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1.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804256

RESUMO

Lysosomal acid phosphatase 2 (Acp2) mutant mice (naked-ataxia, nax) have a severe cerebellar cortex defect with a striking reduction in the number of granule cells. Using a combination of in vivo and in vitro immunohistochemistry, Western blotting, BrdU assays, and RT-qPCR, we show downregulation of MYCN and dysregulation of the SHH signaling pathway in the nax cerebellum. MYCN protein expression is significantly reduced at P10, but not at the peak of proliferation at around P6 when the number of granule cells is strikingly reduced in the nax cerebellum. Despite the significant role of the SHH-MycN pathway in granule cell proliferation, our study suggests that a broader molecular pathway and additional mechanisms regulating granule cell development during the clonal expansion period are impaired in the nax cerebellum. In particular, our results indicate that downregulation of the protein synthesis machinery may contribute to the reduced number of granule cells in the nax cerebellum.


Assuntos
Fosfatase Ácida/genética , Ataxia Cerebelar/genética , Córtex Cerebelar/metabolismo , Proteínas Hedgehog/genética , Proteína Proto-Oncogênica N-Myc/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Ataxia Cerebelar/metabolismo , Ataxia Cerebelar/patologia , Córtex Cerebelar/anormalidades , Córtex Cerebelar/patologia , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lisossomos/genética , Lisossomos/patologia , Camundongos , Mutação , Neurônios/metabolismo , Neurônios/patologia , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Transdução de Sinais/genética
2.
Nat Commun ; 12(1): 1807, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33753743

RESUMO

Mitochondria-lysosome contacts are recently identified sites for mediating crosstalk between both organelles, but their role in normal and diseased human neurons remains unknown. In this study, we demonstrate that mitochondria-lysosome contacts can dynamically form in the soma, axons, and dendrites of human neurons, allowing for their bidirectional crosstalk. Parkinson's disease patient derived neurons harboring mutant GBA1 exhibited prolonged mitochondria-lysosome contacts due to defective modulation of the untethering protein TBC1D15, which mediates Rab7 GTP hydrolysis for contact untethering. This dysregulation was due to decreased GBA1 (ß-glucocerebrosidase (GCase)) lysosomal enzyme activity in patient derived neurons, and could be rescued by increasing enzyme activity with a GCase modulator. These defects resulted in disrupted mitochondrial distribution and function, and could be further rescued by TBC1D15 in Parkinson's patient derived GBA1-linked neurons. Together, our work demonstrates a potential role of mitochondria-lysosome contacts as an upstream regulator of mitochondrial function and dynamics in midbrain dopaminergic neurons in GBA1-linked Parkinson's disease.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Glucosilceramidase/genética , Lisossomos/genética , Mitocôndrias/genética , Mutação , Doença de Parkinson/genética , Células Cultivadas , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/ultraestrutura , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Glucosilceramidase/metabolismo , Humanos , Hidrólise , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Doença de Parkinson/metabolismo , Imagem com Lapso de Tempo/métodos , Proteínas rab de Ligação ao GTP/metabolismo
3.
Nat Cell Biol ; 23(3): 232-242, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33686253

RESUMO

Lysosomes must maintain the integrity of their limiting membrane to ensure efficient fusion with incoming organelles and degradation of substrates within their lumen. Pancreatic cancer cells upregulate lysosomal biogenesis to enhance nutrient recycling and stress resistance, but it is unknown whether dedicated programmes for maintaining the integrity of the lysosome membrane facilitate pancreatic cancer growth. Using proteomic-based organelle profiling, we identify the Ferlin family plasma membrane repair factor Myoferlin as selectively and highly enriched on the membrane of pancreatic cancer lysosomes. Mechanistically, lysosomal localization of Myoferlin is necessary and sufficient for the maintenance of lysosome health and provides an early acting protective system against membrane damage that is independent of the endosomal sorting complex required for transport (ESCRT)-mediated repair network. Myoferlin is upregulated in human pancreatic cancer, predicts poor survival and its ablation severely impairs lysosome function and tumour growth in vivo. Thus, retargeting of plasma membrane repair factors enhances the pro-oncogenic activities of the lysosome.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Biomarcadores Tumorais/genética , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Membranas Intracelulares/patologia , Lisossomos/genética , Lisossomos/patologia , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Musculares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Prognóstico , Transdução de Sinais , Carga Tumoral
4.
PLoS Genet ; 17(1): e1009236, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33465068

RESUMO

The endo-lysosomal two-pore channel (TPC2) has been established as an intracellular cation channel of significant physiological and pathophysiological relevance in recent years. For example, TPC2-/- mice show defects in cholesterol degradation, leading to hypercholesterinemia; TPC2 absence also results in mature-onset obesity, and a role in glucagon secretion and diabetes has been proposed. Infections with bacterial toxins or viruses e.g., cholera toxin or Ebola virus result in reduced infectivity rates in the absence of TPC2 or after pharmacological blockage, and TPC2-/- cancer cells lose their ability to migrate and metastasize efficiently. Finally, melanin production is affected by changes in hTPC2 activity, resulting in pigmentation defects and hair color variation. Here, we analyzed several publicly available genome variation data sets and identified multiple variations in the TPC2 protein in distinct human populations. Surprisingly, one variation, L564P, was found to be the predominant TPC2 isoform on a global scale. By applying endo-lysosomal patch-clamp electrophysiology, we found that L564P is a prerequisite for the previously described M484L gain-of-function effect that is associated with blond hair. Additionally, other gain-of-function variants with distinct geographical and ethnic distribution were discovered and functionally characterized. A meta-analysis of genome-wide association studies was performed, finding the polymorphisms to be associated with both distinct and overlapping traits. In sum, we present the first systematic analysis of variations in TPC2. We functionally characterized the most common variations and assessed their association with various disease traits. With TPC2 emerging as a novel drug target for the treatment of various diseases, this study provides valuable insights into ethnic and geographical distribution of TPC2 polymorphisms and their effects on channel activity.


Assuntos
Canais de Cálcio/genética , Estudo de Associação Genômica Ampla , Cor de Cabelo/genética , Animais , Fibroblastos/metabolismo , Mutação com Ganho de Função/genética , Genoma Humano/genética , Humanos , Lisossomos/genética , Camundongos , Camundongos Knockout , NADP/genética , Pigmentação/genética , Polimorfismo de Nucleotídeo Único/genética , Transdução de Sinais/genética
5.
Dev Cell ; 56(1): 52-66.e7, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33434526

RESUMO

ER tubules form and maintain membrane contact sites (MCSs) with endosomes. How and why these ER-endosome MCSs persist as endosomes traffic and mature is poorly understood. Here we find that a member of the reticulon protein family, Reticulon-3L (Rtn3L), enriches at ER-endosome MCSs as endosomes mature. We show that this localization is due to the long divergent N-terminal cytoplasmic domain of Rtn3L. We found that Rtn3L is recruited to ER-endosome MCSs by endosomal protein Rab9a, which marks a transition stage between early and late endosomes. Rab9a utilizes an FSV region to recruit Rtn3L via its six LC3-interacting region motifs. Consistent with our localization results, depletion or deletion of RTN3 from cells results in endosome maturation and cargo sorting defects, similar to RAB9A depletion. Together our data identify a tubular ER protein that promotes endosome maturation at ER MCSs.


Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico/genética , Motivos de Aminoácidos , Autofagossomos/genética , Autofagossomos/metabolismo , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Endossomos/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nogo/genética , Proteínas Nogo/metabolismo , RNA Interferente Pequeno , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
6.
Adv Exp Med Biol ; 1281: 219-242, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33433878

RESUMO

It has been more than a decade since heterozygous loss-of-function mutations in the progranulin gene (GRN) were first identified as an important genetic cause of frontotemporal lobar degeneration (FTLD). Due to the highly diverse biological functions of the progranulin (PGRN) protein, encoded by GRN, multiple possible disease mechanisms have been proposed. Early work focused on the neurotrophic properties of PGRN and its role in the inflammatory response. However, since the discovery of homozygous GRN mutations in patients with a lysosomal storage disorder, investigation into the possible roles of PGRN and its proteolytic cleavage products granulins, in lysosomal function and dysfunction, has taken center stage. In this chapter, we summarize the GRN mutational spectrum and its associated phenotypes followed by an in-depth discussion on the possible disease mechanisms implicated in FTLD-GRN. We conclude with key outstanding questions which urgently require answers to ensure safe and successful therapy development for GRN mutation carriers.


Assuntos
Degeneração Lobar Frontotemporal , Peptídeos e Proteínas de Sinalização Intercelular , Biologia , Degeneração Lobar Frontotemporal/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Lisossomos/genética , Mutação , Progranulinas/genética
7.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118857, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32949647

RESUMO

Intracellular organelle cross-talk is a new and important research area. Under stress conditions, the coordinated action of the autophagy and endosomal systems in tumor cells is essential for maintaining cellular homeostasis and survival. The activation of the IκB kinase (IKK) complex is also involved in the regulation of stress and homeostasis in tumor cells. Here, we try to explore the effects of constitutively active IKKß subunits (CA-IKKß) on autophagy and endosomal system interactions. We confirm that CA-IKKß induces accumulation of autophagosomes and their fusion with MVBs to form amphisomes in cancer cells, and also drives the release of EVs containing autophagy components through an amphisome-dependent mechanism. We further demonstrate that CA-IKKß inhibits the expression of RAB7, thereby weakening the lysosomal-dependent degradation pathway. CA-IKKß also induces phosphorylation of SNAP23 at Ser95 instead of Ser110, which further promotes amphisome-plasma membrane fusion and sEV secretion. These results indicate that CA-IKKß drives the formation and transport of amphisomes, thereby regulating tumor cell homeostasis, which may illuminate a special survival mechanism in tumor cells under stress.


Assuntos
Autofagia/genética , Quinase I-kappa B/genética , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Proteínas rab de Ligação ao GTP/genética , Autofagossomos/genética , Linhagem Celular Tumoral , Endossomos/genética , Exocitose/genética , Vesículas Extracelulares/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Lisossomos/genética , Fusão de Membrana/genética , Neoplasias/genética , Neoplasias/patologia , Fosforilação/genética , Transdução de Sinais/genética
8.
Sci Rep ; 10(1): 22098, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33328543

RESUMO

Lysosomal dysfunction is an emerging feature in the pathology of Parkinson's disease and Dementia with Lewy bodies. Mutations in the GBA gene, encoding the enzyme Glucocerebrosidase (GCase), have been identified as a genetic risk factor for these synucleinopathies. As a result, there has been a growing interest in the involvement of GCase in these diseases. This GCase activity assay is based on the catalytic hydrolysis of 4-methylumbelliferyl ß-D-glucopyranoside that releases the highly fluorescent 4-methylumbelliferyl (4-MU). The final assay protocol was tested for the following parameters: Lower limit of quantification (LLOQ), precision, parallelism, linearity, spike recovery, number of freeze-thaw events, and sample handling stability. The GCase activity assay is within acceptable criteria for parallelism, precision and spike recovery. The LLOQ of this assay corresponds to an enzymatic activity of generating 0.26 pmol 4-MU/min/ml. The enzymatic activity was stable when samples were processed and frozen at - 80 °C within 4 h after the lumbar puncture procedure. Repetitive freeze-thaw events significantly decreased enzyme activity. We present the validation of an optimized in vitro GCase activity assay, based on commercially available components, to quantify its enzymatic activity in human cerebrospinal fluid and the assessment of preanalytical factors.


Assuntos
Glucosilceramidase/líquido cefalorraquidiano , Corpos de Lewy/enzimologia , Doença de Parkinson/líquido cefalorraquidiano , alfa-Sinucleína/genética , Fluorometria/métodos , Glucosilceramidase/genética , Humanos , Técnicas In Vitro , Corpos de Lewy/patologia , Lisossomos/genética , Lisossomos/patologia , Mutação/genética , Doença de Parkinson/diagnóstico , Doença de Parkinson/patologia , Fatores de Risco , alfa-Sinucleína/deficiência
9.
Sci Rep ; 10(1): 22086, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33328565

RESUMO

Effective phagocytosis is crucial for host defense against pathogens. Macrophages entrap pathogens into a phagosome and subsequently acidic lysosomes fuse to the phagosome. Previous studies showed the pivotal role of actin-remodeling mediated by phosphoinositide-related signaling in phagosome formation, but the mechanisms of phagosome-lysosome fusion remain unexplored. Here we show that in complement-mediated phagocytosis, phagosome-lysosome fusion requires the disappearance of F-actin structure surrounding the phagosome and a tyrosine kinase Syk plays a key role in this process. Using macrophage-like differentiated HL60 and Syk-knockout (Syk-KO) HL60 cells, we found that Syk-KO cells showed insufficient phagosome acidification caused by impaired fusion with lysosomes and permitted the survival of Candida albicans in complement-mediated phagocytosis. Phagosome tracking analysis showed that during phagosome internalization process, F-actin surrounding phagosomes disappeared in both parental and Syk-KO cells but this structure was reconstructed immediately only in Syk-KO cells. In addition, F-actin-stabilizing agent induced a similar impairment of phagosome-lysosome fusion. Collectively, Syk-derived signaling facilitates phagosome-lysosome fusion by regulating actin-remodeling.


Assuntos
Leucemia/genética , Fagocitose/genética , Fagossomos/genética , Quinase Syk/genética , Actinas/genética , Linhagem Celular Tumoral , Proteínas do Sistema Complemento/genética , Regulação Leucêmica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Inativação de Genes , Humanos , Leucemia/patologia , Lisossomos/genética , Macrófagos/metabolismo , Macrófagos/patologia
10.
PLoS Genet ; 16(8): e1008966, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776922

RESUMO

The vacuole of the yeast Saccharomyces cerevisiae plays an important role in nutrient storage. Arginine, in particular, accumulates in the vacuole of nitrogen-replete cells and is mobilized to the cytosol under nitrogen starvation. The arginine import and export systems involved remain poorly characterized, however. Furthermore, how their activity is coordinated by nitrogen remains unknown. Here we characterize Vsb1 as a novel vacuolar membrane protein of the APC (amino acid-polyamine-organocation) transporter superfamily which, in nitrogen-replete cells, is essential to active uptake and storage of arginine into the vacuole. A shift to nitrogen starvation causes apparent inhibition of Vsb1-dependent activity and mobilization of stored vacuolar arginine to the cytosol. We further show that this arginine export involves Ypq2, a vacuolar protein homologous to the human lysosomal cationic amino acid exporter PQLC2 and whose activity is detected only in nitrogen-starved cells. Our study unravels the main arginine import and export systems of the yeast vacuole and suggests that they are inversely regulated by nitrogen.


Assuntos
Arginina/metabolismo , Nitrogênio/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Aminoácidos/genética , Transporte Biológico/genética , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(33): 19970-19981, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32737159

RESUMO

Mitochondrial fission and fusion are highly regulated by energy demand and physiological conditions to control the production, activity, and movement of these organelles. Mitochondria are arrayed in a periodic pattern in Caenorhabditis elegans muscle, but this pattern is disrupted by mutations in the mitochondrial fission component dynamin DRP-1. Here we show that the dramatically disorganized mitochondria caused by a mitochondrial fission-defective dynamin mutation is strongly suppressed to a more periodic pattern by a second mutation in lysosomal biogenesis or acidification. Vitamin B12 is normally imported from the bacterial diet via lysosomal degradation of B12-binding proteins and transport of vitamin B12 to the mitochondrion and cytoplasm. We show that the lysosomal dysfunction induced by gene inactivations of lysosomal biogenesis or acidification factors causes vitamin B12 deficiency. Growth of the C. elegans dynamin mutant on an Escherichia coli strain with low vitamin B12 also strongly suppressed the mitochondrial fission defect. Of the two C. elegans enzymes that require B12, gene inactivation of methionine synthase suppressed the mitochondrial fission defect of a dynamin mutation. We show that lysosomal dysfunction induced mitochondrial biogenesis, which is mediated by vitamin B12 deficiency and methionine restriction. S-adenosylmethionine, the methyl donor of many methylation reactions, including histones, is synthesized from methionine by S-adenosylmethionine synthase; inactivation of the sams-1 S-adenosylmethionine synthase also suppresses the drp-1 fission defect, suggesting that vitamin B12 regulates mitochondrial biogenesis and then affects mitochondrial fission via chromatin pathways.


Assuntos
Caenorhabditis elegans/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Vitamina B 12/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Lisossomos/genética , Mitocôndrias/genética , Dinâmica Mitocondrial , Mutação
12.
Nat Commun ; 11(1): 4286, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855403

RESUMO

Intracellular galectins are carbohydrate-binding proteins capable of sensing and repairing damaged lysosomes. As in the physiological conditions glycosylated moieties are mostly in the lysosomal lumen but not cytosol, it is unclear whether galectins reside in lysosomes, bind to glycosylated proteins, and regulate lysosome functions. Here, we show in gut epithelial cells, galectin-9 is enriched in lysosomes and predominantly binds to lysosome-associated membrane protein 2 (Lamp2) in a Asn(N)-glycan dependent manner. At the steady state, galectin-9 binding to glycosylated Asn175 of Lamp2 is essential for functionality of lysosomes and autophagy. Loss of N-glycan-binding capability of galectin-9 causes its complete depletion from lysosomes and defective autophagy, leading to increased endoplasmic reticulum (ER) stress preferentially in autophagy-active Paneth cells and acinar cells. Unresolved ER stress consequently causes cell degeneration or apoptosis that associates with colitis and pancreatic disorders in mice. Therefore, lysosomal galectins maintain homeostatic function of lysosomes to prevent organ pathogenesis.


Assuntos
Galectinas/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Pâncreas/patologia , Celulas de Paneth/patologia , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Autofagia/fisiologia , Colite/metabolismo , Colite/patologia , Estresse do Retículo Endoplasmático , Galectinas/genética , Células HT29 , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Lisossomos/genética , Lisossomos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas/metabolismo , Pancreatite/metabolismo , Pancreatite/patologia , Celulas de Paneth/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(32): 19266-19275, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32703809

RESUMO

Mitochondria and lysosomes are critical for cellular homeostasis, and dysfunction of both organelles has been implicated in numerous diseases. Recently, interorganelle contacts between mitochondria and lysosomes were identified and found to regulate mitochondrial dynamics. However, whether mitochondria-lysosome contacts serve additional functions by facilitating the direct transfer of metabolites or ions between the two organelles has not been elucidated. Here, using high spatial and temporal resolution live-cell microscopy, we identified a role for mitochondria-lysosome contacts in regulating mitochondrial calcium dynamics through the lysosomal calcium efflux channel, transient receptor potential mucolipin 1 (TRPML1). Lysosomal calcium release by TRPML1 promotes calcium transfer to mitochondria, which was mediated by tethering of mitochondria-lysosome contact sites. Moreover, mitochondrial calcium uptake at mitochondria-lysosome contact sites was modulated by the outer and inner mitochondrial membrane channels, voltage-dependent anion channel 1 and the mitochondrial calcium uniporter, respectively. Since loss of TRPML1 function results in the lysosomal storage disorder mucolipidosis type IV (MLIV), we examined MLIV patient fibroblasts and found both altered mitochondria-lysosome contact dynamics and defective contact-dependent mitochondrial calcium uptake. Thus, our work highlights mitochondria-lysosome contacts as key contributors to interorganelle calcium dynamics and their potential role in the pathophysiology of disorders characterized by dysfunctional mitochondria or lysosomes.


Assuntos
Cálcio/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Mucolipidoses/metabolismo , Canais de Receptores Transientes de Potencial/metabolismo , Transporte Biológico , Humanos , Lisossomos/genética , Mitocôndrias/genética , Dinâmica Mitocondrial , Mucolipidoses/genética , Canais de Receptores Transientes de Potencial/genética
14.
PLoS One ; 15(6): e0234180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511278

RESUMO

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes. Dysfunction of this system is associated with many pathological conditions such as cancer, inflammation and neurodegenerative diseases. Therefore, monitoring the cellular autophagy-endolysosomal activity is crucial for studies on the pathogenesis as well as therapeutics of such disorders. To this end, we here sought to create a novel means exploiting Keima, an acid-stable fluorescent protein possessing pH-dependent fluorescence excitation spectra, for precisely monitoring the autophagy-endolysosomal system. First, we generated three lines of transgenic (tg) mouse expressing monomeric Keima-fused MAP1LC3B (mKeima-LC3B). Then, these tg mice were subjected to starvation by food-restriction, and also challenged to neurodegeneration by genetically crossing with a mouse model of amyotrophic lateral sclerosis; i.e., SOD1H46R transgenic mouse. Unexpectedly, despite that a lipidated-form of endogenous LC3 (LC3-II) was significantly increased, those of mKeima-LC3B (mKeima-LC3B-II) were not changed under both stressed conditions. It was also noted that mKeima-LC3B-positive aggregates were progressively accumulated in the spinal cord of SOD1H46R;mKeima-LC3B double-tg mice, suggestive of acid-resistance and aggregate-prone natures of long-term overexpressed mKeima-LC3B in vivo. Next, we characterized mouse embryonic fibroblasts (MEFs) derived from mKeima-LC3B-tg mice. In contrast with in vivo, levels of mKeima-LC3B-I were decreased under starved conditions. Furthermore, when starved MEFs were treated with chloroquine (CQ), the abundance of mKeima-LC3B-II was significantly increased. Remarkably, when cultured medium was repeatedly changed between DMEM (nutrient-rich) and EBSS (starvation), acidic/neutral signal ratios of mKeima-LC3B-positive compartments were rapidly and reversibly shifted, which were suppressed by the CQ treatment, indicating that intraluminal pH of mKeima-LC3B-positive vesicles was changeable upon nutritional conditions of culture media. Taken together, although mKeima-LC3B-tg mice may not be an appropriate tool to monitor the autophagy-endolysosomal system in vivo, mKeima-LC3B must be one of the most sensitive reporter molecules for monitoring this system under in vitro cultured conditions.


Assuntos
Autofagia/fisiologia , Endossomos/metabolismo , Proteínas Luminescentes/genética , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Animais , Células Cultivadas , Meios de Cultura/farmacologia , Endossomos/genética , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/metabolismo , Lisossomos/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Inanição , Superóxido Dismutase-1/genética , Imagem com Lapso de Tempo
15.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376619

RESUMO

HIV-1 assembly occurs principally at the plasma membrane (PM) of infected cells. Gag polyprotein precursors (Pr55Gag) are targeted to the PM, and their binding is mediated by the interaction of myristoylated matrix domain and a PM-specific phosphoinositide, the phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The major synthesis pathway of PI(4,5)P2 involves the activity of phosphatidylinositol-4-phosphate 5-kinase family type 1 composed of three isoforms (PIP5K1α, PIP5K1ß, and PIP5K1γ). To examine whether the activity of a specific PIP5K1 isoform determines proper Pr55Gag localization at the PM, we compared the cellular behavior of Pr55Gag in the context of PIP5K1 inhibition using siRNAs that individually targeted each of the three isoforms in TZM-bl HeLa cells. We found that downregulation of PIP5K1α and PIP5K1γ strongly impaired the targeting of Pr55Gag to the PM with a rerouting of the polyprotein within intracellular compartments. The efficiency of Pr55Gag release was thus impaired through the silencing of these two isoforms, while PIP5K1ß is dispensable for Pr55Gag targeting to the PM. The PM mistargeting due to the silencing of PIP5K1α leads to Pr55Gag hydrolysis through lysosome and proteasome pathways, while the silencing of PIP5K1γ leads to Pr55Gag accumulation in late endosomes. Our findings demonstrated that, within the PIP5K1 family, only the PI(4,5)P2 pools produced by PIP5K1α and PIP5K1γ are involved in the Pr55Gag PM targeting process.IMPORTANCE PM specificity of Pr55Gag membrane binding is mediated through the interaction of PI(4,5)P2 with the matrix (MA) basic residues. It was shown that overexpression of a PI(4,5)P2-depleting enzyme strongly impaired PM localization of Pr55Gag However, cellular factors that control PI(4,5)P2 production required for Pr55Gag-PM targeting have not yet been characterized. In this study, by individually inhibiting PIP5K1 isoforms, we elucidated a correlation between PI(4,5)P2 metabolism pathways mediated by PIP5K1 isoforms and the targeting of Pr55Gag to the PM of TZM-bl HeLa cells. Confocal microscopy analyses of cells depleted from PIP5K1α and PIP5K1γ show a rerouting of Pr55Gag to various intracellular compartments. Notably, Pr55Gag is degraded by the proteasome and/or by the lysosomes in PIP5K1α-depleted cells, while Pr55Gag is targeted to endosomal vesicles in PIP5K1γ-depleted cells. Thus, our results highlight, for the first time, the roles of PIP5K1α and PIP5K1γ as determinants of Pr55Gag targeting to the PM.


Assuntos
Membrana Celular/metabolismo , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , HIV-1/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Precursores de Proteínas/metabolismo , Membrana Celular/genética , Membrana Celular/virologia , Endossomos/genética , Endossomos/metabolismo , Endossomos/virologia , HIV-1/genética , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/virologia , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Precursores de Proteínas/genética , Proteólise
16.
Platelets ; 31(4): 544-547, 2020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-32436471

RESUMO

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by defective biogenesis of lysosome-related organelles. Clinical manifestations include a bleeding diathesis due to a platelet delta storage pool deficiency, oculocutaneous albinism, inflammatory bowel disease, neutropenia, and pulmonary fibrosis. Ten genes associated with HPS are identified to date, and each gene encodes a protein subunit of either Biogenesis of Lysosome-related Organelles Complex (BLOC)-1, BLOC-2, BLOC-3, or the Adaptor Protein-3 complex. Several genetic variants and phenotypic heterogeneities are reported in individuals with HPS, who generally exhibit easy bruisability and increased bleeding. Desmopressin, pro-coagulants, or platelet transfusion may be used as prophylaxis or treatment for excessive bleeding in patients with HPS. However, response to desmopressin can be variable. Platelets are effective in preventing or treating bleeding in individuals with HPS, but platelets should be transfused judiciously to limit alloimmunization in patients with HPS who are at risk of developing pulmonary fibrosis and may be potential candidates for lung transplantation. The discovery of new genes associated with HPS in people with excessive bleeding and hypopigmentation of unknown etiology may be facilitated by the use of next-generation sequencing or panel-based genetic testing.


Assuntos
Plaquetas/metabolismo , Síndrome de Hermanski-Pudlak/genética , Lisossomos/genética , Ácido Aminocaproico/farmacologia , Antifibrinolíticos/farmacologia , Plaquetas/ultraestrutura , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Contusões/genética , Desamino Arginina Vasopressina/uso terapêutico , Hemorragia/genética , Síndrome de Hermanski-Pudlak/tratamento farmacológico , Síndrome de Hermanski-Pudlak/fisiopatologia , Humanos , Hipopigmentação/genética , Lisossomos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas/genética , Proteínas/metabolismo , Ácido Tranexâmico/farmacologia
18.
Am J Physiol Cell Physiol ; 318(6): C1166-C1177, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32320289

RESUMO

Suboptimal lactation is a common, yet underappreciated cause for early cessation of breastfeeding. Molecular regulation of mammary gland function is critical to the process lactation; however, physiological factors underlying insufficient milk production are poorly understood. The zinc (Zn) transporter ZnT2 is critical for regulation of mammary gland development and maturation during puberty, lactation, and postlactation gland remodeling. Numerous genetic variants in the gene encoding ZnT2 (SLC30A2) are associated with low milk Zn concentration and result in severe Zn deficiency in exclusively breastfed infants. However, the functional impacts of genetic variation in ZnT2 on key mammary epithelial cell functions have not yet been systematically explored at the cellular level. Here we determined a common mutation in SLC30A2/ZnT2 substituting serine for threonine at amino acid 288 (Thr288Ser) was found in 20% of women producing low milk volume (n = 2/10) but was not identified in women producing normal volume. Exploration of cellular consequences in vitro using phosphomimetics showed the serine substitution promoted preferential phosphorylation of ZnT2, driving localization to the lysosome and increasing lysosome biogenesis and acidification. While the substitution did not initiate lysosome-mediated cell death, cellular ATP levels were significantly reduced. Our findings demonstrate the Thr288Ser mutation in SLC30A2/ZnT2 impairs critical functions of mammary epithelial cells and suggest a role for genetic variation in the regulation of milk production and lactation performance.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Metabolismo Energético , Células Epiteliais/metabolismo , Lactação/metabolismo , Lisossomos/metabolismo , Glândulas Mamárias Humanas/metabolismo , Leite Humano/metabolismo , Mutação , Trifosfato de Adenosina/metabolismo , Adulto , Estudos de Casos e Controles , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Metabolismo Energético/genética , Feminino , Humanos , Concentração de Íons de Hidrogênio , Lactação/genética , Lisossomos/genética , Biogênese de Organelas , Fosforilação , Adulto Jovem
19.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118714, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32246947

RESUMO

Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P). CerK is highly expressed in the brain, and its association with the neuronal function has been reported. Previous reports showed that the activity of CerK is regulated by post-translational modifications including phosphorylation, whereas the cellular fate of CerK protein and its role in neuronal functions have not been clearly elucidated. Therefore, we investigated these issues in PC12 cells. Treatment with nerve growth factor (NGF) for 6 h increased the formation of C1P but not CerK mRNA. Knockdown of CerK and overexpression of HA-tagged CerK down- and up-regulated the formation of C1P, respectively. In PC12-CerK-HA cells, serum withdrawal caused ubiquitination of CerK-HA protein and down-regulated both CerK-HA protein and C1P formation within 6 h, and these down-regulations were abolished by co-treatments with NGF or proteasome inhibitors such as MG132 and clasto-lactacystin. Microscopic analysis showed that treatment with the proteasome inhibitors increased CerK-HA in puncture structures, possibly endosomes and/or vesicles, in cells. Treatment with the lysosome inhibitors reduced serum withdrawal-induced down-regulation of CerK-HA protein but not C1P formation. When knockdown or overexpression of CerK was performed, Ca2+-induced release of [3H] noradrenaline was reduced or enhanced, respectively, but neurite extension was not modified. There was a positive correlation between noradrenaline release and formation of C1P and/or CerK-HA levels in NGF- and clasto-lactacystin-treated cells. These results suggest that levels of CerK were down-regulated by the ubiquitin/proteasome and lysosome pathways and the former pathway-sensitive pool of CerK was suggested to be linked with exocytosis in PC12 cells.


Assuntos
Exocitose/genética , Fator de Crescimento Neural/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Ciclo Celular , Proliferação de Células , Ceramidas , Lisossomos/genética , Lisossomos/metabolismo , Redes e Vias Metabólicas/genética , Fator de Crescimento Neural/metabolismo , Células PC12 , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos
20.
Nat Commun ; 11(1): 1416, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184389

RESUMO

The kinase mTOR complex 1 (mTORC1) promotes cellular growth and is frequently dysregulated in cancers. In response to nutrients, mTORC1 is activated on lysosomes by Rag and Rheb guanosine triphosphatases (GTPases) and drives biosynthetic processes. How limitations in nutrients suppress mTORC1 activity remains poorly understood. We find that when amino acids are limited, the Rap1-GTPases confine lysosomes to the perinuclear region and reduce lysosome abundance, which suppresses mTORC1 signaling. Rap1 activation, which is independent of known amino acid signaling factors, limits the lysosomal surface available for mTORC1 activation. Conversely, Rap1 depletion expands the lysosome population, which markedly increases association between mTORC1 and its lysosome-borne activators, leading to mTORC1 hyperactivity. Taken together, we establish Rap1 as a critical coordinator of the lysosomal system, and propose that aberrant changes in lysosomal surface availability can impact mTORC1 signaling output.


Assuntos
Aminoácidos/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Humanos , Lisossomos/enzimologia , Lisossomos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Transdução de Sinais , Proteínas rap de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/genética
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