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1.
Int J Mol Sci ; 20(18)2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505809

RESUMO

Many neurodegenerative disorders have lysosomal impediments, and the list of proposed treatments targeting lysosomes is growing. We investigated the role of lysosomes in Alzheimer's disease (AD) and other age-related disorders, as well as in a strategy to compensate for lysosomal disturbances. Comprehensive immunostaining was used to analyze brains from wild-type mice vs. amyloid precursor protein/presenilin-1 (APP/PS1) mice that express mutant proteins linked to familial AD. Also, lysosomal modulation was evaluated for inducing synaptic and behavioral improvements in transgenic models of AD and Parkinson's disease, and in models of mild cognitive impairment (MCI). Amyloid plaques were surrounded by swollen organelles positive for the lysosome-associated membrane protein 1 (LAMP1) in the APP/PS1 cortex and hippocampus, regions with robust synaptic deterioration. Within neurons, lysosomes contain the amyloid ß 42 (Aß42) degradation product Aß38, and this indicator of Aß42 detoxification was augmented by Z-Phe-Ala-diazomethylketone (PADK; also known as ZFAD) as it enhanced the lysosomal hydrolase cathepsin B (CatB). PADK promoted Aß42 colocalization with CatB in lysosomes that formed clusters in neurons, while reducing Aß deposits as well. PADK also reduced amyloidogenic peptides and α-synuclein in correspondence with restored synaptic markers, and both synaptic and cognitive measures were improved in the APP/PS1 and MCI models. These findings indicate that lysosomal perturbation contributes to synaptic and cognitive decay, whereas safely enhancing protein clearance through modulated CatB ameliorates the compromised synapses and cognition, thus supporting early CatB upregulation as a disease-modifying therapy that may also slow the MCI to dementia continuum.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Disfunção Cognitiva/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/patologia , Disfunção Cognitiva/genética , Disfunção Cognitiva/patologia , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/genética , Lisossomos/patologia , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Sinapses/metabolismo , Sinapses/patologia
2.
Genes (Basel) ; 10(10)2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31557940

RESUMO

The autophagy-lysosome pathway, which involves many crucial genes and proteins, plays crucial roles in the maintenance of intracellular homeostasis by the degradation of damaged components. At present, some of these genes and proteins have been identified but their specific functions are largely unknown. This study was performed to clone and characterize the full-length cDNA sequences of nine key autolysosome-related genes (vps11, vps16, vps18, vps33b, vps41, lamp1, mcoln1, ctsd1 and tfeb) from yellow catfish Pelteobagrus fulvidraco. The expression of these genes and the transcriptional responses to a high-fat diet and fatty acids (FAs) (palmitic acid (PA) and oleic acid (OA)) were investigated. The mRNAs of these genes could be detected in heart, liver, muscle, spleen, brain, mesenteric adipose tissue, intestine, kidney and ovary, but varied with the tissues. In the liver, the mRNA levels of the nine autolysosome-related genes were lower in fish fed a high-fat diet than those fed the control, indicating that a high-fat diet inhibited formation of autolysosomes. Palmitic acid (a saturated FA) significantly inhibited the formation of autolysosomes at 12 h, 24 h and 48 h incubation. In contrast, oleic acid (an unsaturated FA) significantly induced the formation of autolysosomes at 12 h, but inhibited them at 24 h. At 48 h, the effects of OA incubation on autolysosomes were OA concentration-dependent in primary hepatocytes of P. fulvidraco. The results of flow cytometry and laser confocal observations confirmed these results. PA and OA incubation also increased intracellular non-esterified fatty acid (NEFA) concentration at 12 h, 24 h and 48 h, and influenced mRNA levels of fatty acid binding protein (fabp) and fatty acid transport protein 4 (fatp4) which facilitate FA transport in primary hepatocytes of P. fulvidraco. The present study demonstrated the molecular characterization of the nine autolysosome-related genes and their transcriptional responses to fat and FAs in fish, which provides the basis for further exploring their regulatory mechanism in vertebrates.


Assuntos
Autofagossomos/metabolismo , Peixes-Gato/metabolismo , Gorduras na Dieta/farmacologia , Ácidos Graxos/farmacologia , Fígado/metabolismo , Lisossomos/metabolismo , Animais , Gorduras na Dieta/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Fígado/efeitos dos fármacos , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
3.
Nat Commun ; 10(1): 3521, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387993

RESUMO

The intracellular transport of cholesterol is subject to tight regulation. The structure of the lysosomal integral membrane protein type 2 (LIMP-2, also known as SCARB2) reveals a large cavity that traverses the molecule and resembles the cavity in SR-B1 that mediates lipid transfer. The detection of cholesterol within the LIMP-2 structure and the formation of cholesterol-like inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes. We present results of molecular modeling, crosslinking studies, microscale thermophoresis and cell-based assays that support a role of LIMP-2 in cholesterol transport. We show that the cavity in the luminal domain of LIMP-2 can bind and deliver exogenous cholesterol to the lysosomal membrane and later to lipid droplets. Depletion of LIMP-2 alters SREBP-2-mediated cholesterol regulation, as well as LDL-receptor levels. Our data indicate that LIMP-2 operates in parallel with Niemann Pick (NPC)-proteins, mediating a slower mode of lysosomal cholesterol export.


Assuntos
Antígenos CD36/metabolismo , LDL-Colesterol/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/metabolismo , Receptores Depuradores/metabolismo , Animais , Antígenos CD36/genética , Células CHO , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cricetulus , Fibroblastos , Técnicas de Inativação de Genes , Células HeLa , Humanos , Gotículas Lipídicas/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Domínios Proteicos , RNA Interferente Pequeno/metabolismo , Receptores Depuradores/genética
4.
Molecules ; 24(16)2019 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-31426598

RESUMO

We previously reported on a polyhistidine peptide, His16 peptide, as a new cell-penetrating peptide. This peptide is anticipated to be a new carrier for drug delivery systems (DDSs) for targeting intracellular lysosomes because it can transport macromolecules (e.g., liposomes) into these organelles. In the present study, we examined the application of His16 peptide as a DDS carrier against lysosomal storage disease (LSD) cells. LSDs are metabolic disorders caused by loss of specific lysosomal enzymes. For the treatment of LSD cells, we devised a system designated organelle replacement therapy (ORT). ORT is a strategy for transporting exogenous lysosomes containing all kinds of lysosomal enzymes from normal cells into endogenous lysosomes in LSD cells using His16 peptide. To develop the ORT system, we prepared His16 peptide-modified healthy lysosomes (His16-Lyso) by insertion of a stearyl-His16 peptide into a hydrophobic region in the lysosomal membrane. His16-Lyso showed cellular uptake and localization to endogenous lysosomes in LSD cells. His16-Lyso also restored the proliferation of LSD cells, which otherwise showed slower proliferation than normal cells. These results suggested that His16-Lyso replenished deficient lysosomal enzymes in LSD cells. The results further suggest that His16-Lyso are promising candidates as a treatment tool for LSD cells and to establish a foundation for ORT.


Assuntos
Engenharia Celular/métodos , Peptídeos Penetradores de Células/metabolismo , Portadores de Fármacos , Histidina/metabolismo , Lisossomos/transplante , Transporte Biológico , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Peptídeos Penetradores de Células/síntese química , Doença de Fabry/patologia , Doença de Fabry/terapia , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Histidina/síntese química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/química , Lisossomos/metabolismo , Modelos Biológicos , Terapia de Alvo Molecular/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
5.
Invest Ophthalmol Vis Sci ; 60(8): 3046-3053, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31319418

RESUMO

Purpose: Accumulation of lysosomal waste is linked to neurodegeneration in multiple diseases, and pharmacologic enhancement of lysosomal activity is hypothesized to reduce pathology. An excessive accumulation of lysosomal-associated lipofuscin waste and an elevated lysosomal pH occur in retinal pigment epithelial cells of the ABCA4-/- mouse model of Stargardt's retinal degeneration. As treatment with the P2Y12 receptor antagonist ticagrelor was previously shown to lower lysosomal pH and lipofuscin-like autofluorescence in these cells, we asked whether oral delivery of ticagrelor also prevented photoreceptor loss. Methods: Moderate light exposure was used to accelerate photoreceptor loss in albino ABCA4-/- mice as compared to BALB/c controls. Ticagrelor (0.1%-0.15%) was added to mouse chow for between 1 and 10 months. Photoreceptor function was determined with electroretinograms, while cell survival was determined using optical coherence tomography and histology. Results: Protection by ticagrelor was demonstrated functionally by using the electroretinogram, as ticagrelor-treated ABCA4-/- mice had increased a- and b-waves compared to untreated mice. Mice receiving ticagrelor treatment had a thicker outer nuclear layer, as measured with both optical coherence tomography and histologic sections. Ticagrelor decreased expression of LAMP1, implicating enhanced lysosomal function. No signs of retinal bleeding were observed after prolonged treatment with ticagrelor. Conclusions: Oral treatment with ticagrelor protected photoreceptors in the ABCA4-/- mouse, which is consistent with enhanced lysosomal function. As mouse ticagrelor exposure levels were clinically relevant, the drug may be of benefit in preventing the loss of photoreceptors in Stargardt's disease and other neurodegenerations associated with lysosomal dysfunction.


Assuntos
Degeneração Retiniana/prevenção & controle , Epitélio Pigmentado da Retina/patologia , Ticagrelor/administração & dosagem , Administração Oral , Animais , Modelos Animais de Doenças , Eletrorretinografia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicoproteínas de Membrana Associadas ao Lisossomo/biossíntese , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias , Antagonistas do Receptor Purinérgico P2Y/administração & dosagem , RNA/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/fisiopatologia , Tomografia de Coerência Óptica , Resultado do Tratamento
6.
Virol Sin ; 34(5): 508-520, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31215001

RESUMO

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic tick-borne bunyavirus that causes lethal infectious disease and severe fever with thrombocytopenia syndrome (SFTS) in humans. The molecular mechanisms and host cellular factors required for SFTSV infection remain uncharacterized. Using a genome-wide CRISPR-based screening strategy, we identified a host cellular protein, sorting nexin 11 (SNX11) which is involved in the intracellular endosomal trafficking pathway, as an essential cell factor for SFTSV infection. An SNX11-KO HeLa cell line was established, and SFTSV replication was significantly reduced. The glycoproteins of SFTSV were detected and remained in later endosomal compartments but were not detectable in the endoplasmic reticulum (ER) or Golgi apparatus. pH values in the endosomal compartments of the SNX11-KO cells increased compared with the pH of normal HeLa cells, and lysosomal-associated membrane protein 1 (LAMP1) expression was significantly elevated in the SNX11-KO cells. Overall, these results indicated that penetration of SFTSV from the endolysosomes into the cytoplasm of host cells was blocked in the cells lacking SNX11. Our study for the first time provides insight into the important role of the SNX11 as an essential host factor in the intracellular trafficking and penetrating process of SFTSV infection via potential regulation of viral protein sorting, membrane fusion, and other endocytic machinery.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Interações Hospedeiro-Patógeno/genética , Phlebovirus/fisiologia , Nexinas de Classificação/genética , Linhagem Celular , Endossomos/química , Endossomos/virologia , Técnicas de Inativação de Genes , Complexo de Golgi/química , Complexo de Golgi/virologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Phlebovirus/genética , Proteínas Virais/genética
7.
Neurosci Lett ; 706: 164-168, 2019 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-31116970

RESUMO

Mutations in the glucocerebrosidase (GCase) gene (GBA) and GCase deficiency are major risk factors for Lewy body diseases. Decreased GCase activity enhances alpha-synuclein aggregation and disease development. Lysosomal integral membrane protein type 2, encoded by SCARB2, binds GCase targeting it to lysosomes and transcription factor EB (Tfeb) regulates lysosomal proteostasis. Our aim was to find out if GCase deficiency in Lewy body diseases is accompanied by SCARB2 and TFEB deregulation at the transcriptional level involving alternative splicing as well. Relative mRNA expression of two SCARB2 and two TFEB transcripts was studied by real-time PCR in post-mortem brain samples of cases with pure Lewy body pathology (LBP), cases with concomitant LBP and Alzheimer disease-like pathology, and controls. TFEB expression was increased in the temporal cortex and caudate nucleus of LBP cases, and SCARB2 was differentially expressed. Female-gender associated overexpression of all transcripts was found in the caudate nucleus, and disease duration associated TFEB expression changes in the temporal cortex. SCARB2 and TFEB expression correlated negatively with GBA mRNA expression in the temporal cortex. Our findings show disease-specific deregulation of TFEB and SCARB2 expression affecting alternative promoter usage and alternative splicing in Lewy body diseases.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Encéfalo/metabolismo , Doença por Corpos de Lewy/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Receptores Depuradores/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Encéfalo/patologia , Feminino , Humanos , Doença por Corpos de Lewy/genética , Doença por Corpos de Lewy/patologia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Masculino , Pessoa de Meia-Idade , Receptores Depuradores/genética , Fatores Sexuais , Ativação Transcricional , Regulação para Cima
8.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067781

RESUMO

Despite the constantly updated knowledge regarding the alterations occurring in the cells of patients with psoriasis, the status and the role of the lysosome, a control center of cell metabolism, remain to be elucidated. The architecture of the epidermis is largely regulated by the action of lysosomes, possibly activating signaling pathways in the cellular crosstalk of keratinocytes-epidermal cells-with infiltrating immune cells. Thus, in the present study, lysosome alterations were examined in vitro and in situ using a two-dimensional (2D) keratinocyte model of HaCaT cells with "psoriasis-like" inflammation and skin specimens, respectively. Specific fluorescence and immunohistochemical staining showed an augmented level of acidic organelles in response to keratinocyte activation (mimicking a psoriatic condition while maintaining the membrane integrity of these structures) as compared with the control, similar to that seen in skin samples taken from patients. Interestingly, patients with the most pronounced PASI (Psoriasis Area and Severity Index), BSA (Body Surface Area), and DLQI (Dermatology Life Quality Index) scores suffered a high incidence of positive lysosomal-associated membrane protein 1 (LAMP1) expression. Moreover, it was found that the gene deregulation pattern was comparable in lesioned (PP) and non-lesioned (PN) patient-derived skin tissue, which may indicate that these alterations occur prior to the onset of the characteristic phenotype of the disease. Changes in the activity of genes encoding the microphthalmia family (MiT family) of transcription factors and mammalian target of rapamycin complex 1 (MTORC1) were also observed in the in vitro psoriasis model, indicating that the biogenesis pathway of this arm is inhibited. Interestingly, in contrast to the keratinocytes of HaCaT with "psoriasis-like" inflammation, LAMP1 was up-regulated in both PP and PN skin, which can be a potential sign of an alternative mechanism of lysosome formation. Defining the molecular profile of psoriasis in the context of "the awesome lysosome" is not only interesting, but also desired; therefore, it is believed that this paper will serve to encourage other researchers to conduct further studies on this subject.


Assuntos
Queratinócitos/metabolismo , Lisossomos/metabolismo , Psoríase/metabolismo , Pele/metabolismo , Adulto , Idoso , Linhagem Celular , Feminino , Humanos , Queratinócitos/ultraestrutura , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/ultraestrutura , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Pessoa de Meia-Idade , Psoríase/patologia , Pele/ultraestrutura
9.
Gene ; 701: 146-151, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30922709

RESUMO

Circular RNAs (circRNAs) act as pivotal functions in tumor progression. Nevertheless, the functions and mechanism of circRNAs in T-cell lymphoblastic lymphoma (T-LBL) remain unclear. In this work, we first screened the differentially expressed circRNAs between T-LBL tissues and normal infantile thymus and circ-LAMP1 was identified the highest expressed circRNA in cancerous tissues. qRT-PCR further verified its upregulation in T-LBL tissues and cell lines. Cell counting kit-8 (CCK-8) experiment proved the cell proliferation-promoting role of circ-LAMP1. This effect is partially dependent on its inhibition on cell apoptosis proved by flow cytometric assay. Dual-luciferase reporter system further identified that miR-615-5p could be sponged by circ-LAMP1 and discoidin domain receptor tyrosine kinase 2 (DDR2) 3'-UTR is the direct target of miR-615-5p. Rescue assays demonstrated that the biological function of circ-LAMP1 is partly attributed to the modulation of miR-615-5p/DDR2 signaling. In summary, these findings documented that circ-LAMP1 might be an oncogene in T-LBL, which might be useful in developing promising therapies for T-LBL.


Assuntos
Receptor com Domínio Discoidina 2 , Regulação Neoplásica da Expressão Gênica , Linfoma de Células T , Glicoproteínas de Membrana Associadas ao Lisossomo , MicroRNAs , Proteínas de Neoplasias , RNA Neoplásico , Regiões 3' não Traduzidas , Receptor com Domínio Discoidina 2/biossíntese , Receptor com Domínio Discoidina 2/genética , Feminino , Humanos , Células Jurkat , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo
10.
J Biol Chem ; 294(16): 6405-6415, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30733336

RESUMO

Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein-lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways.


Assuntos
Proteínas de Bactérias/metabolismo , Endossomos/metabolismo , Legionella pneumophila/metabolismo , Doença dos Legionários/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Vacúolos/metabolismo , Proteínas de Bactérias/genética , Endossomos/genética , Endossomos/ultraestrutura , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/genética , Legionella pneumophila/patogenicidade , Doença dos Legionários/genética , Doença dos Legionários/patologia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/genética , Lisossomos/ultraestrutura , Macrófagos/microbiologia , Macrófagos/ultraestrutura , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/antagonistas & inibidores , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Células U937 , Vacúolos/genética , Vacúolos/microbiologia , Vacúolos/ultraestrutura , Wortmanina/farmacologia , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
11.
Clin Neuropathol ; 38(4): 157-167, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30738494

RESUMO

Neutral lipid storage disease with myopathy (NLSDM) is a triglyceride metabolic disorder caused by defects of adipose triglyceride lipases (ATGL). The coexistence of lipid vacuoles and rimmed vacuoles in the myofibers is a characteristic pathological change in some NLSDM cases. However, it has not been explored whether autophagic abnormalities exist in the NLSDM myofibers with rimmed vacuole. Herein, we report that 5 patients with NLSDM initially presented with muscle weakness in the right arm related to long-term physical efforts, then developed muscle weakness of other limbs. Pathogenic mutations in the PNPLA2 gene were identified in all patients. Myopathological analysis showed a coexistence of massive lipid vacuoles and rimmed vacuoles, which was not associated with the age of onset or mutation sites, but closely related to the severity of muscle degeneration. The rimmed vacuoles showed strong immunopositivity to autophagic markers, but were negative to apoptotic markers. Significant immunoreactivity of p62 was observed in the rimmed vacuoles, while the lysosomal marker LAMP1 was severely decreased. Our study expanded the clinical and genetic spectrum of NLSDM. Loss of ATGL activity in muscle fibers with rimmed vacuoles induced a marked increase in autophagic formation, but lowered down the turnover of autolysosomes due to malfunction of lysosomes.


Assuntos
Lipase/genética , Erros Inatos do Metabolismo Lipídico/genética , Músculo Esquelético/patologia , Doenças Musculares/patologia , Adulto , Apoptose/fisiologia , Autofagia , Feminino , Humanos , Eritrodermia Ictiosiforme Congênita/genética , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/patologia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Masculino , Pessoa de Meia-Idade , Doenças Musculares/diagnóstico , Doenças Musculares/genética , Mutação/genética , Vacúolos/genética
12.
Molecules ; 24(4)2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30769789

RESUMO

The in vitro efficacy of cancer prodrugs varies significantly between malignant cell lines. The most commonly identified problems relate to delivery: uptake mechanism, endosomal entrapment, and drug release. Here we present the study of collagen/cell penetrating hybrid (COL/CPP) peptide carriers intended to deliver paclitaxel to the hypopharyngeal carcinoma (FaDu) cells. Confocal microscopy imaging revealed the surprising response of FaDu cell to COL/CPP in comparison to previously studied cancer cell lines: hybrid peptides that carry both COL and CPP domain adsorb on the FaDu cell surface. While the CPP domain was design to facilitate the cellular uptake, in the case of FaDu cells, it also induced detrimental interactions with the cell membrane. Despite surface adsorption, the colocalization study with endosomal markers EEA1 and LAMP1 reveals that COL/CPP is internalized via endosomal pathway, peptides are able to escape before lysosome formation and release paclitaxel. Therefore, the main obstacle for paclitaxel delivery to FaDu cells appears to be related to cell surface properties. This behavior seems specific to FaDu cells, and could be linked to previously reported overexpression of T5, heparanase splice variants that produces protein lacking enzymatic activity of heparanase. This results in increased concentration of HSPG on FaDu cell surface, and possibly creates a barrier for cellular uptake of highly charged COL/CPP.


Assuntos
Peptídeos Penetradores de Células/farmacologia , Portadores de Fármacos/farmacologia , Proteoglicanas de Heparan Sulfato/química , Neoplasias Hipofaríngeas/tratamento farmacológico , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Colágeno/química , Colágeno/farmacologia , Portadores de Fármacos/química , Liberação Controlada de Fármacos/genética , Endossomos/química , Endossomos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hipofaríngeas/genética , Neoplasias Hipofaríngeas/patologia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Paclitaxel/química , Paclitaxel/farmacologia , Propriedades de Superfície , Proteínas de Transporte Vesicular/genética
13.
J Assist Reprod Genet ; 36(4): 777-786, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30659447

RESUMO

OBJECTIVE: To study the location and expression of receptors (SR-BI/CLA-1, SR-BII, and LDLr) and transporter (ABCA1) involved in uptake and efflux of cholesterol in human spermatozoa and assess whether obesity alters its location/expression and whether this could be related to infertility. DESIGN: Observational study. SETTING: None PATIENT(S): Ten controls and 20 obese patients. INTERVENTION(S): Anthropometric parameters. Serum and semen samples were collected. MAIN OUTCOME MEASURE(S): Spermatozoon concentration, immunolocalization, and protein expression in semen. RESULTS: Spermatozoon concentration and motility was decreased in morbidly obese patients. SR-BI/CLA-1, SR-BII, LDLr, and ABCA1 are located in the spermatozoon cell membrane and the localization does not change between obese patients and controls. Control spermatozoa showed high SR-BI expression, and less expression for the rest of the receptors analyzed, indicating that SR-BI/CLA-1 is relevant in human spermatozoon cholesterol uptake/efflux. On the contrary, spermatozoa of obese patients showed less SR-BI/CLA-1 expression than controls, and more intense positive staining for SR-BII, LDLr, and ABCA1. Finally, human sperm expresses the 130- and 82-kDa hormone-sensitive lipase (HSL) isoforms. The 130-kDa isoform is expressed in the control sperm, and the expression disappears in the obese patients. CONCLUSION(S): The presence of lipid receptors/transporters and HSL in human spermatozoa suggests their role in the process of maturation/capacitation. The changes in the expression of lipid receptors/transporters and the lack of the 130-kDa HSL isoform in obese patients prevent the hydrolysis of cholesterol esters internalized by these receptors, and favor their accumulation in the cytoplasm of the spermatozoa that could contribute to lipotoxicity and infertility.


Assuntos
Infertilidade Masculina/genética , Obesidade Mórbida/genética , Sêmen/metabolismo , Espermatozoides/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Adulto , Antígenos CD36/genética , Membrana Celular/genética , Colesterol/genética , Colesterol/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Infertilidade Masculina/complicações , Infertilidade Masculina/patologia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Masculino , Obesidade Mórbida/complicações , Obesidade Mórbida/patologia , Isoformas de Proteínas/genética , Receptores de LDL/genética , Receptores Depuradores/genética , Capacitação Espermática/genética , Espermatozoides/patologia , Esterol Esterase/genética
14.
J Cell Sci ; 132(2)2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651381

RESUMO

The pivotal role of lysosomes in cellular processes is increasingly appreciated. An understanding of the balanced interplay between the activity of acidic hydrolases, lysosomal membrane proteins and cytosolic proteins is required. Lysosomal storage diseases (LSDs) are characterized by disturbances in this network and by intralysosomal accumulation of substrates, often only in certain cell types. Even though our knowledge of these diseases has increased and therapies have been established, many aspects of the molecular pathology of LSDs remain obscure. This Review aims to discuss how lysosomal storage affects functions linked to lysosomes, such as membrane repair, autophagy, exocytosis, lipid homeostasis, signalling cascades and cell viability. Therapies must aim to correct lysosomal storage not only morphologically, but reverse its (patho)biochemical consequences. As different LSDs have different molecular causes, this requires custom tailoring of therapies. We will discuss the major advantages and drawbacks of current and possible future therapies for LSDs. Study of the pathological molecular mechanisms underlying these 'experiments of nature' often yields information that is relevant for other conditions found in the general population. Therefore, more common diseases may profit from a correction of impaired lysosomal function.


Assuntos
Autofagia , Exocitose , Doenças por Armazenamento dos Lisossomos/metabolismo , Lisossomos , Animais , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/patologia , Doenças por Armazenamento dos Lisossomos/terapia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/patologia , Doenças Raras/genética , Doenças Raras/metabolismo , Doenças Raras/patologia , Doenças Raras/terapia
15.
J Cell Sci ; 132(3)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30617110

RESUMO

The dipeptide glycyl-l-phenylalanine 2-naphthylamide (GPN) is widely used to perturb lysosomes because its cleavage by the lysosomal enzyme cathepsin C is proposed to rupture lysosomal membranes. We show that GPN evokes a sustained increase in lysosomal pH (pHly), and transient increases in cytosolic pH (pHcyt) and Ca2+ concentration ([Ca2+]c). None of these effects require cathepsin C, nor are they accompanied by rupture of lysosomes, but they are mimicked by structurally unrelated weak bases. GPN-evoked increases in [Ca2+]c require Ca2+ within the endoplasmic reticulum (ER), but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. We conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER in a manner that is independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Citosol/efeitos dos fármacos , Dipeptídeos/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Transporte Biológico , Sistemas CRISPR-Cas , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Catepsina C/genética , Catepsina C/metabolismo , Linhagem Celular Tumoral , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Edição de Genes , Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Ploidias , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
16.
Proc Natl Acad Sci U S A ; 116(2): 512-521, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30610181

RESUMO

Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered a class of cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine (penta-Arg) motif that traffics readily to the cytosol and nucleus of mammalian cells with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs containing a penta-Arg motif are released efficiently from endosomes, while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using an RNA interference screen, fluorescence correlation spectroscopy, and confocal imaging, we identify VPS39-a gene encoding a subunit of the homotypic fusion and protein-sorting (HOPS) complex-as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. CPMPs localize within the lumen of Rab7+ and Lamp1+ endosomes and their transport requires HOPS activity. Overall, our results identify Lamp1+ late endosomes and lysosomes as portals for passing proteins into the cytosol and suggest that this environment is prerequisite for endosomal escape.


Assuntos
Proteínas de Transporte/genética , Peptídeos Penetradores de Células , Endossomos/metabolismo , Fusão de Membrana/efeitos dos fármacos , Motivos de Aminoácidos , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/farmacocinética , Peptídeos Penetradores de Células/farmacologia , Citosol/metabolismo , Endossomos/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
17.
Genet Med ; 21(1): 224-232, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29875425

RESUMO

PURPOSE: Evaluation standards and treatment initiation timing have been debated for a long time, particularly for late-onset Fabry disease (FD), because of its slow progression. However, early initiation of enzyme replacement therapy (ERT) for FD could be effective in stabilizing the disease progression and potentially preventing irreversible organ damage. We aimed to examine globotriaosylceramide (Gb3) deposits in patients' endomyocardial biopsies to understand the early pathogenesis of FD cardiomyopathy. METHODS: Immunofluorescent (IF) staining of Gb3 and lysosomal-associated membrane protein 1 (LAMP-1) was performed on endomyocardial biopsies of patients suspected of Fabry cardiomyopathy who had negative or only slight Gb3 accumulation determined by toluidine blue staining and electron microscopic examination. RESULTS: The IF staining results revealed that all patients examined had abundant Gb3 accumulation in their cardiomyocytes, including the ones who are negative for inclusion bodies. Furthermore, we found that early Gb3 deposits were mostly confined within lysosomes, while they appeared extralysosomally at a later stage. CONCLUSION: A significant amount of lysosomal Gb3 deposits could be detected by IF staining in cardiac tissue before the formation of inclusion bodies, suggesting the cardiomyocytes might have been experiencing cellular stress and damage early on, before the appearance of typical pathological changes of FD during the disease progression.


Assuntos
Doença de Fabry/diagnóstico , Globosídeos/metabolismo , Lisossomos/metabolismo , Miocárdio/metabolismo , Triexosilceramidas/metabolismo , Adulto , Biópsia , Progressão da Doença , Terapia de Reposição de Enzimas , Doença de Fabry/diagnóstico por imagem , Doença de Fabry/metabolismo , Doença de Fabry/patologia , Imunofluorescência , Globosídeos/genética , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Lisossomos/patologia , Masculino , Pessoa de Meia-Idade , Miocárdio/patologia , Triexosilceramidas/genética
18.
J Cell Biol ; 218(1): 267-284, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30538141

RESUMO

Mutations in ATP13A2 cause Kufor-Rakeb syndrome, an autosomal recessive form of juvenile-onset atypical Parkinson's disease (PD). Recent work tied ATP13A2 to autophagy and other cellular features of neurodegeneration, but how ATP13A2 governs numerous cellular functions in PD pathogenesis is not understood. In this study, the ATP13A2-deficient mouse developed into aging-dependent phenotypes resembling those of autophagy impairment. ATP13A2 deficiency impaired autophagosome-lysosome fusion in cultured cells and in in vitro reconstitution assays. In ATP13A2-deficient cells or Drosophila melanogaster or mouse tissues, lysosomal localization and activity of HDAC6 were reduced, with increased acetylation of tubulin and cortactin. Wild-type HDAC6, but not a deacetylase-inactive mutant, restored autophagosome-lysosome fusion, antagonized cortactin hyperacetylation, and promoted lysosomal localization of cortactin in ATP13A2-deficient cells. Mechanistically, ATP13A2 facilitated recruitment of HDAC6 and cortactin to lysosomes. Cortactin overexpression in cultured cells reversed ATP13A2 deficiency-associated impairment of autophagosome-lysosome fusion. PD-causing ATP13A2 mutants failed to rescue autophagosome-lysosome fusion or to promote degradation of protein aggregates and damaged mitochondria. These results suggest that ATP13A2 recruits HDAC6 to lysosomes to deacetylate cortactin and promotes autophagosome-lysosome fusion and autophagy. This study identifies ATP13A2 as an essential molecular component for normal autophagy flux in vivo and implies potential treatments targeting HDAC6-mediated autophagy for PD.


Assuntos
Autofagossomos/metabolismo , Cortactina/genética , Desacetilase 6 de Histona/genética , Lisossomos/metabolismo , Doença de Parkinson Secundária/genética , ATPases Translocadoras de Prótons/genética , Sequência de Aminoácidos , Anilidas/farmacologia , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Autofagia/genética , Cortactina/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster , Regulação da Expressão Gênica , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Leupeptinas/farmacologia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Masculino , Fusão de Membrana/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Doença de Parkinson Secundária/metabolismo , Doença de Parkinson Secundária/patologia , ATPases Translocadoras de Prótons/deficiência , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
19.
Emerg Microbes Infect ; 7(1): 205, 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518755

RESUMO

Enterovirus A71 (EV-A71) is a major etiological agent of human hand, foot and mouth disease, and it can cause severe neurological complications. Although several genotypes of EV-A71 strains are prevalent in different regions of the world, the genotype C4 has circulated in mainland China for more than 20 years. The pathogenicity of different EV-A71 clinical isolates varies and needs to be explored. In this study, hSCARB2 knock-in mice (N = 181) with a wide range of ages were tested for their susceptibility to two EV-A71 strains with the subgenotypes C4 and C2, and two infection routes (intracranial and venous) were compared. The clinical manifestations and pathology and their relationship to the measured viral loads in different tissues were monitored. We observed that 3 weeks is a crucial age, as mice younger than 3-week-old that were infected became extremely ill. However, mice older than 3 weeks displayed diverse clinical symptoms. Significant differences were observed in the pathogenicity of the two strains with respect to clinical signs, disease incidence, survival rate, and body weight change. We concluded that hSCARB2 knock-in mice are a sensitive model for investigating the clinical outcomes resulting from infection by different EV-A71 strains. The intracranial infection model appears to be suitable for evaluating EV-A71 neurovirulence, whereas the venous infection model is appropriate for studying the pathogenicity of EV-A71.


Assuntos
Encéfalo/virologia , Modelos Animais de Doenças , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/virologia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Receptores Depuradores/genética , Administração Intravenosa , Fatores Etários , Animais , Antígenos Virais/genética , Enterovirus Humano A/genética , Infecções por Enterovirus/sangue , Técnicas de Introdução de Genes , Genótipo , Humanos , Camundongos , Crânio/virologia , Carga Viral , Virulência
20.
PLoS One ; 13(11): e0206769, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30395634

RESUMO

OBJECTIVE: To evaluate the association of enterovirus 71 (EV71) susceptibility and clinical severity with polymorphisms in EV71 receptors, including human scavenger receptor class B member 2 (SCARB2), P-selectin glycoprotein ligand-1 (PSGL-1), and annexin II (ANXA2). METHODS: We enrolled laboratory-confirmed EV71 cases and healthy age- and gender-matched controls in Taiwan from 2000 to 2012. We detected genetic polymorphisms in SCARB2, PSGL-1, and ANXA2 and correlated the results with EV71 susceptibility and severity. RESULTS: We collected 599 EV71 cases and 98 controls. Among EV71 patients, the male to female ratio was 1.61, and the mean age was 2.99±2.47 years. For clinical severity, 117 (19.6%) had severe central nervous system involvement with or without cardiopulmonary failure. For outcomes, 46 (7.7%) had sequelae, and 14 (2.3%) died. SCARB2 polymorphisms (rs6824953 and rs11097262) were associated with susceptibility to EV71 infection (OR 1.60, 95% CI 1.07-2.39; and OR 1.64, 95% CI 1.09-2.47, respectively). PSGL-1 polymorphisms (rs7137098 and rs8179137) were significantly associated with severe EV71 infection (OR 1.46, 95% CI 1.1-1.96; and OR 1.47, 95% CI 1.07-2.03, respectively). CONCLUSIONS: SCARB2 polymorphisms (rs6824953 and rs11097262) might be associated with EV71 susceptibility. PSGL-1 polymorphisms (rs7137098 and rs8179137) were associated with severe EV71 infection.


Assuntos
Anexina A2/genética , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , Receptores Depuradores/genética , Receptores Virais/genética , Estudos de Casos e Controles , Pré-Escolar , Infecções por Enterovirus/virologia , Feminino , Predisposição Genética para Doença , Humanos , Lactente , Masculino , Índice de Gravidade de Doença , Taiwan
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