Targeted protein degradation (TPD) represents a revolutionary approach to drug discovery, offering a novel mechanism that outperforms traditional inhibitors. This approach employs small molecule drugs to induce the ubiquitination and subsequent degradation of target protein via the proteasome or lysosomal pathways. Key strategies within TPD include proteolysis targeting chimeras (PROTACs), hydrophobic tag tethering degraders (HyTTDs), and molecular glue degraders (MGDs). PROTACs have been undergone clinical evaluations, MGDs have been used in the clinic, and HyTTDs have shown significant progress in cancer treatment. Each of these strategies presents unique advantages and approaches to target protein degradation. This review summarizes five years of research on PROTACs, HyTTDs, and MGDs, highlighting their design principles, advantages, limitations, and future challenges to provide clear guidance and in-depth insights for advancing drug development.

Fucosyltransferase 2 (FUT2) is an enzyme that adds fucose to proteins or lipids via α-1,2-fucosylation in the intestinal mucosa. While FUT2 deficiency is linked to increased susceptibility to inflammatory bowel disease (IBD), its role in colorectal cancer (CRC) is unclear, and the molecular mechanisms involved remain largely unknown. We established an azoxymethane (AOM) and dextran sulfate sodium (DSS) model to induce CRC. FUT2 expression was assessed in human CRC tissues, AOM/DSS-induced mouse models, and CRC cell lines using qRT-PCR, western blotting, and UEA-I staining. FUT2 knockout (FUT2△IEC) mice were treated with AOM/DSS, and FUT2-overexpressing CRC cells were created to evaluate the effects of FUT2 on apoptosis in both in vitro and in vivo settings through Western blot analyses and functional assays. N-glycoproteomics, UEA-I chromatography, and co-immunoprecipitation were utilized to identify regulatory mechanisms and target fucosylated proteins. FUT2 expression and α-1,2-fucosylation were significantly decreased in CRC. FUT2 deficiency worsened AOM/DSS-induced CRC and reduced tumor apoptosis, while FUT2 overexpression induced apoptosis and inhibited proliferation in CRC cells and xenografts. Mechanistically, FUT2 appears to suppress autophagy by impairing lysosomal function and directly targeting and fucosylating LAMP1, contributing to lysosomal dysfunction. Our study reveals a fucosylation-dependent antitumor mechanism of FUT2 in CRC, suggesting potential therapeutic strategies for CRC treatment.

Parkinson's Disease is characterized by selective degeneration of dopaminergic neurons, primarily in the substantia nigra pars compacta, as well as accumulation of alpha-synuclein enriched protein aggregates within neurons. The pathogenesis of PD is still not completely understood, and no treatments exist that alter disease progression. Obvious genetic causes are detected in only a small number of PD patients (5-10 %), suggesting that environmental factors play a significant role the development of PD. Correlative studies suggest that the microbiota could be an important environmental modifier of neurodegeneration. We identified a microbiotal isolate, Actinomyces viscosus, that reduced neurodegeneration in C. elegans expressing a pathological mutant form (G2019S) of leucine-rich repeat kinase 2 (LRRK2) in dopaminergic neurons. A. viscosus also suppressed autophagic dysfunction in these animals and reduced alpha-synuclein aggregation in a synucleinopathy model. Global gene expression analysis revealed increased expression of aspartic cathepsins in response to A. viscosus. Consistent with the involvement of these proteins in neuroprotection, we found that reducing aspartic cathepsin function increased neurodegeneration in the LRRK2 transgenic model. Our findings contribute to the current understanding of how the gut microbiota may influence PD, elucidating one potential mechanism of microbiota-mediated neuroprotection.

The harmful environmental impact of plastic waste has justifiably received substantial attention from the scientific community. In contrast, the toxicological effects of nanoplastics (NP) on aquatic organisms, as well as the potential implications for human health, remain largely unexplored and poorly understood. Despite the growing awareness of plastic pollution, the risks associated with the ubiquitous presence of nanoplastics in our food and beverages are not yet fully recognized. NPs, which are smaller than 1 µm, along with a mixture of MPs and plastic fragments, can find their way into water bodies through various sources and may easily be taken up by aquatic organisms. This paper summarizes the existing literature on NPs bioavailability, their accumulation patterns within the tissues of fish, shellfish, and zooplankton, as well as the influence of biological and environmental factors on NPs absorption from water and diet. Study indicated that the NPs pose significant risks to both aquatic ecosystems and human health due to their ability to bioaccumulate in marine organisms and biomagnify through the food web. It highlighted that various aquatic species can ingest NPs, leading to their distribution across different tissues, which may result in toxic effects such as oxidative stress, DNA damage, and inflammation, as well as impacts on growth and reproduction. The identified critical gaps in current research, particularly regarding the long-term effects of low-dose NP exposure and the need for standardized testing methodologies to ensure comparability across studies. Furthermore, the necessity for further research to understand the pathways through which humans may be exposed to NPs, their toxicokinetics, and the potential implications for chronic health issues. Therefore, more studies are required which employ rigorous and uniform methodologies to fully address NPs as an emerging threat within aquatic ecosystems and food chains; accurately assess related risks with human health together with cumulative toxicity perhaps when combined with other pollutants.

The endo-lysosomal system plays a crucial role in cellular homeostasis by continuously turning over organelles, proteins, and other cargo of intra- or extracellular origin. Moreover, it senses the nutrient status within the cell and can ignite cellular responses by activating or repressing signaling pathways. To enable these roles, lysosomes are fueled by the biosynthetic pathway and receive cargo for degradation by endocytosis and autophagy. Tight regulation and coordination of these distinct trafficking pathways to lysosomes are critical for cellular health. In this review, we explore how these pathways converge at the late stages of the endo-lysosomal system and highlight the role of the HOPS complex as a unifying gatekeeper for trafficking to the lysosome.

Lysosomes and the endoplasmic reticulum (ER) are vital for cellular homeostasis, degradation, and signaling, making them key imaging targets. However, existing fluorescent probes suffer from limitations such as pH sensitivity, poor photostability, and cytotoxicity. To overcome these challenges, we developed two red-emitting fluorophores, DM and MM, based on a rigid DCM scaffold with morpholine linkers. DM rapidly localizes to lysosomes within 10 minutes, exhibiting exceptional photostability, pH insensitivity, and resilience in live and fixed cells. MM initially targets the ER before redistributing to lysosomes, enabling studies of inter-organelle dynamics and lysosomal maturation. Both probes, excitable at 561 nm, emit in the red spectral region, reducing autofluorescence and phototoxicity while allowing deep tissue imaging. DM efficiently tracks lysosomal dynamics under normal and stressed conditions, including mitophagy and lysosome-mitochondria interactions. MM's dual-targeting behavior provides insights into ER-lysosome crosstalk, crucial for cellular signaling. Both dyes exhibit negligible cytotoxicity (up to 100 μM), ensuring prolonged imaging without disrupting the cellular function. Their rigid scaffold imparts high stability, making them versatile tools for studying lysosomal and ER-associated processes. DM and MM set a new standard for dynamic organelle imaging, advancing biomedical research on lysosomal biology and disease mechanisms.

Atherosclerosis, a major cause of global mortality, involves the transformation of macrophages into foam cells, which is a key pathological process. This study aims to elucidate the molecular mechanisms that contribute to foam cell formation and the progression of atherosclerosis.

We performed a comprehensive bioinformatics analysis of transcriptome data to identify differentially expressed genes (DEGs) associated with atherosclerosis. Using the human acute monocytic leukemia cell line THP-1, we established in vitro models of macrophages and foam cells to simulate the atherosclerotic microenvironment. Functional studies were conducted using siRNA-mediated knockdown, real-time PCR, Western blotting, and immunofluorescence imaging. Our results showed that ATP8B2 was significantly down-regulated in atherosclerotic foam cells. The downregulation of ATP8B2 led to impaired lysosomal membrane fusion, evidenced by an increase in CD63-positive compartments without a change in CD63 protein levels. Additionally, under starvation conditions, there was a significant accumulation of autophagosomes, indicating a defect in the autophagy-lysosomal pathway.

This study, for the first time, demonstrates that the downregulation of ATP8B2 exacerbates atherosclerosis by disrupting lysosomal membrane fusion, leading to lipid accumulation and foam cell formation. These findings provide novel insights into the pathogenesis of atherosclerosis and suggest that ATP8B2 could be a potential therapeutic target for the prevention or treatment of this disease.

Macromolecules are incorporated by eukaryotic cells through a process known as endocytosis, intensely analyzed in mammalian and yeast cells, but still lacking deep studies in pathogenic protists. Here we present what is presently known on endocytic activity carried out in some members of the Apicomplexa group (mainly concentrated in Plasmodium and Toxoplasma, but with references to Eimeria, Babesia, and Theileria). In most cells, endocytic activity takes place throughout the cell's surface. In apicomplexans, it is restricted to a special surface structure known as a micropore (also called cytostome). Recent studies indicate that several proteins are in the micropore, playing a role in macromolecule uptake from the medium via budding vesicles.

Tuberculosis is a widely spread disease caused by Mycobacterium tuberculosis (Mtb). The pathogenicity of the pathogen is closely associated with the immune defense mechanisms of the host cells. As key cellular degradation and metabolic centers, lysosomes critically regulate tuberculosis infection. When Mtb invades the host, it is taken up by macrophages and enters phagosomes. Subsequently, the phagosomes fuse with lysosomes and form phagolysosomes, which eliminate the pathogenic bacteria through the acidic environment and hydrolytic enzymes within lysosomes. However, Mtb can interfere with the normal functions of lysosomes through various strategies. It can secrete specific factors (such as ESAT-6, ppk-1, and AcpM) to inhibit the acidification of lysosomes, enzyme activity, and the fusion of phagosomes and lysosomes, thereby enabling Mtb proliferation within host cells. An in-depth exploration of the mechanism of the interaction between Mtb and lysosomes will both uncover bacterial immune evasion strategies and identify novel anti-tuberculosis therapeutic targets.

Neurodegeneration is a devastating manifestation in most lysosomal storage disorders (LSDs). Loss-of-function mutations in CLN1, encoding palmitoyl-protein thioesterase-1 (PPT1), cause CLN1 disease, a devastating neurodegenerative LSD that has no curative treatment. Numerous proteins in the brain require dynamic S-palmitoylation (palmitoylation-depalmitoylation) for trafficking to their destination. Although PPT1 depalmitoylates S-palmitoylated proteins and its deficiency causes CLN1 disease, the underlying pathogenic mechanism has remained elusive. We report that Niemann-Pick C1 (NPC1), a polytopic membrane protein mediating lysosomal cholesterol egress, requires dynamic S-palmitoylation for trafficking to the lysosome. In Cln1-/- mice, Ppt1 deficiency misroutes NPC1-dysregulating lysosomal cholesterol homeostasis. Along with this defect, increased oxysterol-binding protein (OSBP) promotes cholesterol-mediated activation of mechanistic target of rapamycin C1 (mTORC1), which inhibits autophagy contributing to neurodegeneration. Pharmacological inhibition of OSBP suppresses mTORC1 activation, rescues autophagy, and ameliorates neuropathology in Cln1-/- mice. Our findings reveal a previously unrecognized role of CLN1/PPT1 in lysosomal cholesterol homeostasis and suggest that suppression of mTORC1 activation may be beneficial for CLN1 disease.

TNBC remains the most aggressive and therapy-resistant type of breast cancer, for which efficient targeted therapies have not been developed yet. Here, we identified TRPML3 (ML3) as a potential therapeutic target in TNBC. Our data showed that ML3 is significantly upregulated in TNBC cells compared with nontumorigenic control cells. ML3 knockdown (KD) impairs TNBC cell proliferation by inducing cell cycle arrest and caspase-dependent apoptosis. ML3 KD also inhibits TNBC cell migration and invasion. Mechanistically, ML3 KD reduces lysosomal number and enhances lysosomal acidification, which in turn activates mTORC1, thereby inhibiting autophagy initiation and flux. This disruption negatively impacts mitochondrial function, as evidenced by reduced ATP production, increased ROS and NO production, and mitochondrial fragmentation. Importantly, ML3 KD enhances TNBC cell sensitivity to doxorubicin and paclitaxel. The finding suggests that targeting ML3 disrupts lysosomal and mitochondrial homeostasis and enhance chemosensitivity, presenting ML3 as a potential therapeutic vulnerability in TNBC enhancing chemosensitivity.

Membrane-intercalating conjugated oligoelectrolytes (COEs) are lipid-bilayer-spanning molecules that serve as fluorescent dyes for bioimaging. However, COE emission has thus far only been capable of visualizing dye location and their preferential accumulation in different membrane-bound intracellular compartments. Herein, we report the first example of environmentally sensitive COEs for visualizing temporal changes in live cells, providing information on the physical properties of intracellular lipid bilayer membranes. The new COE-BY series is designed around a BODIPY central unit with a membrane-spanning topology and six cationic pendant groups ensuring solubility in aqueous media. These reporters feature high two-photon absorption cross section, NIR-II excitation capabilities under multiphoton excitation, and high dye brightness; all highly desirable photophysical features for bioimaging. The emission lifetime of the probes was sensitive to changes to both the lipid composition of model vesicle systems and membrane tension within cells, induced by either mechanical or osmotic stress. Using two-photon fluorescence lifetime imaging microscopy, it is possible to use the most efficient emitter, namely, COE-BYPhOC4, to image changes in the mechanical properties of intracellular membranes. We show that these COEs remain stably vesicle-bound within the endolysosomal pathway over extended periods, allowing for long-term monitoring of the associated biophysical changes of these vesicles over time.

Adenosine 5'-triphosphate (ATP) plays a pivotal role as an essential intermediate in energy metabolism, influencing nearly all biological metabolic processes. Cancer cells predominantly rely on glycolysis for ATP production, differing significantly from normal cells. Real-time in situ monitoring and rapid response to intracellular ATP levels offers more valuable insights into cancer cell physiology. Herein, we report a novel ratiometric luminescent probe, Ru-Rho, comprised of a ruthenium(II)-based complex and rhodamine 6G (Rho 6G) with excellent water solubility and photostability. Notably, Ru-Rho selectively responds to ATP at acidic conditions, matching the need of monitoring ATP under the acidic intracellular environment of cancer cells. Moreover, the fast ratiometric detection and imaging of ATP under single wavelength excitation improve the detection accuracy. Ru-Rho has been effectively utilized not only for ratio imaging ATP in cells and zebrafish, but also for assessing the efficacy of glycolysis-inhibiting anticancer drugs in intracellular levels, which accelerates the screening process for anticancer drugs and supports the development of new therapeutic agents. The design strategy based on transition metal ruthenium(II) complexes opens a new pathway for constructing ATP luminescent probes, allowing for better adaptation to complex detection requirements.

The aberrant accumulation of cytotoxic protein aggregates is a hallmark of various neurodegenerative and non-neurodegenerative ailments, necessitating the development of sensitive and selective tools for their detection. Herein, we report a series of morpholine-anchored fluorescent probes, denoted as SC-nmor (n = 2, 4, 6), designed for facile visualization of protein aggregates. These probes display notable changes in their photophysical properties upon binding with protein aggregates, owing to their high sensitivity to the fibrillar microenvironment. Specifically, the SC-4mor probe demonstrates strong selectivity for aggregated insulin proteins over native insulin, accompanied by a significant enhancement in fluorescence lifetime. Live-cell imaging reveals an exclusive localization at the lysosomal compartment. This feature enables the visualization of lysosomal accumulated protein fibrils induced with pepstatin A. Additionally, in vivo assessments on genetically mutated and dietary-modified Drosophila melanogaster, representing neurodegenerative and non-neurodegenerative disease models, demonstrate staining of protein aggregates. The enhanced emission from the eye lobes of Aβ-mutated and HSD brain samples, suggesting that SC-4mor can exhibit adequate retention in the brain with minimal biological toxicity. SC-4mor also shows its capability to cross the blood-brain barrier in mice model. Consequently, SC-4mor emerges as a promising marker for detecting and monitoring neurotoxic protein fibrillation in live cells and animal models, offering potential insights into the pathogenesis and progression of protein aggregation.

As an autophagy receptor, SQSTM1/p62 facilitates the degradation of various cytoplasmic components, including proteins, organelles, and pathogens, by mediating interactions between polyubiquitination cargo and autophagosomes. Our study observed an increase in the expression level of SQSTM1/p62 during autophagy induced by Vibrio alginolyticus (V. alginolyticus) in Penaeus vannamei (P. vannamei), contrary to expectations, which promoted an investigation into the role of SQSTM1/p62 in infectious diseases of aquatic animals. Using silencing techniques, we examined the function and regulatory mechanism of SQSTM1/p62 during V. alginolyticus infection. Silencing the Pvp62 gene in P. vannamei and infecting them with V. alginolyticus led to a significant decrease in the survival rate of P. vannamei, indicating its importance in the infection process. Furthermore, Pvp62 silencing was found to affect the lysosome function of P. vannamei. Immunofluorescence analysis showed that silences of Pvp62 inhibited co-localization of LC3 and lamp1 after infection, while overexpression of Pvp62 promoted this process, suggesting that Pvp62 was a necessary condition for autophagosome-lysosome fusion after infection by V. alginolyticus. Importantly, the overexpression of Pvp62 counteracted the inhibitory effect of the autophagy inhibitor chloroquine on autophagosome-lysosome fusion in primary hemocytes of shrimp after infection, underscoring the protective role of Pvp62-mediated autophagosome-lysosome fusion pathway during V. alginolyticus infection.

Cells may be intrinsically fated to die to sculpt tissues during development or to maintain homeostasis. Cells can also die in response to various stressors, injury or pathological conditions. Additionally, cells of the metazoan body are often highly specialized with distinct domains that differ both structurally and with respect to their neighbors. Specialized cells can also die, as in normal brain development or pathological states and their different regions may be eliminated via different programs. Clearance of different types of cell debris must be performed quickly and efficiently to prevent autoimmunity and secondary necrosis of neighboring cells. Moreover, all cells, including those programmed to die, may be subject to various stressors. Some largely unexplored questions include whether predestined cell elimination during development could be altered by stress, if adaptive stress responses exist and if polarized cells may need compartment-specific stress-adaptive programs. We leveraged Compartmentalized Cell Elimination (CCE) in the nematode C. elegans to explore these questions. CCE is a developmental cell death program whereby three segments of two embryonic polarized cell types are eliminated differently. We have previously employed this in vivo genetic system to uncover a cell compartment-specific, cell non-autonomous clearance function of the fusogen EFF-1 in phagosome closure during corpse internalization. Here, we introduce an adaptive response that serves to aid developmental phagocytosis as a part of CCE during stress. We employ a combination of forward and reverse genetics, CRISPR/Cas9 gene editing, stress response assays and advanced fluorescence microscopy. Specifically, we report that, under heat stress, the selective autophagy receptor SQST-1/p62 promotes the nuclear translocation of the oxidative stress-related transcription factor SKN-1/Nrf via negative regulation of WDR-23. This in turn allows SKN-1/Nrf to transcribe lyst-1/LYST (lysosomal trafficking associated gene) which subsequently promotes the phagocytic resolution of the developmentally-killed internalized cell even under stress conditions.

Lysosomal dysfunction has emerged as a central contributor to the pathogenesis of cardiovascular diseases (CVDs), particularly due to its involvement in chronic inflammation, lipid dysregulation, and oxidative stress. This review highlights the multifaceted roles of lysosomes in CVD pathophysiology, focusing on key mechanisms such as NLRP3 inflammasome activation, TFEB-mediated autophagy regulation, ferroptosis, and the role of apolipoprotein M (ApoM) in preserving lysosomal integrity. Additionally, we discuss how impaired lysosomal acidification, mediated by V-ATPase, contributes to lipid-induced cardiac dysfunction. Therapeutically, several pharmacological agents, such as statins, SGLT2 inhibitors, TRPML1 agonists, resveratrol, curcumin, and ferroptosis modulators (e.g., GLS1 activators and icariin), have demonstrated promise in restoring lysosomal function, enhancing autophagic flux, and reducing inflammatory and oxidative injury in both experimental models and early clinical settings. However, key challenges remain, including limitations in drug delivery systems, the absence of lysosome-specific biomarkers, and insufficient clinical validation of these strategies. Future research should prioritize the development of reliable diagnostic tools for lysosomal dysfunction, the optimization of targeted drug delivery, and large-scale clinical trials to validate therapeutic efficacy. Incorporating lysosome-modulating approaches into standard cardiovascular care may offer a new precision medicine paradigm for managing CVD progression.

Manila clam plays a crucial role in China's marine aquaculture industry. However, frequent vibriosis outbreaks severely hinder sustainable and healthy development of the shellfish aquaculture industry. This study indicated markedly decreased clam survival rates after 48 h of Vibrio alginolyticus challenge. Gill and hepatopancreas damage was investigated through histological observation. The activity of lysozyme in the gills and hepatopancreas peaked at 12 and 24 h, respectively. V. alginolyticus showed a maximum bacterial load in the gills and hepatopancreas at 12 and 24 h, respectively. Additionally, transcriptome sequencing of hepatopancreas revealed ten differentially expressed miRNAs in Va and Cn after 48 h infection with V. alginolyticus, corresponding to 100 target genes, with eight upregulated and two downregulated DE miRNAs. Gene ontology (GO) enrichment analysis identified 50 known miRNAs and 111 novel miRNAs, thereby predicting a total of 1840 target genes. KEGG analysis revealed significant changes in multiple signaling pathways, involving lysosomes, apoptosis, amino acid metabolism, and endocytosis, in response to V. alginolyticus stimulation. This study provided new information regarding the immune regulation mechanisms of R. philippinarum in response to V. alginolyticus stress.

Lysosomal dysfunction has been implicated in the progression of colon adenocarcinoma (COAD), yet the prognostic significance and therapeutic potential of lysosome-related genes (LRGs) remain underexplored. In this study, we construct a 6-LRG-based prognostic risk stratification model (DPP7, ADAM8, CD1B, LRP2, ATP6V1C2, and PLAAT3) by integrating LASSO and Cox regression analyses. Stratifying patients based on median risk scores, we demonstrate that high-risk patients exhibit significantly worse clinical outcomes across the TCGA cohort and five independent GEO datasets. Furthermore, this panel outperforms 136 previously published models in terms of predictive accuracy for 1-, 3-, and 5-year survival rates. Validation multiplex immunofluorescence using an in-house tissue microarray cohort confirms the 6-LRG signature serves as an independent prognostic factor. Additionally, high-risk patients exhibit distinct immunosuppressive tumor microenvironment and aggressive malignancy characteristics. Functional depletion of DPP7 significantly inhibits tumor cell proliferation, migration, and metastasis in both in vitro and in vivo settings. Moreover, DPP7 silencing attenuates epithelial-mesenchymal transition, as evidenced by the upregulation of E-cadherin and downregulation of N-cadherin, Vimentin, and Snail. In conclusion, this study establishes an LRG-based model for COAD prognostic prediction and nominates DPP7 as a promising therapeutic target for COAD treatment.

Lysosomes, as crucial organelles within cells, carry out diverse biological functions such as waste degradation, regulation of the cellular environment, and precise control of cell signaling. This paper reviews the core functions and structural characteristics of lysosomes, and delves into the current research status of lysosomes damage repair mechanisms. Subsequently, we explore in depth the close association between lysosomes and various diseases, including but not limited to age-related chronic diseases, neuro-degenerative diseases, tumors, inflammation, and immune imbalance. Additionally, we also provide a detailed discussion of the application of lysosome-targeted substances in the field of regenerative medicine, especially the enormous potential demonstrated in key areas such as stem cell regulation and therapy, and myocardial cell repair. Though the integration of multidisciplinary research efforts, we believe that lysosomes damage repair mechanisms will demonstrate even greater application value in disease treatment and regenerative medicine.

The chemotherapeutic agent cisplatin is widely utilized for the treatment of various solid tumors. However, its clinical utility is limited by nephrotoxicity. Although numerous studies have demonstrated the potential of enhancing macroautophagy/autophagy in alleviating cisplatin-induced acute kidney injury (AKI), the dynamics of the autophagic process during renal tubular injury remain to be elucidated. In our investigation, we observed that cisplatin treatment leads to increased expression of LC3-II, GABARAPL1, SQSTM1/p62 and NBR1 in mouse renal tubular epithelial cells and BUMPT cells. Moreover, ultrastructurally, there is extensive accumulation of autophagic vacuoles in AKI mice. These findings imply that cisplatin-induced AKI results in impaired autophagic flow within renal tubular cells. Furthermore, LGALS3 (galectin 3) was found to be enriched in lysosomes after cisplatin treatment, revealing a close association between autophagy dysfunction and impaired lysosomal membrane integrity. Given the damaging contents of lysosomes, lysosomal membrane permeabilization must be rapidly resolved. Our findings showed that ESCRT III subunit CHMP4A-mediated lysosomal membrane repair significantly ameliorates autophagic defects and protects against lysosomal damage-induced cell death in a cisplatin-induced AKI model. In conclusion, our study indicates that ESCRT III-mediated lysosomal repair can relieve cisplatin-induced cell apoptosis and restore normal autophagy function in renal tubular epithelial cells. This mechanism plays a protective role against cisplatin-induced AKI.Abbreviations: AAV: adeno-associated virus; AKI: acute kidney injury; CQ: chloroquine; ESCRT: endosomal sorting complex required for transport; LMP: lysosomal membrane permeabilization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PAS: periodic acid Schiff; PTECs: proximal renal tubule epithelial cells; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Cathepsin B is a member of the cysteine protease family and plays an important role in the innate immunity of aquatic invertebrates. A previous study identified that Cathepsin B-like (CTSB-l) may be involved in the response of alkali treatment in Macrobrachium nipponense. The present study aims to identify the potential regulatory roles of CTSB-l in the response of alkali treatment in M. nipponense through performing the quantitative real-time PCR analysis (qPCR), in situ hybridization (ISH) analysis, and RNA interference (RNAi) analysis. The full length of the MnCTSB-l cDNA was 1272 bp with an open reading frame of 987 bp, encoding 328 amino acids. Phylogenetic tree analysis indicated that the amino acid sequence of MnCTSB-l is highly homologous to those of crustacean cathepsin B-like. qPCR analysis showed that MnCTSB-l mRNA is expressed in all tested tissues with the highest level of expression in hepatopancreas in both male and female prawns. The expressions of MnCTSB-l were significantly stimulated in gills under the alkali concentration of both 5 mmol/L and 10 mmol/L, predicting that this gene may be involved in the response of alkali treatment in M. nipponense, which was consistent with the previous study. ISH showed that MnCTSB-l signals were mainly observed in the hemolymph vessels and membranes of gills, as well as in the basement membranes of hepatopancreas, in both male and female prawns. RNAi analysis revealed that the injection of double-stranded RNA of CTSB (dsCTSB) resulted in a significant decrease in MnCTSB-l expressions. In addition, prawn cumulative mortality was significantly higher in the dsCTSB-injected group, compared to that of dsGFP-injected group, under alkali treatments of both 5 mmol/L and 10 mmol/L, indicating CTSB-l plays an essential role in regulating alkalinity acclimation in M. nipponense. The present study identifies the regulatory functions of CTSB-l in the response of alkali treatment in M. nipponense, promoting the survival rate and aquaculture of this species in a water environment with high alkalinity.

Ischemic brain injury occurs in many clinical settings, including stroke, cardiac arrest, hypovolemic shock, cardiac surgery, cerebral edema, and cerebral vasospasm. Decades of work have revealed many important mechanisms related to ischemic brain injury. However, there remain significant gaps in the scientific knowledge to reconcile many ischemic brain injury events. Brain ischemia leads to protein misfolding and aggregation, and damages almost all types of subcellular organelles including mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, etc. Irreparably damaged organelles and insoluble protein aggregates are normally removed by autophagy. The build-up of common autophagic components, such as LC3, p62, and ubiquitinated proteins, are generally observed in brain tissue samples in animal models of both global and focal brain ischemia, but the interpretation of the role of these autophagy-related changes in ischemic brain injury in the literature has been controversial. Many pathological events or mechanisms underlying dysfunctional autophagy after brain ischemia remain unknown. This review aims to provide an update of the current knowledge and future research directions regarding the critical role of dysfunctional autophagy in ischemic brain injury.

Vacuolar protein sorting 41 (VPS41), a component of the homotypic fusion and protein sorting (HOPS) complex for lysosomal fusion, is essential for the trafficking of lysosomal membrane proteins via lysosome-associated membrane protein (LAMP) carriers from the trans-Golgi network (TGN) to endo/lysosomes. However, the molecular mechanisms underlying this pathway and VPS41's role herein remain poorly understood. Here, we investigated the effects of ectopically localizing VPS41 to mitochondria on LAMP distribution. Using electron microscopy, we identified that mitochondrial-localized VPS41 recruited LAMP1- and LAMP2A-positive vesicles resembling LAMP carriers. The retention using selective hooks (RUSH) system further revealed that newly synthesized LAMPs were specifically recruited by mitochondrial VPS41, a function not shared by other HOPS subunits. Notably, we identified the small GTPase Arl8b as a critical factor for LAMP carrier trafficking. Arl8b was present on LAMP carriers and bound to the WD40 domain of VPS41, enabling their recruitment. These findings reveal a unique role of VPS41 in recruiting TGN-derived LAMP carriers and expand our understanding of VPS41-Arl8b interactions beyond endosome-lysosome fusion, providing new insights into lysosomal trafficking mechanisms.

3-Phosphoinositides (3-PIs), phosphatidylinositol (3,4) bisphosphate [PI(3,4)P2] and phosphatidylinositol (3,4,5) trisphosphate (PIP3), are important lipid second messengers in the Phosphoinositide 3-Kinase (PI3K)/Akt signaling pathway, which is crucial to cell growth and frequently dysregulated in cancer. Emerging evidence suggests these lipid second messengers may be present in membranes beyond the plasma membrane, yet their spatial regulation within other membrane compartments is not well understood. To dissect the spatial regulation of specific 3-PI species, we developed genetically encodable biosensors with selectivity for PIP3 or PI(3,4)P2. Using these biosensors, we showed that PIP3 significantly accumulated at the lysosome upon growth factor stimulation, in contrast to the conventional view that PIP3 is exclusively present in the plasma membrane. Furthermore, we showed that lysosomal PIP3 originates from the plasma membrane and relies on dynamin-dependent endocytosis for lipid internalization. Thus, PIP3 can exploit dynamic trafficking pathways to access subcellular compartments and regulate signaling in a spatially selective manner.

Benzo[ghi] perylene (b[ghi]p) is classified as non-carcinogenic to humans, and there are currently no occupational exposure models available to identify its effects. The aim of this work was to evaluate the effect of b[ghi]p on the lysosomes of NL-20 cells (a human bronchial cell line) exposed to 4.5 μM for 3 h. The effect was evaluated through an ultrastructural evaluation, morphological changes, and acridine orange staining of lysosomes. Superoxide was quantified; and SOD1, cathepsin B, LAMP1, galectin-3 and LC3α/β, and Rab7 expression was evaluated by immunocytochemistry. The expression of genes related to oxidative stress responses (NRF2, NQO1, HMOX1 and PRDX1) and genes related to autophagy (ULK1, ATG9, BCN1, VMP1, TMEM41B and p62) were quantified by RT-qPCR. The ultrastructural evaluation revealed an increase in autophagic vesicles and phagophores in cells exposed to b[ghi]p, as well as vesicles derived from mitochondria. Based on morphology, there were vesicles in the cytoplasm. B[ghi]p significantly decreased the number of lysosomes (p < 0.05), and NAC reverse this effect (p < 0.05). Superoxide production was observed from 30 min to 3 h (p < 0.05). Immunocytochemistry revealed increased galectin-3 and LC3α/β. All oxidative stress-related genes showed high expression (p < 0.05), and the expression of ATG9 gene was decreased (p < 0.05). These results demonstrate that b[ghi]p induces oxidative stress, responsible for producing the toxic effects in the lysosomes of NL-20 cells.

Gene delivery systems must overcome multiple barriers, with endosomal escape representing a prominent obstacle. We have previously shown that a bacterial phospholipase C (PLC) enabled endosomal escape of a non-viral protein-based DNA delivery system termed TFAMoplex. Building upon this, this work introduces a calcium-responsive system designed to enhance endosomal escape through non-covalent capturing of PLC to the TFAMoplex followed by its release within endosomes and nanobody-mediated targeting to the endosomal membrane. This approach leads to improved TFAMoplexes enabling transfection of HeLa cells in full serum with a half maximal effective concentration (EC50) of less than 200 ng DNA per mL serum, using only 5 nM PLC. Particularly, the modular capture, release and targeting system could potentially be adapted to other delivery agents previously constrained by poor endosomal escape. These findings present a promising strategy to achieve efficient endosomal escape, offering prospects for improved delivery of macromolecules, in particular nucleic acids.

Metarhizium spp. have emerged as an alternative to chemical pesticides for protecting crops from insect pest. Here, we investigated midgut microbial community and metabolites of Spodoptera litura at three different timepoints after infection with Metarhizium flavoviride. The innate immune system of S. litura was activated with levels of polyphenol oxidase, carboxylesterase, multifunctional oxidase, and glutathione S-transferase activity significantly increasing. Exposure to the fungal pathogen also altered bacterial abundance and diversity in host's midgut, and these changes varied depending on the time elapsed since exposure. We identified more operational taxonomic units in the treated samples as compared to the control samples at all tested time points. A total of 372 metabolites were identified, and 88, 149, and 142 differentially accumulated metabolites (DAMs) were identified between the treatment and control groups at 3 timepoints after treatment, respectively. Based on the changes of DAMs in response to M. flavoviride infection at different timepoints and significantly enriched KEGG pathways, we speculated that "tyrosine metabolism," "galactose metabolism," "ATP-binding cassette transporters," "neuroactive ligand-receptor interaction," "purine metabolism," "arginine and proline metabolism," "beta-alanine metabolism," "lysosome," and "carbon metabolism" may participate in the metabolic-level defense response. An integrated pathway-level analysis of the 16S-rDNA and metabolomic data illustrated the connections and interdependencies between the metabolic responses of S. litura and the midgut microorganisms to M. flavoviride infection. This work emphasizes the value of integrated analyses of insect-pathogen interactions, provides a framework for future studies of critical microorganisms and metabolic determinants of these interactions, establishes a theoretical basis for the sustainable use of M. flavoviride.

Lysosomal enzyme replacement therapy (ERT) holds potential for treating lysosomal storage disorders, but achieving targeted delivery of deficient therapeutic enzymes remains a significant challenge. This study presents a novel approach for the lysosome-specific delivery of the β-glucosidase (B8CYA8) enzyme by covalently conjugating lysosome-targeting mannose-6-phosphate functionalized glycopolypeptides (M6P-GP). We used a protein-glycopolypeptide conjugate developed through advanced protein engineering and bioconjugation techniques. By conjugating β-glucosidase to M6P-GP that has a high affinity for the cation-independent mannose-6-phosphate receptors (CI-MPR) and lysosomal receptors, we enhance the enzyme's selective intracellular uptake and lysosome-specific localization. To attain maximum activity of the near-native enzyme after delivery, we have designed and synthesized an acetal linkage containing the pH-responsive linker maleimide-acetal-azide (MAA), which will cleave in the lysosomal acidic pH to detach the glycopolypeptide from the protein backbone. We demonstrated the efficient cellular uptake of the protein-glycopolypeptide conjugate and showed targeted lysosome delivery, leading to increased enzymatic activity compared to untreated cells. Our results proved that the approach mainly improves the specificity and efficiency of enzyme delivery, particularly into lysosomes, which may enable new methods for ERT. These findings suggest that protein-glycopolypeptide conjugates could represent a class of bioconjugates to design targeted enzyme therapies, offering a pathway to the effective treatment of Gaucher disease (GD) and potentially other related lysosomal storage disorders.

Iridium(III) complexes, particularly those with piano-stool structures, have drawn a lot of interest recently as possible anticancer drugs. These complexes, which have displayed enhanced cytotoxicity and cytoselectivity compared with clinically approved drugs like cisplatin, oxaliplatin, and carboplatin, hold promising prospects for further anticancer research. Our review aims to explore the complex interplay between cytotoxic properties, cellular uptake efficiency, and intracellular distribution properties of this class of Ir(III) complexes, considering the variation of the coordination site atoms. We provide an overview of the majority of research on mono- and polynunclear half-sandwich Ir(III) complexes with mono- and bidentate ligands, focusing on the impact of altering the leaving group, tethers, substituents on the cyclopentadienyl ring and ligand, spacers, and counter ions on the cytotoxicity and mode of action.

Proteins are involved in multiple biological functions. High-throughput technologies have allowed the measurement of thousands of proteins in population biobanks. In this study, we aimed to identify proteins related to Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis by leveraging large-scale genetic and proteomic data. We performed a two-sample cis Mendelian randomization study by selecting instrumental variables for the abundance of >2700 proteins measured by either Olink or SomaScan platforms in plasma from the UK Biobank and the deCODE Health Study. We also used the latest publicly available genome-wide association studies for the neurodegenerative diseases of interest. The potentially causal effect of proteins on neurodegenerative diseases was estimated based on the Wald ratio. We tested 13 377 protein-disease associations, identifying 169 associations that were statistically significant (5% false discovery rate). Evidence of co-localization between plasma protein abundance and disease risk (posterior probability > 0.80) was identified for 61 protein-disease pairs, leading to 50 unique protein-disease associations. Notably, 23 of 50 protein-disease associations corresponded to genetic loci not previously reported by genome-wide association studies. The two-sample Mendelian randomization and co-localization analysis also showed that APOE abundance in plasma was associated with three subcortical volumes (hippocampus, amygdala and nucleus accumbens) and white matter hyper-intensities, whereas PILRA and PILRB abundance in plasma was associated with caudate nucleus volume. Our study provided a comprehensive assessment of the effect of the human proteome that is currently measurable through two different platforms on neurodegenerative diseases. The newly associated proteins indicated the involvement of complement (C1S and C1R), microglia (SIRPA, SIGLEC9 and PRSS8) and lysosomes (CLN5) in Alzheimer's disease; the interleukin-6 pathway (CTF1) in Parkinson's disease; lysosomes (TPP1), blood-brain barrier integrity (MFAP2) and astrocytes (TNFSF13) in amyotrophic lateral sclerosis; and blood-brain barrier integrity (VEGFB), oligodendrocytes (PARP1), node of Ranvier and dorsal root ganglion (NCS1, FLRT3 and CDH15) and the innate immune system (CR1, AHSG and WARS) in multiple sclerosis. Our study demonstrates how harnessing large-scale genomic and proteomic data can yield new insights into the role of the plasma proteome in the pathogenesis of neurodegenerative diseases.

Cells and tissues turn over their aged and damaged components in order to adapt to a changing environment and maintain homeostasis. These functions rely on lysosomes, dynamic and heterogeneous organelles that play essential roles in nutrient redistribution, metabolism, signaling, gene regulation, plasma membrane repair, and immunity. Because of metabolic fluctuations and pathogenic threats, lysosomes must adapt in the short and long term to maintain functionality. In response to such challenges, lysosomes deploy a variety of mechanisms that prevent the breaching of their membrane and escape of their contents, including pathogen-associated molecules and hydrolases. While transient permeabilization of the lysosomal membrane can have acute beneficial effects, supporting inflammation and antigen cross-presentation, sustained or repeated lysosomal perforations have adverse metabolic and transcriptional consequences and can lead to cell death. This review outlines factors contributing to lysosomal stress and damage perception, as well as remedial processes aimed at addressing lysosomal disruptions. We conclude that lysosomal stress plays widespread roles in human physiology and pathology, the understanding and manipulation of which can open the door to novel therapeutic strategies.

Lipid based nanocarriers are a commonly used drug delivery system with cargos ranging from small molecules to complex RNA-based therapies. There are several hypotheses how such carriers can enter the cell, in which organelles they reside, and how they cross or escape the endo-lysosomal system. To provide additional insights, the cell-nanocarrier interplay was visualized exemplarily with lipid-based nanocarriers and macrophage-like cultured cells (J774A.1 cells) using high resolution electron microscopy. Nanocarrier uptake into J774A.1 cells was detectable after the first 15 min by intracellular accumulation of electron-dense material. These accumulations were identified as lysosomes and lipid droplets, indicating complete degradation and a subsequent formation of storage organelles as early as 15 min. Inhibition of lysosomal acid lipase did not block lipid droplet formation, but rather resulted in accumulation of lipid droplets within lysosomes. This suggests that other cellular lipases already degrade acylglycerols before they reach lysosomes. Chloroquine co-treatment allowed visualization of nanocarriers inside endosomal vesicles, multivesicular bodies, and lysosomes.

Healthy cells need functional lysosomes to degrade cargo delivered by autophagy and endocytosis. Defective lysosomes can lead to severe conditions such as lysosomal storage diseases (LSDs) and neurodegeneration. To maintain lysosome integrity and functionality, cells have evolved multiple quality control pathways corresponding to different types of stress and damage. These can be divided into five levels: regulation, reformation, repair, removal, and replacement. The different levels of lysosome quality control often work together to maintain the integrity of the lysosomal network. This review summarizes the different quality control pathways and discusses the less-studied area of lysosome membrane protein regulation and degradation, highlighting key unanswered questions in the field.Abbreviation: ALR: autophagic lysosome reformation; CASM: conjugation of ATG8 to single membranes: ER: endoplasmic reticulum; ESCRT: endosomal sorting complexes required for transport; ILF: intralumenal fragment; LSD: lysosomal storage disease; LYTL: lysosomal tubulation/sorting driven by LRRK2; PITT: phosphoinositide-initiated membrane tethering and lipid transport; PE: phosphatidylethanolamine; PLR: phagocytic lysosome reformation; PS: phosphatidylserine; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; PtdIns(4,5)P2: phosphatidylinositol-4,5-bisphosphate; V-ATPase: vacuolar-type H+-translocating ATPase.

In the dynamic realm of translational nanorobotics, the endeavor to develop nanorobots carrying therapeutics in rational in vivo applications necessitates a profound understanding of the biological landscape of the human body and its complexity. Within this landscape, biological membranes stand as critical barriers to the successful delivery of therapeutic cargo to the target site. Their crossing is not only a challenge for nanorobotics but also a pivotal criterion for the clinical success of therapeutic-carrying nanorobots. Nevertheless, despite their urgency, strategies for membrane crossing in translational nanorobotics remain relatively underrepresented in the scientific literature, signaling an opportunity for further research and innovation. This review focuses on nanorobots with various propulsion mechanisms from chemical and physical to hybrid mechanisms, and it identifies and describes four essential biological membranes that represent the barriers needed to be crossed in the therapeutic journey of nanorobots in in vivo applications. First is the entry point into the blood stream, which is the skin or mucosa or intravenous injection; next is the exit from the bloodstream across the endothelium to the target site; further is the entry to the cell through the plasma membrane and, finally, the escape from the lysosome, which otherwise destroys the cargo. The review also discusses design challenges inherent in translating nanorobot technologies to real-world applications and provides a critical overview of documented membrane crossings. The aim is to underscore the need for further interdisciplinary collaborations between chemists, materials scientists and chemical biologists in this vital domain of translational nanorobotics that has the potential to revolutionize the field of precision medicine.

The role of hypoxia-inducible factor 2α (HIF2α) in clear cell Renal Cell Carcinoma (ccRCC) is still not fully understood. In this study, we identified that urinary prolegumain levels positively correlated with the malignant characteristics of ccRCC. In cultured 786-O and OSRC-2 cells, HIF2α downregulation reduced prolegumain secretion. RNA sequencing assay revealed that HIF2α induces methylation-controlled J (MCJ), a negative regulator on the mitochondrial respiratory chain. Silencing MCJ reduced prolegumain secretion, and MCJ overexpression restored prolegumain secretion inhibited by HIF2α downregulation. Chromatin immunoprecipitation and luciferase assay confirmed MCJ as a transcription target of HIF2α. Furthermore, we showed the ectopic MCJ overexpression reversed the improved mitochondrial damage resulting from HIF2α downregulation, as evidenced by electron microscope, ATP level, GSSG/GSH ratio, MitoSOX, and DHE staining. Through mass spectrometry analysis, we identified oxidation site His343 on the legumain sequence as contributing to the prolegumain secretion. Therapeutically, silencing MCJ or HIF2α or using ROS scavengers Vitamin C or MitoQ alleviated MMP2 activation as well as cell migration and tube formation. In a mouse orthotopic xenograft model of ccRCC, silencing MCJ or administration of MitoQ significantly protected against mitochondrial damage and subsequently reduced the lung metastasis of tumors. Overall, our study identified MCJ as a target molecule of HIF2α in ccRCC. Silencing MCJ or using ROS scavengers like MitoQ can suppress oxidation site His343 in legumain, preventing prolegumain secretion and subsequently reducing metastasis of ccRCC.

Drug discovery plays a crucial role in medicinal chemistry, serving as the cornerstone for developing new treatments to address a wide range of diseases. This review emphasizes the significance of advanced strategies, such as Click Chemistry, Targeted Protein Degradation (TPD), DNA-Encoded Libraries (DELs), and Computer-Aided Drug Design (CADD), in boosting the drug discovery process. Click Chemistry streamlines the synthesis of diverse compound libraries, facilitating efficient hit discovery and lead optimization. TPD harnesses natural degradation pathways to target previously undruggable proteins, while DELs enable high-throughput screening of millions of compounds. CADD employs computational methods to refine candidate selection and reduce resource expenditure. To demonstrate the utility of these methodologies, we highlight exemplary small molecules discovered in the past decade, along with a summary of marketed drugs and investigational new drugs that exemplify their clinical impact. These examples illustrate how these techniques directly contribute to advancing medicinal chemistry from the bench to bedside. Looking ahead, Artificial Intelligence (AI) technologies and interdisciplinary collaboration are poised to address the growing complexity of drug discovery. By fostering a deeper understanding of these transformative strategies, this review aims to inspire innovative research directions and further advance the field of medicinal chemistry.

BORC (BLOC-one-related complex) is a hetero-octameric complex, consisting of eight coiled-coil proteins (BORCS1-8). BORC controls lysosomal and synaptic vesicle transport and positioning by recruiting ARL8. The structural mechanisms underlying BORC assembly and ARL8 activation remain unclear. Here, we reconstitute and construct the structural model of this hetero-octameric complex. We find that BORC adopts an extended, rod-like structure made of coiled coils. Two hemicomplexes, each containing four subunits, are joined end-to-end to form the holocomplex. Within each hemicomplex, BORCS1/4/6/8 or BORCS2/3/5/7 assembles into similar helical bundles. We further study how BORC is built and discover a hierarchical assembly process in which BORCS1/2/3/5 forms the core scaffold and recruits other subunits. Mutations in the inter-hemicomplex interfaces result in two hemicomplexes. The association of ARL8 may require the proper assembly of BORC and is primarily mediated by BORCS5. These results provide guidance for further understanding of the biology of BORC.

Myocardial ischemia/reperfusion (IR) injury is a common adverse event in the clinical treatment of myocardial ischemic disease. Autosis is a form of cell death that occurs when autophagy is excessive in cells, and it has been associated with cardiac IR damage. This study aimed to investigate the regulatory mechanism of circRNA CDR1AS on autosis in cardiomyocytes under IR. The expression of CDR1AS increases after myocardial IR, and overexpression of CDR1AS detrimentally affects cardiac function, increases infarct area, promotes excessive autophagy, and blocks the flow of autophagy to induce autosis after IR. Conversely, knockdown of CDR1AS reversed the autophagy-related markers caused by IR, increasing cardiomyocyte activity, improving cardiac dysfunction and infarct area, and restoring the flow of autophagy. Further analysis of RNA sequencing and validation experiments revealed that CDR1AS aggravated autophagic damage, increased autophagosome accumulation, and promoted autosis by inhibiting the levels of LAMP2 and mTORC1 proteins. Additionally, RIP and pull-down assays showed that CDR1AS interacts with LAMP2 or mTORC1. First-time evidence reveals that circRNA CDR1AS regulates lysosomal membrane proteins by regulating the mTORC1/ULK1 pathway during myocardial IR-induced autosis. This suggests that maintaining moderate autophagy is a crucial part of the fight against myocardial IR damage. KEY MESSAGES: CDR1AS expression was significantly increased in myocardium following IR. CDR1AS can increase the occurrence of autosis after IR. CDR1AS reduces the phosphorylation of ULK1, promoting the formation of autophagosomes. CDR1AS binds to LAMP2 and blocks the autophagosome clearance pathway. The specific mechanism of CDR1AS regulating IR is achieved by regulating autosis.

Lysophagy eliminates damaged lysosomes and is crucial to cellular homeostasis; however, its underlying mechanisms are not entirely understood. We screen a ubiquitination-related compound library and determine that the substrate recognition component of the SCF-type E3 ubiquitin ligase complex, SCFFBXO3(FBXO3), which is a critical lysophagy regulator. Inhibition of FBXO3 reduces lysophagy and lysophagic flux in response to L-leucyl-L-leucine methyl ester (LLOMe). Furthermore, FBXO3 interacts with TMEM192, leading to its ubiquitination in LLOMe-treated cells. We also identify TAX1BP1 as a critical autophagic adaptor that recognizes ubiquitinated TMEM192 during lysophagy and find that TBK1 activation is crucial for lysophagy, as it phosphorylates FBXO3 in response to lysosomal damage. Knockout of FBXO3 significantly impairs lysophagy, and its reconstitution with a loss-of-function mutant (V221I) further confirms its essential role in lysophagy regulation. Collectively, our findings highlight the significance of the TBK1-FBXO3-TMEM192-TAX1BP1 axis in lysophagy and emphasize the critical role of FBXO3 in lysosomal integrity.

Pathological cardiac remodeling is a maladaptive response that leads to changes in the size, structure, and function of the heart. These changes occur due to an acute or chronic stress on the heart and involve a complex interplay of hemodynamic, neurohormonal and molecular factors. As a critical regulator of cell growth, protein synthesis and autophagy mechanistic target of rapamycin complex 1 (mTORC1) is an important mediator of pathological cardiac remodeling. The tumor suppressor folliculin (FLCN) is part of the network regulating non-canonical mTORC1 activity. FLCN activates mTORC1 by functioning as a guanosine triphosphatase activating protein (GAP). Our work has identified a regulatory upstream open reading frame (uORF) localized in the 5'UTR of the FLCN mRNA. These small genetic elements are important regulators of protein expression. They are particularly important for the regulation of stress-responsive protein synthesis. We have studied the relevance of the FLCN uORF in the regulation of FLCN translation. We show that FLCN downregulation through the uORF is linked to cardiomyocyte growth and increased lysosomal activity. In summary, we have identified uORF-mediated control of RNA translation as another layer of regulation in the complex molecular network controlling cardiomyocyte hypertrophy.