TFEB regulates dendritic cell antigen presentation to modulate immune balance in asthma.

OBJECTIVE: Asthma stands as one of the most prevalent chronic respiratory conditions in children, with its pathogenesis tied to the actived antigen presentation by dendritic cells (DCs) and the imbalance within T cell subgroups. This study seeks to investigate the role of the transcription factor EB (TFEB) in modulating the antigen presentation process of DCs and its impact on the differentiation of T cell subgroups. METHODS: Bone marrow dendritic cells (BMDCs) were activated using house dust mites (HDM) and underwent RNA sequencing (RNA-seq) to pinpoint differentially expressed genes. TFEB mRNA expression levels were assessed in the peripheral blood mononuclear cells (PBMCs) of both healthy children and those diagnosed with asthma. In an asthma mouse model induced by HDM, the TFEB expression in lung tissue DCs was evaluated. Further experiments involved LV-shTFEB BMDCs co-cultured with T cells to explore the influence of TFEB on DCs' antigen presentation, T cell subset differentiation, and cytokine production. RESULTS: Transcriptomic sequencing identified TFEB as a significantly differentially expressed gene associated with immune system pathways and antigen presentation. Notably, TFEB expression showed a significant increase in the PBMCs of children diagnosed with asthma compared to healthy counterparts. Moreover, TFEB exhibited heightened expression in lung tissue DCs of HDM-induced asthmatic mice and HDM-stimulated BMDCs. Silencing TFEB resulted in the downregulation of MHC II, CD80, CD86, and CD40 on DCs. This action reinstated the equilibrium among Th1/Th2 and Th17/Treg cell subgroups, suppressed the expression of pro-inflammatory cytokines like IL-4, IL-5, IL-13, and IL-17, while augmenting the expression of the anti-inflammatory cytokine IL-10. CONCLUSION: TFEB might have a vital role in asthma's development by impacting the antigen presentation of DCs, regulating T cell subgroup differentiation, and influencing cytokine secretion. Its involvement could be pivotal in rebalancing the immune system in asthma. These research findings could potentially unveil novel therapeutic avenues for treating asthma.

Genome-wide identification of ZmMYC2 binding sites and target genes in maize.

BACKGROUND: Jasmonate (JA) is the important phytohormone to regulate plant growth and adaption to stress signals. MYC2, an bHLH transcription factor, is the master regulator of JA signaling. Although MYC2 in maize has been identified, its function remains to be clarified. RESULTS: To understand the function and regulatory mechanism of MYC2 in maize, the joint analysis of DAP-seq and RNA-seq is conducted to identify the binding sites and target genes of ZmMYC2. A total of 3183 genes are detected both in DAP-seq and RNA-seq data, potentially as the directly regulating genes of ZmMYC2. These genes are involved in various biological processes including plant growth and stress response. Besides the classic cis-elements like the G-box and E-box that are bound by MYC2, some new motifs are also revealed to be recognized by ZmMYC2, such as nGCATGCAnn, AAAAAAAA, CACGTGCGTGCG. The binding sites of many ZmMYC2 regulating genes are identified by IGV-sRNA. CONCLUSIONS: All together, abundant target genes of ZmMYC2 are characterized with their binding sites, providing the basis to construct the regulatory network of ZmMYC2 and better understanding for JA signaling in maize.

Dimorphic effect of TFE3 in determining mitochondrial and lysosomal content in muscle following denervation.

BACKGROUND: Muscle atrophy is a common consequence of the loss of innervation and is accompanied by mitochondrial dysfunction. Mitophagy is the adaptive process through which damaged mitochondria are removed via the lysosomes, which are regulated in part by the transcription factor TFE3. The role of lysosomes and TFE3 are poorly understood in muscle atrophy, and the effect of biological sex is widely underreported. METHODS: Wild-type (WT) mice, along with mice lacking TFE3 (KO), a transcriptional regulator of lysosomal and autophagy-related genes, were subjected to unilateral sciatic nerve denervation for up to 7 days, while the contralateral limb was sham-operated and served as an internal control. A subset of animals was treated with colchicine to capture mitophagy flux. RESULTS: WT females exhibited elevated oxygen consumption rates during active respiratory states compared to males, however this was blunted in the absence of TFE3. Females exhibited higher mitophagy flux rates and greater lysosomal content basally compared to males that was independent of TFE3 expression. Following denervation, female mice exhibited less muscle atrophy compared to male counterparts. Intriguingly, this sex-dependent muscle sparing was lost in the absence of TFE3. Denervation resulted in 45% and 27% losses of mitochondrial content in WT and KO males respectively, however females were completely protected against this decline. Decreases in mitochondrial function were more severe in WT females compared to males following denervation, as ROS emission was 2.4-fold higher. In response to denervation, LC3-II mitophagy flux was reduced by 44% in females, likely contributing to the maintenance of mitochondrial content and elevated ROS emission, however this response was dysregulated in the absence of TFE3. While both males and females exhibited increased lysosomal content following denervation, this response was augmented in females in a TFE3-dependent manner. CONCLUSIONS: Females have higher lysosomal content and mitophagy flux basally compared to males, likely contributing to the improved mitochondrial phenotype. Denervation-induced mitochondrial adaptations were sexually dimorphic, as females preferentially preserve content at the expense of function, while males display a tendency to maintain mitochondrial function. Our data illustrate that TFE3 is vital for the sex-dependent differences in mitochondrial function, and in determining the denervation-induced atrophy phenotype.

Autophagic-lysosomal damage induced by swainsonine is protected by trehalose through activation of TFEB-regulated pathway in renal tubular epithelial cells.

Swainsonine (SW) is the main toxic component of locoweed. Previous studies have shown that kidney damage is an early pathologic change in locoweed poisoning in animals. Trehalose induces autophagy and alleviates lysosomal damage, while its protective effect and mechanism against the toxic injury induced by SW is not clear. Based on the published literature, we hypothesize that transcription factor EB(TFEB) -regulated is targeted by SW and activating TFEB by trehalose would reverse the toxic effects. In this study, we investigate the mechanism of protective effects of trehalose using renal tubular epithelial cells. The results showed that SW induced an increase in the expression level of microtubule-associated protein light chain 3-II and p62 proteins and a decrease in the expression level of ATPase H+ transporting V1 Subunit A, Cathepsin B, Cathepsin D, lysosome-associated membrane protein 2 and TFEB proteins in renal tubular epithelial cells in a time and dose-dependent manner suggesting TFEB-regulated lysosomal pathway is adversely affected by SW. Conversely, treatment with trehalose, a known activator of TFEB promote TFEB nuclear translocation suggesting that TFEB plays an important role in protection against SW toxicity. We demonstrated in lysosome staining that SW reduced the number of lysosomes and increased the luminal pH, while trehalose could counteract these SW-induced effects. In summary, our results demonstrated for the first time that trehalose could alleviate the autophagy degradation disorder and lysosomal damage induced by SW. Our results provide an interesting method for reversion of SW-induced toxicity in farm animals and furthermore, activation of TFEB by trehalose suggesting novel mechanism of treating lysosomal storage diseases.

Altered TFEB subcellular localization in nigral neurons of subjects with incidental, sporadic and GBA-related Lewy body diseases.

Transcription factor EB (TFEB) is a master regulator of genes involved in the maintenance of autophagic and lysosomal homeostasis, processes which have been implicated in the pathogenesis of GBA-related and sporadic Parkinson's disease (PD), and dementia with Lewy bodies (DLB). TFEB activation results in its translocation from the cytosol to the nucleus. Here, we investigated TFEB subcellular localization and its relation to intracellular alpha-synuclein (aSyn) accumulation in post-mortem human brain of individuals with either incidental Lewy body disease (iLBD), GBA-related PD/DLB (GBA-PD/DLB) or sporadic PD/DLB (sPD/DLB), compared to control subjects. We analyzed nigral dopaminergic neurons using high-resolution confocal and stimulated emission depletion (STED) microscopy and semi-quantitatively scored the TFEB subcellular localization patterns. We observed reduced nuclear TFEB immunoreactivity in PD/DLB patients compared to controls, both in sporadic and GBA-related cases, as well as in iLBD cases. Nuclear depletion of TFEB was more pronounced in neurons with Ser129-phosphorylated (pSer129) aSyn accumulation in all groups. Importantly, we observed previously-unidentified TFEB-immunopositive perinuclear clusters in human dopaminergic neurons, which localized at the Golgi apparatus. These TFEB clusters were more frequently observed and more severe in iLBD, sPD/DLB and GBA-PD/DLB compared to controls, particularly in pSer129 aSyn-positive neurons, but also in neurons lacking detectable aSyn accumulation. In aSyn-negative cells, cytoplasmic TFEB clusters were more frequently observed in GBA-PD/DLB and iLBD patients, and correlated with reduced GBA enzymatic activity as well as increased Braak LB stage. Altered TFEB distribution was accompanied by a reduction in overall mRNA expression levels of selected TFEB-regulated genes, indicating a possible early dysfunction of lysosomal regulation. Overall, we observed cytoplasmic TFEB retention and accumulation at the Golgi in cells without apparent pSer129 aSyn accumulation in iLBD and PD/DLB patients. This suggests potential TFEB impairment at the early stages of cellular disease and underscores TFEB as a promising therapeutic target for synucleinopathies.

[Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway].

Objective: To investigate the mechanism of proanthocyanidin (PA) in regulating the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs), and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB (TFEB) and on the autophagy-lysosome pathway. Methods: PDLSCs were divided into control group and PA group, which were subjected to RNA sequencing analysis (RNA Seq) to detect differentially expressed genes. The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR (RT-qPCR) analysis, alkaline phosphatase (ALP) staining and transmission electron microscope (TEM), respectively. Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs. Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes. The total protein expression of transcription factor EB (TFEB) as well as that in cytoplasm and nucleus were detected by Western blotting. Confocal laser scanning microscope (CLSM) was used to observe the nuclear translocation of TFEB. The PDLSCs were treated with small interfering RNA (siRNA) technology to knock down the expression levels of TFEB gene with or without PA treatment. Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3 (LC3B), as well as osteogenic-related proteins runt-related transcription factor 2 (RUNX2), ALP, and osteocalcin in PDLSCs. Results: Compared with the control group, the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment (P<0.05). The mRNA expression levels of osteogenic-related genes RUNX2 (2.32±0.15) and collagen type Ⅰ alpha 1 (COL1α1) (1.80±0.18), as well as the autophagy related genes LC3B (1.87±0.08) and Beclin1 (1.63±0.08) were significantly increased in the PA group, compared with the control group (1.01±0.16, 1.00±0.10, 1.00±0.07, 1.00±0.06, respectively, all P<0.01). Compared with the control group, the PA group had higher ALP activity, and more autophagosomes and autophagolysosomes observed by TEM. PA promoted the migration of PDLSCs (P<0.05) and the increased number of lysosomes and the expression of lysosomal associated membrane protein 1 (LAMP1). In the PA group, the relative expression level of total TFEB protein (1.49±0.07) and the nuclear/cytoplasmic expression of TFEB protein (1.52±0.12) were significantly higher than the control group (1.00±0.11, 1.00±0.13, respectively) (t=6.43, P<0.01; t=5.07, P<0.01). The relative nuclear/cytoplasmic fluorescence intensity of TFEB in the PA group (0.79±0.09) was increased compared with the control group (0.11±0.08) (t=8.32, P<0.01). Knocking down TFEB significantly reduced the expression of TFEB (1.00±0.15 vs 0.64±0.04), LAMP1 (1.00±0.10 vs 0.69±0.09), Beclin1 (1.00±0.05 vs 0.60±0.05), and LC3B Ⅱ/Ⅰ (1.00±0.06 vs 0.73±0.07) in PDLSCs (P<0.05, P<0.05, P<0.01, P<0.01). When TFEB gene was knocked down, the expression levels of Beclin1 (1.05±0.11), LC3B Ⅱ/Ⅰ (1.02±0.09), RUNX2 (1.04±0.10), ALP (1.04±0.16), and osteocalcin (1.03±0.15) proteins were significantly decreased in the PA group compared with the pre-knockdown period (1.28±0.03, 1.44±0.11, 1.38±0.11, 1.62±0.11, 1.65±0.17, respectively) (P<0.05, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). Conclusions: PA promotes the osteogenic differentiation of PDLSCs through inducing the expression and nuclear translocation of TFEB and activating the autophagy-lysosome pathway.

mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells.

The mechanistic target of rapamycin (RAPA) complex 1 (mTORC1) - transcription factor EB (TFEB) pathway plays a crucial role in response to nutritional status, energy and environmental stress for maintaining cellular homeostasis. But there is few reports on its role in the toxic effects of arsenic exposure and the related mechanisms. Here, we show that the exposure of bronchial epithelial cells (BEAS-2B) to sodium arsenite promoted the activation of mTORC1 (p-mTORC1) and the inactivation of TFEB (p-TFEB), the number and activity of lysosomes decreased, the content of reduced glutathione (GSH) and superoxide dismutase (SOD) decreased, the content of malondialdehyde (MDA) increased, the DNA and chromosome damage elevated. Further, when mTORC1 was inhibited with RAPA, p-mTORC1 and p-TFEB down-regulated, GSH and SOD increased, MDA decreased, the DNA and chromosome damage reduced significantly, as compared with the control group. Our data revealed for the first time that mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells, and it may be a potential intervention target for the toxic effects of arsenic.

Butylated hydroxyanisole induces vascular endothelial injury via TFEB-mediated degradation of GPX4 and FTH1.

Butylated hydroxyanisole (BHA) is one of the most commonly used antioxidants and is widely used in food, but whether it causes vascular damage has not been clearly studied. The present study demonstrated for the first time that BHA reduced the viability of human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (BEND3) in a dose- and time-dependent manner. Moreover, BHA inhibited the migration and proliferation of vascular endothelial cells (ECs). Further analysis revealed that in ECs, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the BHA-induced increase in Fe2+ and malonaldehyde (MDA) levels. Acridine orange staining demonstrated that BHA increased lysosomal permeability. At the protein level, BHA increased the expression of transcription factor EB (TFEB) and decreased the expression of glutathione peroxidase (GPX4), solute carrier family 7 member 11 (SLC7A11, xCT), and ferritin heavy chain 1 (FTH1). Moreover, these effects of BHA could be reversed by knocking down TFEB. In vivo experiments confirmed that BHA caused elevated pulse wave velocity (PWV) and reduced acetylcholine-dependent vascular endothelial diastole. In conclusion, BHA degrades GPX4, xCT, and FTH1 through activation of the TFEB-mediated lysosomal pathway and promotes ferroptosis, ultimately leading to vascular endothelial cell injury.

Liver ChREBP deficiency inhibits fructose-induced insulin resistance in pregnant mice and female offspring.

High fructose intake during pregnancy increases insulin resistance (IR) and gestational diabetes mellitus (GDM) risk. IR during pregnancy primarily results from elevated hormone levels. We aim to determine the role of liver carbohydrate response element binding protein (ChREBP) in insulin sensitivity and lipid metabolism in pregnant mice and their offspring. Pregnant C57BL/6J wild-type mice and hepatocyte-specific ChREBP-deficient mice were fed with a high-fructose diet (HFrD) or normal chow diet (NC) pre-delivery. We found that the combination of HFrD with pregnancy excessively activates hepatic ChREBP, stimulating progesterone synthesis by increasing MTTP expression, which exacerbates IR. Increased progesterone levels upregulated hepatic ChREBP via the progesterone-PPARγ axis. Placental progesterone activated the progesterone-ChREBP loop in female offspring, contributing to IR and lipid accumulation. In normal dietary conditions, hepatic ChREBP modestly affected progesterone production and influenced IR during pregnancy. Our findings reveal the role of hepatic ChREBP in regulating insulin sensitivity and lipid homeostasis in both pregnant mice consuming an HFrD and female offspring, and suggest it as a potential target for managing gestational metabolic disorders, including GDM.

Impaired TFEB-mediated autophagy-lysosome fusion promotes tubular cell cycle G2/M arrest and renal fibrosis by suppressing ATP6V0C expression and interacting with SNAREs.

Increasing evidence suggests that autophagy plays a major role during renal fibrosis. Transcription factor EB (TFEB) is a critical regulator of autophagy- and lysosome-related gene transcription. However, the pathophysiological roles of TFEB in renal fibrosis and fine-tuned mechanisms by which TFEB regulates fibrosis remain largely unknown. Here, we found that TFEB was downregulated in unilateral ureteral obstruction (UUO)-induced human and mouse fibrotic kidneys, and kidney-specific TFEB overexpression using recombinant AAV serotype 9 (rAAV9)-TFEB in UUO mice alleviated renal fibrosis pathogenesis. Mechanically, we found that TFEB's prevention of extracellular matrix (ECM) deposition depended on autophagic flux integrity and its subsequent blockade of G2/M arrest in tubular cells, rather than the autophagosome synthesis. In addition, we together RNA-seq with CUT&Tag analysis to determine the TFEB targeted gene ATP6V0C, and revealed that TFEB was directly bound to the ATP6V0C promoter only at specific site to promote its expression through CUT&Run-qPCR and luciferase reporter assay. Interestingly, TFEB induced autophagic flux integrity, mainly dependent on scaffold protein ATP6V0C-mediated autophagosome-lysosome fusion by bridging with STX17 and VAMP8 (major SNARE complex) by co-immunoprecipitation analysis, rather than its mediated lysosomal acidification and degradation function. Moreover, we further investigated the underlying mechanism behind the low expression of TEFB in UUO-induced renal fibrosis, and clearly revealed that TFEB suppression in fibrotic kidney was due to DNMT3a-associated TFEB promoter hypermethylation by utilizing methylation specific PCR (MSP) and bisulfite-sequencing PCR (BSP), which could be effectively recovered by 5-Aza-2'-deoxycytidine (5A-za) to alleviate renal fibrosis pathogenesis. These findings reveal for the first time that impaired TFEB-mediated autophagosome-lysosome fusion disorder, tubular cell G2/M arrest and renal fibrosis appear to be sequentially linked in UUO-induced renal fibrosis and suggest that DNMT3a/TFEB/ATP6V0C may serve as potential therapeutic targets to prevent renal fibrosis.

Itaconic acid regulation of TFEB-mediated autophagy flux alleviates hyperoxia-induced bronchopulmonary dysplasia.

BACKGROUND: Premature infants often require oxygen supplementation, which can elicit bronchopulmonary dysplasia (BPD) and lead to mitochondrial dysfunction. Mitochondria play important roles in lung development, in both normal metabolism and apoptosis. Enhancing our comprehension of the underlying mechanisms in BPD development can facilitate the effective treatments. METHODS: Plasma samples from BPD and non-BPD infants were collected at 36 weeks post-menstrual age and used for metabolomic analysis. Based on hyperoxia-induced animal and cell models, changes in mitophagy and apoptosis were evaluated following treatment with itaconic acid (ITA). Finally, the mechanism of action of ITA in lung development was comprehensively demonstrated through rescue strategies and administration of corresponding inhibitors. RESULTS: An imbalance in the tricarboxylic acid (TCA) cycle significantly affected lung development, with ITA serving as a significant metabolic marker for the outcomes of lung development. ITA improved the morphological changes in BPD rats, promoted SP-C expression, and inhibited the degree of alveolar type II epithelial cells (AEC II) apoptosis. Mechanistically, ITA mainly promotes the nuclear translocation of transcription factor EB (TFEB) to facilitate dysfunctional mitochondrial clearance and reduces apoptosis in AEC II cells by regulating autophagic flux. CONCLUSION: The metabolic imbalance in the TCA cycle is closely related to lung development. ITA can improve lung development by regulating autophagic flux and promote the nuclear translocation of TFEB, implying its potential therapeutic utility in the treatment of BPD.

BMSC-Exosomes attenuate ALP dysfunction by restoring lysosomal function via the mTOR/TFEB Axis to reduce cerebral ischemia-reperfusion injury.

BACKGROUND: The complex pathophysiological changes following cerebral ischemia-reperfusion injury (CIRI) include the accumulation of defective proteins and damaged organelles, which cause massive neuron demise. To preserve cellular homeostasis, the autophagy-lysosomal pathway (ALP) is crucial for neurons to dispose of these substances. Many studies have shown that bone mesenchymal stem cell exosomes (BMSC-Exos) can reduce CIRI. However, the specific mechanisms have not been well elucidated, a fact that limits its widespread clinical use. This study aimed to clarify whether BMSC-Exos could attenuate ALP dysfunction by restoring lysosomal function after CIRI via inhibiting mTOR and then activating TFEB nucleus translocation. METHODS: In this study, Flow cytometry, Nanoparticle tracking analysis (NTA), Transmission electron microscope (TEM), and Western blot were used to identify the BMSCs and BMSC-Exos used in this experiment as conforming to the requirements. In vivo experiments, SD rats were modeled with temporary middle cerebral artery occlusion (tMCAO), and BMSC-Exos was injected into the tail vein 2 h after modeling. Triphenyl tetrazolium chloride (TTC) staining, modified neurological severity scores (mNSS), corner turn test, and rotating rod test were used to detect neurological deficits in rats after BMSC-Exos intervention. Western blot and Immunofluorescence were used to detect ALP, transcription factor EB(TFEB) nucleus translocation, and mammalian target of rapamycin (mTOR) change at different time points after modeling and after BMSC-Exos intervention. In vitro experiments, pheochromocytoma cells (PC12) cells were subjected to oxygen-glucose deprivation and reperfusion (OGD/R) modeling to mimic CIRI, and were respectively intervened with BMSC-Exos, BMSC-Exos + MHY 1485 (the mTOR agonist), Rapamycin (the mTOR inhibitor). CCK8, Western blot, and Immunofluorescence were used to detect PC12 cell survival, TFEB nucleus translocation, and cathepsin B(CTSB) Immunofluorescence intensity. RESULTS: We found that ALP dysfunction occurred 72 h after tMCAO, and BMSC-Exos can attenuate ALP dysfunction by restoring lysosomal function. Next, we examined TFEB nucleus translocation and the expression of mTOR, a key regulator of translocation. We found that BMSC-Exos could inhibit mTOR and activate TFEB nucleus translocation. Additional in vitro tests revealed that BMSC-Exos could increase PC12 cell survival after OGD/R, activating TFEB nucleus translocation and enhancing the fluorescence intensity of CTSB, which in turn could be reversed by the mTOR agonist, MHY1485. This effect was similar to another mTOR inhibitor, Rapamycin. CONCLUSION: BMSC-Exos could attenuate ALP dysfunction by restoring lysosomal function after CIRI by inhibiting mTOR and then promoting TFEB nucleus translocation.

Transcription factor EB-mediated autophagy affects cell migration and inhibits apoptosis to promote endometriosis.

Autophagy has emerged as an important process of cell metabolism. With continuous in-depth research on autophagy, TFEB has been a key transcription factor regulating autophagy levels in recent years. Studies have established that TFEB regulates autophagy and apoptosis in various diseases. However, the relationship between TFEB and the pathogenesis of endometriosis remains unclear. This study aimed to investigate the effect of TFEB on the mechanism of endometriosis progression. The results showed that TFEB and autophagy-related protein LC3 are highly expressed in ectopic endometrium of patients with endometriosis, overexpression of TFEB in cultured human endometrial stromal cells (HESCs) by lentivirus not only promoted autophagy but also inhibited apoptosis. In addition, the migration and invasion ability of HESCs were enhanced by TFEB overexpression. Furthermore, inhibiting autophagy with specific inhibitors can attenuate migration and invasion of HESCs induced by TFEB. The rat models of endometriosis show that TFEB knockdown can suppress lesion growth in vivo. Our results suggest that autophagy may be involved in the progression mechanism of endometriosis, and the mechanism of autophagy disorder in endometriosis is probably related to TFEB. TFEB may be a key molecule in promoting endometriosis.

The GATOR2 complex maintains lysosomal-autophagic function by inhibiting the protein degradation of MiT/TFEs.

Lysosomes are central to metabolic homeostasis. The microphthalmia bHLH-LZ transcription factors (MiT/TFEs) family members MITF, TFEB, and TFE3 promote the transcription of lysosomal and autophagic genes and are often deregulated in cancer. Here, we show that the GATOR2 complex, an activator of the metabolic regulator TORC1, maintains lysosomal function by protecting MiT/TFEs from proteasomal degradation independent of TORC1, GATOR1, and the RAG GTPase. We determine that in GATOR2 knockout HeLa cells, members of the MiT/TFEs family are ubiquitylated by a trio of E3 ligases and are degraded, resulting in lysosome dysfunction. Additionally, we demonstrate that GATOR2 protects MiT/TFE proteins in pancreatic ductal adenocarcinoma and Xp11 translocation renal cell carcinoma, two cancers that are driven by MiT/TFE hyperactivation. In summary, we find that the GATOR2 complex has independent roles in TORC1 regulation and MiT/TFE protein protection and thus is central to coordinating cellular metabolism with control of the lysosomal-autophagic system.

A SPLICS reporter reveals [Formula: see text]-synuclein regulation of lysosome-mitochondria contacts which affects TFEB nuclear translocation.

Mitochondrial and lysosomal activities are crucial to maintain cellular homeostasis: optimal coordination is achieved at their membrane contact sites where distinct protein machineries regulate organelle network dynamics, ions and metabolites exchange. Here we describe a genetically encoded SPLICS reporter for short- and long- juxtapositions between mitochondria and lysosomes. We report the existence of narrow and wide lysosome-mitochondria contacts differently modulated by mitophagy, autophagy and genetic manipulation of tethering factors. The overexpression of α-synuclein (α-syn) reduces the apposition of mitochondria/lysosomes membranes and affects their privileged Ca2+ transfer, impinging on TFEB nuclear translocation. We observe enhanced TFEB nuclear translocation in α-syn-overexpressing cells. We propose that α-syn, by interfering with mitochondria/lysosomes tethering impacts on local Ca2+ regulated pathways, among which TFEB mediated signaling, and in turn mitochondrial and lysosomal function. Defects in mitochondria and lysosome represent a common hallmark of neurodegenerative diseases: targeting their communication could open therapeutic avenues.

MYC the oncogene from hell: Novel opportunities for cancer therapy.

Cancer comprises a heterogeneous disease, characterized by diverse features such as constitutive expression of oncogenes and/or downregulation of tumor suppressor genes. MYC constitutes a master transcriptional regulator, involved in many cellular functions and is aberrantly expressed in more than 70 % of human cancers. The Myc protein belongs to a family of transcription factors whose structural pattern is referred to as basic helix-loop-helix-leucine zipper. Myc binds to its partner, a smaller protein called Max, forming an Myc:Max heterodimeric complex that interacts with specific DNA recognition sequences (E-boxes) and regulates the expression of downstream target genes. Myc protein plays a fundamental role for the life of a cell, as it is involved in many physiological functions such as proliferation, growth and development since it controls the expression of a very large percentage of genes (∼15 %). However, despite the strict control of MYC expression in normal cells, MYC is often deregulated in cancer, exhibiting a key role in stimulating oncogenic process affecting features such as aberrant proliferation, differentiation, angiogenesis, genomic instability and oncogenic transformation. In this review we aim to meticulously describe the fundamental role of MYC in tumorigenesis and highlight its importance as an anticancer drug target. We focus mainly on the different categories of novel small molecules that act as inhibitors of Myc function in diverse ways hence offering great opportunities for an efficient cancer therapy. This knowledge will provide significant information for the development of novel Myc inhibitors and assist to the design of treatments that would effectively act against Myc-dependent cancers.

Evaluation of TRIM63 RNA in situ hybridization (RNA-ISH) as a potential biomarker for alveolar soft-part sarcoma (ASPS).

Alveolar soft-part sarcoma (ASPS) is a rare soft tissue tumor with a broad morphologic differential diagnosis. While histology and immunohistochemistry can be suggestive, diagnosis often requires exclusion of other entities followed by confirmatory molecular analysis for its characteristic ASPSCR1-TFE3 fusion. Current stain-based biomarkers (such as immunohistochemistry for cathepsin K and TFE3) show relatively high sensitivity but may lack specificity, often showing staining in multiple other entities under diagnostic consideration. Given the discovery of RNA in situ hybridization (RNA-ISH) for TRIM63 as a sensitive and specific marker of MiTF-family aberration renal cell carcinomas, we sought to evaluate its utility in the workup of ASPS. TRIM63 RNA-ISH demonstrated high levels (H-score greater than 200) of expression in 19/20 (95%) cases of ASPS (average H-score 330) and was weak or negative in cases of paraganglioma, clear cell sarcoma, rhabdomyosarcoma, malignant epithelioid hemangioendothelioma, as well as hepatocellular and adrenal cortical carcinomas. Staining was also identified in tumors with known subsets characterized by TFE3 alterations such as perivascular epithelioid cell neoplasm (PEComa, average H-score 228), while tumors known to exhibit overexpression of TFE3 protein without cytogenetic alterations, such as melanoma and granular cell tumor, generally showed less TRIM63 ISH staining (average H-scores 147 and 96, respectively). Quantitative assessment of TRIM63 staining by RNA-ISH is potentially a helpful biomarker for tumors with molecular TFE3 alterations such as ASPS.

Comparative genomics incorporating translocation renal cell carcinoma mouse model reveals molecular mechanisms of tumorigenesis.

Translocation renal cell carcinoma (tRCC) most commonly involves an ASPSCR1-TFE3 fusion, but molecular mechanisms remain elusive and animal models are lacking. Here, we show that human ASPSCR1-TFE3 driven by Pax8-Cre (a credentialed clear cell RCC driver) disrupted nephrogenesis and glomerular development, causing neonatal death, while the clear cell RCC failed driver, Sglt2-Cre, induced aggressive tRCC (as well as alveolar soft part sarcoma) with complete penetrance and short latency. However, in both contexts, ASPSCR1-TFE3 led to characteristic morphological cellular changes, loss of epithelial markers, and an epithelial-mesenchymal transition. Electron microscopy of tRCC tumors showed lysosome expansion, and functional studies revealed simultaneous activation of autophagy and mTORC1 pathways. Comparative genomic analyses encompassing an institutional human tRCC cohort (including a hitherto unreported SFPQ-TFEB fusion) and a variety of tumorgraft models (ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3, RBM10-TFE3, and MALAT1-TFEB) disclosed significant convergence in canonical pathways (cell cycle, lysosome, and mTORC1) and less established pathways such as Myc, E2F, and inflammation (IL-6/JAK/STAT3, interferon-γ, TLR signaling, systemic lupus, etc.). Therapeutic trials (adjusted for human drug exposures) showed antitumor activity of cabozantinib. Overall, this study provides insight into MiT/TFE-driven tumorigenesis, including the cell of origin, and characterizes diverse mouse models available for research.

The MYC2-PUB22-JAZ4 module plays a crucial role in jasmonate signaling in tomato.

Jasmonates (JAs), a class of lipid-derived stress hormones, play a crucial role across an array of plant physiological processes and stress responses. Although JA signaling is thought to rely predominantly on the degradation of specific JAZ proteins by SCFCOI1, it remains unclear whether other pathways are involved in the regulation of JAZ protein stability. Here, we report that PUB22, a plant U-box type E3 ubiquitin ligase, plays a critical role in the regulation of plant resistance against Helicoverpa armigera and other JA responses in tomato. Whereas COI1 physically interacts with JAZ1/2/5/7, PUB22 physically interacts with JAZ1/3/4/6. PUB22 ubiquitinates JAZ4 to promote its degradation via the 26S proteasome pathway. Importantly, we observed that pub22 mutants showreduced resistance to H. armigera, whereas jaz4 single mutants and jaz1 jaz3 jaz4 jaz6 quadruple mutants have enhanced resistance. The hypersensitivity of pub22 mutants to herbivores could be partially rescued by JAZ4 mutation. Moreover, we found that expression of PUB22 can be transcriptionally activated by MYC2, thus forming a positive feedback circuit in JA signaling. We noticed that the PUB22-JAZ4 module also regulates other JA responses, including defense against B. cinerea, inhibition of root elongation, and anthocyanin accumulation. Taken together, these results indicate that PUB22 plays a crucial role in plant growth and defense responses, together with COI1-regulated JA signaling, by targeting specific JAZs.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.