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1.
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
2.
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
3.
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
4.
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
5.
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
6.
Cell Death Dis ; 13(11): 953, 2022 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-36371383

RESUMO

Macroautophagy/autophagy is an evolutionarily conserved and tightly regulated catabolic process involved in the maintenance of cellular homeostasis whose dysregulation is implicated in several pathological processes. Autophagy begins with the formation of phagophores that engulf cytoplasmic cargo and mature into double-membrane autophagosomes; the latter fuse with lysosomes/vacuoles for cargo degradation and recycling. Here, we report that yeast Set2, a histone lysine methyltransferase, and its mammalian homolog, SETD2, both act as positive transcriptional regulators of autophagy. However, whereas Set2 regulates the expression of several autophagy-related (Atg) genes upon nitrogen starvation, SETD2 effects in mammals were found to be more restricted. In fact, SETD2 appears to primarily regulate the differential expression of protein isoforms encoded by the ATG14 gene. SETD2 promotes the expression of a long ATG14 isoform, ATG14L, that contains an N-terminal cysteine repeats domain, essential for the efficient fusion of the autophagosome with the lysosome, that is absent in the short ATG14 isoform, ATG14S. Accordingly, SETD2 loss of function decreases autophagic flux, as well as the turnover of aggregation-prone proteins such as mutant HTT (huntingtin) leading to increased cellular toxicity. Hence, our findings bring evidence to the emerging concept that the production of autophagy-related protein isoforms can differentially affect core autophagy machinery bringing an additional level of complexity to the regulation of this biological process in more complex organisms.


Assuntos
Autofagossomos , Macroautofagia , Animais , Autofagossomos/metabolismo , Lisossomos/metabolismo , Autofagia/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Mamíferos
7.
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
8.
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
9.
Cell Rep ; 41(5): 111583, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36323251

RESUMO

Mitochondrial malfunction and autophagy defects are often concurrent phenomena associated with neurodegeneration. We show that Miga, a mitochondrial outer-membrane protein that regulates endoplasmic reticulum-mitochondrial contact sites (ERMCSs), is required for autophagy. Loss of Miga results in an accumulation of autophagy markers and substrates, whereas PI3P and Syx17 levels are reduced. Further experiments indicated that the fusion between autophagosomes and lysosomes is defective in Miga mutants. Miga binds to Atg14 and Uvrag; concordantly, Miga overexpression results in Atg14 and Uvrag recruitment to mitochondria. The heightened PI3K activity induced by Miga requires Uvrag, whereas Miga-mediated stabilization of Syx17 is dependent on Atg14. Miga-regulated ERMCSs are critical for PI3P formation but are not essential for the stabilization of Syx17. In summary, we identify a mitochondrial protein that regulates autophagy by recruiting two alternative components of the PI3K complex present at the ERMCSs.


Assuntos
Autofagia , Proteínas Mitocondriais , Proteínas Mitocondriais/metabolismo , Autofagia/fisiologia , Lisossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo
10.
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
11.
Cells ; 11(21)2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36359798

RESUMO

Lysosomes are highly dynamic organelles involved in the breakdown and recycling of macromolecules, cell cycle, cell differentiation, and cell death, among many other functions in eukaryotic cells. Recently, lysosomes have been identified as cellular hubs for the modulation of intracellular signaling pathways, such as the Wnt/beta-catenin pathway. Here we analyzed morphological and functional characteristics of lysosomes in muscle and non-muscle cells during chick myogenesis, as well as their modulation by the Wnt/beta-catenin pathway. Our results show that (i) muscle and non-muscle cells show differences in lysosomal size and its distribution, (ii) lysosomes are found in spherical structures in myoblasts and fibroblasts and tubular structures in myotubes, (iii) lysosomes are found close to the plasma membrane in fibroblasts and close to the nucleus in myoblasts and myotubes, (iv) lysosomal distribution and size are dependent on the integrity of microtubules and microfilaments in myogenic cells, (v) alterations in lysosomal function, in the expression of LAMP2, and in Wnt/beta-catenin pathway affect the distribution and size of lysosomes in myogenic cells, (vi) the effects of the knockdown of LAMP2 on myogenesis can be rescued by the activation of the Wnt/beta-catenin pathway, and (vii) the chloroquine Lys05 is a potent inhibitor of both the Wnt/beta-catenin pathway and lysosomal function. Our data highlight the involvement of the Wnt/beta-catenin pathway in the regulation of the positioning, size, and function of lysosomes during chick myogenesis.


Assuntos
Desenvolvimento Muscular , beta Catenina , beta Catenina/metabolismo , Desenvolvimento Muscular/fisiologia , Via de Sinalização Wnt , Fibras Musculares Esqueléticas/metabolismo , Citoesqueleto/metabolismo , Lisossomos/metabolismo
12.
Int J Mol Sci ; 23(21)2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36361823

RESUMO

BACKGROUND: An emerging body of evidence indicates an association between anthropogenic particulate matter (PM) and neurodegeneration. Although the historical focus of PM toxicity has been on the cardiopulmonary system, ultrafine PM particles can also exert detrimental effects in the brain. However, only a few studies are available on the harmful interaction between PM and CNS and on the putative pathomechanisms. METHODS: Ultrafine PM particles with a diameter < 0.1 µm (PM0.1) and nanoparticles < 20 nm (NP20) were sampled in a lab-scale combustion system. Their effect on cell tracking in the space was studied by time-lapse and high-content microscopy in NSC-34 motor neurons while pHrodo™ Green conjugates were used to detect PM endocytosis. Western blotting analysis was used to quantify protein expression of lysosomal channels (i.e., TRPML1 and TPC2) and autophagy markers. Current-clamp electrophysiology and Fura2-video imaging techniques were used to measure membrane potential, intracellular Ca2+ homeostasis and TRPML1 activity in NSC-34 cells exposed to PM0.1 and NP20. RESULTS: NP20, but not PM0.1, reduced NSC-34 motor neuron movement in the space. Furthermore, NP20 was able to shift membrane potential of motor neurons toward more depolarizing values. PM0.1 and NP20 were able to enter into the cells by endocytosis and exerted mitochondrial toxicity with the consequent stimulation of ROS production. This latter event was sufficient to determine the hyperactivation of the lysosomal channel TRPML1. Consequently, both LC3-II and p62 protein expression increased after 48 h of exposure together with AMPK activation, suggesting an engulfment of autophagy. The antioxidant molecule Trolox restored TRPML1 function and autophagy. CONCLUSIONS: Restoring TRPML1 function by an antioxidant agent may be considered a protective mechanism able to reestablish autophagy flux in motor neurons exposed to nanoparticles.


Assuntos
Material Particulado , Canais de Potencial de Receptor Transitório , Material Particulado/toxicidade , Material Particulado/análise , Antioxidantes/farmacologia , Lisossomos/metabolismo , Autofagia , Neurônios Motores/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo
13.
Curr Top Membr ; 90: 37-63, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36368874

RESUMO

Lysosomal acid ceramidase (AC) has been reported to determine multivesicular body (MVB) fate and exosome secretion in different mammalian cells including coronary arterial endothelial cells (CAECs). However, this AC-mediated regulation of exosome release from CAECs and associated underlying mechanism remain poorly understood. In the present study, we hypothesized that AC controls lysosomal Ca2+ release through TRPML1 channel to regulate exosome release in murine CAECs. To test this hypothesis, we isolated and cultured CAECs from WT/WT and endothelial cell-specific Asah1 gene (gene encoding AC) knockout mice. Using these CAECs, we first demonstrated a remarkable increase in exosome secretion and significant reduction of lysosome-MVB interaction in CAECs lacking Asah1 gene compared to those cells from WT/WT mice. ML-SA1, a TRPML1 channel agonist, was found to enhance lysosome trafficking and increase lysosome-MVB interaction in WT/WT CAECs, but not in CAECs lacking Asah1 gene. However, sphingosine, an AC-derived sphingolipid, was able to increase lysosome movement and lysosome-MVB interaction in CAECs lacking Asah1 gene, leading to reduced exosome release from these cells. Moreover, Asah1 gene deletion was shown to substantially inhibit lysosomal Ca2+ release through suppression of TRPML1 channel activity in CAECs. Sphingosine as an AC product rescued the function of TRPML1 channel in CAECs lacking Asah1 gene. These results suggest that Asah1 gene defect and associated deficiency of AC activity may inhibit TRPML1 channel activity, thereby reducing MVB degradation by lysosome and increasing exosome release from CAECs. This enhanced exosome release from CAECs may contribute to the development of coronary arterial disease under pathological conditions.


Assuntos
Exossomos , Canais de Potencial de Receptor Transitório , Camundongos , Animais , Ceramidase Ácida/genética , Ceramidase Ácida/metabolismo , Exossomos/metabolismo , Células Endoteliais/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Esfingosina/metabolismo , Lisossomos/metabolismo , Camundongos Knockout , Mamíferos/metabolismo
14.
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
15.
FEBS J ; 289(22): 6822-6831, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36377590

RESUMO

The major criterion that distinguishes eukaryotes from prokaryotes is the presence of organelles in the former. Organelles provide a compartment in which biochemical processes are corralled within bespoke biophysical conditions and act as storage depots, powerhouses, waste storage/recycling units and innate immune signalling hubs. A key challenge faced by organelles is to define, and then retain, their identity; this is mediated by complex proteostasis mechanisms including the import of an organelle-specific proteome, the exclusion of non-organellar proteins and the removal of misfolded proteins via dedicated quality control mechanisms. This Special Issue on Organelle Homeostasis provides an engaging, eclectic, yet integrative, perspective on organelle homeostasis in a range of organelles including those from the secretory and endocytic pathways, mitochondria, the autophagy-lysosomal pathway and the nucleus and its sub-compartments. Some lesser-known organelles including migrasomes (organelles that are released by migrating cells) and GOMED (a Golgi-specific form of autophagy) are also introduced. In the spirit of the principles of organelle biology, we hope you find the reviews in this Issue both encapsulating and captivating, and we thank the authors for their excellent contributions.


Assuntos
Retículo Endoplasmático , Organelas , Retículo Endoplasmático/metabolismo , Organelas/metabolismo , Complexo de Golgi/metabolismo , Lisossomos/metabolismo , Mitocôndrias , Homeostase
16.
Proc Natl Acad Sci U S A ; 119(45): e2207402119, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36322752

RESUMO

The intracellular metabolism of organelles, like lysosomes and mitochondria, is highly coordinated spatiotemporally and functionally. The activities of lysosomal enzymes significantly rely on the cytoplasmic temperature, and heat is constantly released by mitochondria as the byproduct of adenosine triphosphate (ATP) generation during active metabolism. Here, we developed temperature-sensitive LysoDots and MitoDots to monitor the in situ thermal dynamics of lysosomes and mitochondria. The design is based on upconversion nanoparticles (UCNPs) with high-density surface modifications to achieve the exceptionally high sensitivity of 2.7% K-1 and low uncertainty of 0.8 K for nanothermometry to be used in living cells. We show the measurement is independent of the ion concentrations and pH values. With Ca2+ ion shock, the temperatures of both lysosomes and mitochondria increased by ∼2 to 4 °C. Intriguingly, with chloroquine (CQ) treatment, the lysosomal temperature was observed to decrease by up to ∼3 °C, while mitochondria remained relatively stable. Lastly, with oxidative phosphorylation inhibitor treatment, we observed an ∼3 to 7 °C temperature increase and a thermal transition from mitochondria to lysosomes. These observations indicate different metabolic pathways and thermal transitions between lysosomes and mitochondria inside HeLa cells. The nanothermometry probes provide a powerful tool for multimodality functional imaging of subcellular organelles and interactions with high spatial, temporal, and thermal dynamics resolutions.


Assuntos
Lisossomos , Nanopartículas , Humanos , Temperatura , Células HeLa , Lisossomos/metabolismo , Organelas/metabolismo , Mitocôndrias/metabolismo
17.
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
18.
J Cell Biol ; 221(11)2022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36219209

RESUMO

Lysosomes mediate hydrolase-catalyzed macromolecule degradation to produce building block catabolites for reuse. Lysosome function requires an osmo-sensing machinery that regulates osmolytes (ions and organic solutes) and water flux. During hypoosmotic stress or when undigested materials accumulate, lysosomes become swollen and hypo-functional. As a membranous organelle filled with cargo macromolecules, catabolites, ions, and hydrolases, the lysosome must have mechanisms that regulate its shape and size while coordinating content exchange. In this review, we discussed the mechanisms that regulate lysosomal fusion and fission as well as swelling and condensation, with a focus on solute and water transport mechanisms across lysosomal membranes. Lysosomal H+, Na+, K+, Ca2+, and Cl- channels and transporters sense trafficking and osmotic cues to regulate both solute flux and membrane trafficking. We also provide perspectives on how lysosomes may adjust the volume of themselves, the cytosol, and the cytoplasm through the control of lysosomal solute and water transport.


Assuntos
Canais Iônicos , Lisossomos , Água , Citoplasma , Citosol , Hidrolases/metabolismo , Canais Iônicos/metabolismo , Íons/metabolismo , Lisossomos/metabolismo , Água/metabolismo
19.
Nat Commun ; 13(1): 6023, 2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36224168

RESUMO

Changes in sub-cellular pH play a key role in metabolism, membrane transport, and triggering cargo release from therapeutic delivery systems. Most methods to measure pH rely on intensity changes of pH sensitive fluorophores, however, these measurements are hampered by high uncertainty in the inferred pH and the need for multiple fluorophores. To address this, here we combine pH dependant fluorescent lifetime imaging microscopy (pHLIM) with deep learning to accurately quantify sub-cellular pH in individual vesicles. We engineer the pH sensitive protein mApple to localise in the cytosol, endosomes, and lysosomes, and demonstrate that pHLIM can rapidly detect pH changes induced by drugs such as bafilomycin A1 and chloroquine. We also demonstrate that polyethylenimine (a common transfection reagent) does not exhibit a proton sponge effect and had no measurable impact on the pH of endocytic vesicles. pHLIM is a simple and quantitative method that will help to understand drug action and disease progression.


Assuntos
Técnicas Biossensoriais , Polietilenoimina , Cloroquina/farmacologia , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Polietilenoimina/metabolismo , Prótons
20.
Nat Commun ; 13(1): 5924, 2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36207292

RESUMO

Haploinsufficiency of GRN causes frontotemporal dementia (FTD). The GRN locus produces progranulin (PGRN), which is cleaved to lysosomal granulin polypeptides. The function of lysosomal granulins and why their absence causes neurodegeneration are unclear. Here we discover that PGRN-deficient human cells and murine brains, as well as human frontal lobes from GRN-mutation FTD patients have increased levels of gangliosides, glycosphingolipids that contain sialic acid. In these cells and tissues, levels of lysosomal enzymes that catabolize gangliosides were normal, but levels of bis(monoacylglycero)phosphates (BMP), lipids required for ganglioside catabolism, were reduced with PGRN deficiency. Our findings indicate that granulins are required to maintain BMP levels to support ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. Lysosomal ganglioside accumulation may contribute to neuroinflammation and neurodegeneration susceptibility observed in FTD due to PGRN deficiency and other neurodegenerative diseases.


Assuntos
Demência Frontotemporal , Gangliosidoses , Progranulinas/metabolismo , Animais , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Gangliosídeos/metabolismo , Gangliosidoses/metabolismo , Granulinas/metabolismo , Humanos , Lisossomos/metabolismo , Camundongos , Ácido N-Acetilneuramínico/metabolismo , Fosfatos/metabolismo , Progranulinas/genética
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