Amino Acids Enhance Polyubiquitination of Rheb and Its Binding to mTORC1 by Blocking Lysosomal ATXN3 Deubiquitinase Activity.

Amino-acid-induced lysosomal mechanistic target of rapamycin complex 1 (mTORC1) localization through the Rag GTPases is a critical step for its activation by Rheb GTPase. However, how the mTORC1 interacts with Rheb on the lysosome remains elusive. We report that amino acids enhance the polyubiquitination of Rheb (Ub-Rheb), which shows a strong binding preference for mTORC1 and supports its activation, while the Ub-Rheb is subjected to subsequent degradation. Mechanistically, we identified ATXN3 as a Ub-Rheb deubiquitinase whose lysosomal localization is blocked by active Rag heterodimer in response to amino acid stimulation. Consistently, cells lacking functional Rag heterodimer on the lysosome accumulate Ub-Rheb, and blockade of its degradation instigates robust lysosomal mTORC1 localization and its activation without the Ragulator-Rag system. Thus, polyubiquitination of Rheb is an important post-translational modification, which facilitates the binding of mTORC1 to Rheb on the lysosome and is another crosstalk between the amino acid and growth factor signaling for mTORC1 activation.

Myeloid-specific ablation of Basp1 ameliorates diet-induced NASH in mice by attenuating pro-inflammatory signaling.

BACKGROUND AND AIMS: NASH represents a severe stage of fatty liver disease characterized by hepatocyte injury, inflammation, and liver fibrosis. Myeloid-derived innate immune cells, such as macrophages and dendritic cells, play an important role in host defense and disease pathogenesis. Despite this, the nature of transcriptomic reprogramming of myeloid cells in NASH liver and its contribution to disease progression remain incompletely defined. APPROACH AND RESULTS: In this study, we performed bulk and single-cell RNA sequencing (sc-RNA seq) analysis to delineate the landscape of macrophage and dendritic cell transcriptomes in healthy and NASH livers. Our analysis uncovered cell type-specific patterns of transcriptomic reprogramming on diet-induced NASH. We identified brain-abundant membrane-attached signal protein 1 (Basp1) as a myeloid-enriched gene that is markedly induced in mouse and human NASH liver. Myeloid-specific inactivation of Basp1 attenuates the severity of diet-induced NASH pathologies, as shown by reduced hepatocyte injury and liver fibrosis in mice. Mechanistically, cultured macrophages lacking Basp1 exhibited a diminished response to pro-inflammatory stimuli, impaired NLRP3 inflammasome activation, and reduced cytokine secretion. CONCLUSIONS: Together, these findings uncover Basp1 as a critical regulator of myeloid inflammatory signaling that underlies NASH pathogenesis.

The molecular and metabolic program by which white adipocytes adapt to cool physiologic temperatures.

Although visceral adipocytes located within the body's central core are maintained at approximately 37°C, adipocytes within bone marrow, subcutaneous, and dermal depots are found primarily within the peripheral shell and generally exist at cooler temperatures. Responses of brown and beige/brite adipocytes to cold stress are well studied; however, comparatively little is known about mechanisms by which white adipocytes adapt to temperatures below 37°C. Here, we report that adaptation of cultured adipocytes to 31°C, the temperature at which distal marrow adipose tissues and subcutaneous adipose tissues often reside, increases anabolic and catabolic lipid metabolism, and elevates oxygen consumption. Cool adipocytes rely less on glucose and more on pyruvate, glutamine, and, especially, fatty acids as energy sources. Exposure of cultured adipocytes and gluteal white adipose tissue (WAT) to cool temperatures activates a shared program of gene expression. Cool temperatures induce stearoyl-CoA desaturase-1 (SCD1) expression and monounsaturated lipid levels in cultured adipocytes and distal bone marrow adipose tissues (BMATs), and SCD1 activity is required for acquisition of maximal oxygen consumption at 31°C.

Identification of circular dorsal ruffles as signal platforms for the AKT pathway in glomerular podocytes.

Circular dorsal ruffles (CDRs) are rounded membrane ruffles induced by growth factors to function as precursors of the large-scale endocytosis called macropinocytosis. In addition to their role in cellular uptake, recent research using cell line systems has shown that CDRs/macropinocytosis regulate the canonical AKT-mTORC1 growth factor signaling pathway. However, as CDRs have not been observed in tissues, their physiological relevance has remained unclear. Here, utilizing ultrahigh-resolution scanning electron microscopy, we first report that CDRs are expressed in glomerular podocytes ex vivo and in vivo, and we visually captured the transformation process to macropinocytosis. Moreover, through biochemical and imaging analyses, we show that AKT phosphorylation localized to CDRs upstream of mTORC1 activation in podocyte cell lines and isolated glomeruli. These results demonstrate the physiological role of CDRs as signal platforms for the AKT-mTORC1 pathway in glomerular podocytes at the tissue level. As mTORC1 plays critical roles in podocyte metabolism, and aberrant activation of mTORC1 triggers podocytopathies, our results strongly suggest that targeting CDR formation could represent a potential therapeutic approach for these diseases.

ER-associated degradation preserves hematopoietic stem cell quiescence and self-renewal by restricting mTOR activity.

Hematopoietic stem cells (HSC) self-renew to sustain stem cell pools and differentiate to generate all types of blood cells. HSCs remain in quiescence to sustain their long-term self-renewal potential. It remains unclear whether protein quality control is required for stem cells in quiescence when RNA content, protein synthesis, and metabolic activities are profoundly reduced. Here, we report that protein quality control via endoplasmic reticulum-associated degradation (ERAD) governs the function of quiescent HSCs. The Sel1L/Hrd1 ERAD genes are enriched in the quiescent and inactive HSCs, and conditional knockout of Sel1L in hematopoietic tissues drives HSCs to hyperproliferation, which leads to complete loss of HSC self-renewal and HSC depletion. Mechanistically, ERAD deficiency via Sel1L knockout leads to activation of mammalian target of rapamycin (mTOR) signaling. Furthermore, we identify Ras homolog enriched in brain (Rheb), an activator of mTOR, as a novel protein substrate of Sel1L/Hrd1 ERAD, which accumulates upon Sel1L deletion and HSC activation. Importantly, inhibition of mTOR, or Rheb, rescues HSC defects in Sel1L knockout mice. Protein quality control via ERAD is, therefore, a critical checkpoint that governs HSC quiescence and self-renewal by Rheb-mediated restriction of mTOR activity.

Rapamycin directly activates lysosomal mucolipin TRP channels independent of mTOR.

Rapamycin (Rap) and its derivatives, called rapalogs, are being explored in clinical trials targeting cancer and neurodegeneration. The underlying mechanisms of Rap actions, however, are not well understood. Mechanistic target of rapamycin (mTOR), a lysosome-localized protein kinase that acts as a critical regulator of cellular growth, is believed to mediate most Rap actions. Here, we identified mucolipin 1 (transient receptor potential channel mucolipin 1 [TRPML1], also known as MCOLN1), the principle Ca2+ release channel in the lysosome, as another direct target of Rap. Patch-clamping of isolated lysosomal membranes showed that micromolar concentrations of Rap and some rapalogs activated lysosomal TRPML1 directly and specifically. Pharmacological inhibition or genetic inactivation of mTOR failed to mimic the Rap effect. In vitro binding assays revealed that Rap bound directly to purified TRPML1 proteins with a micromolar affinity. In both healthy and disease human fibroblasts, Rap and rapalogs induced autophagic flux via nuclear translocation of transcription factor EB (TFEB). However, such effects were abolished in TRPML1-deficient cells or by TRPML1 inhibitors. Hence, Rap and rapalogs promote autophagy via a TRPML1-dependent mechanism. Given the demonstrated roles of TRPML1 and TFEB in cellular clearance, we propose that lysosomal TRPML1 may contribute a significant portion to the in vivo neuroprotective and anti-aging effects of Rap via an augmentation of autophagy and lysosomal biogenesis.

Inhibition of AMPK catabolic action by GSK3.

AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic and activating catabolic processes. While AMPK activation has been extensively studied, mechanisms that inhibit AMPK remain elusive. Here we report that glycogen synthase kinase 3 (GSK3) inhibits AMPK function. GSK3 forms a stable complex with AMPK through interactions with the AMPK ß regulatory subunit and phosphorylates the AMPK α catalytic subunit. This phosphorylation enhances the accessibility of the activation loop of the α subunit to phosphatases, thereby inhibiting AMPK kinase activity. Surprisingly, PI3K-Akt signaling, which is a major anabolic signaling and normally inhibits GSK3 activity, promotes GSK3 phosphorylation and inhibition of AMPK, thus revealing how AMPK senses anabolic environments in addition to cellular energy levels. Consistently, disrupting GSK3 function within the AMPK complex sustains higher AMPK activity and cellular catabolic processes even under anabolic conditions, indicating that GSK3 acts as a critical sensor for anabolic signaling to regulate AMPK.

Macropinocytosis, mTORC1 and cellular growth control.

The growth and proliferation of metazoan cells are driven by cellular nutrient status and by extracellular growth factors. Growth factor receptors on cell surfaces initiate biochemical signals that increase anabolic metabolism and macropinocytosis, an actin-dependent endocytic process in which relatively large volumes of extracellular solutes and nutrients are internalized and delivered efficiently into lysosomes. Macropinocytosis is prominent in many kinds of cancer cells, and supports the growth of cells transformed by oncogenic K-Ras. Growth factor receptor signaling and the overall metabolic status of the cell are coordinated in the cytoplasm by the mechanistic target-of-rapamycin complex-1 (mTORC1), which positively regulates protein synthesis and negatively regulates molecular salvage pathways such as autophagy. mTORC1 is activated by two distinct Ras-related small GTPases, Rag and Rheb, which associate with lysosomal membranes inside the cell. Rag recruits mTORC1 to the lysosomal surface where Rheb directly binds to and activates mTORC1. Rag is activated by both lysosomal luminal and cytosolic amino acids; Rheb activation requires phosphoinositide 3-kinase, Akt, and the tuberous sclerosis complex-1/2. Signals for activation of Rag and Rheb converge at the lysosomal membrane, and several lines of evidence support the idea that growth factor-dependent endocytosis facilitates amino acid transfer into the lysosome leading to the activation of Rag. This review summarizes evidence that growth factor-stimulated macropinocytosis is essential for amino acid-dependent activation of mTORC1, and that increased solute accumulation by macropinocytosis in transformed cells supports unchecked cell growth.

Loss of AMPKα2 Impairs Hedgehog-Driven Medulloblastoma Tumorigenesis.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that has a dual role in cancer, i.e., pro- or anti-tumorigenic, depending on the context. In medulloblastoma, the most frequent malignant pediatric brain tumor, several in vitro studies previously showed that AMPK suppresses tumor cell growth. The role of AMPK in this disease context remains to be tested in vivo. Here, we investigate loss of AMPKα2 in a genetically engineered mouse model of sonic hedgehog (SHH)-medulloblastoma. In contrast to previous reports, our study reveals that AMPKα2 KO impairs SHH medulloblastoma tumorigenesis. Moreover, we performed complementary molecular and genomic analyses that support the hypothesis of a pro-tumorigenic SHH/AMPK/CNBP axis in medulloblastoma. In conclusion, our observations further underline the context-dependent role of AMPK in cancer, and caution is warranted for the previously proposed hypothesis that AMPK agonists may have therapeutic benefits in medulloblastoma patients. Note: an abstract describing the project was previously submitted to the American Society for Investigative Pathology PISA 2018 conference and appears in The American Journal of Pathology (Volume 188, Issue 10, October 2018, Page 2433).

Role of Ragulator in the Regulation of Mechanistic Target of Rapamycin Signaling in Podocytes and Glomerular Function.

Aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1) in glomerular podocytes leads to glomerular insufficiency and may contribute to the development of glomerular diseases, including diabetic nephropathy. Thus, an approach for preventing mTORC1 activation may allow circumvention of the onset and progression of mTORC1-dependent podocyte injury and glomerular diseases. mTORC1 activation requires inputs from both growth factors and nutrients that inactivate the tuberous sclerosis complex (TSC), a key suppressor of mTORC1, on the lysosome. Previous studies in mice revealed that the growth factor-phosphatidylinositol 3-kinase pathway and mTORC1 are essential for maintaining normal podocyte function, suggesting that direct inhibition of the phosphatidylinositol 3-kinase pathway or mTORC1 may not be an ideal approach to sustaining physiologic podocyte functions under certain disease conditions. Here, we report the role of the Ragulator complex, which recruits mTORC1 to lysosomes in response to nutrient availability in podocytes. Notably, podocytes lacking Ragulator maintain basal mTORC1 activity. Unlike podocyte-specific mTORC1-knockout mice, mice lacking functional Ragulator in podocytes did not show abnormalities in podocyte or glomerular function. However, aberrant mTORC1 activation induced by active Rheb in podocyte-specific TSC1-knockout (podo-TSC1 KO) mice did require Ragulator. Moreover, ablation of Ragulator in the podocytes of podo-TSC1 KO mice or streptozotocin-induced diabetic mice significantly blocked the development of pathologic renal phenotypes. These observations suggest that the blockade of mTORC1 recruitment to lysosomes may be a useful clinical approach to attenuate aberrant mTORC1 activation under certain disease conditions.

Aberrant mTORC1 activation kills tubular cells by inactivating miR148b-3p.

A new study by Kuwagata et al. now shows that aberrant activation of the mechanistic target of rapamycin complex 1 in renal tubular cells causes their injury and cell death by enhancing endoplasmic reticulum stress and cell death pathway under diabetic conditions. The study has revealed a novel molecular mechanism in which mechanistic target of rapamycin complex 1 stimulates the tumor necrosis factor signaling by attenuating miR-148b-3p, thereby deteriorating renal tubular cell dysfunction under diabetic conditions.

The role of mechanistic target of rapamycin in maintenance of glomerular epithelial cells.

PURPOSE OF REVIEW: Recent studies have emerged to reveal the pivotal roles of mechanistic target of rapamycin (mTOR) signaling not only in the maintenance of the physiological functions of renal cells but also in the pathogenesis of renal cell dysfunctions and kidney diseases. We introduce the current understanding of mTOR signaling, and its crucial roles in glomerular epithelial cell biology and the pathophysiology related to kidney diseases. RECENT FINDINGS: mTOR, a Ser/Thr kinase, forms two distinct functional complexes, mTORC1 and mTORC2. Recent studies revealed that physiologic levels of mTORC1 and mTORC2 activity play key roles in maintaining podocyte and glomerular functions. However, aberrant activation of mTORC1 or loss of mTORC2 activity in podocytes may underlie the pathogenesis of glomerular disorders, including diabetic kidney disease. SUMMARY: An effective treatment for mTORC1-associated podocyte and glomerular dysfunction may require the attenuation of mTORC1 activity in the setting of both an intact mTORC2 pathway and normal basal mTORC1 activity in order to preserve physiologic podocyte functions.

Cross-talk between sirtuin and mammalian target of rapamycin complex 1 (mTORC1) signaling in the regulation of S6 kinase 1 (S6K1) phosphorylation.

p70 ribosomal S6 kinase (S6K1), a major substrate of the mammalian target of rapamycin (mTOR) kinase, regulates diverse cellular processes including protein synthesis, cell growth, and survival. Although it is well known that the activity of S6K1 is tightly coupled to its phosphorylation status, the regulation of S6K1 activity by other post-translational modifications such as acetylation has not been well understood. Here we show that the acetylation of the C-terminal region (CTR) of S6K1 blocks mTORC1-dependent Thr-389 phosphorylation, an essential phosphorylation site for S6K1 activity. The acetylation of the CTR of S6K1 is inhibited by the class III histone deacetylases, SIRT1 and SIRT2. An S6K1 mutant lacking acetylation sites in its CTR shows enhanced Thr-389 phosphorylation and kinase activity, whereas the acetylation-mimetic S6K1 mutant exhibits decreased Thr-389 phosphorylation and kinase activity. Interestingly, relative to the acetylation-mimetic S6K1 mutant, the acetylation-defective mutant displays higher affinity toward Raptor, an essential scaffolding component of mTORC1 that recruits mTORC1 substrates. These observations indicate that sirtuin-mediated regulation of S6K1 acetylation is an additional important regulatory modification that impinges on the mechanisms underlying mTORC1-dependent S6K1 activation.

Liver clock protein BMAL1 promotes de novo lipogenesis through insulin-mTORC2-AKT signaling.

The clock protein BMAL1 (brain and muscle Arnt-like protein 1) participates in circadian regulation of lipid metabolism, but its contribution to insulin AKT-regulated hepatic lipid synthesis is unclear. Here we used both Bmal1(-/-) and acute liver-specific Bmal1-depleted mice to study the role of BMAL1 in refeeding-induced de novo lipogenesis in the liver. Both global deficiency and acute hepatic depletion of Bmal1 reduced lipogenic gene expression in the liver upon refeeding. Conversely, Bmal1 overexpression in mouse liver by adenovirus was sufficient to elevate the levels of mRNA of lipogenic enzymes. Bmal1(-/-) primary mouse hepatocytes displayed decreased levels of de novo lipogenesis and lipogenic enzymes, supporting the notion that BMAL1 regulates lipid synthesis in hepatocytes in a cell-autonomous manner. Both refed mouse liver and insulin-treated primary mouse hepatocytes showed impaired AKT activation in the case of either Bmal1 deficiency or Bmal1 depletion by adenoviral shRNA. Restoring AKT activity by a constitutively active mutant of AKT nearly normalized de novo lipogenesis in Bmal1(-/-) hepatocytes. Finally, Bmal1 deficiency or knockdown decreased the protein abundance of RICTOR, the key component of the mTORC2 complex, without affecting the gene expression of key factors of insulin signaling. Thus, our study uncovered a novel metabolic function of hepatic BMAL1 that promotes de novo lipogenesis via the insulin-mTORC2-AKT signaling during refeeding.

AMPK and mTOR in cellular energy homeostasis and drug targets.

The mammalian target of rapamycin (mTOR) is a central controller of cell growth and proliferation. mTOR forms two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is regulated by multiple signals such as growth factors, amino acids, and cellular energy and regulates numerous essential cellular processes including translation, transcription, and autophagy. The AMP-activated protein kinase (AMPK) is a cellular energy sensor and signal transducer that is regulated by a wide array of metabolic stresses. These two pathways serve as a signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth, and dysregulation of each pathway may contribute to the development of metabolic disorders such as obesity, type 2 diabetes, and cancer. This review focuses on our current understanding of the relationship between AMPK and mTORC1 signaling and discusses their roles in cellular and organismal energy homeostasis.

Ume6 transcription factor is part of a signaling cascade that regulates autophagy.

Autophagy has been implicated in a number of physiological processes important for human heath and disease. Autophagy involves the formation of a double-membrane cytosolic vesicle, an autophagosome. Central to the formation of the autophagosome is the ubiquitin-like protein autophagy-related (Atg)8 (microtubule-associated protein 1 light chain 3/LC3 in mammalian cells). Following autophagy induction, Atg8 shows the greatest change in expression of any of the proteins required for autophagy. The magnitude of autophagy is, in part, controlled by the amount of Atg8; thus, controlling Atg8 protein levels is one potential mechanism for modulating autophagy activity. We have identified a negative regulator of ATG8 transcription, Ume6, which acts along with a histone deacetylase complex including Sin3 and Rpd3 to regulate Atg8 levels; deletion of any of these components leads to an increase in Atg8 and a concomitant increase in autophagic activity. A similar regulatory mechanism is present in mammalian cells, indicating that this process is highly conserved.

GSK3ß-dependent inhibition of AMPK potentiates activation of neutrophils and macrophages and enhances severity of acute lung injury.

Although AMP-activated protein kinase (AMPK) is involved in regulating carbohydrate and lipid metabolism, activated AMPK also plays an anti-inflammatory role in many cell populations. However, despite the ability of AMPK activation to diminish the severity of inflammatory responses, previous studies have found that AMPK activity is diminished in LPS-treated neutrophils and also in lungs of mice with LPS-induced acute lung injury (ALI). Since GSK3ß participates in regulating AMPK activity, we examined potential roles for GSK3ß in modulating LPS-induced activation of neutrophils and macrophages and in influencing severity of ALI. We found that GSK3ß-dependent phosphorylation of T479-AMPK was associated with pT172 dephosphorylation and inactivation of AMPK following TLR4 engagement. GSK3ß inhibitors BIO (6-bromoindirubin-3'-oxime), SB216763, or siRNA knockdown of GSK3ß, but not the PI3K/AKT inhibitor LY294002, prevented Thr172-AMPK dephosphorylation. Exposure to LPS resulted in rapid binding between IKKß and AMPKα, and phosphorylation of S485-AMPK by IKKß. These results suggest that IKKß-dependent phosphorylation of S485-AMPK was an essential step in subsequent phosphorylation and inactivation AMPK by GSK3ß. Inhibition of GSK3ß activity delayed IκBα degradation and diminished expression of the proinflammatory TNF-α in LPS-stimulated neutrophils and macrophages. In vivo, inhibition of GSK3ß decreased the severity of LPS-induced lung injury as assessed by development of pulmonary edema, production of TNF-α and MIP-2, and release of the alarmins HMGB1 and histone 3 in the lungs. These results show that inhibition of AMPK by GSK3ß plays an important contributory role in enhancing LPS-induced inflammatory responses, including worsening the severity of ALI.