSK3β Serine 389 Phosphorylation Modulates Cardiomyocyte Hypertrophy and Ischemic Injury

Prior studies show that glycogen synthase kinase 3β (GSK3β) contributes to cardiac ischemic injury and cardiac hypertrophy. GSK3β is constitutionally active and phosphorylation of GSK3β at serine 9 (S9) inactivates the kinase and promotes cellular growth. GSK3β is also phosphorylated at serine 389 (S389), but the significance of this phosphorylation in the heart is not known. We analyzed GSK3β S389 phosphorylation in diseased hearts and utilized overexpression of GSK3β carrying ser→ala mutations at S9 (S9A) and S389 (S389A) to study the biological function of constitutively active GSK3β in primary cardiomyocytes. We found that phosphorylation of GSK3β at S389 was increased in left ventricular samples from patients with dilated cardiomyopathy and ischemic cardiomyopathy, and in hearts of mice subjected to thoracic aortic constriction. Overexpression of either GSK3β S9A or S389A reduced the viability of cardiomyocytes subjected to hypoxia–reoxygenation. Overexpression of double GSK3β mutant (S9A/S389A) further reduced cardiomyocyte viability. Determination of protein synthesis showed that overexpression of GSK3β S389A or GSK3β S9A/S389A increased both basal and agonist-induced cardiomyocyte growth. Mechanistically, GSK3β S389A mutation was associated with activation of mTOR complex 1 signaling. In conclusion, our data suggest that phosphorylation of GSK3β at S389 enhances cardiomyocyte survival and protects from cardiomyocyte hypertrophy.

Regulation of human mTOR complexes by DEPTOR

The vertebrate-specific DEP domain-containing mTOR interacting protein (DEPTOR), an oncoprotein or tumor suppressor, has important roles in metabolism, immunity, and cancer. It is the only protein that binds and regulates both complexes of mammalian target of rapamycin (mTOR), a central regulator of cell growth. Biochemical analysis and cryo-EM reconstructions of DEPTOR bound to human mTOR complex 1 (mTORC1) and mTORC2 reveal that both structured regions of DEPTOR, the PDZ domain and the DEP domain tandem (DEPt), are involved in mTOR interaction. The PDZ domain binds tightly with mildly activating effect, but then acts as an anchor for DEPt association that allosterically suppresses mTOR activation. The binding interfaces of the PDZ domain and DEPt also support further regulation by other signaling pathways. A separate, substrate-like mode of interaction for DEPTOR phosphorylation by mTOR complexes rationalizes inhibition of non-stimulated mTOR activity at higher DEPTOR concentrations. The multifaceted interplay between DEPTOR and mTOR provides a basis for understanding the divergent roles of DEPTOR in physiology and opens new routes for targeting the mTOR-DEPTOR interaction in disease.

Lamin regulates the dietary restriction response via the mTOR pathway in Caenorhabditis elegans

Animals subjected to dietary restriction (DR) have reduced body size, low fecundity, slower development, lower fat content and longer life span. We identified lamin as a regulator of multiple dietary restriction phenotypes. Downregulation of lmn-1, the single Caenorhabditis elegans lamin gene, increased animal size and fat content, specifically in DR animals. The LMN-1 protein acts in the mTOR pathway, upstream to RAPTOR and S6K, key component and target of mTOR complex 1 (mTORC1), respectively. DR excludes the mTORC1 activator RAGC-1 from the nucleus. Downregulation of lmn-1 restores RAGC-1 to the nucleus, a necessary step for the activation of the mTOR pathway. These findings further link lamin to metabolic regulation.

TSC2-extracellular matrix crosstalk controls pulmonary vascular proliferation and pulmonary hypertension

Increased proliferation and survival of resident cells in small pulmonary arteries (PA) are important drivers of pulmonary hypertension (PH). Tuberous sclerosis complex 2 (TSC2) is a negative regulator of mTOR complex 1 and cell growth. Here we show that TSC2 is deficient in small remodeled PA/PA vascular smooth muscle cells (PAVSMC) from human PAH and experimental PH lungs. TSC2 deficiency was reproduced in vitro by maintaining PAVSMC on pathologically stiff substrates and was required for stiffness-induced proliferation, accumulation of transcriptional co-activators YAP/TAZ and up-regulation of mTOR. Depletion of TSC2 reproduced PH features in vitro in human PAVSMC and in vivo in SM22-Tsc2+/- mice. TSC2 loss in PAVSMC was supported by YAP and led to the up-regulation of YAP/TAZ and mTOR via modulating the extracellular matrix (ECM) composition. ECM, produced by TSC2-deficient PAVSMC, promoted growth of non-diseased PA adventitial fibroblasts and PAVSMC, which, in turn, was prevented by α5β1 integrin receptor antagonist ATN161. In vitro, molecular and pharmacological (SRT2104) restoration of TSC2 down-regulated YAP/TAZ, mTOR, and ECM pro-duction, inhibited proliferation and induced apoptosis in human PAH PAVSMC. In vivo, orally administrated SRT2104 restored TSC2, resolved pulmonary vascular remodeling, PH, and improved right heart in two rodent models of PH. Thus, PAVSMC TSC2 is a critical integrator of ECM composition and stiffness with pro-proliferative signaling and PH, and the restoration of functional TSC2 could be an attractive therapeutic option to treat PH.

The mTOR–Autophagy Axis and the Control of Metabolism

The mechanistic target of rapamycin (mTOR), master regulator of cellular metabolism, exists in two distinct complexes: mTOR complex 1 and mTOR complex 2 (mTORC1 and 2). MTORC1 is a master switch for most energetically onerous processes in the cell, driving cell growth and building cellular biomass in instances of nutrient sufficiency, and conversely, allowing autophagic recycling of cellular components upon nutrient limitation. The means by which the mTOR kinase blocks autophagy include direct inhibition of the early steps of the process, and the control of the lysosomal degradative capacity of the cell by inhibiting the transactivation of genes encoding structural, regulatory, and catalytic factors. Upon inhibition of mTOR, autophagic recycling of cellular components results in the reactivation of mTORC1; thus, autophagy lies both downstream and upstream of mTOR. The functional relationship between the mTOR pathway and autophagy involves complex regulatory loops that are significantly deciphered at the cellular level, but incompletely understood at the physiological level. Nevertheless, genetic evidence stemming from the use of engineered strains of mice has provided significant insight into the overlapping and complementary metabolic effects that physiological autophagy and the control of mTOR activity exert during fasting and nutrient overload.

Mitogenic and Metabolic signals regulate mTOR complex 1 function to control GSK3β nuclear program

Mitogenic and metabolic signalling are two cell pathways that control different aspects of cellular physiology including, growth, proliferation, metabolism, and transcription. Mitogenic signalling involves mitogens and growth factors to stimulate various receptor signalling pathways such as epidermal growth factor receptor (EGFR), while metabolic signalling involves proteins that sense changes in abundance of specific nutrients or metabolites such as amino acids and ATP. Here, I have uncovered that EGFR signalling is controlled by clathrin nanodomains at the plasma membrane, yet this requirement for clathrin does not reflect a role for receptor internalization in EGFR signalling. Specifically, I found that clathrin is required for activation of the key signaling intermediate Akt by EGFR upon EGF stimulation. Furthermore, I have also resolved a series of signals including Phospholipase C γ1 (PLCγ1) that may control EGF stimulated Akt activation by modulating the assembly of clathrin into plasma membrane nanodomains. These findings suggest that clathrin nanodomains at the plasma membrane are important for controlling EGFR signalling, thus impacting mitogenic signaling. A downstream signalling pathway controlled by Akt is the Glycogen synthase kinase 3 (GSK3) pathway. GSK3 phosphorylates and thereby regulates a wide range of protein substrates involved in diverse cellular functions. Some GSK3 substrates, such as c-Myc and Snail, are nuclear transcription factors, suggesting the possibility that GSK3 function is controlled through regulation of its nuclear localization. I found that perturbations in mTOR complex 1 (mTORC1) leads to partial redistribution of GSK3 from the cytosol to the nucleus and to a GSK3 dependent reduction of the levels of both c-Myc and Snail. In addition to conditional nuclear localization, GSK3 was also detected on several distinct endomembrane compartments, including lysosomes. Consistently, disruption of various aspects of the function and regulation late endosomes/lysosomes resulted in perturbation of GSK3 nucleocytoplasmic shuttling and activity. Furthermore, I found that DEPDC5, a subunit of the lysosomal amino-acid sensing GATOR1 complex, controls amino acid sensing mechanisms to regulate GSK3 nucleocytoplasmic shuttling. These findings uncover a new signalling axis that is controlled by specific aspects of both mitogenic and metabolic signalling, which may interface with the nucleus to reprogram transcriptional cellular networks for growth and proliferation. Understanding how mTORC1- GSK3 signalling impacts transcriptional networks may be an important target for different therapies and treatments against diverse forms of cancer.

Mitogenic and Metabolic signals regulate mTOR complex 1 function to control GSK3β nuclear program

Mitogenic and metabolic signalling are two cell pathways that control different aspects of cellular physiology including, growth, proliferation, metabolism, and transcription. Mitogenic signalling involves mitogens and growth factors to stimulate various receptor signalling pathways such as epidermal growth factor receptor (EGFR), while metabolic signalling involves proteins that sense changes in abundance of specific nutrients or metabolites such as amino acids and ATP. Here, I have uncovered that EGFR signalling is controlled by clathrin nanodomains at the plasma membrane, yet this requirement for clathrin does not reflect a role for receptor internalization in EGFR signalling. Specifically, I found that clathrin is required for activation of the key signaling intermediate Akt by EGFR upon EGF stimulation. Furthermore, I have also resolved a series of signals including Phospholipase C γ1 (PLCγ1) that may control EGF stimulated Akt activation by modulating the assembly of clathrin into plasma membrane nanodomains. These findings suggest that clathrin nanodomains at the plasma membrane are important for controlling EGFR signalling, thus impacting mitogenic signaling. A downstream signalling pathway controlled by Akt is the Glycogen synthase kinase 3 (GSK3) pathway. GSK3 phosphorylates and thereby regulates a wide range of protein substrates involved in diverse cellular functions. Some GSK3 substrates, such as c-Myc and Snail, are nuclear transcription factors, suggesting the possibility that GSK3 function is controlled through regulation of its nuclear localization. I found that perturbations in mTOR complex 1 (mTORC1) leads to partial redistribution of GSK3 from the cytosol to the nucleus and to a GSK3 dependent reduction of the levels of both c-Myc and Snail. In addition to conditional nuclear localization, GSK3 was also detected on several distinct endomembrane compartments, including lysosomes. Consistently, disruption of various aspects of the function and regulation late endosomes/lysosomes resulted in perturbation of GSK3 nucleocytoplasmic shuttling and activity. Furthermore, I found that DEPDC5, a subunit of the lysosomal amino-acid sensing GATOR1 complex, controls amino acid sensing mechanisms to regulate GSK3 nucleocytoplasmic shuttling. These findings uncover a new signalling axis that is controlled by specific aspects of both mitogenic and metabolic signalling, which may interface with the nucleus to reprogram transcriptional cellular networks for growth and proliferation. Understanding how mTORC1- GSK3 signalling impacts transcriptional networks may be an important target for different therapies and treatments against diverse forms of cancer.

Dual mTORC1/mTORC2 inhibition as a Host-Directed Therapeutic Target in Pathologically Distinct Mouse Models of Tuberculosis

Efforts to develop more effective and shorter-course therapies for tuberculosis have included a focus on host-directed therapy (HDT). The goal of HDT is to modulate the host response to infection, thereby improving immune defenses to reduce the duration of antibacterial therapy and/or the amount of lung damage. As a mediator of innate and adaptive immune responses involved in eliminating intracellular pathogens, autophagy is a potential target for HDT in tuberculosis. Because Mycobacterium tuberculosis modulates mammalian target of rapamycin (mTOR) signaling to impede autophagy, pharmacologic mTOR inhibition could provide effective HDT. mTOR exists within two distinct multiprotein complexes, mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2). Rapamycin and its analogs only partially inhibit mTORC1. We hypothesized that novel mTOR kinase inhibitors blocking both complexes would have expanded therapeutic potential. We compared the effects of two mTOR inhibitors: rapamycin and the orally available mTOR kinase domain inhibitor CC214-2, which blocks both mTORC1 and mTORC2, as adjunctive therapies against murine TB, when added to the first-line regimen (RHZE) or the novel bedaquiline-pretomanid-linezolid (BPaL) regimen. Neither mTOR inhibitor affected lung CFU counts after 4-8 weeks of treatment when combined with BPaL or RHZE. However, addition of CC214-2 to BPaL and RHZE was associated with significantly fewer relapses in C3HeB/FeJ compared to addition of rapamycin and, in RHZE-treated mice, resulted in fewer relapses compared to RHZE alone. Therefore, CC214-2 and related mTOR kinase inhibitors may be more effective candidates for HDT than rapamycin analogs and may have the potential to shorten the duration of TB treatment.

Effects of L-Glutamine Supplementation during the Gestation of Gilts and Sows on the Offspring Development in a Traditional Swine Breed

The use of amino acids during pregnancy, such as glutamine (Gln), seems to be a promising strategy in selected swine breeds to improve the offspring prenatal development. The main goal of the current study was to assess the development of the offspring from parity 1–3 sows of a traditional breed, which were supplemented with 1% glutamine after Day 35 of gestation, under farm conditions. A total of 486 (288 treated) piglets from 78 (46 treated) Iberian sows were used. At birth and slaughterhouse, fatty acid composition, metabolism, and mTOR pathway gene expression were analyzed. At birth, treated newborns showed greater amounts of specific amino acids in plasma, such as glutamine, asparagine, or alanine, and Σn-3 fatty acids in cellular membranes than control newborns. The expression of genes belonging to mTOR Complex 1 was also higher in treated piglets with normal birth-weight. However, these findings did not improve productive traits at birth or following periods in litters from supplemented gilts (parity 1) or sows (parities 2–3). Thus, further research is needed to properly understand the effects of prenatal glutamine supplementation, particularly in traditional swine breeds.