Ionophore polyether antibiotics (IPAs) exhibit remarkable therapeutic potential in combating parasitic diseases and cancer, yet their clinical utility is significantly hampered by severe hepatotoxicity. Despite widespread documentation of IPAs-induced hepatotoxicity, the precise molecular mechanisms underlying this phenomenon remain elusive. This study elucidates the role of ferroptosis in IPAs-induced liver injury and delineates the associated regulatory pathways. Through comprehensive in vitro (HepG2 cells) and in vivo (mice) investigations, we demonstrate that IPAs, particularly the highly toxic maduramicin (Mad), induce hepatocyte ferroptosis. Mechanistic studies employing lipid reactive oxygen species (ROS) quantification, intracellular Fe2+ assays, and western blot analysis revealed that IPAs-induced ferroptosis occurs through an autophagy-dependent pathway. Surface plasmon resonance (SPR) and molecular docking analyses confirmed direct binding and regulation of transcription factor EB (TFEB) by maduramicin. This interaction activates TFEB, subsequently mediating nuclear receptor coactivator 4 (NCOA4)-regulated lysosomal degradation processes that culminate in ferroptosis-mediated hepatotoxicity. Importantly, our findings extend beyond maduramicin, as other IPAs including monensin and salinomycin similarly targeted TFEB, triggering hepatocyte ferroptosis. Crucially, adeno-associated virus serotype 8 (AAV8)-mediated TFEB knockdown in mice conferred protection against IPAs-induced liver injury and attenuated hepatocyte ferroptosis. These findings establish TFEB-mediated NCOA4-dependent ferritinophagy and ferroptosis as central mechanisms in IPAs-induced hepatotoxicity, thereby identifying TFEB as a promising therapeutic target for mitigating IPAs-induced liver damage. This study provides critical insights into the molecular mechanisms of IPAs-induced liver injury and offers a novel strategy for therapeutic intervention.

The maintenance of a properly folded proteome is critical for cellular function and organismal health, and its age-dependent collapse is associated with a wide range of diseases. Here, we find that despite the central role of Coenzyme A as a molecular cofactor in hundreds of cellular reactions, inhibition of the first and rate-limiting step in CoA biosynthesis can be beneficial and promote proteostasis. Impairment of the cytosolic iron-sulfur cluster formation pathway, which depends on Coenzyme A, similarly promotes proteostasis and acts in the same pathway. Proteostasis improvement by interference with the Coenzyme A/iron-sulfur cluster biosynthesis pathways is dependent on the conserved HLH-30/TFEB transcription factor. Strikingly, under these conditions, HLH-30 promotes proteostasis by potentiating the expression of select chaperone genes, providing a chaperone-mediated proteostasis shield, rather than by its established role as an autophagy and lysosome biogenesis-promoting factor. This reflects the versatile nature of this conserved transcription factor, which can transcriptionally activate a wide range of protein quality control mechanisms, including chaperones and stress response genes alongside autophagy and lysosome biogenesis genes. These results highlight TFEB as a key proteostasis-promoting transcription factor and underscore it and its upstream regulators as potential therapeutic targets in proteostasis-related diseases.

The endoplasmic reticulum (ER) is a large organelle, found in all eukaryotes, that is essential for normal cellular function. This function encompasses protein folding and quality control, post-translational modifications, lipid regulation, and the storage of intracellular calcium, among others. These diverse processes are essential for maintaining proteome stability. Therefore, a robust surveillance system is established under stress to ensure cell homeostasis. Sources of stress can originate from the cellular environment, including nutrient deprivation, hypoxia, and low pH, as well as from endogenous signals within the cell, such as metabolic challenges and increased demands for protein production. When cellular homeostasis is altered by one of these triggers, ER primary functions are altered which leads to the accumulation of misfolded proteins. These impaired proteins trigger the activation of the Unfolded Protein Response (UPR) pathway. This response aims at reducing ER stress by implementing the induction of complex programs to restore cell homeostasis. However, extended ER stress can modify the UPR response, shifting its signals from promoting survival to triggering pathways that reprogram or eliminate affected cells.

STING1 is an essential component of the innate immune defense against a wide variety of pathogens. Whereas induction of type I interferon (IFN) responses is one of the best-defined functions of STING1, our transcriptomic analysis revealed IFN-independent activities of STING1 in macrophages, including transcriptional upregulation of numerous lysosomal and autophagic genes. This upregulation was mediated by the STING1-induced activation of the transcription factors TFEB and TFE3, and led to increased autophagy, lysosomal biogenesis, and lysosomal acidification. TFEB and TFE3 also modulated IFN-dependent STING1 signaling by controlling IRF3 activation. IFN production and cell death were increased in TFEB- and TFE3-depleted iBMDMs. Conversely, TFEB overexpression led to reduced IRF3 activation and an almost complete inhibition of IFN synthesis and secretion, resulting in decreased CASP3 activation and increased cell survival. Our study reveals a key role of TFEB and TFE3 as regulators of STING1-mediated innate antiviral immunity.Abbreviation: ACOD1/IRG1, aconitate decarboxylase 1; cGAMP, cyclic guanosine monophosphate-adenosine monophosphate; CGAS, cyclic GMP-AMP synthase; DMXAA, 5,6-dimethylxanthenone-4-acetic acid; EIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1; GABARAP, GABA type A receptor-associated protein; HSV-1, herpes simplex virus type; iBMDMs, immortalized bone marrow-derived macrophages; IFN, type I interferon; IFNB, interferon beta; IKBKE, inhibitor of nuclear factor kappa B kinase subunit epsilon; IRF3, interferon regulatory factor 3; LAMP1, lysosomal associated membrane protein 1; LAMP2, lysosomal associated membrane protein 2; MTORC1, mechanistic target of rapamycin kinase complex 1; RPS6, ribosomal protein S6; STING1, stimulator of interferon response cGAMP interactor 1; TBK1, TANK binding kinase 1; TFE3, transcription factor binding to IGHM enhancer 3; TFEB, transcription factor EB.

Macrophages act to defend against infection, but can fail to completely prevent bacterial replication and dissemination in an immunocompetent host. Recent studies have shown that activation of a host transcription factor, TFEB, a regulator of lysosomal biogenesis, could restrict intramacrophage replication of the human pathogen Mycobacterium tuberculosis and synergize with suboptimal levels of the antibiotic rifampin to reduce bacterial loads. Currently available small molecule TFEB activators lack selectivity and potency, but could be potentially useful in a variety of pathological conditions with suboptimal lysosomal activity. TFEB nuclear translocation and activation depend on its phosphorylation status which is controlled by multiple cellular pathways. We devised a whole cell, high throughput screening assay to identify small molecules that activate TFEB by establishing a stably transfected HEK293T reporter cell line for ATF4, a basic leucine zipper transcription factor induced by stress response and activated in parallel to TFEB. We optimized its use in vitro using compounds that target endoplasmic reticulum stress and intracellular calcium signaling. We report results from screening the commercially available LOPAC library and the Selleck Chemicals library modified to include only FDA-approved drugs and clinical research compounds. We identified twenty-one compounds across six clinical use categories that activate ATF4, and confirmed that two proteasome inhibitors promote TFEB activation. The results of this study provide an assay that could be used to screen for small molecules that activate ATF4 and TFEB and a potential list of compounds identified as activators of the ATF4 transcription factor in response to cellular stress.

To investigate the effects of Huayu Qutan recipe (, HYQT) on the atherosclerosis (AS) model of ApoE-/- mice with a high-fat diet and to illustrate the underlying mechanisms from modern patho-physiological conceptualizations.

High performance liquid chromatography of quadrupole time of flight-tandem mass spectrometry (HPLC-Q-TOF-MS/MS) analysis was used to identify the active compounds in the recipe. The mice were randomly allocated into 7 groups: control (CTRL) group, normal diet (ND) group, high-fat diet (HFD) group, HYQT groups (low dose, medium dose, and high dose), and simvastatin (SIM) group. Deferent doses of HYQT were gavaged twice a day, and then the protective effect of HYQT on plaque formation in ApoE-/- mice with a high-fat diet was verified viahematoxylin-eosin (HE) staining and oil red o (ORO) staining. We observed the co-localization in aortic macrophages and lipid droplets (LDs) by CD68 and the Bodipy fluorescence probe. Light chain 3 phosphoprotein class Ⅱ/light chain 3 phosphoprotein class Ⅰ (LC3Ⅱ/LC3Ⅰ) was examined by western blotting, and sequestosome 1 (SQSTM1/p62), Beclin1, Lamp1, mammalian target of rapamycin (mTOR), phosphorylated mammalian target of rapamycin (p-mTOR), and ATP-binding cassette transporter A1 (ABCA1) were examined by real-time polymerase chain reaction (RT-PCR) and Western blotting. Transcription factor EB (TFEB) nuclear translocation was determined by immunofluorescence analysis.

Five active compounds were identified using HPLC-Q-TOF-MS/MS analysis: ferulic acid, chlorogenic acid, calycosin, formononetin, and 8,2'-dihydroxy-7,4'-dimethoxy-isoflavane. The effect of HYQT on atherosclerotic plaque formation in ApoE-/- mice was investigated. These findings showed that HYQT decreased the co-localization of CD68 and Bodipy and increased the co-localization of CD68 and LC3B. Medium and high doses of HYQT increased autophagosome formation and promoted the maturation of LC3Ⅱ/LC3Ⅰ. Additionally, HYQT decreased the expression of SQSTM1/p62. Medium and high doses of HYQT also increased the expression of Beclin1 and Lamp1. RT-PCR and Western blot results suggested that HYQT enhanced the expression of ABCA1 mRNA and protein and regulated the mTORC1/TFEB signaling pathway.

The results indicate that HYQT is an effective traditional Chinese herbal remedy for the treatment of AS. HYQT mitigates macrophage-derived foam cell formation by activating autophagy in atherosclerosis. The mTOR/TFEB signaling pathway and ABCA1 are therapeutic targets of HYQT for the treatment of AS.

Transcription Factor EB (TFEB) controls lysosomal biogenesis and autophagy in response to nutritional status and other stress factors. Although its regulation by nuclear translocation is known to involve a complex network of well-studied regulatory processes, the precise contribution of each of these mechanisms is unclear. Using microfluidics technology and real-time imaging coupled with mathematical modelling, we explored the dynamic regulation of TFEB under different conditions. We found that TFEB nuclear translocation upon nutrient deprivation happens in two phases: a fast one characterised by a transient boost in TFEB dephosphorylation dependent on transient calcium release mediated by mucolipin 1 (MCOLN1) followed by activation of the Calcineurin phosphatase, and a slower one driven by inhibition of mTORC1-dependent phosphorylation of TFEB. Upon refeeding, TFEB cytoplasmic relocalisation kinetics are determined by Exportin 1 (XPO1). Collectively, our results show how different mechanisms interact to regulate TFEB activation and the power of microfluidics and quantitative modelling to elucidate complex biological mechanisms.

TFE3 rearranged renal cell carcinoma (TFE3 rRCC), classified as a distinct entity of RCCs, exhibits aggressive biological behavior and possesses unique metabolic characteristics. In the present study, TFE3 rRCC with high expression of TFE3 fusion proteins was employed to investigate the features of lipid metabolism and its underlying mechanism in cancer progression.

Fluorescence microscope and flow cytometry were employed to detect lipid droplets (LDs). GPO-PAP method and Oil Red O staining were used to quantify triacylglycerol levels. The data for bioinformatics analysis were sourced from GEO and iProX. The biological roles of TFE3 and LAMP2A were investigated by CCK8 assay, EdU staining, seahorse, transwell assay, colony, and sphere formation assay. The regulatory mechanisms involving TFE3, LAMP2A and Hsc70 were investigated using western blotting, immunohistochemistry, qRT-PCR, luciferase assays, Co-IP techniques, and ChIP analyses.

The level of LDs accumulation in TFE3 rRCC was relatively low, and the knockdown of TFE3 led to an increase in LDs accumulation while inhibiting tumor progression. The underlying mechanism revealed that TFE3 fusion proteins inhibited the biosynthesis of LDs within the endoplasmic reticulum by promoting the degradation of DGAT1 and DGAT2 via autophagy. Furthermore, TFE3 fusion proteins upregulated LAMP2A, thereby enhancing chaperone-mediated autophagy pathways. The process facilitated the degradation of LDs and promoted oxidative metabolism of long-chain fatty acids in mitochondria.

TFE3 fusion proteins facilitated the progression of TFE3 rRCC through enhancing the degradation of LDs via chaperone-mediated lipophagy. LAMP2A could serve as a novel potential prognostic biomarker and therapeutic targets.

Breast cancer stem cells (CSCs) are difficult to therapeutically target, but continued efforts are critical given their contribution to tumor heterogeneity and treatment resistance in triple-negative breast cancer. CSC properties are influenced by metabolic stress, but specific mechanisms are lacking for effective drug intervention. Our previous work on TFEB suggested a key function in CSC metabolism. Indeed, TFEB knockdown (KD) inhibited mammosphere formation in vitro and tumor initiation/growth in vivo. These phenotypic effects were accompanied by a decline in CD44high/CD24low cells. Glycolysis inhibitor 2-deoxy-D-glucose (2-DG) induced TFEB nuclear translocation, indicative of TFEB transcriptional activity. TFEB KD blunted, whereas TFEB (S142A) augmented 2-DG-driven unfolded protein response (UPR) mediators, notably BiP/HSPA5 and CHOP. Like TFEB KD, silencing BiP/HSPA5 inhibited CSC self-renewal, suggesting that TFEB augments UPR-related survival. Further studies showed that TFEB KD attenuated 2-DG-directed autophagy, suggesting a mechanism whereby TFEB protects CSCs against 2-DG-induced stress. Our data indicate that TFEB modulates CSC metabolic stress response via autophagy and UPR. These findings reveal the novel role of TFEB in regulating CSCs during metabolic stress in triple-negative breast cancer.

Melanin pigmentation in oocytes is a critical feature for both the esthetic and developmental aspects of oocytes, influencing their polarity and overall development. Despite substantial knowledge of melanogenesis in melanocytes and retinal pigment epithelium cells, the molecular mechanisms underlying oocyte melanogenesis remain largely unknown.

Here, we compare the oocytes of wild-type, tyr-/- and mitf-/- Xenopus tropicalis and found that mitf-/- oocytes exhibit normal melanin deposition at the animal pole, whereas tyr-/- oocytes show no melanin deposition at this site. Transmission electron microscopy confirmed that melanogenesis in mitf-/- oocytes proceeds normally, similar to wild-type oocytes. Transcriptomic analysis revealed that mitf-/- oocytes still express melanogenesis-related genes, enabling them to complete melanogenesis. Additionally, in Xenopus tropicalis oocytes, the expression of the MiT subfamily factor tfe3 is relatively high, while tfeb, mitf, and tfec levels are extremely low. The expression pattern of tfe3 is similar to that of tyr and other melanogenesis-related genes. Thus, melanogenesis in Xenopus tropicalis oocytes is independent of Mitf and may be regulated by other MiT subfamily factors such as Tfe3, which control the expression of genes like tyr, dct, and tyrp1. Furthermore, transcriptomic data revealed that changes in the expression of genes related to mitochondrial cloud formation represent the most significant molecular changes during oocyte development.

Overall, these findings suggest that further elucidation of Tyr-dependent and Mitf-independent mechanisms of melanin deposition at the animal pole will enhance our understanding of melanogenesis and Oogenesis.

The protein interactome of p65/RELA, the most active subunit of the transcription factor (TF) NF-κB, has not been previously determined in living cells. Using p65-miniTurbo fusion proteins and biotin tagging, we identify >350 RELA interactors from untreated and IL-1α-stimulated cells, including many TFs (47% of all interactors) and >50 epigenetic regulators belonging to different classes of chromatin remodeling complexes. A comparison with the interactomes of two point mutants of p65 reveals that the interactions primarily require intact dimerization rather than DNA-binding properties. A targeted RNAi screen for 38 interactors and subsequent functional transcriptome and bioinformatics studies identify gene regulatory (sub)networks, each controlled by RELA in combination with one of the TFs ZBTB5, GLIS2, TFE3/TFEB, or S100A8/A9. The large, dynamic and versatile high-resolution interactome of RELA and its gene regulatory logics provides a rich resource and a new framework for explaining how RELA cooperativity determines gene expression patterns.

Cancer cells rely on invasive growth to survive in a hostile microenvironment; this growth is characterised by interconnected processes such as epithelial-to-mesenchymal transition and migration. A master regulator of these events is the MET oncogene, which is overexpressed in the majority of cancers; however, since mutations in the MET oncogene are seen only rarely in cancers and are relatively infrequent, the mechanisms that cause this widespread MET overexpression remain obscure. Here, we show that the 5' untranslated region (5'UTR) of MET mRNA harbours two functional stress-responsive elements, conferring translational regulation by the integrated stress response (ISR), regulated by phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) at serine 52. ISR activation by serum starvation, leucine deprivation, hypoxia, irradiation, thapsigargin or gemcitabine is followed by MET protein overexpression. We mechanistically link MET translation to the ISR by (i) mutation of the two uORFs within the MET 5'UTR, (ii) CRISPR/Cas9-mediated mutation of eIF2α (S52A), or (iii) the application of ISR pathway inhibitors. All of these interventions reduce stress-induced MET overexpression. Finally, we show that blocking stress-induced MET translation blunts MET-dependent invasive growth. These findings indicate that upregulation of the MET oncogene is a functional requirement linking integrated stress response to cancer progression.

Eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, which regulates all 3 unfolded protein response pathways, helps maintain cellular homeostasis and overcome endoplasmic reticulum (ER) stress through transcriptional and translational reprogramming. However, transcriptional regulation of mitochondrial homeostasis by eIF2α phosphorylation during ER stress is not fully understood. Here, we report that the eIF2α phosphorylation-activating transcription factor 4 (ATF4) axis is required for the expression of multiple transcription factors, including nuclear factor erythroid 2-related factor 2 and its target genes responsible for mitochondrial homeostasis during ER stress. eIF2α phosphorylation-deficient (A/A) cells displayed dysregulated mitochondrial dynamics and mitochondrial DNA replication, decreased expression of oxidative phosphorylation complex proteins, and impaired mitochondrial functions during ER stress. ATF4 overexpression suppressed impairment of mitochondrial homeostasis in A/A cells during ER stress by promoting the expression of downstream transcription factors and their target genes. Our findings underscore the importance of the eIF2α phosphorylation-ATF4 axis for maintaining mitochondrial homeostasis through transcriptional reprogramming during ER stress.

Dysregulated eIF4E-dependent translation is a central driver of tumorigenesis and therapy resistance. eIF4E-binding proteins (4E-BP1/2/3) are major negative regulators of eIF4E-dependent translation that are inactivated in tumors through inhibitory phosphorylation or downregulation. Previous studies have linked PP2A phosphatase(s) to activation of 4E-BP1. Here, we leveraged biased small-molecule activators of PP2A (SMAPs) to explore the role of B56-PP2A(s) in 4E-BP regulation and the potential of B56-PP2A activation for restoring translational control in tumors. SMAP treatment promoted PP2A-dependent hypophosphorylation of 4E-BP1/2, supporting a role for B56-PP2As (e.g., B56α-PP2A) as 4E-BP phosphatases. Unexpectedly, SMAPs induced transcriptional upregulation of 4E-BP1 through a B56-PP2A→TFE3/TFEB→ATF4 axis. Cap-binding and coimmunoprecipitation assays showed that B56-PP2A(s) activation blocks assembly of the eIF4F translation initiation complex, and cap-dependent translation assays confirmed the translation-inhibitory effects of SMAPs. Thus, B56-PP2A(s) orchestrate a translation-repressive program involving transcriptional induction and activation of 4E-BP1. Notably, SMAPs promoted 4E-BP1-dependent apoptosis in tumor cells and potentiated 4E-BP1 function in the presence of ERK or mTOR inhibitors, agents that rely on inhibition of eIF4E-dependent translation for antitumor activity. These findings, combined with the ability of SMAPs to regulate 4E-BP1 in vivo, highlight the potential of PP2A activators for cancer therapy and overcoming therapy resistance.

The integrated stress response (ISR) regulates cell fate during conditions of stress by leveraging the cell's capacity to endure sustainable and efficient adaptive stress responses. Protein phosphatase 2A (PP2A) activity modulation has been shown to be successful in achieving both therapeutic efficacy and safety across various cancer models. However, the molecular mechanisms driving its selective antitumor effects remain unclear. Here, we show for the first time that ISR plasticity relies on PP2A activation to regulate drug response and dictate cellular survival under conditions of chronic stress. We demonstrate that genetic and chemical modulation of the PP2A leads to chronic proteolytic stress and triggers an ISR to dictate whether the cell lives or dies. More specifically, we uncovered that the PP2A-TFE3-ATF4 pathway governs ISR cell plasticity during endoplasmic reticular and cellular stress independent of the unfolded protein response. We further show that normal cells reprogram their genetic signatures to undergo ISR-mediated adaptation and homeostatic recovery thereby avoiding toxicity following PP2A-mediated stress. Conversely, oncogenic specific cytotoxicity induced by chemical modulation of PP2A is achieved by activating chronic and irreversible ISR in cancer cells. Our findings propose that a differential response to chemical modulation of PP2A is determined by intrinsic ISR plasticity, providing a novel biological vulnerability to selectively induce cancer cell death and improve targeted therapeutic efficacy.

Mitophagy is a selective cellular process critical for the removal of damaged mitochondria. It is essential in regulating mitochondrial number, ensuring mitochondrial functionality, and maintaining cellular equilibrium, ultimately influencing cell destiny. Numerous pathologies, such as neurodegenerative diseases, cardiovascular disorders, cancers, and various other conditions, are associated with mitochondrial dysfunctions. Thus, a detailed exploration of the regulatory mechanisms of mitophagy is pivotal for enhancing our understanding and for the discovery of novel preventive and therapeutic options for these diseases. Nanomaterials have become integral in biomedicine and various other sectors, offering advanced solutions for medical uses including biological imaging, drug delivery, and disease diagnostics and therapy. Mitophagy is vital in managing the cellular effects elicited by nanomaterials. This review provides a comprehensive analysis of the molecular mechanisms underpinning mitophagy, underscoring its significant influence on the biological responses of cells to nanomaterials. Nanoparticles can initiate mitophagy via various pathways, among which the PINK1-Parkin pathway is critical for cellular defense against nanomaterial-induced damage by promoting mitophagy. The role of mitophagy in biological effects was induced by nanomaterials, which are associated with alterations in Ca2+ levels, the production of reactive oxygen species, endoplasmic reticulum stress, and lysosomal damage.

The transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy. We identify a distinct nuclear interactome of TFEB, with ubiquitin-specific protease 7 (USP7) emerging as a key post-translational modulator of TFEB. Genetic depletion and inhibition of USP7 reveal its critical role in preserving TFEB stability within both nuclear and cytoplasmic compartments. Specifically, USP7 is identified as the deubiquitinase responsible for removing the K48-linked polyubiquitination signal from TFEB at lysine residues K116, K264, and K274, thereby preventing its proteasomal degradation. Functional assays demonstrate the involvement of USP7 in preserving TFEB-mediated transcriptional responses to nutrient deprivation while also modulating autophagy flux and lysosome biogenesis. As USP7 is a deubiquitinase that protects TFEB from proteasomal degradation, these findings provide the foundation for therapeutic targeting of the USP7-TFEB axis in conditions characterized by TFEB dysregulation and metabolic abnormalities, particularly in certain cancers.

Lysosomal dysfunction is causally linked to neurodegeneration in many lysosomal storage disorders (LSDs) and is associated with various age-related neurodegenerative diseases 1,2 , but there is limited understanding of the mechanisms by which altered lysosomal function leads to changes in gene expression that drive pathogenic cellular phenotypes. To investigate this question, we performed systematic imaging, transcriptomic, and epigenetic studies of major brain cell types in Sgsh null (KO) mice, a preclinical mouse model for Sanfilippo syndrome (Mucopolysaccharidosis Type IIIA, MPS-IIIA) 3,4 . MPS-IIIA is a neurodegenerative LSD caused by homozygous loss-of-function (LoF) mutations in SGSH which results in severe early-onset developmental, behavioral, and neurocognitive impairment 5-15 . Electron microscopy, immunohistochemistry, and single-nucleus RNA-sequencing analysis revealed microglia as the cell type exhibiting the most dramatic phenotypic alterations in Sgsh KO mice. Further temporal analysis of microglia gene expression showed dysregulation of genes associated with lysosomal function and immune signaling pathways beginning early in the course of the disease. Sgsh deficiency similarly resulted in increases in open chromatin and histone acetylation at thousands of putative microglia-specific enhancers associated with upregulated genes but had much less impact on the epigenetic landscapes of neurons or oligodendrocytes. We provide evidence for dominant and context-dependent roles of members of the MITF/TFE family as major drivers of microglia-specific epigenetic and transcriptional changes resulting from lysosomal stress that are dependent on collaborative interactions with PU.1/ETS and C/EBP transcription factors. Lastly, we show that features of the transcriptomic and epigenetic alterations observed in murine Sgsh deficiency are also observed in microglia derived from mouse models of age-related neurodegeneration and in human Alzheimer's disease patients, revealing common and disease-specific transcriptional mechanisms associated with disease-associated microglia phenotypes.

The transcription factor TFEB is a major regulator of lysosomal biogenesis and autophagy. There is growing evidence that posttranslational modifications play a crucial role in regulating TFEB activity. Here, we show that lactate molecules can covalently modify TFEB, leading to its lactylation and stabilization. Mechanically, lactylation at K91 prevents TFEB from interacting with E3 ubiquitin ligase WWP2, thereby inhibiting TFEB ubiquitination and proteasome degradation, resulting in increased TFEB activity and autophagy flux. Using a specific antibody against lactylated K91, enhanced TFEB lactylation was observed in clinical human pancreatic cancer samples. Our results suggest that lactylation is a novel mode of TFEB regulation and that lactylation of TFEB may be associated with high levels of autophagy in rapidly proliferating cells, such as cancer cells.

Lipidated ATG8/LC3 proteins are recruited to single membrane compartments as well as autophagosomes, supporting their functions. Although recent studies have shown that Golgi-LC3 lipidation follows Golgi damage, its molecular mechanism and function under Golgi stress remain unknown. Here, by combining DLK1 overexpression as a new strategy for induction of Golgi-specific LC3 lipidation, and the application of Golgi-damaging reagents, we unravel the mechanism and role of Golgi-LC3 lipidation. Upon DLK1 overexpression, LC3 is lipidated on the Golgi apparatus in an ATG12-ATG5-ATG16L1 complex-dependent manner; a post-Golgi trafficking blockade is the primary cause of this lipidation. During Golgi stress, ATG16L1 is recruited through its interaction with V-ATPase for Golgi-LC3 lipidation. After post-Golgi trafficking inhibition, TFE3, a key regulator of the Golgi stress response, is translocated to the nucleus. Defects in LC3 lipidation disrupt this translocation, leading to an attenuation of the Golgi stress response. Together, our results reveal the mechanism and unexplored function of Golgi-LC3 lipidation in the Golgi stress response.

The endoplasmic reticulum (ER) is the organelle mainly involved in maintaining cellular homeostasis and driving correct protein folding. ER-dependent defects or dysfunctions are associated with the genesis/progression of several pathological conditions, including cancer, inflammation, and neurodegenerative disorders, that are directly or indirectly correlated to a wide set of events collectively named under the term "ER stress". Despite the recent increase in interest concerning ER activity, further research studies are needed to highlight all the mechanisms responsible for ER failure. In this field, recent discoveries paved the way for the comprehension of the strong interaction between ER stress development and the endocannabinoid system. The activity of the endocannabinoid system is mediated by the activation of cannabinoid receptors (CB), G protein-coupled receptors that induce a decrease in cAMP levels, with downstream anti-inflammatory effects. CB activation drives, in most cases, the recovery of ER homeostasis through the regulation of ER stress hallmarks PERK, ATF6, and IRE1. In this review, we focus on the CB role in modulating ER stress, with particular attention to the cellular processes leading to UPR activation and oxidative stress response extinguishment, and to the mechanisms underlying natural cannabinoids' modulation of this complex cellular machine.

Background: Identical translocations involving the TFE3 gene and various partners have been found in both renal and soft tissue tumors, like alveolar soft part sarcoma (ASPSCR1), ossifying fibromyxoid tumor (PHF1), epithelioid hemangioendothelioma, and the clear cell stromal tumor of the lung (YAP1). Methods: Herein, we review in detail the clinicopathologic and molecular data of TFE3-rearranged renal tumors and propose our perspective, which may shed light on this emerging conundrum. Results: Among the kidney tumors carrying TFE3 translocations, most are morphologically heterogeneous carcinomas labeling for the tubular marker PAX8. The others are mesenchymal neoplasms known as PEComas, characterized by epithelioid cells co-expressing smooth muscle actin, cathepsin-K, melanogenesis markers, and sometimes melanin pigment deposition. Over the past 30 years, numerous TFE3 fusion partners have been identified, with ASPL/ASPSCR1, PRCC, SFPQ/PSF, and NONO being the most frequent. Conclusions: It is not well understood why similar gene fusions can give rise to renal tumors with different morpho-immunophenotypes, which may contribute to the recent disagreement regarding their classification. However, as these two entities, respectively, epithelial and mesenchymal in nature, are widely recognized by the pathology community and their clinicopathologic features well established, we overall believe it is still better to retain the names TFE3-rearranged renal cell carcinoma and TFE3-rearranged PEComa.

Small cell lung cancer (SCLC) is characterized by rapid development of chemoresistance and poor outcomes. Cyclin-dependent kinase 4/6 inhibitors (CDK4/6is) are widely used in breast cancer and other cancer types. However, the molecular mechanisms of CDK4/6 in SCLC chemoresistance remain poorly understood. Here, Rb1flox/flox, Trp53flox/flox, Ptenflox/flox (RTP) and Rb1flox/flox, Trp53flox/flox, MycLSL/LSL (RPM) spontaneous SCLC mouse models, SCLC cell line-derived xenograft (CDX) models, and SCLC patient-derived xenograft (PDX) models are established to reveal the potential effects of CDK4/6is on SCLC chemoresistance. In this study, it is found that CDK4/6is palbociclib (PD) or ribociclib (LEE) combined with chemotherapeutic drugs significantly inhibit SCLC tumor growth. Mechanistically, CDK4/6is do not function through the classic Retionblastoma1 (RB) dependent axis in SCLC. CDK4/6is induce impair autophagy through the AMBRA1-lysosome signaling pathway. The upregulated AMBRA1 protein expression leads to CDK6 degradation via autophagy,  and the following TFEB and TFE3 nuclear translocation inhibition leading to the lysosome-related genes levels downregulation. Moreover, it is found that the expression of CDK6 is higher in SCLC tumors than in normal tissue and it is associated with the survival and prognosis of SCLC patients. Finally, these findings demonstrate that combining CDK4/6is with chemotherapy treatment may serve as a potential therapeutic option for SCLC patients.

A coding variant in Phospholipase D3 ( PLD3 ) increases the risk of Alzheimer's disease (AD). PLD3 is a lysosomal protein, and endosomal and lysosomal abnormalities are linked to AD; however, the role of PLD3 in lysosomal homeostasis and its implications in AD remain poorly understood. To address this knowledge gap, we conducted comprehensive studies integrating transcriptomics, proteomics, and cell biology approaches. We observed significant enlargement of lysosomes in neurons lacking PLD3, accompanied by increased endocytosis and autophagy, but a decline in lysosomal proteolytic activity. Lysosomes of PLD3-deficient cells underwent proteome remodeling, manifested by an enrichment of proteins involved in lysosomal biogenesis, endocytosis and calcium signaling. Mechanistically, we discovered that PLD3 mediates TFEB/TFE3 degradation through the proteasome, and as a result, PLD3 deficiency leads to increased TFEB/TFE3 levels, nuclear translocation, and transcriptional activities. Notably, variants in PLD3, e.g., V232M or K486R, do not alter its impact on TFEB/TFE3 metabolism. Transcriptomic profiling further confirmed the enrichment of transcripts involved in lysosomal biogenesis, endocytosis, autophagy, mTOR signaling and AD in response to PLD3 loss. Additionally, PLD3 ablation has synergistic effects with β-amyloid in causing lysosomal abnormalities and modifying TFEB/TFE3 signaling. In conclusion, our findings demonstrate that PLD3 is involved in regulating lysosomal biogenesis via TFEB/TFE3 signaling, and lysosomal abnormalities resulting from PLD3 deficiency are potentially a risk factor for AD.

Acetaminophen (APAP) overdose can induce hepatocyte necrosis and acute liver failure in experimental rodents and humans. APAP is mainly metabolized via hepatic cytochrome P450 enzymes to generate the highly reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which forms acetaminophen protein adducts (APAP-adducts) and damages mitochondria, triggering necrosis. APAP-adducts and damaged mitochondria can be selectively removed by autophagy. Increasing evidence implies that the activation of autophagy may be beneficial for APAP-induced liver injury (AILI). In this minireview, we briefly summarize recent progress on autophagy, in particular, the pharmacological targeting of SQSTM1/p62 and TFEB in AILI.

TFEB and TFE3 (TFEB/3), key regulators of lysosomal biogenesis and autophagy, play diverse roles depending on cell type. This study highlights a hitherto unrecognized role of TFEB/3 crucial for peripheral nerve repair. Specifically, they promote the generation of progenitor-like repair Schwann cells after axonal injury. In Schwann cell-specific TFEB/3 double knock-out mice of either sex, the TFEB/3 loss disrupts the transcriptomic reprogramming that is essential for the formation of repair Schwann cells. Consequently, mutant mice fail to populate the injured nerve with repair Schwann cells and exhibit defects in axon regrowth, target reinnervation, and functional recovery. TFEB/3 deficiency inhibits the expression of injury-responsive repair Schwann cell genes, despite the continued expression of c-jun, a previously identified regulator of repair Schwann cell function. TFEB/3 binding motifs are enriched in the enhancer regions of injury-responsive genes, suggesting their role in repair gene activation. Autophagy-dependent myelin breakdown is not impaired despite TFEB/3 deficiency. These findings underscore a unique role of TFEB/3 in adult Schwann cells that is required for proper peripheral nerve regeneration.

Protein biogenesis within the endoplasmic reticulum (ER) is crucial for organismal function. Errors during protein folding necessitate the removal of faulty products. ER-associated protein degradation and ER-phagy target misfolded proteins for proteasomal and lysosomal degradation. The mechanisms initiating ER-phagy in response to ER proteostasis defects are not well understood. By studying mouse primary cells and patient samples as a model of ER storage disorders (ERSDs), we show that accumulation of faulty products within the ER triggers a response involving SESTRIN2, a nutrient sensor controlling mTORC1 signaling. SESTRIN2 induction by XBP1 inhibits mTORC1's phosphorylation of TFEB/TFE3, allowing these transcription factors to enter the nucleus and upregulate the ER-phagy receptor FAM134B along with lysosomal genes. This response promotes ER-phagy of misfolded proteins via FAM134B-Calnexin complex. Pharmacological induction of FAM134B improves clearance of misfolded proteins in ERSDs. Our study identifies the interplay between nutrient signaling and ER quality control, suggesting therapeutic strategies for ERSDs.

TFE3 and TFEB, as the master regulators of lysosome biogenesis and autophagy, are well characterized to enhance the synaptic protein α-synuclein degradation in protecting against Parkinson's disease (PD) and their levels are significantly decreased in the brain of PD patients. However, how TFE3 and TFEB are regulated during PD pathogenesis remains largely vague. Herein, we identified that programmed cell death 4 (PDCD4) promoted pathologic α-synuclein accumulation to facilitate PD development via suppressing both TFE3 and TFEB translation. Conversely, PDCD4 deficiency significantly augmented global and nuclear TFE3 and TFEB distributions to alleviate neurodegeneration in a mouse model of PD with overexpressing α-synuclein in the striatum. Mechanistically, like TFEB as we reported before, PDCD4 also suppressed TFE3 translation, rather than influencing its transcription and protein stability, to restrain its nuclear translocation and lysosomal functions, eventually leading to α-synuclein aggregation. We proved that the two MA3 domains of PDCD4 mediated the translational suppression of TFE3 through binding to its 5'-UTR of mRNA in an eIF-4A dependent manner. Based on this, we developed a blood-brain barrier penetrating RVG polypeptide modified small RNA drug against pdcd4 to efficiently prevent α-synuclein neurodegeneration in improving PD symptoms by intraperitoneal injections. Together, we suggest PDCD4 as a PD-risk protein to facilitate α-synuclein neurodegeneration via suppressing TFE3 and TFEB translation and further provide a potential small RNA drug against pdcd4 to treat PD by intraperitoneal injections.

TFEB and TFE3 belong to the MiT/TFE family of transcription factors that bind identical DNA responsive elements in the regulatory regions of target genes. They are involved in regulating lysosomal biogenesis, function, exocytosis, autophagy, and lipid catabolism. Precise control of TFEB and TFE3 activity is crucial for processes such as senescence, stress response, energy metabolism, and cellular catabolism. Dysregulation of these factors is implicated in various diseases, thus researchers have explored pharmacological approaches to modulate MiT/TFE activity, considering these transcription factors as potential therapeutic targets. However, the physiological complexity of their functions and the lack of suitable in vivo tools have limited the development of selective MiT/TFE modulating agents. Here, we have created a reporter-based biosensor, named CLEARoptimized, facilitating the pharmacological profiling of TFEB- and TFE3-mediated transcription. This innovative tool enables the measurement of TFEB and TFE3 activity in living cells and mice through imaging and biochemical techniques. CLEARoptimized consists of a promoter with six coordinated lysosomal expression and regulation motifs identified through an in-depth bioinformatic analysis of the promoters of 128 TFEB-target genes. The biosensor drives the expression of luciferase and tdTomato reporter genes, allowing the quantification of TFEB and TFE3 activity in cells and in animals through optical imaging and biochemical assays. The biosensor's validity was confirmed by modulating MiT/TFE activity in both cell culture and reporter mice using physiological and pharmacological stimuli. Overall, this study introduces an innovative tool for studying autophagy and lysosomal pathway modulation at various biological levels, from individual cells to the entire organism.Abbreviations: CLEAR: coordinated lysosomal expression and regulation; MAR: matrix attachment regions; MiT: microphthalmia-associated transcription factor; ROI: region of interest; TBS: tris-buffered saline; TF: transcription factor; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TH: tyrosine hydroxylase; TK: thymidine kinase; TSS: transcription start site.

Cancer resistance to therapy is still an unsolved scientific and clinical problem. In 2022, the hallmarks of cancer have been expanded to include four new features, including cellular senescence. Therapy-induced senescence (TIS) is a stressor-based response to conventional treatment methods, e.g. chemo- and radiotherapy, but also to non-conventional targeted therapies. Since TIS reinforces resistance in cancers, new strategies for sensitizing cancer cells to therapy are being adopted. These include macroautophagy as a potential target for inhibition due to its potential cytoprotective role in many cancers. The mechanism of late-stage autophagy inhibitors is based on blockage of autophagolysosome formation or an increase in lysosomal pH, resulting in disrupted cargo degradation. Such inhibitors are relevant candidates for increasing anticancer therapy effectiveness. In particular, 4-aminoquoline derivatives: chloroquine/hydroxychloroquine (CQ/HCQ) have been tested in multiple clinical trials in combination with senescence-inducing anti-cancer drugs. In this review, we summarize the properties of selected late-autophagy inhibitors and their role in the regulation of autophagy and senescent cell phenotype in vitro and in vivo models of cancer as well as treatment response in clinical trials on oncological patients. Additionally, we point out that, although these compounds increase the effectiveness of treatment in some cases, their practical usage might be hindered due to systemic toxicity, hypoxic environment, dose- ant time-dependent inhibitory effects, as well as a possible contribution to escaping from TIS.

We have reported that an environmental pollutant, cadmium, promotes cell death in the human renal tubular cells (RTCs) through hyperactivation of a serine/threonine kinase Akt. However, the molecular mechanisms downstream of Akt in this process have not been elucidated. Cadmium has a potential to accumulate misfolded proteins, and proteotoxicity is involved in cadmium toxicity. To clear the roles of Akt in cadmium exposure-induced RTCs death, we investigated the possibility that Akt could regulate proteotoxicity through autophagy in cadmium chloride (CdCl2)-exposed HK-2 human renal proximal tubular cells. CdCl2 exposure promoted the accumulation of misfolded or damaged proteins, the formation of aggresomes (pericentriolar cytoplasmic inclusions), and aggrephagy (selective autophagy to degrade aggresome). Pharmacological inhibition of Akt using MK2206 or Akti-1/2 enhanced aggrephagy by promoting dephosphorylation and nuclear translocation of transcription factor EB (TFEB)/transcription factor E3 (TFE3), lysosomal transcription factors. TFEB or TFE3 knockdown by siRNAs attenuated the protective effects of MK2206 against cadmium toxicity. These results suggested that aberrant activation of Akt attenuates aggrephagy via TFEB or TFE3 to facilitate CdCl2-induced cell death. Furthermore, these roles of Akt/TFEB/TFE3 were conserved in CdCl2-exposed primary human RTCs. The present study shows the molecular mechanisms underlying Akt activation that promotes cadmium-induced RTCs death.

The global prevalence of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is increasing, now affecting 25%-30% of the population worldwide. MASLD, characterized by hepatic steatosis, results from an imbalance in lipid metabolism, leading to oxidative stress, lipoperoxidation, and inflammation. The activation of autophagy, particularly lipophagy, alleviates hepatic steatosis by regulating intracellular lipid levels. Lutein, a carotenoid with antioxidant and anti-inflammatory properties, protects against liver damage, and individuals who consume high amounts of lutein have a lower risk of developing MASLD. Evidence suggests that lutein could modulate autophagy-related signaling pathways, such as the transcription factor EB (TFEB). TFEB plays a crucial role in regulating lipid homeostasis by linking autophagy to energy metabolism at the transcriptional level, making TFEB a potential target against MASLD. STARD3, a transmembrane protein that binds and transports cholesterol and sphingosine from lysosomes to the endoplasmic reticulum and mitochondria, has been shown to transport and bind lutein with high affinity. This protein may play a crucial role in the uptake and transport of lutein in the liver, contributing to the decrease in hepatic steatosis and the regulation of oxidative stress and inflammation. This review summarizes current knowledge on the role of lutein in lipophagy, the pathways it is involved in, its relationship with STARD3, and its potential as a pharmacological strategy to treat hepatic steatosis.

Mitochondria and the endoplasmic reticulum (ER) have a synergistic relationship and are key regulatory hubs in maintaining cell homeostasis. Communication between these organelles is mediated by mitochondria ER contact sites (MERCS), allowing the exchange of material and information, modulating calcium homeostasis, redox signalling, lipid transfer and the regulation of mitochondrial dynamics. MERCS are dynamic structures that allow cells to respond to changes in the intracellular environment under normal homeostatic conditions, while their assembly/disassembly are affected by pathophysiological conditions such as ageing and disease. Disruption of protein folding in the ER lumen can activate the Unfolded Protein Response (UPR), promoting the remodelling of ER membranes and MERCS formation. The UPR stress receptor kinases PERK and IRE1, are located at or close to MERCS. UPR signalling can be adaptive or maladaptive, depending on whether the disruption in protein folding or ER stress is transient or sustained. Adaptive UPR signalling via MERCS can increase mitochondrial calcium import, metabolism and dynamics, while maladaptive UPR signalling can result in excessive calcium import and activation of apoptotic pathways. Targeting UPR signalling and the assembly of MERCS is an attractive therapeutic approach for a range of age-related conditions such as neurodegeneration and sarcopenia. This review highlights the emerging evidence related to the role of redox mediated UPR activation in orchestrating inter-organelle communication between the ER and mitochondria, and ultimately the determination of cell function and fate.

The maintenance of a properly folded proteome is critical for cellular function and organismal health, and its age-dependent collapse is associated with a wide range of diseases. Here, we find that despite the central role of Coenzyme A as a molecular cofactor in hundreds of cellular reactions, limiting Coenzyme A levels in C. elegans and in human cells, by inhibiting the conserved pantothenate kinase, promotes proteostasis. Impairment of the cytosolic iron-sulfur clusters formation pathway, which depends on Coenzyme A, similarly promotes proteostasis and acts in the same pathway. Proteostasis improvement by Coenzyme A/iron-sulfur cluster deficiencies are dependent on the conserved HLH-30/TFEB transcription factor. Strikingly, under these conditions, HLH-30 promotes proteostasis by potentiating the expression of select chaperone genes providing a chaperone-mediated proteostasis shield, rather than by its established role as an autophagy and lysosome biogenesis promoting factor. This reflects the versatile nature of this conserved transcription factor, that can transcriptionally activate a wide range of protein quality control mechanisms, including chaperones and stress response genes alongside autophagy and lysosome biogenesis genes. These results highlight TFEB as a key proteostasis-promoting transcription factor and underscore it and its upstream regulators as potential therapeutic targets in proteostasis-related diseases.

This study aims to investigate the mechanism of the anti-atherosclerosis effect of Huayu Qutan Recipe (HYQT) on the inhibition of foam cell formation. In vivo, the mice were randomly divided into three groups: CTRL group, MOD group and HYQT group. The HYQT group received HYQT oral administration twice a day (20.54 g/kg/d), and the plaque formation in ApoE-/- mice was observed using haematoxylin-eosin (HE) staining and oil red O (ORO) staining. The co-localization of aortic macrophages and lipid droplets (LDs) was examined using fluorescent labelling of CD11b and BODIPY fluorescence probe. In vitro, RAW 264.7 cells were exposed to 50 μg/mL ox-LDL for 48 h and then treated with HYQT for 24 h. The accumulation of LDs was evaluated using ORO and BODIPY. Cell viability was assessed using the CCK-8 assay. The co-localization of LC3b and BODIPY was detected via immunofluorescence and fluorescence probe. LysoTracker Red and BODIPY 493/503 were used as markers for lysosomes and LDs, respectively. Autophagosome formation were observed via transmission electron microscopy. The levels of LC3A/B II/LC3A/B I, p-mTOR/mTOR, p-4EBP1/4EBP1, p-P70S6K/P70S6K and TFEB protein level were examined via western blotting, while SQSTM1/p62, Beclin1, ABCA1, ABCG1 and SCARB1 were examined via qRT-PCR and western blotting. The nuclear translocation of TFEB was detected using immunofluorescence. The components of HYQT medicated serum were determined using Q-Orbitrap high-resolution MS analysis. Molecular docking was employed to identify the components of HYQT medicated serum responsible for the mTOR signalling pathway. The mechanism of taurine was illustrated. HYQT has a remarkable effect on atherosclerotic plaque formation and blood lipid level in ApoE-/- mice. HYQT decreased the co-localization of CD11b and BODIPY. HYQT (10% medicated serum) reduced the LDs accumulation in RAW 264.7 cells. HYQT and RAPA (rapamycin, a mTOR inhibitor) could promote cholesterol efflux, while chloroquine (CQ, an autophagy inhibitor) weakened the effect of HYQT. Moreover, MHY1485 (a mTOR agonist) also mitigated the effects of HYQT by reduced cholesterol efflux. qRT-PCR and WB results suggested that HYQT improved the expression of the proteins ABCA1, ABCG1 and SCARB1.HYQT regulates ABCA1 and SCARB1 protein depending on the mTORC1/TFEB signalling pathway. However, the activation of ABCG1 does not depend on this pathway. Q-Orbitrap high-resolution MS analysis results demonstrated that seven core compounds have good binding ability to the mTOR protein. Taurine may play an important role in the mechanism regulation. HYQT may reduce cardiovascular risk by promoting cholesterol efflux and degrading macrophage-derived foam cell formation. It has been observed that HYQT and ox-LDL regulate lipophagy through the mTOR/TFEB signalling pathway, rather than the mTOR/4EBP1/P70S6K pathway. Additionally, HYQT is found to regulate cholesterol efflux through the mTORC1/TFEB/ABCA1-SCARB1 signal axis, while taurine plays a significant role in lipophagy.