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1.
Methods Mol Biol ; 2566: 37-43, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152240

RESUMO

Lysosomes play key roles in different cellular processes such as autophagy, phagocytosis, and apoptosis. Lysosomal dysfunction is related to many diseases. Fluorescence lysosome staining strategy is valuable for the researches on the lysosome involvement in different pathological diagnosis. Here we describe fluorescence lysosome staining methods with carbon dots for the identification of lysosomes in living and fixed cells.


Assuntos
Carbono , Corantes Fluorescentes , Lisossomos , Microscopia Confocal , Coloração e Rotulagem
2.
Methods Mol Biol ; 2566: 141-147, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152248

RESUMO

Autophagy is crucial for maintaining cellular homeostasis and its deregulation is involved in disease development, including cancer. The key players of chaperone-mediated autophagy (CMA), a particular selective subtype of autophagy, are HSPA8 and LAMP2A. Both proteins can be immunohistochemically detected in formalin-fixed paraffin-embedded (FFPE) tissue. LAMP2A is frequently overexpressed in a variety of cancers where it likely supports cancer cell survival and resistance to anti-cancer therapies in a context-dependent manner. Here we present the immunohistochemical staining protocol of antibodies against LAMP2A and HSPA8, using an automated staining system, suitable for routine diagnostics. Additionally, we also suggest a staining evaluation method.


Assuntos
Autofagia Mediada por Chaperonas , Autofagia/fisiologia , Formaldeído/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Chaperonas Moleculares/metabolismo , Inclusão em Parafina
3.
Talanta ; 251: 123770, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35961081

RESUMO

Iron oxide nanoparticles (IONPs) have been widely used as a nanoscale tool in biomedical research. However, it remains largely unknown how IONPs are transformed at a subcellular level to elicit distinct biological effects. In the present study, we prepared three different IONPs, including two IONPs targeting mitochondria (IONP-TPP) and lysosomes (IONP-APM), respectively, and a control with no specified target (IONP). By MTT assay and JC-1 staining, mitochondria-targeted IONP-TPP was found to produce significant cytotoxicity and severe mitochondrial membrane depolarization in MCF-7 cells. Furthermore, X-ray absorption spectroscopy (XAS) analysis revealed that IONP-TPP underwent remarkable edge defects and oxidation inside the cell. These findings suggest that IONPs are prone to the chemical transformation at mitochondria, and mitochondria are vulnerable to IONPs accumulation in the cell.


Assuntos
Nanopartículas de Magnetita , Nanopartículas , Compostos Férricos/toxicidade , Lisossomos , Nanopartículas de Magnetita/química , Mitocôndrias , Coloração e Rotulagem
4.
Semin Cell Dev Biol ; 133: 3-9, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-35227625

RESUMO

Axon growth and guidance in the developing nervous system rely on intracellular membrane dynamics that involve endosome maturation and transport, as well as its regulated tethering to the endoplasmic reticulum (ER). Recent studies have identified several key molecules, such as protrudin, which plays a dynamic role at membrane contact sites between the ER and endosomes/lysosomes, and myosin Va, which acts as a sensor for ER-derived Ca2+ that triggers peri-ER membrane export. These molecules form different types of multiprotein complexes at the interface of organelles and, in response to their surrounding microenvironments, such as Ca2+ concentrations and lipid contents, regulate the directional movement of endosomal vesicles in extending axons. Here, we review the molecular mechanisms underlying membrane dynamics and inter-organelle interactions during neuronal morphogenesis.


Assuntos
Retículo Endoplasmático , Endossomos , Lisossomos , Membranas Mitocondriais , Axônios
5.
Spectrochim Acta A Mol Biomol Spectrosc ; 285: 121884, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36179563

RESUMO

The AIE bio-probes have attracted extensive attention because of their good brightness, long-term in situ retention ability, photostability and low cytotoxicity. Recently, the transformation of ACQ to AIE has become very popular, which is very important for the further development of AIE probes. Herein, a series of novel dyes (NR-Lyso-Ⅰ, NR-Lyso-Ⅱ, NR-Lyso-III, NR-Lyso-IV) were designed and synthesized. It was found that alkylation of 4-aminonaphthalimide could achieve the transformation of the dye from ACQ to AIE effect due to the growth of carbon chain. Moreover, the AIE probe NR-Lyso-IV exhibited dual-state emission (DSE) and large Stokes shift (>100 nm), excellent selectivity, photostability, and low cytotoxicity, which was able to simultaneous visualize the lipid droplets (LDs) and lysosomes of HeLa cells and zebrafish.


Assuntos
Corantes Fluorescentes , Gotículas Lipídicas , Humanos , Animais , Células HeLa , Peixe-Zebra , Lisossomos
6.
Spectrochim Acta A Mol Biomol Spectrosc ; 286: 122027, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36323089

RESUMO

Lysosome, an organelle which contains a number of hydrolases and hydrogen ions, plays a crucial role in cellular survival and apoptosis. If selectively destroy lysosomes membrane, inner hydrolases and hydrogen ions will leak and induce cell death. In this work, three lysosome-targeting fluorescent probes (HCL 1-3, heptamethine cyanine lysosomal-targeting probe) were designed, synthesized and developed for photodynamic therapy. Piperazine and N, N-dimethyl structures made HCL 1-3 have good lysosome targeting ability while Pearson's correlation coefficients reached 0.85, 0.87 and 0.78. It can be concluded from MTT test, HCL 1-3 have high photo cytotoxicity and low dark cytotoxicity from MTT test. Calcein/PI staining assays also supported cytotoxicity of HCL 1-3 under light conditions. In vivo experiments, HCL 2 accumulated in tumor and a strong fluorescence signal was observed at 12 h post injection. All results showed that our experiments provide help and new ideas for cyanine dyes in cancer treatment.


Assuntos
Fotoquimioterapia , Fotoquimioterapia/métodos , Prótons , Lisossomos/metabolismo , Corantes Fluorescentes/química , Hidrolases/análise , Hidrolases/metabolismo
7.
Spectrochim Acta A Mol Biomol Spectrosc ; 286: 121966, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36252305

RESUMO

Hypochlorous acid (HOCl), as an important biological reactive oxygen species (ROS), plays an important role in microbial immune defense and inflammatory response. Abnormal levels of HOCl in lysosomes can cause lysosomal membrane rupture and release of various hydrolases, leading to a variety of diseases, including cancer. In order to better monitor the level of HOCl in lysosomes, phenothiazine was chosen as fluorophore to construct a NIR fluorescent probe PMM with intramolecular change transfer process (ICT). PMM is a colorimetric and ratiometric fluorescent probe, which has high sensitivity with a low detection limit (20 nM), high selectivity and anti-interference. PMM has good stability in the weakly acidic environment of pH 4.0-5.5. PPM has good localization ability for lysosomes and has been successfully used for fluorescence imaging of exogenous and endogenous HOCl in HepG2 cells. Moreover, nude mouse imaging also demonstrated that PMM could be used to detect HOCl in vivo.


Assuntos
Corantes Fluorescentes , Ácido Hipocloroso , Camundongos , Animais , Humanos , Ácido Hipocloroso/química , Corantes Fluorescentes/química , Lisossomos , Imagem Óptica , Células Hep G2
8.
Bioorg Chem ; 130: 106199, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36370648

RESUMO

Due to the diverse H2O2 distribution in organelles, fluorescent probes were usually required to be prepared separately, which limited the convenience and practicability. Herein, we reported a flexible strategy to in-situ construct H2O2 fluorescent probes in different organelles. A tetrazine fused probe TP was developed with rapid click reaction capacity and sensitive H2O2 response. When treated with H2O2, the turn-on fluorescence was effectively quenched by the tetrazine part. Only after click reaction with dienophiles, the fluorescence resumed. In application, cells were firstly treated with triphenylphosphorus tagged norbornene (TPP-NB) to label mitochondria, which was followed by the introduction of probe TP to trigger click reaction. The in-situ constructed probe P1 served as a local H2O2 sensor. In a similar way, probe P2 was in-situ constructed in lysosomes via probe TP and morpholine tagged norbornene (MP-NB). With this on-demand modular assembling and double turn-on features, our strategy to construct fluorescent probes presented high flexibility and anti-interference performance, which was expected to inspired more applications in biological studies.


Assuntos
Corantes Fluorescentes , Peróxido de Hidrogênio , Humanos , Corantes Fluorescentes/metabolismo , Peróxido de Hidrogênio/metabolismo , Células HeLa , Lisossomos/metabolismo , Mitocôndrias , Norbornanos/metabolismo
9.
Biochim Biophys Acta Mol Cell Res ; 1870(1): 119386, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36302466

RESUMO

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1fl/fl/Podocre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether Ac deficiency impairs autophagic flux in podocytes through blockade of transient receptor potential mucolipin 1 (TRPML1) channel as a potential pathogenic mechanism of podocytopathy in Asah1fl/fl/Podocre mice. We first demonstrated that impairment of autophagic flux occurred in podocytes lacking Asah1 gene, which was evidenced by autophagosome accumulation and reduced lysosome-autophagosome interaction. TRPML1 channel agonists recovered lysosome-autophagosome interaction and attenuated autophagosome accumulation in podocytes from Asah1fl/fl/Podocre mice, while TRPML1 channel inhibitors impaired autophagic flux in WT/WT podocytes and worsened autophagic deficiency in podocytes lacking Asah1 gene. The effects of TRPML1 channel agonist were blocked by dynein inhibitors, indicating a critical role of dynein activity in the control of lysosome movement due to TRPML1 channel-mediated Ca2+ release. It was also found that there is an enhanced phenotypic transition to dedifferentiation status in podocytes lacking Asah1 gene in vitro and in vivo. Such podocyte phenotypic transition was inhibited by TRPML1 channel agonists but enhanced by TRPML1 channel inhibitors. Moreover, we found that TRPML1 gene silencing induced autophagosome accumulation and dedifferentiation in podocytes. Based on these results, we conclude that Ac activity is essential for autophagic flux and maintenance of differentiated status of podocytes. Dysfunction or deficiency of Ac may impair autophagic flux and induce podocyte dedifferentiation, which may be an important pathogenic mechanism of podocytopathy and associated nephrotic syndrome.


Assuntos
Síndrome Nefrótica , Podócitos , Animais , Camundongos , Ceramidase Ácida/farmacologia , Autofagia , Dineínas/farmacologia , Lisossomos/genética
10.
J Ethnopharmacol ; 301: 115766, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36183948

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Zhachong Shisanwei Pill (ZSP) is a commonly used Mongolian medicine in treating cerebrovascular diseases and plays a role in the clinical treatment of ischemic stroke (IS). AIM OF THE STUDY: Based on determining the protective effect of ZSP on cerebral ischemia, they adopted the proteomics method to explore the mechanism of ZSP against IS. MATERIALS AND METHODS: Rats with middle cerebral artery occlusion (MCAO) model were prepared by wire embolization method, and divided into sham group, model group, ZSP high-dose group, medium-dose group, low-dose group and positive drug group. We collected the brain tissue of rats for 12 h after modeling. Neurological deficit score and cerebral infarction volume ratio evaluated pharmacodynamics, and we selected the optimal dose for subsequent experiments. Proteomics was used to screen out possible ZSP anti-IS mediated pathways and differentially expression proteins. Network pharmacology was used to verify the correlation between diseases and drugs. Hematoxylin-eosin (HE) staining and transmission electron microscope (TEM) were used to explore further the pharmacodynamic effect of ZSP against IS and its possible mechanism. RESULTS: The cerebral infarction rate and neurological function score in rats showed that the medium-dose ZSP group had the best efficacy. Proteomics results showed that the anti-IS action of ZSP was mainly through lysosome pathway. LAMP2, AP3M1, and SCARB2 were the differentially changed proteins in this pathway. Network pharmacology verified this. HE staining and TEM results showed that ZSP could improve the pathological state of neurons in MCAO rats and reduce the number of lysosomes in MCAO rats. Western blot (WB) results showed that compared with the model group, the protein expression levels of LAMP2 and AP3M1 in the ZSP group were significantly down-regulated, and the protein expression levels of SCARB2 were significantly up-regulated. CONCLUSION: This study confirms that ZSP regulates the lysosomal pathway, which may protect IS by down-regulating LAMP2 and AP3M1 and up-regulating SCARB2.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Animais , Ratos , Proteômica , Ratos Sprague-Dawley , Biologia Computacional , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Lisossomos/metabolismo , Acidente Vascular Cerebral/patologia
11.
Sci Total Environ ; 857(Pt 3): 159626, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36280083

RESUMO

Cadmium (Cd) pollution in environment is toxic to birds. This study aimed to assess antagonistic effect of honokiol (HNK) on Cd-induced quail (Coturnix japonica) liver tissue damage and Cd-induced vacuolation in hepatocytes. We found that HNK alleviated Cd-induced liver pathological damage marked by elevated serum liver biochemical indicators, disordered antioxidant levels and trace elements in quails. HNK reduced Cd-induced liver cell apoptosis as assessed by western blotting and TUNEL staining. The ultrastructure of hepatocytes under transmission electron microscope revealed that Cd induced mitochondrial damage in addition to abnormal enlargement and increased vacuolar structure of cells. Mitochondrial damage and vacuolization were reduced in the HNK + Cd group. Cd induced an increase in the levels of endosomal/lysosomal-related genes, while HNK treatment reversed this effect. Finally, we demonstrated that vacuolation in buffalo rat liver 3A (BRL 3A) cells occurred primarily due to Cd-induced oxidative stress damage that reduces mitochondrial ATP content and indirectly led to dysfunction of ATP-dependent lipid kinase PIKfyve complex. In summary, we are the first to report that Cd induces abnormal enlargement of endosome/lysosomes in quail liver cells and HNK alleviated this phenomenon by reducing mitochondrial damage and increasing intracellular ATP level. This study demonstrated the toxic effect of Cd pollution on birds and how HNK mitigated these effect at the cellular level. Overall, more research on Cd pollution and HNK use in animal husbandry is warranted.


Assuntos
Cádmio , Coturnix , Ratos , Animais , Cádmio/metabolismo , Codorniz , Estresse Oxidativo , Mitocôndrias , Fígado/metabolismo , Lisossomos , Endossomos , Trifosfato de Adenosina/metabolismo
12.
J Cell Biol ; 222(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36239631

RESUMO

At the trans-Golgi, complex traffic connections exist to the endolysosomal system additional to the main Golgi-to-plasma membrane secretory route. Here, we investigated three hits in a Drosophila screen displaying secretory cargo accumulation in autophagic vesicles: ESCRT-III component Vps20, SNARE-binding Rop, and lysosomal pump subunit VhaPPA1-1. We found that Vps20, Rop, and lysosomal markers localize near the trans-Golgi. Furthermore, we document that the vicinity of the trans-Golgi is the main cellular location for lysosomes and that early, late, and recycling endosomes associate as well with a trans-Golgi-associated degradative compartment where basal microautophagy of secretory cargo and other materials occurs. Disruption of this compartment causes cargo accumulation in our hits, including Munc18 homolog Rop, required with Syx1 and Syx4 for Rab11-mediated endosomal recycling. Finally, besides basal microautophagy, we show that the trans-Golgi-associated degradative compartment contributes to the growth of autophagic vesicles in developmental and starvation-induced macroautophagy. Our results argue that the fly trans-Golgi is the gravitational center of the whole endomembrane system.


Assuntos
Autofagia , Endossomos , Complexo de Golgi , Lisossomos , Animais , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Lisossomos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteínas rab de Ligação ao GTP
13.
J Cell Biol ; 222(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36282215

RESUMO

Arl8b, an Arf-like GTP-binding protein, regulates cargo trafficking and positioning of lysosomes. However, it is unknown whether Arl8b regulates lysosomal cargo sorting. Here, we report that Arl8b binds to the Rab4 and Rab14 interaction partner, RUN and FYVE domain-containing protein (RUFY) 1, a known regulator of cargo sorting from recycling endosomes. Arl8b determines RUFY1 endosomal localization through regulating its interaction with Rab14. RUFY1 depletion led to a delay in CI-M6PR retrieval from endosomes to the TGN, resulting in impaired delivery of newly synthesized hydrolases to lysosomes. We identified the dynein-dynactin complex as an RUFY1 interaction partner, and similar to a subset of activating dynein adaptors, the coiled-coil region of RUFY1 was required for interaction with dynein and the ability to mediate dynein-dependent organelle clustering. Our findings suggest that Arl8b and RUFY1 play a novel role on recycling endosomes, from where this machinery regulates endosomes to TGN retrieval of CI-M6PR and, consequently, lysosomal cargo sorting.


Assuntos
Fatores de Ribosilação do ADP , Proteínas Adaptadoras de Transdução de Sinal , Dineínas , Endossomos , Lisossomos , Proteínas rab de Ligação ao GTP , Humanos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Endossomos/metabolismo , Células HeLa , Lisossomos/metabolismo , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo
14.
Biol Pharm Bull ; 45(11): 1609-1615, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36328496

RESUMO

Autophagy is a highly conserved intracellular degrading system and its dysfunction is considered related to the cause of neurodegenerative disorders. A previous study showed that the inhibition of endocytosis transport attenuates soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein transport to lysosomes and block autophagy. The other studies demonstrated oxidative stress, one of the inducers of neurodegenerative diseases inhibits endocytosis transport. Thus, we hypothesized that oxidative stress-induced endocytosis inhibition causes alteration of SNARE protein transport to lysosomes and impairs autophagy. Here, we demonstrated that oxidative stress inhibits endocytosis and decreased the lysosomal localization of VAMP8, one of the autophagy-related SNARE proteins in a human neuroblastoma cell line. Moreover, this oxidative stress induction blocked the autophagosome-lysosome fusion step. Since we also observed decreased lysosomal localization of VAMP8 and inhibition of autophagosome-lysosome fusion in endocytosis inhibitor-treated cells, oxidative stress may inhibit VAMP8 trafficking by suppressing endocytosis and impair autophagy. Our findings suggest that oxidative stress-induced inhibition of VAMP8 trafficking to lysosomes is associated with the development of neurodegenerative diseases due to the blocked autophagosome-lysosome fusion, and may provide a new therapeutic target for restoring the autophagic activity.


Assuntos
Autofagia , Lisossomos , Humanos , Autofagia/fisiologia , Lisossomos/metabolismo , Fusão de Membrana , Estresse Oxidativo , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Transporte Biológico
15.
Nat Commun ; 13(1): 6623, 2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36333297

RESUMO

Activities of dendritic cells (DCs) that present tumor antigens are often suppressed in tumors. Here we report that this suppression is induced by tumor microenvironment-derived factors, which activate the activating transcription factor-3 (ATF3) transcription factor and downregulate cholesterol 25-hydroxylase (CH25H). Loss of CH25H in antigen presenting cells isolated from human lung tumors is associated with tumor growth and lung cancer progression. Accordingly, mice lacking CH25H in DCs exhibit an accelerated tumor growth, decreased infiltration and impaired activation of intratumoral CD8+ T cells. These mice do not establish measurable long-term immunity against malignant cells that undergo chemotherapy-induced immunogenic cell death. Mechanistically, downregulation of CH25H stimulates membrane fusion between endo-phagosomes and lysosomes, accelerates lysosomal degradation and restricts cross-presentation of tumor antigens in the intratumoral DCs. Administration of STING agonist MSA-2 reduces the lysosomal activity in DCs, restores antigen cross presentation, and increases therapeutic efficacy of PD-1 blockade against tumour challenge in a CH25H-dependent manner. These studies highlight the importance of downregulation of CH25H in DCs for tumor immune evasion and resistance to therapy.


Assuntos
Apresentação Cruzada , Neoplasias Pulmonares , Camundongos , Humanos , Animais , Antígenos de Neoplasias , Linfócitos T CD8-Positivos , Células Dendríticas , Neoplasias Pulmonares/metabolismo , Lisossomos , Camundongos Endogâmicos C57BL , Microambiente Tumoral
16.
Nano Lett ; 22(22): 9163-9173, 2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36374537

RESUMO

Maximizing the therapeutic capacity of drugs by allowing them to escape lysosomal degradation is a long-term challenge for nanodrug delivery. Japanese encephalitis virus (JEV) has evolved the ability to escape the endosomal region to avoid degradation of internal genetic material by lysosomes and further induce upregulation of cellular autophagy for the purpose of their mass reproduction. In this work, to exploit the lysosome escape and autophagy-inducing properties of JEV for cancer therapy, we constructed a virus-mimicking nanodrug consisting of anti-PDL1 antibody-decorated JEV-mimicking virosome encapsulated with a clinically available autophagy inhibitor, hydroxychloroquine (HCQ). Our study indicated that the nanodrug can upregulate the autophagy level and inhibit the autophagic flux, thereby inducing the apoptosis of tumor cells, and further activating the immune response, which can greatly improve the antitumor and tumor metastasis suppression effects and provide a potential therapeutic strategy for tumor treatment.


Assuntos
Nanopartículas , Neoplasias , Autofagia , Lisossomos/metabolismo , Apoptose , Hidroxicloroquina/farmacologia , Hidroxicloroquina/uso terapêutico , Nanopartículas/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo
17.
PLoS One ; 17(11): e0277058, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36409725

RESUMO

Isomeric lysosphingolipids, galactosylsphingosine (GalSph) and glucosylsphingosine (GlcSph), are present in only minute levels in healthy cells. Due to defects in their lysosomal hydrolysis, they accumulate at high levels and cause cytotoxicity in patients with Krabbe and Gaucher diseases, respectively. Here, we show that GalSph and GlcSph induce lysosomal membrane permeabilization, a hallmark of lysosome-dependent cell death, in human breast cancer cells (MCF7) and primary fibroblasts. Supporting lysosomal leakage as a causative event in lysosphingolipid-induced cytotoxicity, treatment of MCF7 cells with lysosome-stabilizing cholesterol prevented GalSph- and GlcSph-induced cell death almost completely. In line with this, fibroblasts from a patient with Niemann-Pick type C disease, which is caused by defective lysosomal cholesterol efflux, were significantly less sensitive to lysosphingolipid-induced lysosomal leakage and cell death. Prompted by the data showing that MCF7 cells with acquired resistance to lysosome-destabilizing cationic amphiphilic drugs (CADs) were partially resistant to the cell death induced by GalSph and GlcSph, we compared these cell death pathways with each other. Like CADs, GalSph and GlcSph activated the cyclic AMP (cAMP) signalling pathway, and cAMP-inducing forskolin sensitized cells to cell death induced by low concentrations of lysosphingolipids. Contrary to CADs, lysosphingolipid-induced cell death was independent of lysosomal Ca2+ efflux through P2X purinerigic receptor 4. These data reveal GalSph and GlcSph as lysosome-destabilizing lipids, whose putative use in cancer therapy should be further investigated. Furthermore, the data supports the development of lysosome stabilizing drugs for the treatment of Krabbe and Gaucher diseases and possibly other sphingolipidoses.


Assuntos
Doença de Gaucher , Neoplasias , Humanos , Psicosina/metabolismo , Lisossomos/metabolismo , Morte Celular , Doença de Gaucher/metabolismo , AMP Cíclico/metabolismo , Colesterol/metabolismo , Neoplasias/metabolismo
18.
Proc Natl Acad Sci U S A ; 119(45): e2200477119, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36322753

RESUMO

IGF2BP2 binds to a number of RNA transcripts and has been suggested to function as a tumor promoter, although little is known regarding the mechanisms that regulate its roles in RNA metabolism. Here we demonstrate that IGF2BP2 binds to the 3' untranslated region of the transcript encoding ATP6V1A, a catalytic subunit of the vacuolar ATPase (v-ATPase), and serves as a substrate for the NAD+-dependent deacetylase SIRT1, which regulates how IGF2BP2 affects the stability of the ATP6V1A transcript. When sufficient levels of SIRT1 are expressed, it catalyzes the deacetylation of IGF2BP2, which can bind to the ATP6V1A transcript but does not mediate its degradation. However, when SIRT1 expression is low, the acetylated form of IGF2BP2 accumulates, and upon binding to the ATP6V1A transcript recruits the XRN2 nuclease, which catalyzes transcript degradation. Thus, the stability of the ATP6V1A transcript is significantly compromised in breast cancer cells when SIRT1 expression is low or knocked-down. This leads to a reduction in the expression of functional v-ATPase complexes in cancer cells and to an impairment in their lysosomal activity, resulting in the production of a cellular secretome consisting of increased numbers of exosomes enriched in ubiquitinated protein cargo and soluble hydrolases, including cathepsins, that together combine to promote tumor cell survival and invasiveness. These findings describe a previously unrecognized role for IGF2BP2 in mediating the degradation of a messenger RNA transcript essential for lysosomal function and highlight how its sirtuin-regulated acetylation state can have significant biological and disease consequences.


Assuntos
Neoplasias , ATPases Vacuolares Próton-Translocadoras , Humanos , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Sirtuína 1/metabolismo , RNA/metabolismo , Processos Neoplásicos , Lisossomos/genética , Lisossomos/metabolismo , Neoplasias/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
19.
EMBO J ; 41(22): e111476, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36394115

RESUMO

Retrograde transport of lysosomes is recognised as a critical autophagy regulator. Here, we found that acrolein, an aldehyde that is significantly elevated in Parkinson's disease patient serum, enhances autophagy by promoting lysosomal clustering around the microtubule organising centre via a newly identified JIP4-TRPML1-ALG2 pathway. Phosphorylation of JIP4 at T217 by CaMK2G in response to Ca2+ fluxes tightly regulated this system. Increased vulnerability of JIP4 KO cells to acrolein indicated that lysosomal clustering and subsequent autophagy activation served as defence mechanisms against cytotoxicity of acrolein itself. Furthermore, the JIP4-TRPML1-ALG2 pathway was also activated by H2 O2 , indicating that this system acts as a broad mechanism of the oxidative stress response. Conversely, starvation-induced lysosomal retrograde transport involved both the TMEM55B-JIP4 and TRPML1-ALG2 pathways in the absence of the JIP4 phosphorylation. Therefore, the phosphorylation status of JIP4 acts as a switch that controls the signalling pathways of lysosoma l distribution depending on the type of autophagy-inducing signal.


Assuntos
Acroleína , Canais de Potencial de Receptor Transitório , Humanos , Acroleína/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Lisossomos/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo
20.
Nat Commun ; 13(1): 6808, 2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36357396

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) integrates inputs from growth factors and nutrients, but how mTORC1 autoregulates its activity remains unclear. The MiT/TFE transcription factors are phosphorylated and inactivated by mTORC1 following lysosomal recruitment by RagC/D GTPases in response to amino acid stimulation. We find that starvation-induced lysosomal localization of the RagC/D GAP complex, FLCN:FNIP2, is markedly impaired in a mTORC1-sensitive manner in renal cells with TSC2 loss, resulting in unexpected TFEB hypophosphorylation and activation upon feeding. TFEB phosphorylation in TSC2-null renal cells is partially restored by destabilization of the lysosomal folliculin complex (LFC) induced by FLCN mutants and is fully rescued by forced lysosomal localization of the FLCN:FNIP2 dimer. Our data indicate that a negative feedback loop constrains amino acid-induced, FLCN:FNIP2-mediated RagC activity in renal cells with constitutive mTORC1 signaling, and the resulting MiT/TFE hyperactivation may drive oncogenesis with loss of the TSC2 tumor suppressor.


Assuntos
Aminoácidos , Lisossomos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Retroalimentação , Lisossomos/metabolismo , Aminoácidos/metabolismo , Politetrafluoretileno/metabolismo
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