A dual-channel fluorescent probe targeting lysosomes for differential detection of Cys/Hcy and GSH: Applications in food, pharmaceutical analysis and bioimaging.

Thiols function as antioxidants in food, prolonging shelf life and enhancing flavor. Moreover, thiols are vital biomolecules involved in enzyme activity, cellular signal transduction, and protein folding among critical biological processes. In this paper, the fluorescent probe PYL-NBD was designed and synthesized, which utilized the fluorescent molecule pyrazoline, the lysosome-targeted morpholine moiety, and the sensing moiety NBD. Probe PYL-NBD was tailored for the recognition of biothiols through single-wavelength excitation, yielding distinct fluorescence emission signals: blue for Cys, Hcy, and GSH; green for Cys, Hcy. Probe PYL-NBD exhibited rapid reaction kinetics (<10 min), distinct fluorescence response signals, and low detection limits (15.7 nM for Cys, 14.4 nM for Hcy, and 12.6 nM for GSH). Probe PYL-NBD enabled quantitative determination of Cys content in food samples and L-cysteine capsules. Furthermore, probe PYL-NBD had been successfully applied for confocal imaging with dual-channel detection of biothiols in various biological specimens, including HeLa cells, zebrafish, tumor sections, and Arabidopsis thaliana.

GSK-3ß inhibitor amplifies autophagy-lysosomal pathways by regulating TFEB in Parkinson's disease models.

Parkinson's disease (PD), a common neurodegenerative disorder characterized by degeneration of the substantia nigra and a marked increase in Lewy bodies in the brain, primarily manifests as motor dysfunction. Glycogen synthase kinase-3 beta (GSK-3ß) is known to play a critical role in various pathological processes of neurodegenerative diseases. However, the impact of GSK-3ß inhibitors on PD progression and the underlying molecular mechanisms responsible for the effects have not been fully elucidated. Using in vitro and mouse models of 1-methyl-4-phenylpyridine (MPP+)-or methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD, we found that inhibition of GSK-3ß activity alleviated mitochondrial damage, cell apoptosis, and neuronal cell loss by promoting the nuclear translocation of transcription factor EB (TFEB), thereby amplifying the autophagy-lysosomal pathway (ALP). Importantly, siRNA silencing of the TFEB gene impaired the GSK-3ß inhibitor-mediated activation of the ALP pathway, thus negating the metabolic support required for neuronal functional improvement. Short-term treatment with the GSK-3ß inhibitor significantly ameliorated motor dysfunction and improved motor coordination in model mice with MPTP-induced PD. GSK-3ß inhibition increased the ALP and TFEB activities in the mice, thereby reducing α-synuclein aggregation and neuronal damage. In conclusion, our study demonstrates that inhibition of GSK-3ß activity can delay the pathological processes of PD via promotion of the TFEB-ALP pathway, potentially providing a novel therapeutic target for this neurodegenerative disorder.

The European reference network for metabolic diseases (MetabERN) clinical pathway recommendations for Pompe disease (acid maltase deficiency, glycogen storage disease type II).

Clinical pathway recommendations (CPR) are based on existing guidelines and deliver a short overview on how to deal with a specific diagnosis, resulting therapy and follow-up. In this paper we propose a methodology for developing CPRs for Pompe disease, a metabolic myopathy caused by deficiency of lysosomal acid alpha-glucosidase. The CPR document was developed within the activities of the MetabERN, a non-profit European Reference Network for Metabolic Diseases established by the European Union. A working group was selected among members of the MetabERN lysosomal storage disease subnetwork, with specific expertise in the care of Pompe disease, and patient support group representatives. The working strategy was based on a systematic literature search to develop a database, followed by quality assessment of the studies selected from the literature, and by the development of the CPR document according to a matrix provided by MetabERN. Quality assessment of the literature and collection of citations was conducted according to the AGREE II criteria and Grading of Recommendations, Assessment, Development and Evaluation methodology. General aspects were addressed in the document, including pathophysiology, genetics, frequency, classification, manifestations and clinical approach, laboratory diagnosis and multidisciplinary evaluation, therapy and supportive measures, follow-up, monitoring, and pregnancy. The CPR document that was developed was intended to be a concise and easy-to-use tool for standardization of care for patients among the healthcare providers that are members of the network or are involved in the care for Pompe disease patients.

From LAL-D to MASLD: Insights into the role of LAL and Kupffer cells in liver inflammation and lipid metabolism.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent liver pathology worldwide, closely associated with obesity and metabolic disorders. Increasing evidence suggests that macrophages play a crucial role in the development of MASLD. Several human studies have shown an inverse correlation between circulating lysosomal acid lipase (LAL) activity and MASLD. LAL is the sole enzyme known to degrade cholesteryl esters (CE) and triacylglycerols in lysosomes. Consequently, these substrates accumulate when their enzymatic degradation is impaired due to LAL deficiency (LALD). This study aimed to investigate the role of hepatic LAL activity and liver-resident macrophages (i.e., Kupffer cells (KC)) in MASLD. To this end, we analyzed lipid metabolism in hepatocyte-specific (hep)Lal-/- mice and depleted KC with clodronate treatment. When fed a high-fat/high-cholesterol diet (HF/HCD), hepLal-/- mice exhibited CE accumulation and an increased number of macrophages in the liver and significant hepatic inflammation. KC were depleted upon clodronate administration, whereas infiltrating/proliferating CD68-expressing macrophages were less affected. This led to exacerbated hepatic CE accumulation and dyslipidemia, as evidenced by increased LDL-cholesterol concentrations. Along with proteomic analysis of liver tissue, these findings indicate that hepatic LAL-D in HF/HCD-fed mice leads to macrophage infiltration into the liver and that KC depletion further exacerbates hepatic CE concentrations and dyslipidemia.

The Age pigment lipofuscin causes oxidative stress, lysosomal dysfunction, and pyroptotic cell death.

Accumulation of the age pigment lipofuscin represents a ubiquitous hallmark of the aging process. However, our knowledge about cellular effects of lipofuscin accumulation is potentially flawed, because previous research mainly utilized highly artificial methods of lipofuscin generation. In order to address this tremendous problem, we developed a convenient protocol for isolation of authentic lipofuscin from human and equine cardiac tissue in high purity and quantity. Isolated lipofuscin aggregates contained elevated concentrations of proline and metals such as calcium or iron. The material was readily incorporated by fibroblasts and caused cell death at low concentrations (LC50 = 5.0 µg/mL) via a pyroptosis-like pathway. Lipofuscin boosted mitochondrial ROS production and caused lysosomal dysfunction by lysosomal membrane permeabilization leading to reduced lysosome quantity and impaired cathepsin D activity. In conclusion, this is the first study utilizing authentic lipofuscin to experimentally validate the concept of the lysosomal-mitochondrial axis theory of aging and cell death.

Intelligent nanocatalyst mediated lysosomal ablation pathway to coordinate the amplification of tumor treatment.

The production of reactive oxygen species (ROS) is susceptible to external excitation or insufficient supply of related participants (e.g., hydrogen peroxide (H2O2) and sensitizer), liming ROS-driven tumor treatment. Additionally, the lysosomal retention effect severely hinders the utilization of ROS-based nanosystems and severely restricted the therapeutic effect of tumors. Therefore, first reported herein an intelligent nanocatalyst, TCPP-Cu@MnOx ((MnII)1(MnIII)2.1(MnIV)2.6O9.35), and proposed a programmed ROS amplification strategy to treat tumors. Initially, the acidity-unlocked nanocatalyst was voluntarily triggered to generate abundant singlet oxygen (1O2) to mediate acid lysosomal ablation to assist nanocatalyst escape and partially induce lysosomal death, a stage known as lysosome-driven therapy. More unexpectedly, the high-yielding production of 1O2 in acid condition (pH 5.0) was showed compared to neutral media (pH 7.4), with a difference of about 204 times between the two. Subsequently, the escaping nanocatalyst further activated H2O2-mediated 1O2 and hydroxyl radical (•OH) generation and glutathione (GSH) consumption for further accentuation tumor therapy efficiency, which is based on the Fenton-like reaction and Russell reaction mechanisms. Therefore, in this system, a program-activatable TCPP-Cu@MnOx nanocatalyst, was proposed to efficiently destruct organelle-lysosome via 1O2 inducing, and stimulated H2O2 conversion into highly toxic 1O2 and •OH in cytoplasm, constituting an attractive method to overcome limitations of current ROS treatment.

Cipaglucosidase alfa plus miglustat: linking mechanism of action to clinical outcomes in late-onset Pompe disease.

Enzyme replacement therapy (ERT) is the only approved disease-modifying treatment modality for Pompe disease, a rare, inherited metabolic disorder caused by a deficiency in the acid α-glucosidase (GAA) enzyme that catabolizes lysosomal glycogen. First-generation recombinant human GAA (rhGAA) ERT (alglucosidase alfa) can slow the progressive muscle degeneration characteristic of the disease. Still, most patients experience diminished efficacy over time, possibly because of poor uptake into target tissues. Next-generation ERTs aim to address this problem by increasing bis-phosphorylated high mannose (bis-M6P) N-glycans on rhGAA as these moieties have sufficiently high receptor binding affinity at the resultant low interstitial enzyme concentrations after dosing to drive uptake by the cation-independent mannose 6-phosphate receptor on target cells. However, some approaches introduce bis-M6P onto rhGAA via non-natural linkages that cannot be hydrolyzed by natural human enzymes and thus inhibit the endolysosomal glycan trimming necessary for complete enzyme activation after cell uptake. Furthermore, all rhGAA ERTs face potential inactivation during intravenous delivery (and subsequent non-productive clearance) as GAA is an acid hydrolase that is rapidly denatured in the near-neutral pH of the blood. One new therapy, cipaglucosidase alfa plus miglustat, is hypothesized to address these challenges by combining an enzyme enriched with naturally occurring bis-M6P N-glycans with a small-molecule stabilizer. Here, we investigate this hypothesis by analyzing published and new data related to the mechanism of action of the enzyme and stabilizer molecule. Based on an extensive collection of in vitro, preclinical, and clinical data, we conclude that cipaglucosidase alfa plus miglustat successfully addresses each of these challenges to offer meaningful advantages in terms of pharmacokinetic exposure, target-cell uptake, endolysosomal processing, and clinical benefit.

Oncological Aspects of Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) are caused by the deficient activity of a lysosomal hydrolase or the lack of a functional membrane protein, transporter, activator, or other protein. Lysosomal enzymes break down macromolecular compounds, which contribute to metabolic homeostasis. Stored, undegraded materials have multiple effects on cells that lead to the activation of autophagy and apoptosis, including the toxic effects of lyso-lipids, the disruption of intracellular Ca2+ ion homeostasis, the secondary storage of macromolecular compounds, the activation of signal transduction, apoptosis, inflammatory processes, deficiencies of intermediate compounds, and many other pathways. Clinical observations have shown that carriers of potentially pathogenic variants in LSD-associated genes and patients affected with some LSDs are at a higher risk of cancer, although the results of studies on the frequency of oncological diseases in LSD patients are controversial. Cancer is found in individuals affected with Gaucher disease, Fabry disease, Niemann-Pick type A and B diseases, alfa-mannosidosis, and sialidosis. Increased cancer prevalence has also been reported in carriers of a potentially pathogenic variant of an LSD gene, namely CLN3, SGSH, GUSB, NEU1, and, to a lesser extent, in other genes. In this review, LSDs in which oncological events can be observed are described.

LAMTOR1 ablation impedes cGAS degradation caused by chemotherapy and promotes antitumor immunity.

Chemotherapy resistance remains a significant obstacle that limits the long-term efficacy of cancer therapy, necessitating further investigations into the underlying mechanisms. Here, we find that DNA fragments induced by chemotherapeutic agents trigger the degradation of cGAS, a potent double-strand DNA (dsDNA) sensor, by lysosomes. Mechanically, the lysosome-localized protein LAMTOR1 is up-regulated, and the interaction between LAMTOR1 and cGAS is enhanced upon exposure to DNA fragments, boosting the accumulation and digestion of cGAS in lysosomes through the receptor protein p62. LAMTOR1 deficiency increases cGAS abundance and promotes activation of the cGAS-STING pathway, leading to subsequent production of type I interferons induced by cytosolic DNA stimulation. Loss of LAMTOR1 synergizes with immunotherapy and chemotherapy to inhibit tumor growth and prolong the survival time of tumor-bearing mice by promoting the infiltration of effective T lymphocytes. Thus, our study reveals a regulation of cGAS abundance and provides a potential strategy to overcome chemotherapy resistance by targeting LAMTOR1.

NT-proBNP Reflects Left Ventricular Hypertrophy Rather than Left Ventricular Dilatation or Systolic Dysfunction in Patients with Fabry Disease.

Background: The diagnosis and follow-up of cardiac involvement in Fabry disease constitutes an important challenge for clinicians caring for affected patients. Combining cardiac imaging with laboratory biomarkers appears most appropriate for longitudinal monitoring. Therefore, we examined the use of NT-proBNP and its association with imaging findings in patients with Fabry disease. Methods: We analysed cardiac MRI and echocardiography data, as well as laboratory results, from a single-centre prospective registry. Results: Repetitive follow-ups of 38 patients with Fabry disease, of whom 18 presented with left ventricular hypertrophy (LVH), revealed a correlation of NT-proBNP with left ventricular (LV) interventricular septal thickness, LV maximum wall thickness, LV and right ventricular (RV) mass index and trabecular mass in patients with LVH. Patients without LVH did not exhibit any tangible association between NT-proBNP and the mentioned parameters. Conversely, we could not detect an association of NT-proBNP with impairment of LV or RV ejection fraction or diastolic volume. Conclusions: NT-proBNP plays a pivotal role as a biomarker for cardiac involvement in patients with Fabry disease. Interestingly, in this specific population with mostly preserved ejection fraction, it seems to reflect ventricular hypertrophy rather than ventricular dysfunction or dilatation. While strong associations were found in hypertrophic patients, NT-proBNP's prognostic value appears limited in non- or pre-hypertrophic stages.

Transcriptome Analysis Reveals Immune and Antioxidant Defense Mechanisms in the Eriocheir japonica sinensis after Exposure to Ammonia.

As a key species in freshwater aquaculture, Eriocheir japonica sinensis was subjected to ammonia stress to assess its impact on the hepatopancreas. A total of 4007 differentially expressed genes (DEGs) were identified between control and treatment groups, comprising 1838 upregulated and 2169 downregulated genes. Following exposure to 300 mg/L of ammonia, the oxidative phosphorylation pathway was activated, while the lysosomal pathway was suppressed, thereby influencing immune functions. Thirteen DEGs from these pathways were further validated via qRT-PCR, revealing gene expression changes of one- to two-fold. Both acid phosphatase (ACP) and alkaline phosphatase (AKP) levels in the hepatopancreas and hemolymph initially increased and then decreased, indicating a disruption in immune functionality. Additionally, alanine transaminase (ALT) and triglyceride (TG) levels were measured, alongside catalase (CAT) activity, total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content, all of which showed an upward trend, signifying oxidative stress and tissue damage. These results offer critical insights into the antioxidant and immune mechanisms of E. j. sinensis in ammonia-enriched environments.

Nanoplatforms for Magnetic-Photo-Heating of Thermo-Resistant Tumor Cells: Singular Synergic Therapeutic Effects at Mild Temperature.

A self-assemble amphiphilic diblock copolymer that can incorporate iron oxide nanocubes (IONCs) in chain-like assemblies as heat mediators for magnetic hyperthermia (MHT) and tuneable amounts of IR780 dye as agent for photothermal therapy (PTT) is developed. MHT-heating performance of photobeads in viscous media have the same heat performances in water at magnetic field conditions of clinical use. Thanks to IR780, the photobeads are activated by infrared laser light within the first biological window (808 nm) with a significant enhancement of photo-stability of IR780 enabling the raise of the temperature at therapeutic values during multiple PTT cycles and showing unchanged optical features up to 8 days. Moreover, the photobeads fluorescent signal is preserved once internalized by glioblastoma multiforme (GBM) cells. Peculiarly, the photobeads are used as toxic agents to eradicate thermo-resistant GBM cells at mild heat, as low as 41 °C, with MHT and PTT both of clinical use. Indeed, a high U87 GBM cell mortality percentage is obtained only with dual MHT/PTT while each single treatment dose not provide the same cytotoxic effects. Only for the combined treatment, the cell death mechanism is assigned to clear sign of apoptosis as observed by structural/morphological cell studies and enhanced lysosome permeability.

Engineering memory T cells as a platform for long-term enzyme replacement therapy in lysosomal storage disorders.

Enzymopathy disorders are the result of missing or defective enzymes. Among these enzymopathies, mucopolysaccharidosis type I is a rare genetic lysosomal storage disorder caused by mutations in the gene encoding alpha-L-iduronidase (IDUA), which ultimately causes toxic buildup of glycosaminoglycans (GAGs). There is currently no cure and standard treatments provide insufficient relief to the skeletal structure and central nervous system (CNS). Human memory T (Tm) cells migrate throughout the body's tissues and can persist for years, making them an attractive approach for cellular-based, systemic enzyme replacement therapy. Here, we tested genetically engineered, IDUA-expressing Tm cells as a cellular therapy in an immunodeficient mouse model of MPS I. Our results demonstrate that a single dose of engineered Tm cells leads to detectable IDUA enzyme levels in the blood for up to 22 weeks and reduced urinary GAG excretion. Furthermore, engineered Tm cells take up residence in nearly all tested tissues, producing IDUA and leading to metabolic correction of GAG levels in the heart, lung, liver, spleen, kidney, bone marrow, and the CNS, although only minimal improved cognition was observed. Our study indicates that genetically engineered Tm cells hold great promise as a platform for cellular-based enzyme replacement therapy for the treatment of mucopolysaccharidosis type I and potentially many other enzymopathies and protein deficiencies.

USP7 protects TFEB from proteasome-mediated degradation.

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.

Lysosome toxicities induced by nanoparticle exposure and related mechanisms.

Nanoparticles (NPs) have achieved extensive utilization across diverse domains, highlighting their unavoidable impact on health. The internalization of NPs carries the potential to trigger inflammation and instigate ailments by selectively targeting lysosomes, thereby posing significant public health concern. Lysosomes, essential organelles responsible for the degradation of biological macromolecules within cells, are crucial for cellular homeostasis and participate in key biological processes, including inter-organelle communication, signal transduction, plasma membrane repair, and immune responses. Consequently, a thorough understanding of lysosomal function is essential for elucidating the mechanisms underlying NPs-mediated toxicity. NPs-induced lysosomal dysfunction primarily involves disruptions in the acidic microenvironment of lysosomes, lysosomal membrane rupture, and membrane permeabilization. Additionally, potential molecular mechanisms contributing to the increased risk of lysosomal damage caused by NPs have been described, particularly concerning ion channel proteins such as V-ATPase, TRPM2, CLC-7, and LAMPs. This review aims to detail the alterations in lysosomal functionality induced by NPs and their associated mechanisms. By providing a theoretical framework, this review aims to support the potential application of NPs in biomedical fields.

Should it stay or should it go: gap junction protein GJA1/Cx43 conveys damaged lysosomes to the cell periphery to potentiate exocytosis.

GJA1/Cx43 (gap junction protein alpha 1) has long been associated with gap junctions-mediated communication between adjacent cells. However, recent data have defied this concept, with studies implicating GJA1 in other biological processes, such as macroautophagy/autophagy regulation, mitochondrial activity and extracellular vesicles biology. In our recent study we unveiled an additional role played by GJA1 in lysosomal trafficking. We demonstrate that GJA1 promotes the exocytosis of damaged lysosomes, through a mechanism that relies on ACTR2/ARP2-ACTR3/ARP3-dependent actin remodeling. Our findings ascribe to GJA1 an important role during pathogen infection and lysosomal storage disorders, favoring the release of dysfunctional lysosomes.

Assessment of genes involved in lysosomal diseases using the ClinGen clinical validity framework.

Lysosomal diseases (LDs) are a heterogeneous group of rare genetic disorders that result in impaired lysosomal function, leading to progressive multiorgan system dysfunction. Accurate diagnosis is paramount to initiating targeted therapies early in the disease process in addition to providing prognostic information and appropriate support for families. In recent years, genomic sequencing technologies have become the first-line approach in the diagnosis of LDs. Understanding the clinical validity of the role of a gene in a disease is critical for the development of genomic technologies, such as which genes to include on next generation sequencing panels, and the interpretation of results from exome and genome sequencing. To this aim, the ClinGen Lysosomal Diseases Gene Curation Expert Panel utilized a semi-quantitative framework incorporating genetic and experimental evidence to assess the clinical validity of the 56 LD-associated genes on the Lysosomal Disease Network's list. Here, we describe the results, and the key themes and challenges encountered.

Lysosomal activity depends on TRPML1-mediated Ca2+ release coupled to incoming vesicle fusions.

The lysosomal cation channel TRPML1/MCOLN1 facilitates autophagic degradation during amino acid starvation based on studies involving long-term TRMPL1 modulation. Here we show that lysosomal activation (more acidic pH and higher hydrolase activity) depends on incoming vesicle fusions. We identify an immediate, calcium-dependent role of TRPML1 in lysosomal activation through promoting autophagosome-lysosome fusions and lysosome acidification within 10-20 minutes of its pharmacological activation. Lysosomes also become more fusion competent upon TRPML1 activation via increased transport of lysosomal SNARE proteins syntaxin 7 and VAMP7 by SNARE carrier vesicles. We find that incoming vesicle fusion is a prerequisite for lysosomal Ca2+ efflux that leads to acidification and hydrolytic enzyme activation. Physiologically, the first vesicle fusions likely trigger generation of the phospholipid PI(3,5)P2 that activates TRPML1, and allosteric TRPML1 activation in the absence of PI(3,5)P2 restores autophagosome-lysosome fusion and rescues abnormal SNARE sequestration within lysosomes. We thus identify a prompt role of TRPML1-mediated calcium signaling in lysosomal fusions, activation, and SNARE trafficking.

The unlikely combination: Anderson-Fabry disease and congenital dyserythropoietic anemia type II in a pediatric patient.

Key Clinical Message: Anderson-Fabry disease, a rare X-linked lysosomal disorder, and congenital dyserythropoietic anemia (CDA) Type II, an autosomal recessive condition, both have distinct inheritance patterns. Their co-occurrence is extremely rare, never been reported before. Therefore, screening is crucial for early management, and families should seek genetic counseling for children showing unusual presentations. Abstract: Anderson-Fabry disease (AFD) is a rare condition, characterized by a lysosomal storage disorder affecting lipid storage. It manifests in two forms: classic (early-onset) and nonclassic (late-onset). Conversely, congenital dyserythropoietic anemia (CDA) is a rare blood disorder caused by ineffective erythropoiesis, which results in the production of abnormal erythroblasts during the maturation of red blood cells, with CDA type II being the most frequent type. Both disorders have well-understood pathophysiologies, yet they are genetically distinct. AFD is inherited in an X-linked manner, whereas CDA type II follows an autosomal recessive pattern of inheritance. Although both AFD and CDA type II have been reported separately in the literature. The co-existence for both AFD and CDA type II has not been reported. We describe a 10-year-old boy, with both which is believed to be the first documented case.

Proteomic characterization of ubiquitin carboxyl-terminal hydrolase 19 deficient cells reveals a role for USP19 in secretion of lysosomal proteins.

Ubiquitin carboxyl-terminal hydrolase 19 (USP19) is a unique deubiquitinase (DUB), characterized by multiple variants generated by alternative splicing. Several variants bear a C-terminal transmembrane domain that anchors them to the endoplasmic reticulum (ER). Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from ER-associated protein degradation (ERAD) in a catalytic-independent manner, promote autophagy and address proteins to lysosomal degradation via endosomal microautophagy. USP19 has recently emerged as the protein responsible for the unconventional secretion of misfolded proteins including Parkinson's disease-associated protein α-synuclein. Despite mounting evidence that USP19 plays crucial roles in several biological processes, the underlying mechanisms are unclear due to lack of information on the physiological substrates of USP19. Herein, we used high-resolution quantitative proteomics to analyze changes in the secretome and cell proteome induced by loss of USP19 to identify proteins whose secretion or turnover is regulated by USP19. We found that ablation of USP19 induced significant proteomic alterations both in and out of the cell. Loss of USP19 impaired the release of several lysosomal proteins, including legumain (LGMN) and several cathepsins. In order to understand the underlaying mechanism, we dissected the USP19-regulated secretion of LGMN in several cell types. We found that LGMN was not a DUB substrate of USP19 and that its USP19-dependent release did not require their direct interaction. LGMN secretion occurred by a mechanism that involved the Golgi apparatus, autophagosome formation and lysosome function. This mechanism resembled the recently described "lysosomal exocytosis", by which lysosomal hydrolases are secreted, when ubiquitination of p62 is increased in cells lacking deubiquitinases such as USP15 and USP17. In conclusion, our proteomic characterization of USP19 has identified a collection of proteins in the secretome and within the cell that are regulated by USP19, which link USP19 to secretion of lysosomal proteins, including LGMN.