ZNPs reduce epidermal mechanical strain resistance by promoting desmosomal cadherin endocytosis via mTORC1-TFEB-BLOC1S3 axis.

Zinc oxide nanoparticles (ZNPs) are widely used in sunscreens and nanomedicines, and it was recently confirmed that ZNPs can penetrate stratum corneum into deep epidermis. Therefore, it is necessary to determine the impact of ZNPs on epidermis. In this study, ZNPs were applied to mouse skin at a relatively low concentration for one week. As a result, desmosomes in epidermal tissues were depolymerized, epidermal mechanical strain resistance was reduced, and the levels of desmosomal cadherins were decreased in cell membrane lysates and increased in cytoplasmic lysates. This finding suggested that ZNPs promote desmosomal cadherin endocytosis, which causes desmosome depolymerization. In further studies, ZNPs were proved to decrease mammalian target of rapamycin complex 1 (mTORC1) activity, activate transcription factor EB (TFEB), upregulate biogenesis of lysosome-related organelle complex 1 subunit 3 (BLOC1S3) and consequently promote desmosomal cadherin endocytosis. In addition, the key role of mTORC1 in ZNP-induced decrease in mechanical strain resistance was determined both in vitro and in vivo. It can be concluded that ZNPs reduce epidermal mechanical strain resistance by promoting desmosomal cadherin endocytosis via the mTORC1-TFEB-BLOC1S3 axis. This study helps elucidate the biological effects of ZNPs and suggests that ZNPs increase the risk of epidermal fragmentation.

Diffuse Intense 18 F-PSMA-1007 Uptake of the Breasts in a Woman With TFE3-Rearranged Renal Cell Carcinoma.

ABSTRACT: A 23-year-old woman presenting with gross hematuria was found to have a left renal mass suspicious for renal cell carcinoma on abdominal contrast-enhanced CT. An 18 F-PSMA-1007 PET/CT scan was performed for evaluating the renal mass. 18 F-PSMA-1007 PET/CT showed focal activity of the renal mass, which was a transcription factor E3-rearranged renal cell carcinoma proved after nephrectomy. Surprisingly, diffuse heterogeneous intense activity of the bilateral breasts and moderate activity of the right accessory breast was observed. There was no morphological abnormality of the bilateral breasts and right accessory breast on CT images, indicating physiological PSMA uptake.

Mycobacterium tuberculosis resides in lysosome-poor monocyte-derived lung cells during chronic infection.

Mycobacterium tuberculosis (Mtb) infects lung myeloid cells, but the specific Mtb-permissive cells and host mechanisms supporting Mtb persistence during chronic infection are incompletely characterized. We report that after the development of T cell responses, CD11clo monocyte-derived cells harbor more live Mtb than alveolar macrophages (AM), neutrophils, and CD11chi monocyte-derived cells. Transcriptomic and functional studies revealed that the lysosome pathway is underexpressed in this highly permissive subset, characterized by less lysosome content, acidification, and proteolytic activity than AM, along with less nuclear TFEB, a regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in CD11clo monocyte-derived cells but promotes recruitment of monocytes that develop into permissive lung cells, mediated by the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome functions of macrophages in vitro and in vivo, improving control of Mtb infection. Our results suggest that Mtb exploits lysosome-poor lung cells for persistence and targeting lysosome biogenesis is a potential host-directed therapy for tuberculosis.

Impairing hydrolase transport machinery prevents human melanoma metastasis.

Metastases are the major cause of cancer-related death, yet, molecular weaknesses that could be exploited to prevent tumor cells spreading are poorly known. Here, we found that perturbing hydrolase transport to lysosomes by blocking either the expression of IGF2R, the main receptor responsible for their trafficking, or GNPT, a transferase involved in the addition of the specific tag recognized by IGF2R, reduces melanoma invasiveness potential. Mechanistically, we demonstrate that the perturbation of this traffic, leads to a compensatory lysosome neo-biogenesis devoided of degradative enzymes. This regulatory loop relies on the stimulation of TFEB transcription factor expression. Interestingly, the inhibition of this transcription factor playing a key role of lysosome production, restores melanomas' invasive potential in the absence of hydrolase transport. These data implicate that targeting hydrolase transport in melanoma could serve to develop new therapies aiming to prevent metastasis by triggering a physiological response stimulating TFEB expression in melanoma.

A rare case of TFEB/6p21/VEGFA-amplified renal cell carcinoma diagnosed by whole-exome sequencing: clinicopathological and genetic feature report and literature review.

BACKGROUND: TFEB/6p21/VEGFA-amplified renal cell carcinoma (RCC) is rare and difficult to diagnose, with diverse histological patterns and immunohistochemical and poorly defined molecular genetic characteristics. CASE PRESENTATION: We report a case of a 63-year-old male admitted in 2017 with complex histomorphology, three morphological features of clear cell, eosinophilic and papillary RCC and resembling areas of glomerular and tubular formation. The immunophenotype also showed a mixture of CD10 and P504s. RCC with a high suspicion of collision tumors was indicated according to the 2014 WHO classification system; no precise diagnosis was possible. The patient was diagnosed at a different hospital with poorly differentiated lung squamous cell carcinoma one year after RCC surgery. We exploited molecular technology advances to retrospectively investigate the patient's molecular genetic alterations by whole-exome sequencing. The results revealed a 6p21 amplification in VEGFA and TFEB gene acquisition absent in other RCC subtypes. Clear cell, papillary, chromophobe, TFE3-translocation, eosinophilic solid and cystic RCC were excluded. Strong TFEB and Melan-A protein positivity prompted rediagnosis as TFEB/6p21/VEGFA-amplified RCC as per 2022 WHO classification. TMB-L (low tumor mutational load), CCND3 gene acquisition and MRE11A and ATM gene deletion mutations indicated sensitivity to PD-1/PD-L1 inhibitor combinations and the FDA-approved targeted agents Niraparib (Grade C), Olaparib (Grade C), Rucaparib (Grade C) and Talazoparib (Class C). GO (Gene Ontology) and KEGG enrichment analyses revealed major mutations and abnormal CNVs in genes involved in biological processes such as the TGF-ß, Hippo, E-cadherin, lysosomal biogenesis and autophagy signaling pathways, biofilm synthesis cell adhesion substance metabolism regulation and others. We compared TFEB/6p21/VEGFA-amplified with TFEB-translocated RCC; significant differences in disease onset age, histological patterns, pathological stages, clinical prognoses, and genetic characteristics were revealed. CONCLUSION: We clarified the patient's challenging diagnosis and discussed the clinicopathology, immunophenotype, differential diagnosis, and molecular genetic information regarding TFEB/6p21/VEGFA-amplified RCC via exome analysis and a literature review.

A Proteomic Approach Identified TFEB as a Key Player in the Protective Action of Novel CB2R Bitopic Ligand FD22a against the Deleterious Effects Induced by ß-Amyloid in Glial Cells.

Neurodegenerative diseases (NDDs) are progressive multifactorial disorders of the nervous system sharing common pathogenic features, including intracellular misfolded protein aggregation, mitochondrial deficit, and inflammation. Taking into consideration the multifaceted nature of NDDs, development of multitarget-directed ligands (MTDLs) has evolved as an attractive therapeutic strategy. Compounds that target the cannabinoid receptor type II (CB2R) are rapidly emerging as novel effective MTDLs against common NDDs, such as Alzheimer's disease (AD). We recently developed the first CB2R bitopic/dualsteric ligand, namely FD22a, which revealed the ability to induce neuroprotection with fewer side effects. To explore the potential of FD22a as a multitarget drug for the treatment of NDDs, we investigated here its ability to prevent the toxic effect of ß-amyloid (Aß25-35 peptide) on human cellular models of neurodegeneration, such as microglia (HMC3) and glioblastoma (U87-MG) cell lines. Our results displayed that FD22a efficiently prevented Aß25-35 cytotoxic and proinflammatory effects in both cell lines and counteracted ß-amyloid-induced depression of autophagy in U87-MG cells. Notably, a quantitative proteomic analysis of U87-MG cells revealed that FD22a was able to potently stimulate the autophagy-lysosomal pathway (ALP) by activating its master transcriptional regulator TFEB, ultimately increasing the potential of this novel CB2R bitopic/dualsteric ligand as a multitarget drug for the treatment of NDDs.

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.

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.

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.

TXNIP deficiency attenuates renal fibrosis by modulating mTORC1/TFEB-mediated autophagy in diabetic kidney disease.

Thioredoxin-interacting protein (TXNIP) is an important regulatory protein for thioredoxin (TRX) that elicits the generation of reactive oxygen species (ROS) by inhibiting the redox function of TRX. Abundant evidence suggests that TXNIP is involved in the fibrotic process of diabetic kidney disease (DKD). However, the potential mechanism of TXNIP in DKD is not yet well understood. In this study, we found that TXNIP knockout suppressed renal fibrosis and activation of mammalian target of rapamycin complex 1 (mTORC1) and restored transcription factor EB (TFEB) and autophagy activation in diabetic kidneys. Simultaneously, TXNIP interference inhibited epithelial-to-mesenchymal transformation (EMT), collagen I and fibronectin expression, and mTORC1 activation, increased TFEB nuclear translocation, and promoted autophagy restoration in HK-2 cells exposed to high glucose (HG). Rapamycin, an inhibitor of mTORC1, increased TFEB nuclear translocation and autophagy in HK-2 cells under HG conditions. Moreover, the TFEB activators, curcumin analog C1 and trehalose, effectively restored HG-induced autophagy, and abrogated HG-induced EMT and collagen I and fibronectin expression in HK-2 cells. Taken together, these findings suggest that TXNIP deficiency ameliorates renal fibrosis by regulating mTORC1/TFEB-mediated autophagy in diabetic kidney diseases.

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.

A genome-first approach to variants in MLXIPL and their association with hepatic steatosis and plasma lipids.

BACKGROUND: Common variants of the max-like protein X (MLX)-interacting protein-like (MLXIPL) gene, encoding the transcription factor carbohydrate-responsive element-binding protein, have been shown to be associated with plasma triglyceride levels. However, the role of these variants in steatotic liver disease (SLD) is unclear. METHODS: We used a genome-first approach to analyze a variety of metabolic phenotypes and clinical outcomes associated with a common missense variant in MLXIPL, Gln241His, in 2 large biobanks: the UK Biobank and the Penn Medicine Biobank. RESULTS: Carriers of MLXIPL Gln241His were associated with significantly lower serum levels of triglycerides, apolipoprotein-B, gamma-glutamyl transferase, and alkaline phosphatase. Additionally, MLXIPL Gln241His carriers were associated with significantly higher serum levels of HDL cholesterol and alanine aminotransferase. Carriers homozygous for MLXIPL Gln241His showed a higher risk of SLD in 2 unrelated cohorts. Carriers of MLXIPL Gln241His were especially more likely to be diagnosed with SLD if they were female, obese, and/or also carried the PNPLA3 I148M variant. Furthermore, the heterozygous carriage of MLXIPL Gln241His was associated with significantly higher all-cause, liver-related, and cardiovascular mortality rates. Nuclear magnetic resonance metabolomics data indicated that carriage of MLXIPL Gln241His was significantly associated with lower serum levels of VLDL and increased serum levels of HDL cholesterol. CONCLUSIONS: Analyses of the MLXIPL Gln241His polymorphism showed a significant association with a higher risk of SLD diagnosis and elevated serum alanine aminotransferase as well as significantly lower serum triglycerides and apolipoprotein-B levels. MLXIPL might, therefore, be a potential pharmacological target for the treatment of SLD and hyperlipidemia, notably for patients at risk. More mechanistic studies are needed to better understand the role of MLXIPL Gln241His on lipid metabolism and steatosis development.

Low-dose cadmium telluride quantum dots trigger M1 polarization in macrophages through mTOR-mediated transcription factor EB activation.

The increasing application of quantum dots (QDs) increases interactions with organisms. The inflammatory imbalance is a significant manifestation of immunotoxicity. Macrophages maintain inflammatory homeostasis. Using macrophages differentiated by phorbol 12-myristate 13-acetate-induced THP-1 cells as models, the study found that low-dose (5 µM) cadmium telluride QDs (CdTe-QDs) hindered monocyte-macrophage differentiation. CD11b is a surface marker of macrophage, and the addition of CdTe-QDs during induction resulted in a decrease in CD11b expression. Moreover, exposure of differentiated THP-1 macrophage (dTHP-1) to 5 µM CdTe-QDs led to the initiation of M1 polarization. This was indicated by the increased surface marker CD86 expression, along with elevated level of NF-κB and IL-1ß proteins. The potential mechanisms are being explored. The transcription factor EB (TFEB) plays a significant role in immune regulation and serves as a crucial regulator of the autophagic lysosomal pathway. After exposed to CdTe-QDs, TFEB activation-mediated autophagy and M1 polarization were observed to occur simultaneously in dTHP-1. The mTOR signaling pathway contributed to TFEB activation induced by CdTe-QDs. However, mTOR-independent activation of TFEB failed to promote M1 polarization. These results suggest that mTOR-TFEB is an advantageous target to enhance the biocompatibility of CdTe-QDs.

[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.

Assessing the Function of CBF1 in Modulating the Interaction Between Phytochrome B and PIF4.

Phytochromes are red (R) and far-red (FR) light photoreceptors in plants. Upon light exposure, photoactivated phytochromes translocate into the nucleus, where they interact with their partner proteins to transduce light signals. The yeast two-hybrid (Y2H) system is a powerful technique for rapidly identifying and verifying protein-protein interactions, and PHYTOCHROME-INTERACTING FACTOR3 (PIF3), the founding member of the PIF proteins, was initially identified in a Y2H screen for phytochrome B (phyB)-interacting proteins. Recently, we developed a yeast three-hybrid (Y3H) system by introducing an additional vector into this Y2H system, and thus a new regulator could be co-expressed and its role in modulating the interactions between phytochromes and their signaling partners could be examined. By employing this Y3H system, we recently showed that both MYB30 and CBF1, two negative regulators of seedlings photomorphogenesis, act to inhibit the interactions between phyB and PIF4/PIF5. In this chapter, we will use the CBF1-phyB-PIF4 module as an example and describe the detailed procedure for performing this Y3H assay. It will be intriguing and exciting to explore the potential usage of this Y3H system in future research.

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.

Puerarin enhances TFEB-mediated autophagy and attenuates ROS-induced pyroptosis after ischemic injury of random-pattern skin flaps.

BACKGROUND AND AIM: Necrosis of random-pattern flaps restricts their application in clinical practice. Puerarin has come into focus due to its promising therapeutic effects in ischemic diseases. Here, we employed Puerarin and investigated its role and potential mechanisms in flap survival. EXPERIMENTAL PROCEDURE: The effect of Puerarin on the viability of human umbilical vein endothelial cells (HUVECs) was assessed by CCK-8, EdU staining, migration, and scratch assays. Survival area measurement and laser Doppler blood flow (LDBF) were utilized to assess the viability of ischemic injury flaps. Levels of molecules related to oxidative stress, pyroptosis, autophagy, transcription factor EB (TFEB), and the AMPK-TRPML1-Calcineurin signaling pathway were detected using western blotting, immunofluorescence, dihydroethidium (DHE) staining, RT-qPCR and Elisa. KEY RESULTS: The findings demonstrated that Puerarin enhanced the survivability of ischemic flaps. Autophagy, oxidative stress, and pyroptosis were implicated in the ability of Puerarin in improving flap survival. Increased autophagic flux and augmented tolerance to oxidative stress contribute to Puerarin's suppression of pyroptosis. Additionally, Puerarin modulated the activity of TFEB through the AMPK-TRPML1-Calcineurin signaling pathway, thereby enhancing autophagic flux. CONCLUSIONS AND IMPLICATIONS: Puerarin promoted flap survival from ischemic injury through upregulation of TFEB-mediated autophagy and inhibition of oxidative stress. Our findings offered valuable support for the clinical application of Puerarin in the treatment of ischemic diseases, including random-pattern flaps.

TFE3-SLC36A1 axis promotes resistance to glucose starvation in kidney cancer cells.

Higher demand for nutrients including glucose is characteristic of cancer. "Starving cancer" has been pursued to curb tumor progression. An intriguing regime is to inhibit glucose transporter GLUT1 in cancer cells. In addition, during cancer progression, cancer cells may suffer from insufficient glucose supply. Yet, cancer cells can somehow tolerate glucose starvation. Uncovering the underlying mechanisms shall shed insight into cancer progression and benefit cancer therapy. TFE3 is a transcription factor known to activate autophagic genes. Physiological TFE3 activity is regulated by phosphorylation-triggered translocation responsive to nutrient status. We recently reported TFE3 constitutively localizes to the cell nucleus and promotes cell proliferation in kidney cancer even under nutrient replete condition. It remains unclear whether and how TFE3 responds to glucose starvation. In this study, we show TFE3 promotes kidney cancer cell resistance to glucose starvation by exposing cells to physiologically relevant glucose concentration. We find glucose starvation triggers TFE3 protein stabilization through increasing its O-GlcNAcylation. Furthermore, through an unbiased functional genomic study, we identify SLC36A1, a lysosomal amino acid transporter, as a TFE3 target gene sensitive to TFE3 protein level. We find SLC36A1 is overexpressed in kidney cancer, which promotes mTOR activity and kidney cancer cell proliferation. Importantly, SLC36A1 level is induced by glucose starvation through TFE3, which enhances cellular resistance to glucose starvation. Suppressing TFE3 or SLC36A1 significantly increases cellular sensitivity to GLUT1 inhibitor in kidney cancer cells. Collectively, we uncover a functional TFE3-SLC36A1 axis that responds to glucose starvation and enhances starvation tolerance in kidney cancer.

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.

d-mannose targets PD-1 to lysosomal degradation and enhances T cell-mediated anti-tumor immunity.

High expression of programmed cell death protein 1 (PD-1), a typical immune checkpoint, results in dysfunction of T cells in tumor microenvironment. Antibodies and inhibitors against PD-1 or its ligand (PD-L1) have been widely used in various malignant tumors. However, the mechanisms by which PD-1 is regulated are not fully understood. Here, we report a mechanism of PD-1 degradation triggered by d-mannose and the universality of this mechanism in anti-tumor immunity. We show that d-mannose inactivates GSK3ß via promoting phosphorylation of GSK3ß at Ser9, thereby leading to TFE3 translocation to nucleus and subsequent PD-1 proteolysis induced by enhanced lysosome biogenesis. Notably, combination of d-mannose and PD-1 blockade exhibits remarkable tumor growth suppression attributed to elevated cytotoxicity activity of T cells in vivo. Furthermore, d-mannose treatment dramatically improves the therapeutic efficacy of MEK inhibitor (MEKi) trametinib in vivo. Our findings unveil a universally unrecognized anti-tumor mechanism of d-mannose by destabilizing PD-1 and provide strategies to enhance the efficacy of both immune checkpoint blockade (ICB) and MEKi -based therapies.