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1.
Methods Mol Biol ; 2814: 45-53, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38954196

RESUMO

Eukaryotic cells have been constantly challenged throughout their evolution by pathogens, mechanical stresses, or toxic compounds that induce plasma membrane (PM) or endolysosomal membrane damage. The survival of the wounded cells depends on damage detection and repair machineries that are evolutionary conserved between protozoan, plants, and animals. We use the social amoeba Dictyostelium discoideum as a model system to study bacteria, mechanical or sterile membrane damage that allows us to identify and monitor factors involved in PM, endolysosomal damage response (ELDR), and endolysosomal homeostasis. Importantly, the sterile damage techniques presented here homogenously affect cell populations, which allows to phenotype mutant strains and quantify various aspects of cell fitness using live cell microscopy. This is instrumental to functionally assess genes involved in the repair of damaged plasma membrane or intracellular compartments and the degradation of extensively damaged compartments. Here, we describe how to inflict sterile PM or endolysosomal membrane damage, how to monitor the cell-intrinsic response to damage, and how to proxy proton leakage from damaged acidic compartments and quantify cell viability.


Assuntos
Membrana Celular , Dictyostelium , Lisossomos , Dictyostelium/genética , Dictyostelium/metabolismo , Membrana Celular/metabolismo , Lisossomos/metabolismo , Sobrevivência Celular
2.
Methods Mol Biol ; 2814: 55-79, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38954197

RESUMO

Lysosomes are membrane-enclosed organelles that digest intracellular material. They contain more than 50 different enzymes that can degrade a variety of macromolecules including nucleic acids, proteins, polysaccharides, and lipids. In addition to functioning within lysosomes, lysosomal enzymes are also secreted. Alterations in the levels and activities of lysosomal enzymes dysregulates lysosomes, which can lead to the intralysosomal accumulation of biological material and the development of lysosomal storage diseases (LSDs) in humans. Dictyostelium discoideum has a long history of being used to study the trafficking and functions of lysosomal enzymes. More recently, it has been used as a model system to study several LSDs. In this chapter, we outline the methods for assessing the activity of several lysosomal enzymes in D. discoideum (α-galactosidase, ß-galactosidase, α-glucosidase, ß-glucosidase, ß-N-acetylglucosaminidase, α-mannosidase, cathepsin B, cathepsin D, cathepsin F, palmitoyl protein thioesterase 1, and tripeptidyl peptidase 1).


Assuntos
Dictyostelium , Lisossomos , Dictyostelium/enzimologia , Lisossomos/enzimologia , Lisossomos/metabolismo , Tripeptidil-Peptidase 1 , Ensaios Enzimáticos/métodos , Humanos , beta-Galactosidase/metabolismo , Doenças por Armazenamento dos Lisossomos/enzimologia , Doenças por Armazenamento dos Lisossomos/metabolismo , Tioléster Hidrolases/metabolismo
3.
Methods Mol Biol ; 2814: 97-106, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38954200

RESUMO

Autophagy is an intracellular clearance and recycling pathway that delivers different types of cargos to lysosomes for degradation. In recent years, autophagy has attracted considerable medical interest, and many different techniques are being developed to study this process in experimental models such as Dictyostelium. Here we describe the use of different autophagic markers in confocal microscopy, in vivo and also in fixed cells. In particular, we describe the use of the GFP-Atg8-RFP-Atg8ΔG marker and the optimization of the GFP-PgkA cleavage assay to detect small differences in autophagy flux.


Assuntos
Autofagia , Dictyostelium , Microscopia Confocal , Dictyostelium/metabolismo , Dictyostelium/fisiologia , Autofagia/fisiologia , Microscopia Confocal/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Lisossomos/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética
4.
J Immunol ; 213(2): 109-114, 2024 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-38950331

RESUMO

ATPase cation transporting 13A2 (ATP13A2) is an endolysosomal P-type ATPase known to be a polyamine transporter, explored mostly in neurons. As endolysosomal functions are also crucial in innate immune cells, we aimed to explore the potential role of ATP13A2 in the human immunocellular compartment. We found that human plasmacytoid dendritic cells (pDCs), the professional type I IFN-producing immune cells, especially have a prominent enrichment of ATP13A2 expression in endolysosomal compartments. ATP13A2 knockdown in human pDCs interferes with cytokine induction in response to TLR9/7 activation in response to bona fide ligands. ATP13A2 plays this crucial role in TLR9/7 activation in human pDCs by regulating endolysosomal pH and mitochondrial reactive oxygen generation. This (to our knowledge) hitherto unknown regulatory mechanism in pDCs involving ATP13A2 opens up a new avenue of research, given the crucial role of pDC-derived type I IFNs in protective immunity against infections as well as in the immunopathogenesis of myriad contexts of autoreactive inflammation.


Assuntos
Células Dendríticas , Endossomos , Lisossomos , Receptor Toll-Like 9 , Humanos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Lisossomos/metabolismo , Lisossomos/imunologia , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/imunologia , Endossomos/metabolismo , Endossomos/imunologia , ATPases Translocadoras de Prótons/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/imunologia , Células Cultivadas , Interferon Tipo I/metabolismo , Interferon Tipo I/imunologia , Receptor 7 Toll-Like
5.
Elife ; 122024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38953285

RESUMO

We studied lysosomal Ca2+ in inflammasome. Lipopolysaccharide (LPS) + palmitic acid (PA) decreased lysosomal Ca2+ ([Ca2+]Lys) and increased [Ca2+]i through mitochondrial ROS, which was suppressed in Trpm2-KO macrophages. Inflammasome activation and metabolic inflammation in adipose tissue of high-fat diet (HFD)-fed mice were ameliorated by Trpm2 KO. ER→lysosome Ca2+ refilling occurred after lysosomal Ca2+ release whose blockade attenuated LPS + PA-induced inflammasome. Subsequently, store-operated Ca2+entry (SOCE) was activated whose inhibition suppressed inflammasome. SOCE was coupled with K+ efflux whose inhibition reduced ER Ca2+ content ([Ca2+]ER) and impaired [Ca2+]Lys recovery. LPS + PA activated KCa3.1 channel, a Ca2+-activated K+ channel. Inhibitors of KCa3.1 channel or Kcnn4 KO reduced [Ca2+]ER, attenuated increase of [Ca2+]i or inflammasome activation by LPS + PA, and ameliorated HFD-induced inflammasome or metabolic inflammation. Lysosomal Ca2+ release induced delayed JNK and ASC phosphorylation through CAMKII-ASK1. These results suggest a novel role of lysosomal Ca2+ release sustained by ER→lysosome Ca2+ refilling and K+ efflux through KCa3.1 channel in inflammasome activation and metabolic inflammation.


Assuntos
Cálcio , Retículo Endoplasmático , Inflamassomos , Inflamação , Lisossomos , Camundongos Knockout , Potássio , Animais , Inflamassomos/metabolismo , Camundongos , Lisossomos/metabolismo , Cálcio/metabolismo , Potássio/metabolismo , Inflamação/metabolismo , Retículo Endoplasmático/metabolismo , Lipopolissacarídeos , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPM/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Camundongos Endogâmicos C57BL , Macrófagos/metabolismo , Masculino , Dieta Hiperlipídica
6.
Med Microbiol Immunol ; 213(1): 14, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38977511

RESUMO

Mycobacterium tuberculosis, a lethal pathogen in human history, causes millions of deaths annually, which demands the development of new concepts of drugs. Considering this fact, earlier research has explored the anti-tuberculosis potential of a probiotic strain, Lactocaseibacillus rhamnosus PMC203, leading to a subsequent focus on the molecular mechanism involved in its effect, particularly on autophagy. In this current study, immunoblotting-based assay exhibited a remarkable expression of autophagy marker LC3-II in the PMC203 treated group compared to an untreated group. A remarkable degradation of p62 was also noticed within treated cells compared to control. Furthermore, the immunofluorescence-based assay showed significant fold change in fluorescence intensity for alexa-647-LC3 and alexa-488-LC3, whereas p62 was degraded noticeably. Moreover, lysosomal biogenesis generation was elevated significantly in terms of LAMP1 and acidic vesicular organelles. As a result, PMC203-induced autophagy played a vital role in reducing M. tuberculosis burden within the macrophages in treated groups compared to untreated group. A colony -forming unit assay also revealed a significant reduction in M. tuberculosis in the treated cells over time. Additionally, the candidate strain significantly upregulated the expression of autophagy induction and lysosomal biogenesis genes. Together, these results could enrich our current knowledge of probiotics-mediated autophagy in tuberculosis and suggest its implications for innovatively managing tuberculosis.


Assuntos
Autofagia , Lacticaseibacillus rhamnosus , Macrófagos , Mycobacterium tuberculosis , Probióticos , Mycobacterium tuberculosis/genética , Lacticaseibacillus rhamnosus/fisiologia , Lacticaseibacillus rhamnosus/metabolismo , Macrófagos/microbiologia , Humanos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Carga Bacteriana , Tuberculose/microbiologia
7.
Nat Commun ; 15(1): 5715, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38977659

RESUMO

Mitochondria are maternally inherited, but the mechanisms underlying paternal mitochondrial elimination after fertilization are far less clear. Using Drosophila, we show that special egg-derived multivesicular body vesicles promote paternal mitochondrial elimination by activating an LC3-associated phagocytosis-like pathway, a cellular defense pathway commonly employed against invading microbes. Upon fertilization, these egg-derived vesicles form extended vesicular sheaths around the sperm flagellum, promoting degradation of the sperm mitochondrial derivative and plasma membrane. LC3-associated phagocytosis cascade of events, including recruitment of a Rubicon-based class III PI(3)K complex to the flagellum vesicular sheaths, its activation, and consequent recruitment of Atg8/LC3, are all required for paternal mitochondrial elimination. Finally, lysosomes fuse with strings of large vesicles derived from the flagellum vesicular sheaths and contain degrading fragments of the paternal mitochondrial derivative. Given reports showing that in some mammals, the paternal mitochondria are also decorated with Atg8/LC3 and surrounded by multivesicular bodies upon fertilization, our findings suggest that a similar pathway also mediates paternal mitochondrial elimination in other flagellated sperm-producing organisms.


Assuntos
Proteínas de Drosophila , Fertilização , Mitocôndrias , Corpos Multivesiculares , Fagocitose , Espermatozoides , Animais , Mitocôndrias/metabolismo , Masculino , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Feminino , Espermatozoides/metabolismo , Corpos Multivesiculares/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Óvulo/metabolismo , Lisossomos/metabolismo , Cauda do Espermatozoide/metabolismo , Mitofagia
8.
Elife ; 132024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38899618

RESUMO

The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including α-synuclein (αSyn) associated with the pathogenesis of Parkinson's disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble αSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.


Assuntos
Autofagia , Lisossomos , Doença de Parkinson , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Autofagia/efeitos dos fármacos , Humanos , alfa-Sinucleína/metabolismo , Albendazol/farmacologia , Centro Organizador dos Microtúbulos/metabolismo , Autofagossomos/metabolismo , Autofagossomos/efeitos dos fármacos
9.
Proc Natl Acad Sci U S A ; 121(26): e2317945121, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38889154

RESUMO

Chaperone-mediated autophagy (CMA) is part of the mammalian cellular proteostasis network that ensures protein quality control, maintenance of proteome homeostasis, and proteome changes required for the adaptation to stress. Loss of proteostasis is one of the hallmarks of aging. CMA decreases with age in multiple rodent tissues and human cell types. A decrease in lysosomal levels of the lysosome-associated membrane protein type 2A (LAMP2A), the CMA receptor, has been identified as a main reason for declined CMA in aging. Here, we report constitutive activation of CMA with calorie restriction (CR), an intervention that extends healthspan, in old rodent livers and in an in vitro model of CR with cultured fibroblasts. We found that CR-mediated upregulation of CMA is due to improved stability of LAMP2A at the lysosome membrane. We also explore the translational value of our observations using calorie-restriction mimetics (CRMs), pharmacologically active substances that reproduce the biochemical and functional effects of CR. We show that acute treatment of old mice with CRMs also robustly activates CMA in several tissues and that this activation is required for the higher resistance to lipid dietary challenges conferred by treatment with CRMs. We conclude that part of the beneficial effects associated with CR/CRMs could be a consequence of the constitutive activation of CMA mediated by these interventions.


Assuntos
Restrição Calórica , Autofagia Mediada por Chaperonas , Proteína 2 de Membrana Associada ao Lisossomo , Lisossomos , Animais , Camundongos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Lisossomos/metabolismo , Humanos , Envelhecimento/metabolismo , Fibroblastos/metabolismo , Proteostase , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Autofagia
10.
Int J Mol Sci ; 25(11)2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38892390

RESUMO

Aurora kinase A (AURKA) is a serine/threonine-protein kinase that regulates microtubule organization during neuron migration and neurite formation. Decreased activity of AURKA was found in Alzheimer's disease (AD) brain samples, but little is known about the role of AURKA in AD pathogenesis. Here, we demonstrate that AURKA is expressed in primary cultured rat neurons, neurons from adult mouse brains, and neurons in postmortem human AD brains. AURKA phosphorylation, which positively correlates with its activity, is reduced in human AD brains. In SH-SY5Y cells, pharmacological activation of AURKA increased AURKA phosphorylation, acidified endolysosomes, decreased the activity of amyloid beta protein (Aß) generating enzyme ß-site amyloid precursor protein cleaving enzyme (BACE-1), increased the activity of the Aß degrading enzyme cathepsin D, and decreased the intracellular and secreted levels of Aß. Conversely, pharmacological inhibition of AURKA decreased AURKA phosphorylation, de-acidified endolysosomes, decreased the activity of cathepsin D, and increased intracellular and secreted levels of Aß. Thus, reduced AURKA activity in AD may contribute to the development of intraneuronal accumulations of Aß and extracellular amyloid plaque formation.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Aurora Quinase A , Lisossomos , Neurônios , Aurora Quinase A/metabolismo , Animais , Neurônios/metabolismo , Humanos , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Camundongos , Ratos , Lisossomos/metabolismo , Fosforilação , Linhagem Celular Tumoral , Encéfalo/metabolismo , Células Cultivadas , Masculino , Secretases da Proteína Precursora do Amiloide/metabolismo
11.
J Cell Biol ; 223(8)2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38913026

RESUMO

The double-stranded RNA-binding protein Staufen1 (STAU1) regulates a variety of physiological and pathological events via mediating RNA metabolism. STAU1 overabundance was observed in tissues from mouse models and fibroblasts from patients with neurodegenerative diseases, accompanied by enhanced mTOR signaling and impaired autophagic flux, while the underlying mechanism remains elusive. Here, we find that endogenous STAU1 forms dynamic cytoplasmic condensate in normal and tumor cell lines, as well as in mouse Huntington's disease knockin striatal cells. STAU1 condensate recruits target mRNA MTOR at its 5'UTR and promotes its translation both in vitro and in vivo, and thus enhanced formation of STAU1 condensate leads to mTOR hyperactivation and autophagy-lysosome dysfunction. Interference of STAU1 condensate normalizes mTOR levels, ameliorates autophagy-lysosome function, and reduces aggregation of pathological proteins in cellular models of neurodegenerative diseases. These findings highlight the importance of balanced phase separation in physiological processes, suggesting that modulating STAU1 condensate may be a strategy to mitigate the progression of neurodegenerative diseases with STAU1 overabundance.


Assuntos
Autofagia , Biossíntese de Proteínas , Proteínas de Ligação a RNA , Serina-Treonina Quinases TOR , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Animais , Humanos , Autofagia/genética , Camundongos , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/genética , Lisossomos/metabolismo , Lisossomos/genética , Transdução de Sinais , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Doença de Huntington/genética
12.
Cell Death Dis ; 15(6): 424, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38890356

RESUMO

Alterations in the dopamine catabolic pathway are known to contribute to the degeneration of nigrostriatal neurons in Parkinson's disease (PD). The progressive cellular buildup of the highly reactive intermediate 3,4-dihydroxyphenylacetaldehye (DOPAL) generates protein cross-linking, oligomerization of the PD-linked αSynuclein (αSyn) and imbalance in protein quality control. In this scenario, the autophagic cargo sequestome-1 (SQSTM1/p62) emerges as a target of DOPAL-dependent oligomerization and accumulation in cytosolic clusters. Although DOPAL-induced oxidative stress and activation of the Nrf2 pathway promote p62 expression, p62 oligomerization rather seems to be a consequence of direct DOPAL modification. DOPAL-induced p62 clusters are positive for ubiquitin and accumulate within lysosomal-related structures, likely affecting the autophagy-lysosomal functionality. Finally, p62 oligomerization and clustering is synergistically augmented by DOPAL-induced αSyn buildup. Hence, the substantial impact on p62 proteostasis caused by DOPAL appears of relevance for dopaminergic neurodegeneration, in which the progressive failure of degradative pathways and the deposition of proteins like αSyn, ubiquitin and p62 in inclusion bodies represent a major trait of PD pathology.


Assuntos
Dopamina , Proteína Sequestossoma-1 , Animais , Humanos , alfa-Sinucleína/metabolismo , Autofagia , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteína Sequestossoma-1/metabolismo
13.
Mol Neurodegener ; 19(1): 49, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38890703

RESUMO

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people in the developed world, and the number of people affected is expected to almost double by 2040. The retina presents one of the highest metabolic demands in our bodies that is partially or fully fulfilled by mitochondria in the neuroretina and retinal pigment epithelium (RPE), respectively. Together with its post-mitotic status and constant photooxidative damage from incoming light, the retina requires a tightly-regulated housekeeping system that involves autophagy. The natural polyphenol Urolithin A (UA) has shown neuroprotective benefits in several models of aging and age-associated disorders, mostly attributed to its ability to induce mitophagy and mitochondrial biogenesis. Sodium iodate (SI) administration recapitulates the late stages of AMD, including geographic atrophy and photoreceptor cell death. METHODS: A combination of in vitro, ex vivo and in vivo models were used to test the neuroprotective potential of UA in the SI model. Functional assays (OCT, ERGs), cellular analysis (flow cytometry, qPCR) and fine confocal microscopy (immunohistochemistry, tandem selective autophagy reporters) helped address this question. RESULTS: UA alleviated neurodegeneration and preserved visual function in SI-treated mice. Simultaneously, we observed severe proteostasis defects upon SI damage induction, including autophagosome accumulation, that were resolved in animals that received UA. Treatment with UA restored autophagic flux and triggered PINK1/Parkin-dependent mitophagy, as previously reported in the literature. Autophagy blockage caused by SI was caused by severe lysosomal membrane permeabilization. While UA did not induce lysosomal biogenesis, it did restore upcycling of permeabilized lysosomes through lysophagy. Knockdown of the lysophagy adaptor SQSTM1/p62 abrogated viability rescue by UA in SI-treated cells, exacerbated lysosomal defects and inhibited lysophagy. CONCLUSIONS: Collectively, these data highlight a novel putative application of UA in the treatment of AMD whereby it bypasses lysosomal defects by promoting p62-dependent lysophagy to sustain proteostasis.


Assuntos
Cumarínicos , Animais , Camundongos , Cumarínicos/farmacologia , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Retina/metabolismo , Retina/efeitos dos fármacos , Retina/patologia , Mitofagia/efeitos dos fármacos , Mitofagia/fisiologia , Proteína Sequestossoma-1/metabolismo , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Humanos , Modelos Animais de Doenças , Fármacos Neuroprotetores/farmacologia , Camundongos Endogâmicos C57BL , Iodatos/toxicidade
14.
J Hazard Mater ; 474: 134791, 2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38833954

RESUMO

Despite the growing awareness of potential human and environmental risks associated with sunscreens, identifying the specific constituents responsible for their potential toxicity is challenging. In this study, we applied three different types of sunscreens with contrasting compositions and compared the effects of their particulate and soluble fractions based on 15 cellular biomarkers of HaCaT cells. Multilinear regression analysis revealed that the internalized soluble fractions played a primary role in the overall cytotoxicity of sunscreen mixtures, which was primarily attributed to their biotransformation, generating metabolites with higher toxicity. The presence of plastic microspheres in sunscreens either inhibited the internalization of soluble fractions or led to their redistribution toward lysosomes. Conversely, subcellular toxicity resulting from the sunscreen mixture was predominantly influenced by particulates. Bio-transformable particulates such as ZnO dissolved in the organelles and induced higher subcellular toxicity compared to bioinert particulates such as microplastics. Subcellular biomarkers including lysosomal count, lysosomal size, mitochondrial count and mitochondrial shape emerged as the potential predictors of sunscreen presence. Our study provides important understanding of sunscreen toxicity by elucidating the differential impacts of particulate and soluble fractions in mixture contaminants.


Assuntos
Lisossomos , Protetores Solares , Protetores Solares/toxicidade , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Linhagem Celular , Células HaCaT , Biomarcadores/metabolismo , Solubilidade , Óxido de Zinco/toxicidade , Óxido de Zinco/química , Microplásticos/toxicidade , Material Particulado/toxicidade , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Microesferas
15.
Proc Natl Acad Sci U S A ; 121(25): e2322588121, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38861598

RESUMO

The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of biomolecules; lysosome-related organelles known as gut granules are important for many of these functions. Aspects of intestine biology are not well understood, including the export of the nutrients it imports and the molecules it synthesizes, as well as the complete functions and protein content of the gut granules. Here, we report a mass spectrometry (MS)-based proteomic analysis of the intestine of the Caenorhabditis elegans and of its gut granules. Overall, we identified approximately 5,000 proteins each in the intestine and the gonad and showed that most of these proteins can be detected in samples extracted from a single worm, suggesting the feasibility of individual-level genetic analysis using proteomes. Comparing proteomes and published transcriptomes of the intestine and the gonad, we identified proteins that appear to be synthesized in the intestine and then transferred to the gonad. To identify gut granule proteins, we compared the proteome of individual intestines deficient in gut granules to the wild type. The identified gut granule proteome includes proteins known to be exclusively localized to the granules and additional putative gut granule proteins. We selected two of these putative gut granule proteins for validation via immunohistochemistry, and our successful confirmation of both suggests that our strategy was effective in identifying the gut granule proteome. Our results demonstrate the practicability of single-tissue MS-based proteomic analysis in small organisms and in its future utility.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Lisossomos , Proteômica , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteômica/métodos , Lisossomos/metabolismo , Proteoma/metabolismo , Intestinos , Mucosa Intestinal/metabolismo , Gônadas/metabolismo , Espectrometria de Massas/métodos , Organelas/metabolismo
16.
Reprod Domest Anim ; 59(6): e14643, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38877774

RESUMO

Progesterone has been shown to stimulate glycogen catabolism in uterine epithelial cells. Acid α-glucosidase (GAA) is an enzyme that breaks down glycogen within lysosomes. We hypothesized that progesterone may stimulate glycogenolysis in the uterine epithelium via GAA. We found that GAA was more highly expressed in the stroma on Day 1 than on Day 11. However, GAA did not appear to differ in the epithelium on Days 1 and 11. Progesterone (0-10 µM) had no effect on the levels of the full-length inactive protein (110 kDa) or the cleaved (active) peptides present inside the lysosome (70 and 76 kDa) in immortalized bovine uterine epithelial (BUTE) cells. Furthermore, the activity of GAA did not differ between the BUTE cells treated with 10 µM progesterone or control. Overall, we confirmed that GAA is present in the cow endometrium and BUTE cells. However, progesterone did not affect protein levels or enzyme activity.


Assuntos
Endométrio , Progesterona , alfa-Glucosidases , Animais , Bovinos , Feminino , Endométrio/metabolismo , Endométrio/enzimologia , Progesterona/farmacologia , Progesterona/metabolismo , alfa-Glucosidases/metabolismo , alfa-Glucosidases/genética , Células Epiteliais/metabolismo , Glicogenólise , Lisossomos/enzimologia , Lisossomos/metabolismo , Glicogênio/metabolismo
17.
Nat Commun ; 15(1): 5388, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38918376

RESUMO

Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.


Assuntos
Acetiltransferases , Microscopia Crioeletrônica , Lisossomos , Mucopolissacaridose III , Mucopolissacaridose III/genética , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/enzimologia , Humanos , Lisossomos/metabolismo , Lisossomos/enzimologia , Acetiltransferases/metabolismo , Acetiltransferases/química , Acetiltransferases/genética , Domínio Catalítico , Mutação , Heparitina Sulfato/metabolismo , Acetilcoenzima A/metabolismo , Acetilcoenzima A/química , Modelos Moleculares , Glucosamina/metabolismo , Glucosamina/química , Acetilação , Membranas Intracelulares/metabolismo
18.
Chem Commun (Camb) ; 60(53): 6793-6796, 2024 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-38869018

RESUMO

We report an integrated ratiometric lysosomal nitric oxide (NO) nanoprobe based on engineered semiconducting polymer dots (Pdots), LyNO-Pdots, which consist of a newly designed NO-responsive dye, a fluorescent conjugated polymer and two functional polymers. The developed probe LyNO-Pdots exhibit high specificity and stability, good photostability and favorable blood-brain barrier (BBB) penetration ability. The LyNO-Pdots are successfully applied to ratiometric imaging of lysosomal NO variations in brain-derived endothelial cells, brain tissues and mice brains with Alzheimer's disease (AD). The results demonstrate that the NO content in the brains of AD mice is considerably higher than that in normal mice.


Assuntos
Doença de Alzheimer , Encéfalo , Corantes Fluorescentes , Lisossomos , Óxido Nítrico , Imagem Óptica , Animais , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Lisossomos/química , Lisossomos/metabolismo , Camundongos , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Humanos , Polímeros/química , Barreira Hematoencefálica/metabolismo , Pontos Quânticos/química
19.
Cell Death Dis ; 15(6): 414, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38871731

RESUMO

The repurposing of medications developed for central nervous system (CNS) disorders, possessing favorable safety profiles and blood-brain barrier permeability, represents a promising strategy for identifying new therapies to combat glioblastoma (GBM). In this study, we investigated the anti-GBM activity of specific antipsychotics and antidepressants in vitro and in vivo. Our results demonstrate that these compounds share a common mechanism of action in GBM, disrupting lysosomal function and subsequently inducing lysosomal membrane rupture and cell death. Notably, PTEN intact GBMs possess an increased sensitivity to these compounds. The inhibition of lysosomal function synergized with inhibitors targeting the EGFR-PI3K-Akt pathway, leading to an energetic and antioxidant collapse. These findings provide a foundation for the potential clinical application of CNS drugs in GBM treatment. Additionally, this work offers critical insights into the mechanisms and determinants of cytotoxicity for drugs currently undergoing clinical trials as repurposing agents for various cancers, including Fluoxetine, Sertraline, Thioridazine, Chlorpromazine, and Fluphenazine.


Assuntos
Antipsicóticos , Glioblastoma , Lisossomos , PTEN Fosfo-Hidrolase , Transdução de Sinais , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Animais , Linhagem Celular Tumoral , Camundongos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos Nus , Reposicionamento de Medicamentos , Fosfatidilinositol 3-Quinases/metabolismo , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inibidores , Clorpromazina/farmacologia
20.
Biomolecules ; 14(6)2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38927017

RESUMO

Renal interstitial fibrosis (RIF) is a classic pathophysiological process of chronic kidney disease (CKD). However, the mechanisms underlying RIF remain unclear. The present study found that a novel circular RNA, cirInpp5b, might be involved in RIF by high-throughput sequencing. Subsequent experiments revealed that circInpp5b was reduced in UUO mouse kidney tissues and TGF-ß1-treated proximal tubular cells. The overexpression of circInpp5b inhibited RIF in UUO mice and prevented extracellular matrix (ECM) deposition in TGF-ß1-treated proximal tubular cells. Furthermore, overexpression of circInpp5b down-regulated the protein level of DDX1. Mechanistically, circInpp5b bound to the DDX1 protein and promoted its lysosomal degradation. Collectively, the findings of our study demonstrate that circInpp5b ameliorates RIF by binding to the DDX1 protein and promoting its lysosomal degradation.


Assuntos
RNA Helicases DEAD-box , Fibrose , Lisossomos , RNA Circular , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Animais , Camundongos , Lisossomos/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Proteólise , Masculino , Camundongos Endogâmicos C57BL , Humanos , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Nefropatias/genética
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