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1.
Life Sci Alliance ; 6(3)2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36585258

RESUMO

Nutrient deprivation ("starvation") is a major catabolic stress faced by mammalian cells in both pathological and physiological situations. Starvation induces autophagosome biogenesis in the immediate vicinity of ER and leads to lysosome spatial repositioning, but little is known about the consequences of nutritional stress on endosomes. Here, we report that starvation induces tethering of endosomal tubules to ER subregions, fostering autophagosome assembly. We show that this endosomal membrane generation is regulated by sorting nexin 1 (SNX1) protein and is important for the autophagic response. These newly formed SNX1 endosomal tubules establish connections with ER subdomains engaged in early autophagic machinery mobilization. Such endosome-ER transient tethers are regulated by a local dialog between SNX2, an endosomal partner of SNX1, and VAPB, an ER protein associated with autophagy initiation stage regulation. We propose that in a very early response to starvation, SNX1 and SNX2 cooperation induces and regulates endosomal membrane tubulation towards VAPB-positive ER subdomains involved in autophagosome biogenesis, highlighting the contribution of early endosomes in the cellular response to nutritional stress.


Assuntos
Proteínas de Transporte , Proteínas de Transporte Vesicular , Animais , Proteínas de Transporte/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Mamíferos/metabolismo
2.
Adv Neurobiol ; 29: 281-304, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255679

RESUMO

Gangliosides are sialylated glycosphingolipids (GSLs) with essential but enigmatic functions in brain activities and neural stem cell (NSC) maintenance. Our group has pioneered research on the importance of gangliosides for growth factor receptor signaling and epigenetic regulation of NSC activity and differentiation. The primary localization of gangliosides is on cell-surface microdomains and the drastic dose and composition changes during neural differentiation strongly suggest that they are not only important as biomarkers, but also are involved in modulating NSC fate determination. Ganglioside GD3 is the predominant species in NSCs and GD3-synthase knockout (GD3S-KO) revealed reduction of postnatal NSC pools with severe behavioral deficits. Exogenous administration of GD3 significantly restored the NSC pools and enhanced the stemness of NSCs with multipotency and self-renewal. Since morphological changes during neurogenesis require a huge amount of energy, mitochondrial functions are vital for neurogenesis. We discovered that a mitochondrial fission protein, the dynamin-related protein-1 (Drp1), as a novel GD3-binding protein, and GD3 regulates mitochondrial dynamics. Furthermore, we discovered that GM1 ganglioside promotes neuronal differentiation by an epigenetic regulatory mechanism. Nuclear GM1 binds with acetylated histones on the promoters of N-acetylgalactosaminyltransferase (GalNAcT; GM2 synthase) as well as on the NeuroD1 genes in differentiated neurons. In addition, epigenetic activation of the GalNAcT gene was detected as accompanied by an apparent induction of neuronal differentiation in NSCs responding to an exogenous supplement of GM1. GM1 is indeed localized in the nucleus where it can interact with transcriptionally active histones. Interestingly, GM1 could induce epigenetic activation of the tyrosine hydroxylase (TH) gene, with recruitment of nuclear receptor related 1 (Nurr1, also known as NR4A2), a dopaminergic neuron-associated transcription factor, to the TH promoter region. In this way, GM1 epigenetically regulates dopaminergic neuron specific gene expression. GM1 interacts with active chromatin via acetylated histones to recruit transcription factors at the nuclear periphery, resulting in changes in gene expression for neuronal differentiation. The significance is that multifunctional gangliosides modulate lipid microdomains to regulate functions of important molecules on multiple sites: the plasma membrane, mitochondrial membrane, and nuclear membrane. Versatile gangliosides could regulate functional neurons as well as sustain NSC functions via modulating protein and gene activities on ganglioside microdomains.


Assuntos
Gangliosídeo G(M1) , N-Acetilgalactosaminiltransferases , Humanos , Gangliosídeo G(M1)/metabolismo , Epigênese Genética , Histonas/genética , Histonas/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Gangliosídeos/genética , Gangliosídeos/metabolismo , Neurônios/metabolismo , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Glicoesfingolipídeos/metabolismo , Membranas Intracelulares/metabolismo , Biomarcadores/metabolismo , Cromatina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Mol Biol Cell ; 33(14): ar144, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36287829

RESUMO

Membrane remodeling mediated by heteropolymeric filaments composed of ESCRT-III subunits is an essential process that occurs at a variety of organelles to maintain cellular homeostasis. Members of the evolutionarily conserved Lgd/CC2D1 protein family have been suggested to regulate ESCRT-III polymer assembly, although their specific roles, particularly in vivo, remain unclear. Using the Caenorhabditis elegans early embryo as a model system, we show that Lgd/CC2D1 localizes to endosomal membranes, and its loss impairs endolysosomal cargo sorting and degradation. At the ultrastructural level, the absence of Lgd/CC2D1 results in the accumulation of enlarged endosomal compartments that contain a reduced number of intralumenal vesicles (ILVs). However, unlike aberrant endosome morphology caused by depletion of other ESCRT components, ILV size is only modestly altered in embryos lacking Lgd/CC2D1. Instead, loss of Lgd/CC2D1 impairs normal accumulation of ESCRT-III on endosomal membranes, likely slowing the kinetics of ILV formation. Together, our findings suggest a role for Lgd/CC2D1 in the recruitment and/or stable assembly of ESCRT-III subunits on endosomal membranes to facilitate efficient ILV biogenesis.


Assuntos
Endossomos , Corpos Multivesiculares , Animais , Corpos Multivesiculares/metabolismo , Endossomos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Membranas Intracelulares/metabolismo , Transporte Proteico/fisiologia , Caenorhabditis elegans/metabolismo
4.
J Cell Biol ; 221(11)2022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36169638

RESUMO

Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers contribute to the flux of membrane lipids and proteins to acceptor organelles, but how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. Using imaging approaches on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability, and functions of recycling endosomal tubules. In vitro, BLOC-1 binds and tubulates negatively charged membranes, including those containing PI4P. In cells, endosomal PI4P production by type II PI4-kinases is needed to form and stabilize BLOC-1-dependent recycling endosomal tubules. Decreased PI4KIIs expression impairs the recycling of endosomal cargoes and the life cycles of intracellular pathogens such as Chlamydia bacteria and influenza virus that exploit the membrane dynamics of recycling endosomes. This study demonstrates how a phospholipid and a protein complex coordinate the remodeling of cellular membranes into functional tubules.


Assuntos
Endossomos , Membranas Intracelulares , Peptídeos e Proteínas de Sinalização Intracelular , Fosfatos de Fosfatidilinositol , Membrana Celular/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte Proteico
5.
Proc Natl Acad Sci U S A ; 119(35): e2205590119, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-35994655

RESUMO

The endosomal sorting complex required for transport (ESCRT) machinery is centrally involved in the repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca2+-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca2+-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to the membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, and CHMP2A subunits along with the AAA+ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open an avenue for the mechanistic understanding of endolysosomal membrane repair.


Assuntos
Cálcio , Complexos Endossomais de Distribuição Requeridos para Transporte , Membranas Intracelulares , Lisossomos , ATPases Associadas a Diversas Atividades Celulares , Proteínas Reguladoras de Apoptose , Transporte Biológico , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Proteínas de Ciclo Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo
6.
J Biol Chem ; 298(10): 102394, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35988652

RESUMO

Exosomes are small extracellular vesicles of ∼30 to 150 nm that are secreted by all cells, abundant in all biofluids, and play important roles in health and disease. However, details about the mechanism of exosome biogenesis are unclear. Here, we carried out a cargo-based analysis of exosome cargo protein biogenesis in which we identified the most highly enriched exosomal cargo proteins and then followed their biogenesis, trafficking, and exosomal secretion to test different hypotheses for how cells make exosomes. We show that exosome cargo proteins bud from cells (i) in exosome-sized vesicles regardless of whether they are localized to plasma or endosome membranes, (ii) ∼5-fold more efficiently when localized to the plasma membrane, (iii) ∼5-fold less efficiently when targeted to the endosome membrane, (iv) by a stochastic process that leads to ∼100-fold differences in their abundance from one exosome to another, and (v) independently of small GTPase Rab27a, the ESCRT complex-associated protein Alix, or the cargo protein CD63. Taken together, our results demonstrate that cells use a shared, stochastic mechanism to bud exosome cargoes along the spectrum of plasma and endosome membranes and far more efficiently from the plasma membrane than the endosome. Our observations also indicate that the pronounced variation in content between different exosome-sized vesicles is an inevitable consequence of a stochastic mechanism of small vesicle biogenesis, that the origin membrane of exosome-sized extracellular vesicles simply cannot be determined, and that most of what we currently know about exosomes has likely come from studies of plasma membrane-derived vesicles.


Assuntos
Exossomos , Proteínas de Transporte Vesicular , Endossomos/metabolismo , Exossomos/metabolismo , Membranas Intracelulares/metabolismo , Humanos , Proteínas de Transporte Vesicular/metabolismo
7.
Nat Commun ; 13(1): 4685, 2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-35948564

RESUMO

The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1.


Assuntos
Complexo de Golgi , Lisossomos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo de Golgi/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosforilação
8.
Mol Biol Cell ; 33(13): ar124, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36044336

RESUMO

Lysosomes are dynamic organelles that can remodel their membrane as an adaptive response to various cell signaling events including membrane damage. Recently, we have discovered that damaged lysosomes form and sort tubules into moving vesicles. We named this process LYTL for LYsosomal Tubulation/sorting driven by LRRK2, as the Parkinson's disease protein LRRK2 promotes tubulation by recruiting the motor adaptor protein JIP4 to lysosomes via phosphorylated RAB proteins. Here we use spinning-disk microscopy combined with superresolution to further characterize LYTL after membrane damage with LLOMe (l-leucyl-l-leucine methyl ester). We identified the endoplasmic reticulum (ER) colocalizing with sites of fission of lysosome-derived tubules. In addition, modifying the morphology of the ER by reducing ER tubules leads to a decrease in LYTL sorting, suggesting that contact with tubular ER is necessary for lysosomal membrane sorting. Given the central roles of LRRK2 and lysosomal biology in Parkinson's disease, these discoveries are likely relevant to disease pathology and highlight interactions between organelles in this model.


Assuntos
Doença de Parkinson , Dineínas/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Transporte Proteico
9.
Elife ; 112022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35852853

RESUMO

The formation of a clathrin-coated vesicle (CCV) is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment 'hook' to an 'anchor' protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin is sufficient for intracellular CCV formation.


Assuntos
Clatrina , Invaginações Revestidas da Membrana Celular , Membrana Celular/metabolismo , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Dinaminas/metabolismo , Endocitose , Membranas Intracelulares/metabolismo
10.
Methods Mol Biol ; 2473: 309-331, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35819773

RESUMO

Intracellular membrane trafficking is a dynamic and complex cellular process. To study membrane trafficking with a high spatiotemporal resolution, we present an optogenetic method based on a blue-light inducible oligomerization of Rab GTPases, termed light-activated reversible inhibition by assembly trap of intracellular membranes (IM-LARIAT). In this chapter, we focus on the optical disruption of the dynamics and functions of previously studied intracellular membrane trafficking events, including transferrin recycling and growth cone regulation in relation to specific Rab GTPases. To aid general application, we provide a detailed description of transfection, imaging with a confocal microscope, and analysis of data.


Assuntos
Membranas Intracelulares , Optogenética , Cones de Crescimento/metabolismo , Membranas Intracelulares/metabolismo , Membranas/metabolismo , Optogenética/métodos , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
11.
Cell ; 185(13): 2292-2308.e20, 2022 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-35750034

RESUMO

Lysosomes require an acidic lumen between pH 4.5 and 5.0 for effective digestion of macromolecules. This pH optimum is maintained by proton influx produced by the V-ATPase and efflux through an unidentified "H+ leak" pathway. Here we show that TMEM175, a genetic risk factor for Parkinson's disease (PD), mediates the lysosomal H+ leak by acting as a proton-activated, proton-selective channel on the lysosomal membrane (LyPAP). Acidification beyond the normal range potently activated LyPAP to terminate further acidification of lysosomes. An endogenous polyunsaturated fatty acid and synthetic agonists also activated TMEM175 to trigger lysosomal proton release. TMEM175 deficiency caused lysosomal over-acidification, impaired proteolytic activity, and facilitated α-synuclein aggregation in vivo. Mutational and pH normalization analyses indicated that the channel's H+ conductance is essential for normal lysosome function. Thus, modulation of LyPAP by cellular cues may dynamically tune the pH optima of endosomes and lysosomes to regulate lysosomal degradation and PD pathology.


Assuntos
Doença de Parkinson , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Canais de Potássio/metabolismo , Prótons
12.
J Cell Biol ; 221(6)2022 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-35499567

RESUMO

Cellular membranes differ in protein and lipid composition as well as in the protein-lipid ratio. Thus, progression of membranous organelles along traffic routes requires mechanisms to control bilayer lipid chemistry and their abundance relative to proteins. The recent structural and functional characterization of VPS13-family proteins has suggested a mechanism through which lipids can be transferred in bulk from one membrane to another at membrane contact sites, and thus independently of vesicular traffic. Here, we show that SHIP164 (UHRF1BP1L) shares structural and lipid transfer properties with these proteins and is localized on a subpopulation of vesicle clusters in the early endocytic pathway whose membrane cargo includes the cation-independent mannose-6-phosphate receptor (MPR). Loss of SHIP164 disrupts retrograde traffic of these organelles to the Golgi complex. Our findings raise the possibility that bulk transfer of lipids to endocytic membranes may play a role in their traffic.


Assuntos
Endossomos , Complexo de Golgi , Membranas Intracelulares , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipídeos
13.
J Cell Sci ; 135(9)2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35437598

RESUMO

Mammalian PEX16 has been considered essential for generating and maintaining peroxisomal membranes. This view is based primarily on the finding that fibroblasts from several PEX16-deficient patients are devoid of peroxisomal structures but can form peroxisomes upon expression of PEX16. However, unlike these patient-derived cells, pex16 mutants in other model organisms contain partially functional peroxisomes. Here, we report that PEX16-knockout (KO) cells derived from three mammalian cultured cell lines comprise cells containing a fewer number of enlarged peroxisomes and cells lacking peroxisomes. We also suggest that PEX16 accelerates the process by which peroxisome-less cells form peroxisomal membranes and subsequently establish mature peroxisomes, independently of its ability to mediate peroxisomal targeting of PEX3. Nevertheless, PEX16 is not absolutely required for this process. Moreover, a well-known patient-derived PEX16 mutant inhibits the de novo formation of peroxisomal membranes. Our findings suggest that although PEX16 is undoubtedly important for optimal peroxisomal membrane biogenesis, mammalian cells may be able to form peroxisomes de novo and maintain the organelles without the aid of PEX16.


Assuntos
Sistemas CRISPR-Cas , Peroxissomos , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Humanos , Membranas Intracelulares/metabolismo , Mamíferos/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo
14.
Cells ; 11(6)2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35326372

RESUMO

A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.


Assuntos
Canais Iônicos , Doenças por Armazenamento dos Lisossomos , Humanos , Membranas Intracelulares/metabolismo , Canais Iônicos/metabolismo , Íons/metabolismo , Doenças por Armazenamento dos Lisossomos/metabolismo , Lisossomos/metabolismo , Técnicas de Patch-Clamp
15.
Mol Cell ; 82(10): 1836-1849.e5, 2022 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-35338845

RESUMO

mTORC1 controls cellular metabolic processes in response to nutrient availability. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which are localized on the lysosomal surface by the Ragulator complex. The Rag GTPases receive amino acid signals from multiple upstream regulators. One negative regulator, GATOR1, is a GTPase activating protein (GAP) for RagA. GATOR1 binds to the Rag GTPases via two modes: an inhibitory mode and a GAP mode. How these two binding interactions coordinate to process amino acid signals is unknown. Here, we resolved three cryo-EM structural models of the GATOR1-Rag-Ragulator complex, with the Rag-Ragulator subcomplex occupying the inhibitory site, the GAP site, and both binding sites simultaneously. When the Rag GTPases bind to GATOR1 at the GAP site, both Rag subunits contact GATOR1 to coordinate their nucleotide loading states. These results reveal a potential GAP mechanism of GATOR1 during the mTORC1 inactivation process.


Assuntos
Proteínas Ativadoras de GTPase , Proteínas Monoméricas de Ligação ao GTP , Aminoácidos/metabolismo , Microscopia Crioeletrônica , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo
16.
Trends Neurosci ; 45(4): 312-322, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35249745

RESUMO

Neurons rely heavily on properly regulated mitochondrial and lysosomal homeostasis, with multiple neurodegenerative diseases linked to dysfunction in these two organelles. Interestingly, mitochondria-lysosome membrane contact sites have been identified as a key pathway mediating their crosstalk in neurons. Recent studies have further elucidated the regulation of mitochondria-lysosome contact dynamics via distinct tethering/untethering protein machinery. Moreover, this pathway has been shown to have additional functions in regulating organelle network dynamics and metabolite transfer between lysosomes and mitochondria. In this review, we highlight recent advances in the field of mitochondria-lysosome contact sites and their misregulation across multiple neurodegenerative disorders, which further underscore a potential role for this pathway in neuronal homeostasis and disease.


Assuntos
Doenças Neurodegenerativas , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo
17.
J Lipid Res ; 63(3): 100178, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35143844

RESUMO

Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here, we report the development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.


Assuntos
Colesterol , Lisossomos , Animais , Membrana Celular/metabolismo , Colesterol/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Mamíferos/metabolismo
18.
J Biol Chem ; 298(3): 101744, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35183507

RESUMO

The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is activated by intracellular nutritional sufficiency and extracellular growth signals. It has been reported that mTORC1 acts as a hub that integrates these inputs to orchestrate a number of cellular responses, including translation, nucleotide synthesis, lipid synthesis, and lysosome biogenesis. However, little is known about specific control of mTORC1 signaling downstream of this complex. Here, we demonstrate that Ragulator, a heteropentameric protein complex required for mTORC1 activation in response to amino acids, is critical for inhibiting the nuclear translocation of transcription factor EB (TFEB). We established a unique RAW264.7 clone that lacked Ragulator but retained total mTORC1 activity. In a nutrition-sufficient state, the nuclear translocation of TFEB was markedly enhanced in the clone despite total mTORC1 kinase activity. In addition, as a cellular phenotype, the number of lysosomes was increased by tenfold in the Ragulator-deficient clone compared with that of control cells. These findings indicate that mTORC1 essentially requires the Ragulator complex for regulating the subcellular distribution of TFEB. Our findings also suggest that other scaffold proteins may be associated with mTORC1 for the specific regulation of downstream signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Membranas Intracelulares , Lisossomos , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Núcleo Celular/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Transporte Proteico , Células RAW 264.7 , Transdução de Sinais
19.
Biochem Pharmacol ; 197: 114939, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35114188

RESUMO

Kidney injury often causes anemia due to a lack of production of the erythroid growth factor erythropoietin (EPO) in the kidneys. Roxadustat is one of the first oral medicines inducing EPO production in patients with renal anemia by activating hypoxia-inducible factors (HIFs), which are activators of EPO gene expression. In this study, to develop prodrugs of roxadustat with improved permeability through cell membrane, we investigated the effects of 8 types of esterification on the pharmacokinetics and bioactivity of roxadustat using Hep3B hepatoma cells that HIF-dependently produce EPO. Mass spectrometry of cells incubated with the esterified roxadustat derivatives revealed that the designed compounds were deesterified after being taken up by cells and showed low cytotoxicity compared to the original compound. Esterification prolonged the effective duration of roxadustat with respect to EPO gene induction and HIF activation in cells transiently exposed to the compounds. In the kidneys and livers of mice, both of which are unique sites of EPO production, a majority of the methyl-esterified roxadustat was deesterified within 6 h after drug administration. The deesterified roxadustat derivative was continuously detectable in plasma and urine for at least 48 h after administration, while the administered compound became undetectable 24 h after administration. Additionally, we confirmed that methyl-esterified roxadustat activated erythropoiesis in mice by inducing Epo mRNA expression exclusively in renal interstitial cells, which have intrinsic EPO-producing potential. These data suggest that esterification could lead to the development of roxadustat prodrugs with improvements in cell membrane permeability, effective duration and cytotoxicity.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Glicina/análogos & derivados , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Membranas Intracelulares/metabolismo , Isoquinolinas/metabolismo , Isoquinolinas/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Esterificação/efeitos dos fármacos , Esterificação/fisiologia , Glicina/metabolismo , Glicina/farmacologia , Humanos , Membranas Intracelulares/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Fatores de Tempo , Resultado do Tratamento , Células Tumorais Cultivadas
20.
FEBS Open Bio ; 12(4): 694-707, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35203107

RESUMO

For a long time, lysosomes were purely seen as organelles in charge of garbage disposal within the cell. They destroy any cargo delivered into their lumen with a plethora of highly potent hydrolytic enzymes, including various proteases. In case of damage to their limiting membranes, the lysosomes release their soluble content with detrimental outcomes for the cell. In recent years, however, this view of the lysosome changed towards acknowledging it as a platform for integration of manifold intracellular and extracellular signals. Even impaired lysosomal membrane integrity is no longer considered to be a one-way street to cell death. Increasing evidence suggests that lysosomal enzymes, mainly cathepsin proteases, can be released in a spatially and temporarily restricted manner that is compatible with cellular survival. This way, cathepsins can act in the cytosol and the nucleus, where they affect important cellular processes such as cell division. Here, we review this evidence and discuss the routes and molecular mechanisms by which the cathepsins may reach their unusual destination.


Assuntos
Catepsinas , Peptídeo Hidrolases , Catepsinas/metabolismo , Citosol/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Peptídeo Hidrolases/metabolismo
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