Coordinated regulation of the lysosomal and autophagic systems ensures basal catabolism and normal cell physiology, and failure of either system causes disease. Here we describe an epigenetic ...rheostat orchestrated by c-MYC and histone deacetylases that inhibits lysosomal and autophagic biogenesis by concomitantly repressing the expression of the transcription factors MiT/TFE and FOXH1, and that of lysosomal and autophagy genes. Inhibition of histone deacetylases abates c-MYC binding to the promoters of lysosomal and autophagy genes, granting promoter occupancy to the MiT/TFE members, TFEB and TFE3, and/or the autophagy regulator FOXH1. In pluripotent stem cells and cancer, suppression of lysosomal and autophagic function is directly downstream of c-MYC overexpression and may represent a hallmark of malignant transformation. We propose that, by determining the fate of these catabolic systems, this hierarchical switch regulates the adaptive response of cells to pathological and physiological cues that could be exploited therapeutically.
A member of the sirtuin family of NAD+-dependent deacetylases, SIRT3, is identified as one of the major mitochondrial deacetylases located in mammalian mitochondria responsible for deacetylation of ...several metabolic enzymes and components of oxidative phosphorylation. Regulation of protein deacetylation by SIRT3 is important for mitochondrial metabolism, cell survival, and longevity. In this study, we identified one of the Complex II subunits, succinate dehydrogenase flavoprotein (SdhA) subunit, as a novel SIRT3 substrate in SIRT3 knockout mice. Several acetylated Lys residues were mapped by tandem mass spectrometry, and we determined the role of acetylation in Complex II activity in SIRT3 knockout mice. In agreement with SIRT3-dependent activation of Complex I, we observed that deacetylation of the SdhA subunit increased the Complex II activity in wild-type mice. In addition, we treated K562 cell lines with nicotinamide and kaempferol to inhibit deacetylase activity of SIRT3 and stimulate SIRT3 expression, respectively. Stimulation of SIRT3 expression decreased the level of acetylation of the SdhA subunit and increased Complex II activity in kaempherol-treated cells compared to control and nicotinamide-treated cells. Evaluation of acetylated residues in the SdhA crystal structure from porcine and chicken suggests that acetylation of the hydrophilic surface of SdhA may control the entry of the substrate into the active site of the protein and regulate the enzyme activity. Our findings constitute the first evidence of the regulation of Complex II activity by the reversible acetylation of the SdhA subunit as a novel substrate of the NAD+-dependent deacetylase, SIRT3.
A hallmark of cancer cells is the metabolic switch from oxidative phosphorylation (OXPHOS) to glycolysis, a phenomenon referred to as the 'Warburg effect', which is also observed in primed human ...pluripotent stem cells (hPSCs). Here, we report that downregulation of SIRT2 and upregulation of SIRT1 is a molecular signature of primed hPSCs and that SIRT2 critically regulates metabolic reprogramming during induced pluripotency by targeting glycolytic enzymes including aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase. Remarkably, knockdown of SIRT2 in human fibroblasts resulted in significantly decreased OXPHOS and increased glycolysis. In addition, we found that miR-200c-5p specifically targets SIRT2, downregulating its expression. Furthermore, SIRT2 overexpression in hPSCs significantly affected energy metabolism, altering stem cell functions such as pluripotent differentiation properties. Taken together, our results identify the miR-200c-SIRT2 axis as a key regulator of metabolic reprogramming (Warburg-like effect), via regulation of glycolytic enzymes, during human induced pluripotency and pluripotent stem cell function.
ERK is a key coordinator of the epithelial-to-mesenchymal transition (EMT) in that a variety of EMT-inducing factors activate signaling pathways that converge on ERK to regulate EMT transcription ...programs. However, the mechanisms by which ERK controls the EMT program are not well understood. Through an analysis of the global changes of gene expression mediated by ERK2, we identified the transcription factor FoxO1 as a potential mediator of ERK2-induced EMT, and thus we investigated the mechanism by which ERK2 regulates FoxO1. Additionally, our analysis revealed that ERK2 induced the expression of Dock10, a Rac1/Cdc42 GEF, during EMT. We demonstrate that the activation of the Rac1/JNK signaling axis downstream of Dock10 leads to an increase in FoxO1 expression and EMT. Taken together, our study uncovers mechanisms by which epithelial cells acquire less proliferative but more migratory mesenchymal properties and reveals potential therapeutic targets for cancers evolving into a metastatic disease state.
Disruption of myelination during development has been implicated in a range of neurodevelopmental disorders including tuberous sclerosis complex (TSC). TSC patients with autism display impairments in ...white matter integrity. Similarly, mice lacking neuronal
have a hypomyelination phenotype. However, the mechanisms that underlie these phenotypes remain unknown. In this study, we demonstrate that neuronal TSC1/2 orchestrates a program of oligodendrocyte maturation through the regulated secretion of connective tissue growth factor (CTGF). We characterize oligodendrocyte maturation both in vitro and in vivo. We find that neuron-specific
deletion results in an increase in CTGF secretion that non-cell autonomously stunts oligodendrocyte development and decreases the total number of oligodendrocytes. Genetic deletion of CTGF from neurons, in turn, mitigates the TSC-dependent hypomyelination phenotype. These results show that the mechanistic target of rapamycin (mTOR) pathway in neurons regulates CTGF production and secretion, revealing a paracrine mechanism by which neuronal signaling regulates oligodendrocyte maturation and myelination in TSC. This study highlights the role of mTOR-dependent signaling between neuronal and nonneuronal cells in the regulation of myelin and identifies an additional therapeutic avenue for this disease.
The future of safe cell-based therapy rests on overcoming teratoma/tumor formation, in particular when using human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human ...induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation, complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro- and antiapoptotic gene expression profile, we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e., survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors, leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular, a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g., quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast, differentiated cell types (e.g., dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC- or hiPSC-derived cells. Taken together, these results provide the “proof of concept” that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy.
Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous ...materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.
Tuberous sclerosis complex (TSC) is a neurodevelopmental disease caused by TSC1 or TSC2 mutations and subsequent activation of the mTORC1 kinase. Upon mTORC1 activation, anabolic metabolism, which ...requires mitochondria, is induced, yet at the same time the principal pathway for mitochondrial turnover, autophagy, is compromised. How mTORC1 activation impacts mitochondrial turnover in neurons remains unknown. Here, we demonstrate impaired mitochondrial homeostasis in neuronal in vitro and in vivo models of TSC. We find that Tsc1/2-deficient neurons accumulate mitochondria in cell bodies, but are depleted of axonal mitochondria, including those supporting presynaptic sites. Axonal and global mitophagy of damaged mitochondria is impaired, suggesting that decreased turnover may act upstream of impaired mitochondrial metabolism. Importantly, blocking mTORC1 or inducing mTOR-independent autophagy restores mitochondrial homeostasis. Our study clarifies the complex relationship between the TSC-mTORC1 pathway, autophagy, and mitophagy, and defines mitochondrial homeostasis as a therapeutic target for TSC and related diseases.
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•Mitochondria, some of which are dysfunctional, accumulate in Tsc2-deficient neurons•Axonal mitochondria, including those captured at presynaptic sites, are depleted•Spatiotemporal dynamics of axonal mitophagy and global mitophagic flux are impaired•Enhancing mTOR-dependent and -independent autophagy restores mitochondrial turnover
Ebrahimi-Fakhari et al. show that mitochondrial dynamics and mitophagy are impaired in neuronal models of tuberous sclerosis complex. Axonal transport, turnover, and presynaptic capturing of mitochondria are compromised. Enhancing autophagic flux through mTORC1-dependent and -independent mechanisms restores mitochondrial homeostasis.
In recent years, inorganic/organic hybrid solar cell concept has received growing attention for alternative energy solution because of the potential for facile and low-cost fabrication and high ...efficiency. Here, we report highly efficient hybrid solar cells based on silicon nanowires (SiNWs) and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (
PSS) using transfer-imprinted metal mesh front electrodes. Such a structure increases the optical absorption and shortens the carrier transport distance, thus, it greatly increases the charge carrier collection efficiency. Compared with hybrid cells formed using indium tin oxide (ITO) electrodes, we find an increase in power conversion efficiency from 5.95% to 13.2%, which is attributed to improvements in both the electrical and optical properties of the Au mesh electrode. Our fabrication strategy for metal mesh electrode is suitable for the large-scale fabrication of flexible transparent electrodes, paving the way towards low-cost, high-efficiency, flexible solar cells.
Salinity and pH have direct and indirect impacts on the growth and metabolic activities of microorganisms. In this study, the effects of salt and alkaline stresses on the kinetic balance between ...nitrous oxide (N
O) production and consumption in the denitrification pathway of
strain RCB were examined. N
O accumulated transiently only in insignificant amounts at low salinity (≤0.5% NaCl) and circumneutral pH (7.0 and 7.5). As compared to these control conditions, incubation at 0.7% salinity resulted in substantially longer lag phase and slower growth rate, along with the increase in the amounts of transiently accumulated N
O (15.8 ± 2.8 μmoles N
O-N/vessel). Incubation at pH 8.0 severely inhibited growth and resulted in permanent accumulation of 29.9 ± 1.3 μmoles N
O-N/vessel from reduction of 151 ± 20 μmoles NO
/vessel. Monitoring of temporal changes in
,
, and
transcription suggested that the
/(
+
) ratios were indicative of whether N
O was produced or consumed at the time points where measurements were taken. The salt and alkaline stresses altered the N
O consumption kinetics of the resting
cells with expressed nitrous oxide reductases. The N
O consumption rates of the cells subjected to the salt and alkaline stress conditions were significantly reduced from 0.84 ± 0.007 μmoles min
mg protein
of the control to 0.27 ± 0.02 μmoles min
mg protein
and 0.31 ± 0.03 μmoles min
mg protein
, respectively, when the initial dissolved N
O concentration was 0.1 mM. As the rates of N
O production from NO
reduction was not significantly affected by the stresses (0.45-0.55 μmoles min
mg protein
), the N
O consumption rate was lower than the N
O production rate at the stress conditions, but not at the control condition. These results clearly indicate that the altered kinetics of expressed nitrous oxide reductase and the resultant disruption of kinetic balance between N
O production and consumption was another cause of enhanced N
O emission observed under the salt and alkaline stress conditions. These findings suggest that canonical denitrifiers may become a significant N
O source when faced with abrupt environmental changes.
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