Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 37.650
Filtrar
1.
Biol Aujourdhui ; 218(1-2): 41-54, 2024.
Artigo em Francês | MEDLINE | ID: mdl-39007776

RESUMO

The review is focused on recent drug discovery advances based on targeted protein degradation strategies. This new area of research has exploded leading to the development of potential drugs useful in a large variety of human diseases. They first target disease relevant proteins difficult to counteract with other classical strategies and extend now to aggregates, organelles, nucleic acids or lipidic droplets. These degraders engaged either the ubiquitin-proteasome system for PROTACs and molecular glues (first generation), or the lysosomal system via endosome-lysosome degradation (LYTACs) and autophagy-lysosome degradation (ATTEC, AUTAC, AUTOTAC) (following generations of degraders). PROTACs have expanded from the orthodox heterobifunctional ones to new derivatives such as homo-PROTACs, pro-PROTACs, CLIPTACs, HaloPROTACs, PHOTOTACs, Bac-PROTACs, AbTACs, ARN-PROTACs. The small molecular-weight molecular glues induce the formation of new ternary complexes which implicate the targeted protein and an ubiquitin ligase E3 allowing the protein ubiquinitation followed by its proteasomal degradation. Lysosomal degraders (LYTAC, ATTEC, AUTAC, AUTOTAC) specifically recognize extracellular and membrane proteins or dysfunctional organelles and transport them into lysosomes where they are degraded. They overcome the limitations observed with proteasomal degradations induced by PROTAC and molecular glues and demonstrate their potential to treat human diseases, especially neurodegenerative ones. Pharmaceutical companies are engaged at the world level to develop these new potential drugs targeting cancers, immuno-inflammatory and neurodegenerative diseases as well as a variety of other ones. Efficiency and risks for these novel therapeutic strategies are discussed.


Title: Induction de proximité et dégradation de cibles thérapeutiques par les nouveaux dégradeurs : quels concepts, quels développements, quel futur ? Abstract: La recherche dans le domaine de la dégradation ciblée des protéines s'est considérablement développée conduisant à l'élaboration de nouveaux outils chimiques à visée thérapeutique, les dégradeurs, potentiellement utiles dans diverses pathologies. Une grande variété d'objets à dégrader appartenant à divers compartiments intra- ou extracellulaires (protéines, complexes ou agrégats, organelles, acides nucléiques, gouttelettes lipidiques) a été ciblée à l'aide de ligands déjà existants, d'autres restent à découvrir. Les molécules de première génération, PROTAC et colles moléculaires, utilisent le système ubiquitine-protéasome pour détruire spécifiquement des protéines pathogéniques, certaines considérées jusqu'à présent comme inaccessibles en tant que cibles thérapeutiques. Au cours des cinq dernières années, ont été développés de nouveaux types de PROTAC hétéro-bifonctionnels comme les homo-PROTAC, pro-PROTAC, CLIPTAC, HaloPROTAC, PHOTOTAC, Bac-PROTAC, mais aussi des PROTAC macromoléculaires comme les AbTAC et ARN-PROTAC. Du fait de la grande diversité des substrats dégradés par les lysosomes, de nouveaux dégradeurs impliquant deux voies distinctes ont été ensuite produits : les chimères LYTAC pour la voie endosome-lysosome et les chimères ATTEC, AUTAC et AUTOTAC pour la voie autophagie-lysosome, augmentant ainsi considérablement le champ d'action des dégradeurs. Ces nouvelles molécules reconnaissent spécifiquement des protéines et/ou des organelles et permettent leur transport dans les lysosomes où ils sont dégradés. Les succès obtenus, que ce soit par dégradation protéasomale ou lysosomale pour plusieurs dizaines de dégradeurs (preuves de concepts et études cliniques en cours), expliquent l'intérêt quasi mondial des industries pharmaceutiques pour ces nouvelles molécules. Les challenges posés par leur développement et leur utilisation en clinique sont discutés.


Assuntos
Lisossomos , Proteólise , Humanos , Proteólise/efeitos dos fármacos , Lisossomos/metabolismo , Animais , Proteínas/metabolismo , Descoberta de Drogas/tendências , Descoberta de Drogas/métodos , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Autofagia/fisiologia
2.
J Cell Biol ; 223(10)2024 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-39007804

RESUMO

To breach the basement membrane, cells in development and cancer use large, transient, specialized lipid-rich membrane protrusions. Using live imaging, endogenous protein tagging, and cell-specific RNAi during Caenorhabditis elegans anchor cell (AC) invasion, we demonstrate that the lipogenic SREBP transcription factor SBP-1 drives the expression of the fatty acid synthesis enzymes POD-2 and FASN-1 prior to invasion. We show that phospholipid-producing LPIN-1 and sphingomyelin synthase SMS-1, which use fatty acids as substrates, produce lysosome stores that build the AC's invasive protrusion, and that SMS-1 also promotes protrusion localization of the lipid raft partitioning ZMP-1 matrix metalloproteinase. Finally, we discover that HMG-CoA reductase HMGR-1, which generates isoprenoids for prenylation, localizes to the ER and enriches in peroxisomes at the AC invasive front, and that the final transmembrane prenylation enzyme, ICMT-1, localizes to endoplasmic reticulum exit sites that dynamically polarize to deliver prenylated GTPases for protrusion formation. Together, these results reveal a collaboration between lipogenesis and a polarized lipid prenylation system that drives invasive protrusion formation.


Assuntos
Membrana Basal , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Retículo Endoplasmático , Lipogênese , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Membrana Basal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Retículo Endoplasmático/metabolismo , Lipogênese/genética , Prenilação , Peroxissomos/metabolismo , Movimento Celular , Lisossomos/metabolismo
3.
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
4.
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
5.
J Cell Mol Med ; 28(10): e18402, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-39008328

RESUMO

Syntaxin 17 (STX17) has been identified as a crucial factor in mediating the fusion of autophagosomes and lysosomes. However, its specific involvement in the context of atherosclerosis (AS) remains unclear. This study sought to elucidate the role and mechanistic contributions of STX17 in the initiation and progression of AS. Utilizing both in vivo and in vitro AS model systems, we employed ApoE knockout (KO) mice subjected to a high-fat diet and human umbilical vein endothelial cells (HUVECs) treated with oxidized low-density lipoprotein (ox-LDL) to assess STX17 expression. To investigate underlying mechanisms, we employed shRNA-STX17 lentivirus to knock down STX17 expression, followed by evaluating autophagy and inflammation in HUVECs. In both in vivo and in vitro AS models, STX17 expression was significantly upregulated. Knockdown of STX17 exacerbated HUVEC damage, both with and without ox-LDL treatment. Additionally, we observed that STX17 knockdown impaired autophagosome degradation, impeded autophagy flux and also resulted in the accumulation of dysfunctional lysosomes in HUVECs. Moreover, STX17 knockdown intensified the inflammatory response following ox-LDL treatment in HUVECs. Further mechanistic exploration revealed an association between STX17 and STING; reducing STX17 expression increased STING levels. Further knockdown of STING enhanced autophagy flux. In summary, our findings suggest that STX17 knockdown worsens AS by impeding autophagy flux and amplifying the inflammatory response. Additionally, the interaction between STX17 and STING may play a crucial role in STX17-mediated autophagy.


Assuntos
Aterosclerose , Autofagia , Células Endoteliais da Veia Umbilical Humana , Inflamação , Lipoproteínas LDL , Proteínas Qa-SNARE , Autofagia/genética , Animais , Humanos , Aterosclerose/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Camundongos , Lipoproteínas LDL/metabolismo , Técnicas de Silenciamento de Genes , Lisossomos/metabolismo , Camundongos Knockout , Masculino , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Dieta Hiperlipídica/efeitos adversos , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Apolipoproteínas E/deficiência
6.
Molecules ; 29(13)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38999083

RESUMO

The delivery of therapeutic agents faces significant hurdles posed by the endo-lysosomal pathway, a bottleneck that hampers clinical effectiveness. This comprehensive review addresses the urgent need to enhance cellular delivery mechanisms to overcome these obstacles. It focuses on the potential of smart nanomaterials, delving into their unique characteristics and mechanisms in detail. Special attention is given to their ability to strategically evade endosomal entrapment, thereby enhancing therapeutic efficacy. The manuscript thoroughly examines assays crucial for understanding endosomal escape and cellular uptake dynamics. By analyzing various assessment methods, we offer nuanced insights into these investigative approaches' multifaceted aspects. We meticulously analyze the use of smart nanocarriers, exploring diverse mechanisms such as pore formation, proton sponge effects, membrane destabilization, photochemical disruption, and the strategic use of endosomal escape agents. Each mechanism's effectiveness and potential application in mitigating endosomal entrapment are scrutinized. This paper provides a critical overview of the current landscape, emphasizing the need for advanced delivery systems to navigate the complexities of cellular uptake. Importantly, it underscores the transformative role of smart nanomaterials in revolutionizing cellular delivery strategies, leading to a paradigm shift towards improved therapeutic outcomes.


Assuntos
Endossomos , Lisossomos , Lisossomos/metabolismo , Humanos , Endossomos/metabolismo , Sistemas de Liberação de Medicamentos , Portadores de Fármacos/química , Nanoestruturas/química , Animais , Nanopartículas/química
7.
Int J Mol Sci ; 25(13)2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38999927

RESUMO

Docosahexaenoic acid (DHA, C22:6 ω3) may be involved in various neuroprotective mechanisms that could prevent Alzheimer's disease (AD). Its influence has still been little explored regarding the dysfunction of the endolysosomal pathway, known as an early key event in the physiopathological continuum triggering AD. This dysfunction could result from the accumulation of degradation products of the precursor protein of AD, in particular the C99 fragment, capable of interacting with endosomal proteins and thus contributing to altering this pathway from the early stages of AD. This study aims to evaluate whether neuroprotection mediated by DHA can also preserve the endolysosomal function. AD-typical endolysosomal abnormalities were recorded in differentiated human SH-SY5Y neuroblastoma cells expressing the Swedish form of human amyloid precursor protein. This altered phenotype included endosome enlargement, the reduced secretion of exosomes, and a higher level of apoptosis, which confirmed the relevance of the cellular model chosen for studying the associated deleterious mechanisms. Second, neuroprotection mediated by DHA was associated with a reduced interaction of C99 with the Rab5 GTPase, lower endosome size, restored exosome production, and reduced neuronal apoptosis. Our data reveal that DHA may influence protein localization and interactions in the neuronal membrane environment, thereby correcting the dysfunction of endocytosis and vesicular trafficking associated with AD.


Assuntos
Doença de Alzheimer , Ácidos Docosa-Hexaenoicos , Endossomos , Lisossomos , Neurônios , Proteínas rab5 de Ligação ao GTP , Humanos , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Proteínas rab5 de Ligação ao GTP/metabolismo , Endossomos/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Neurônios/efeitos dos fármacos , Lisossomos/metabolismo , Linhagem Celular Tumoral , Precursor de Proteína beta-Amiloide/metabolismo , Apoptose , Fármacos Neuroprotetores/farmacologia , Sobrevivência Celular/efeitos dos fármacos
8.
Dalton Trans ; 53(28): 11836-11849, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-38949269

RESUMO

A novel lysosome-targeted photosensitizer/photoredox catalyst based on cyclometalated Ir(III) complex IrL has been designed and synthesized, which exhibited excellent phosphorescence properties and the ability to generate single oxygen (1O2) and photocatalytically oxidize 1,4-dihydronicotinamide adenine dinucleotide (NADH) under light irradiation. Most importantly, the aforementioned activities are significantly enhanced due to protonation under acidic conditions, which makes them highly attractive in light-activated tumor therapy, especially for acidic lysosomes and tumor microenvironments. The photocytotoxicity of IrL and the mechanism of cell death have been investigated. Additionally, the tumor-killing ability of IrL under light irradiation was evaluated using a 4T1 tumor-bearing mouse model. This work provides a strategy for the development of lysosome-targeted photosensitizers/photoredox catalysts to overcome hypoxic tumors.


Assuntos
Complexos de Coordenação , Irídio , Lisossomos , Oxirredução , Fármacos Fotossensibilizantes , Lisossomos/metabolismo , Irídio/química , Irídio/farmacologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/síntese química , Animais , Catálise , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/síntese química , Camundongos , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Humanos , Fotoquimioterapia , Linhagem Celular Tumoral , Luz , Camundongos Endogâmicos BALB C
9.
Anal Chem ; 96(28): 11581-11587, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-38951996

RESUMO

Ferroptosis is an iron-dependent programmed cell death that is characterized by the dysregulation of lipid reactive oxygen species (ROS) production, causing abnormal changes in hypochlorous acid (HClO) levels in lysosomes. Super-resolution imaging can observe the fine structure of the lysosome at the nanometer level; therefore, it can be used to detect lysosome HClO levels during ferroptosis at the suborganelle level. Herein, we utilize a ratiometric fluorescent probe, SRF-HClO, for super-resolution imaging of lysosome HClO. Structured-illumination microscopy (SIM) improves the accuracy of lysosome targeting and enables the probe SRF-HClO to be successfully applied to rapidly monitor the up-regulated lysosome HClO at the nanoscale during inflammation and ferroptosis. Importantly, the probe SRF-HClO can also detect HClO changes in inflammatory and ferroptosis mice and evaluate the inhibitory effect of ferroptosis on mice tumors.


Assuntos
Ferroptose , Corantes Fluorescentes , Ácido Hipocloroso , Lisossomos , Ferroptose/efeitos dos fármacos , Corantes Fluorescentes/química , Ácido Hipocloroso/análise , Ácido Hipocloroso/metabolismo , Lisossomos/química , Lisossomos/metabolismo , Animais , Camundongos , Humanos , Imagem Óptica , Células RAW 264.7
10.
Cell Mol Life Sci ; 81(1): 304, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-39009859

RESUMO

The autophagy-lysosomal pathway plays a critical role in the clearance of tau protein aggregates that deposit in the brain in tauopathies, and defects in this system are associated with disease pathogenesis. Here, we report that expression of Tau35, a tauopathy-associated carboxy-terminal fragment of tau, leads to lipid accumulation in cell lines and primary cortical neurons. Our findings suggest that this is likely due to a deleterious block of autophagic clearance and lysosomal degradative capacity by Tau35. Notably, upon induction of autophagy by Torin 1, Tau35 inhibited nuclear translocation of transcription factor EB (TFEB), a key regulator of lysosomal biogenesis. Both cell lines and primary cortical neurons expressing Tau35 also exhibited changes in endosomal protein expression. These findings implicate autophagic and endolysosomal dysfunction as key pathological mechanisms through which disease-associated tau fragments could lead to the development and progression of tauopathy.


Assuntos
Autofagia , Endossomos , Metabolismo dos Lipídeos , Lisossomos , Neurônios , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/genética , Lisossomos/metabolismo , Humanos , Neurônios/metabolismo , Animais , Endossomos/metabolismo , Tauopatias/metabolismo , Tauopatias/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Camundongos
11.
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
12.
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
13.
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
14.
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
15.
Cell Death Dis ; 15(7): 496, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38992005

RESUMO

Intracellular organelles support cellular physiology in diverse conditions. In the skin, epidermal keratinocytes undergo differentiation with gradual changes in cellular physiology, accompanying remodeling of lysosomes and the Golgi apparatus. However, it was not known whether changes in Golgi and lysosome morphology and their redistribution were linked. Here, we show that disassembled Golgi is distributed in close physical apposition to lysosomes in differentiated keratinocytes. This atypical localization requires the Golgi tethering protein GRASP65, which is associated with both the Golgi and lysosome membranes. Depletion of GRASP65 results in the loss of Golgi-lysosome apposition and the malformation of lysosomes, defined by their aberrant morphology, size, and function. Surprisingly, a trans-Golgi enzyme and secretory Golgi cargoes are extensively localized to the lysosome lumen and secreted to the cell surface, contributing to total protein secretion of differentiated keratinocytes but not in proliferative precursors, indicating that lysosomes acquire specialization during differentiation. We further demonstrate that the secretory function of the Golgi apparatus is critical to maintain keratinocyte lysosomes. Our study uncovers a novel form of Golgi-lysosome cross-talk and its role in maintaining specialized secretory lysosomes in differentiated keratinocytes.


Assuntos
Diferenciação Celular , Complexo de Golgi , Proteínas da Matriz do Complexo de Golgi , Queratinócitos , Lisossomos , Lisossomos/metabolismo , Queratinócitos/metabolismo , Queratinócitos/citologia , Complexo de Golgi/metabolismo , Humanos , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/genética , Proteínas de Membrana/metabolismo
16.
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
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.
Ann Clin Transl Neurol ; 11(7): 1715-1731, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38837642

RESUMO

OBJECTIVE: Krabbe disease (KD) is a multisystem neurodegenerative disorder with severe disability and premature death, mostly with an infancy/childhood onset. In rare cases of late-onset phenotypes, symptoms are often milder and difficult to diagnose. We here present a translational approach combining diagnostic and biochemical analyses of a male patient with a progressive gait disorder starting at the age of 44 years, with a final diagnosis of late-onset KD (LOKD). METHODS: Additionally to cerebral MRI, protein structural analyses of the ß-galactocerebrosidase protein (GALC) were performed. Moreover, expression, lysosomal localization, and activities of ß-glucocerebrosidase (GCase), cathepsin B (CTSB), and cathepsin D (CTSD) were analyzed in leukocytes, fibroblasts, and lysosomes of fibroblasts. RESULTS: Exome sequencing revealed biallelic likely pathogenic variants: GALC exons 11-17: 33 kb deletion; exon 4: missense variant (c.334A>G, p.Thr112Ala). We detected a reduced GALC activity in leukocytes and fibroblasts. While histological KD phenotypes were absent in fibroblasts, they showed a significantly decreased activities of GCase, CTSB, and CTSD in lysosomal fractions, while expression levels were unaffected. INTERPRETATION: The presented LOKD case underlines the age-dependent appearance of a mildly pathogenic GALC variant and its interplay with other lysosomal proteins. As GALC malfunction results in reduced ceramide levels, we assume this to be causative for the here described decrease in CTSB and CTSD activity, potentially leading to diminished GCase activity. Hence, we emphasize the importance of a functional interplay between the lysosomal enzymes GALC, CTSB, CTSD, and GCase, as well as between their substrates, and propose their conjoined contribution in KD pathology.


Assuntos
Catepsina B , Catepsina D , Galactosilceramidase , Leucodistrofia de Células Globoides , Humanos , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Leucodistrofia de Células Globoides/diagnóstico , Masculino , Catepsina D/genética , Catepsina D/metabolismo , Galactosilceramidase/genética , Adulto , Catepsina B/genética , Catepsina B/metabolismo , Paraplegia/genética , Idade de Início , Glucosilceramidase/genética , Lisossomos , Fibroblastos/metabolismo , Fibroblastos/patologia
19.
Acta Physiol (Oxf) ; 240(8): e14186, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38837572

RESUMO

AIM: Understanding the physiological role of ATP6V1A, a component of the cytosolic V1 domain of the proton pump vacuolar ATPase, in regulating neuronal development and function. METHODS: Modeling loss of function of Atp6v1a in primary murine hippocampal neurons and studying neuronal morphology and function by immunoimaging, electrophysiological recordings and electron microscopy. RESULTS: Atp6v1a depletion affects neurite elongation, stabilization, and function of excitatory synapses and prevents synaptic rearrangement upon induction of plasticity. These phenotypes are due to an overall decreased expression of the V1 subunits, that leads to impairment of lysosomal pH-regulation and autophagy progression with accumulation of aberrant lysosomes at neuronal soma and of enlarged vacuoles at synaptic boutons. CONCLUSIONS: These data suggest a physiological role of ATP6V1A in the surveillance of synaptic integrity and plasticity and highlight the pathophysiological significance of ATP6V1A loss in the alteration of synaptic function that is associated with neurodevelopmental and neurodegenerative diseases. The data further support the pivotal involvement of lysosomal function and autophagy flux in maintaining proper synaptic connectivity and adaptive neuronal properties.


Assuntos
Hipocampo , Plasticidade Neuronal , Neurônios , Sinapses , ATPases Vacuolares Próton-Translocadoras , Animais , Hipocampo/metabolismo , Hipocampo/citologia , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Camundongos , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Sinapses/metabolismo , Sinapses/fisiologia , Células Cultivadas , Autofagia/fisiologia , Lisossomos/metabolismo
20.
Mater Horiz ; 11(14): 3287-3297, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38842407

RESUMO

Eukaryotic cells regulate various cellular processes through membrane-bound and membrane-less organelles, enabling active signal communication and material exchange. Lysosomes and lipid droplets are representative organelles, contributing to cell lipophagy when their interaction and metabolism are disrupted. Our limited understanding of the interacting behaviours and physicochemical properties of different organelles during lipophagy hinders accurate diagnosis and treatment of related diseases. In this contribution, we report a fluorescent probe, PTZ, engineered for dual-targeting of lipid droplets and lysosomes. PTZ can track liquid-liquid phase separation and respond to polarity shifts through ratiometric fluorescence emission, elucidating the lipophagy process from the perspective of organelle behavior and physicochemical properties. Leveraging on the multifunctionality of PTZ, we have successfully tracked the polarity and dynamic changes of lysosomes and lipid droplets during lipophagy. Furthermore, an unknown homogeneous transition of lipid droplets and lysosomes was discovered, which provided a new perspective for understanding lipophagy processes. And this work is expected to serve as a reference for diagnosis and treatment of lipophagy-related diseases.


Assuntos
Corantes Fluorescentes , Gotículas Lipídicas , Lisossomos , Humanos , Lisossomos/metabolismo , Gotículas Lipídicas/metabolismo , Transição de Fase , Autofagia/fisiologia , Células HeLa
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...