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1.
Nat Commun ; 15(1): 6150, 2024 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-39034314

RESUMO

Non-neovascular or dry age-related macular degeneration (AMD) is a multi-factorial disease with degeneration of the aging retinal-pigmented epithelium (RPE). Lysosomes play a crucial role in RPE health via phagocytosis and autophagy, which are regulated by transcription factor EB/E3 (TFEB/E3). Here, we find that increased AKT2 inhibits PGC-1α to downregulate SIRT5, which we identify as an AKT2 binding partner. Crosstalk between SIRT5 and AKT2 facilitates TFEB-dependent lysosomal function in the RPE. AKT2/SIRT5/TFEB pathway inhibition in the RPE induced lysosome/autophagy signaling abnormalities, disrupted mitochondrial function and induced release of debris contributing to drusen. Accordingly, AKT2 overexpression in the RPE caused a dry AMD-like phenotype in aging Akt2 KI mice, as evident from decline in retinal function. Importantly, we show that induced pluripotent stem cell-derived RPE encoding the major risk variant associated with AMD (complement factor H; CFH Y402H) express increased AKT2, impairing TFEB/TFE3-dependent lysosomal function. Collectively, these findings suggest that targeting the AKT2/SIRT5/TFEB pathway may be an effective therapy to delay the progression of dry AMD.


Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Lisossomos , Degeneração Macular , Proteínas Proto-Oncogênicas c-akt , Epitélio Pigmentado da Retina , Transdução de Sinais , Sirtuínas , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sirtuínas/metabolismo , Sirtuínas/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Degeneração Macular/genética , Humanos , Camundongos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Lisossomos/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Modelos Animais de Doenças , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino
2.
ArXiv ; 2024 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-38903737

RESUMO

Deep neural networks have been applied to improve the image quality of fluorescence microscopy imaging. Previous methods are based on convolutional neural networks (CNNs) which generally require more time-consuming training of separate models for each new imaging experiment, impairing the applicability and generalization. Once the model is trained (typically with tens to hundreds of image pairs) it can then be used to enhance new images that are like the training data. In this study, we proposed a novel imaging-transformer based model, Convolutional Neural Network Transformer (CNNT), to outperform the CNN networks for image denoising. In our scheme we have trained a single CNNT based "backbone model" from pairwise high-low SNR images for one type of fluorescence microscope (instance structured illumination, iSim). Fast adaption to new applications was achieved by fine-tuning the backbone on only 5-10 sample pairs per new experiment. Results show the CNNT backbone and fine-tuning scheme significantly reduces the training time and improves the image quality, outperformed training separate models using CNN approaches such as - RCAN and Noise2Fast. Here we show three examples of the efficacy of this approach on denoising wide-field, two-photon and confocal fluorescence data. In the confocal experiment, which is a 5×5 tiled acquisition, the fine-tuned CNNT model reduces the scan time form one hour to eight minutes, with improved quality.

4.
Nat Commun ; 15(1): 93, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38168055

RESUMO

Lysosomes have emerged as critical regulators of cellular homeostasis. Here we show that the lysosomal protein TMEM55B contributes to restore cellular homeostasis in response to oxidative stress by three different mechanisms: (1) TMEM55B mediates NEDD4-dependent PLEKHM1 ubiquitination, causing PLEKHM1 proteasomal degradation and halting autophagosome/lysosome fusion; (2) TMEM55B promotes recruitment of components of the ESCRT machinery to lysosomal membranes to stimulate lysosomal repair; and (3) TMEM55B sequesters the FLCN/FNIP complex to facilitate translocation of the transcription factor TFE3 to the nucleus, allowing expression of transcriptional programs that enable cellular adaptation to stress. Knockout of tmem55 genes in zebrafish embryos increases their susceptibility to oxidative stress, causing early death of tmem55-KO animals in response to arsenite toxicity. Altogether, our work identifies a role for TMEM55B as a molecular sensor that coordinates autophagosome degradation, lysosomal repair, and activation of stress responses.


Assuntos
Autofagia , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Autofagia/genética , Lisossomos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Estresse Oxidativo
5.
Cells ; 12(24)2023 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-38132146

RESUMO

There is growing evidence supporting the role of fibroblasts in all stages of atherosclerosis, from the initial phase to fibrous cap and plaque formation. In the arterial wall, as with macrophages and vascular smooth muscle cells, fibroblasts are exposed to a myriad of LDL lipids, including the lipid species formed during the oxidation of their polyunsaturated fatty acids of cholesteryl esters (PUFA-CEs). Recently, our group identified the final oxidation products of the PUFA-CEs, cholesteryl hemiesters (ChE), in tissues from cardiovascular disease patients. Cholesteryl hemiazelate (ChA), the most prevalent lipid of this family, is sufficient to impact lysosome function in macrophages and vascular smooth muscle cells, with consequences for their homeostasis. Here, we show that the lysosomal compartment of ChA-treated fibroblasts also becomes dysfunctional. Indeed, fibroblasts exposed to ChA exhibited a perinuclear accumulation of enlarged lysosomes full of neutral lipids. However, this outcome did not trigger de novo lysosome biogenesis, and only the lysosomal transcription factor E3 (TFE3) was slightly transcriptionally upregulated. As a consequence, autophagy was inhibited, probably via mTORC1 activation, culminating in fibroblasts' apoptosis. Our findings suggest that the impairment of lysosome function and autophagy and the induction of apoptosis in fibroblasts may represent an additional mechanism by which ChA can contribute to the progression of atherosclerosis.


Assuntos
Aterosclerose , Doenças Cardiovasculares , Humanos , Camundongos , Animais , Ésteres do Colesterol , Lisossomos/fisiologia , Ácidos Graxos , Fibroblastos
6.
Curr Biol ; 33(17): R886-R888, 2023 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-37699340

RESUMO

Contreras and Puertollano introduce TFEB, a transcription factor that orchestrates cellular responses to stress via mechanisms including upregulation of lysosome biogenesis and autophagy.


Assuntos
Autofagia , Lisossomos , Fatores de Transcrição , Ativação Transcricional , Regulação para Cima
7.
bioRxiv ; 2023 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-37609254

RESUMO

Age-related macular degeneration (AMD), the leading cause of geriatric blindness, is a multi-factorial disease with retinal-pigmented epithelial (RPE) cell dysfunction as a central pathogenic driver. With RPE degeneration, lysosomal function is a core process that is disrupted. Transcription factors EB/E3 (TFEB/E3) tightly control lysosomal function; their disruption can cause aging disorders, such as AMD. Here, we show that induced pluripotent stem cells (iPSC)-derived RPE cells with the complement factor H variant [ CFH (Y402H)] have increased AKT2, which impairs TFEB/TFE3 nuclear translocation and lysosomal function. Increased AKT2 can inhibit PGC1α, which downregulates SIRT5, an AKT2 binding partner. SIRT5 and AKT2 co-regulate each other, thereby modulating TFEB-dependent lysosomal function in the RPE. Failure of the AKT2/SIRT5/TFEB pathway in the RPE induced abnormalities in the autophagy-lysosome cellular axis by upregulating secretory autophagy, thereby releasing a plethora of factors that likely contribute to drusen formation, a hallmark of AMD. Finally, overexpressing AKT2 in RPE cells in mice led to an AMD-like phenotype. Thus, targeting the AKT2/SIRT5/TFEB pathway could be a potential therapy for atrophic AMD.

8.
JCI Insight ; 8(16)2023 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-37463048

RESUMO

Gene therapy is under advanced clinical development for several lysosomal storage disorders. Pompe disease, a debilitating neuromuscular illness affecting infants, children, and adults with different severity, is caused by a deficiency of lysosomal glycogen-degrading enzyme acid α-glucosidase (GAA). Here, we demonstrated that adeno-associated virus-mediated (AAV-mediated) systemic gene transfer reversed glycogen storage in all key therapeutic targets - skeletal and cardiac muscles, the diaphragm, and the central nervous system - in both young and severely affected old Gaa-knockout mice. Furthermore, the therapy reversed secondary cellular abnormalities in skeletal muscle, such as those in autophagy and mTORC1/AMPK signaling. We used an AAV9 vector encoding a chimeric human GAA protein with enhanced uptake and secretion to facilitate efficient spread of the expressed protein among multiple target tissues. These results lay the groundwork for a future clinical development strategy in Pompe disease.


Assuntos
Doença de Depósito de Glicogênio Tipo II , alfa-Glucosidases , Criança , Camundongos , Humanos , Animais , alfa-Glucosidases/genética , Doença de Depósito de Glicogênio Tipo II/genética , Doença de Depósito de Glicogênio Tipo II/terapia , Doença de Depósito de Glicogênio Tipo II/patologia , Dependovirus/genética , Dependovirus/metabolismo , Vetores Genéticos/genética , Camundongos Knockout , Glicogênio/metabolismo
9.
Cell Genom ; 3(5): 100290, 2023 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-37228749

RESUMO

Human genetic diversity can reveal critical factors in host-pathogen interactions. This is especially useful for human-restricted pathogens like Salmonella enterica serovar Typhi (S. Typhi), the cause of typhoid fever. One key defense during bacterial infection is nutritional immunity: host cells attempt to restrict bacterial replication by denying bacteria access to key nutrients or supplying toxic metabolites. Here, a cellular genome-wide association study of intracellular replication by S. Typhi in nearly a thousand cell lines from around the world-and extensive follow-up using intracellular S. Typhi transcriptomics and manipulation of magnesium availability-demonstrates that the divalent cation channel mucolipin-2 (MCOLN2 or TRPML2) restricts S. Typhi intracellular replication through magnesium deprivation. Mg2+ currents, conducted through MCOLN2 and out of endolysosomes, were measured directly using patch-clamping of the endolysosomal membrane. Our results reveal Mg2+ limitation as a key component of nutritional immunity against S. Typhi and as a source of variable host resistance.

10.
Traffic ; 24(7): 284-307, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37129279

RESUMO

A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins (LDL) in lysosomes. This event culminates in the loss of cell homeostasis, inflammation, and cell death. Nevertheless, the exact chemical etiology of atherogenesis and the molecular and cellular mechanisms responsible for the impairment of lysosome function in plaque macrophages are still unknown. Here, we demonstrate that macrophages exposed to cholesteryl hemiazelate (ChA), one of the most prevalent products of LDL-derived cholesteryl ester oxidation, exhibit enlarged peripheral dysfunctional lysosomes full of undigested ChA and neutral lipids. Both lysosome area and accumulation of neutral lipids are partially irreversible. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences for the pathology. We further demonstrate that this phenotype is mechanistically linked to the nuclear translocation of the MiT/TFE family of transcription factors. The induction of lysosome biogenesis by ChA appears to partially protect macrophages from lipid-induced cytotoxicity. In sum, our data show that ChA is involved in the etiology of lysosome dysfunction and promotes the exocytosis of these organelles. This latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.


Assuntos
Aterosclerose , Ésteres do Colesterol , Humanos , Ésteres do Colesterol/metabolismo , Macrófagos/metabolismo , Lisossomos/metabolismo , Aterosclerose/metabolismo , Exocitose
11.
iScience ; 26(3): 106169, 2023 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-36785787

RESUMO

Beta-coronaviruses have emerged as a severe threat to global health. Undercovering the interplay between host and beta-coronaviruses is essential for understanding disease pathogenesis and developing efficient treatments. Here we report that the transcription factors TFEB and TFE3 translocate from the cytosol to the nucleus in response to beta-coronavirus infection by a mechanism that requires activation of calcineurin phosphatase. In the nucleus, TFEB and TFE3 bind to the promoter of multiple lysosomal and immune genes. Accordingly, MHV-induced upregulation of immune regulators is significantly decreased in TFEB/TFE3-depleted cells. Conversely, over-expression of either TFEB or TFE3 is sufficient to increase expression of several cytokines and chemokines. The reduced immune response observed in the absence of TFEB and TFE3 results in increased cellular survival of infected cells but also in reduced lysosomal exocytosis and decreased viral infectivity. These results suggest a central role of TFEB and TFE3 in cellular response to beta-coronavirus infection.

12.
EMBO Rep ; 24(2): e55472, 2023 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-36507874

RESUMO

The transcription factor EB (TFEB) regulates energy homeostasis and cellular response to a wide variety of stress conditions, including nutrient deprivation, oxidative stress, organelle damage, and pathogens. Here we identify S401 as a novel phosphorylation site within the TFEB proline-rich domain. Phosphorylation of S401 increases significantly in response to oxidative stress, UVC light, growth factors, and LPS, whereas this increase is prevented by p38 MAPK inhibition or depletion, revealing a new role for p38 MAPK in TFEB regulation. Mutation of S401 in THP1 cells demonstrates that the p38 MAPK/TFEB pathway plays a particularly relevant role during monocyte differentiation into macrophages. TFEB-S401A monocytes fail to upregulate the expression of multiple immune genes in response to PMA-induced differentiation, including critical cytokines, chemokines, and growth factors. Polarization of M0 macrophages into M1 inflammatory macrophages is also aberrant in TFEB-S401A cells. These results indicate that TFEB-S401 phosphorylation links differentiation signals to the transcriptional control of monocyte differentiation.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Diferenciação Celular , Macrófagos , Monócitos , Proteínas Quinases p38 Ativadas por Mitógeno , Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fosforilação
13.
Mol Cell ; 82(15): 2732-2734, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35931037

RESUMO

Zhang et al. (2022) report that itaconate, a mitochondrial metabolite produced by macrophages upon inflammatory stimuli, activates the master regulator of lysosomal biogenesis TFEB to facilitate clearance of invading bacteria and efficient immune response.


Assuntos
Macrófagos , Succinatos , Antibacterianos/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Succinatos/metabolismo
14.
Autophagy ; 18(10): 2333-2349, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35230915

RESUMO

TFEB (transcription factor EB) and TFE3 (transcription factor binding to IGHM enhancer 3) orchestrate the cellular response to a variety of stressors, including nutrient deprivation, oxidative stress and pathogens. Here we describe a novel interaction of TFEB and TFE3 with the FAcilitates Chromatin Transcription (FACT) complex, a heterodimeric histone chaperone consisting of SSRP1 and SUPT16H that mediates nucleosome disassembly and assembly, thus facilitating transcription. Extracellular stimuli, such as nutrient deprivation or oxidative stress, induce nuclear translocation and activation of TFEB and TFE3, which then associate with the FACT complex to regulate stress-induced gene transcription. Depletion of FACT does not affect TFEB activation, stability, or binding to the promoter of target genes. In contrast, reduction of FACT levels by siRNA or treatment with the FACT inhibitor curaxin, severely impairs induction of numerous antioxidant and lysosomal genes, revealing a crucial role of FACT as a regulator of cellular homeostasis. Furthermore, upregulation of antioxidant genes induced by TFEB over-expression is significantly reduced by curaxin, consistent with a role of FACT as a TFEB transcriptional activator. Together, our data show that chromatin remodeling at the promoter of stress-responsive genes by FACT is important for efficient expression of TFEB and TFE3 targets, thus providing a link between environmental changes, chromatin modifications and transcriptional regulation.Abbreviations: ADNP2, ADNP homeobox 2; ATP6V0D1, ATPase H+ transporting V0 subunit d1; ATP6V1A, ATPase H+ transporting V1 subunit A; ATP6V1C1, ATPase H+ transporting V1 subunit C1; CSNK2/CK2, casein kinase 2; CLCN7, chloride voltage-gated channel 7; CTSD, cathepsin D; CTSZ, cathepsin Z; EBSS, earle's balanced salt solution; FACT complex, facilitates chromatin transcription complex; FOXO3, forkhead box O3; HEXA, hexosaminidase subunit alpha; HIF1A, hypoxia inducible factor 1 subunit alpha; HMOX1, heme oxygenase 1; LAMP1, lysosomal associated membrane protein 1; MAFF, MAF bZIP transcription factor F; MAFG, MAF bZIP transcription factor G; MCOLN1, mucolipin TRP cation channel 1; MTORC1, mechanistic target of rapamycin kinase complex 1; NaAsO2, sodium arsenite; POLR2, RNA polymerase II; PPARGC1A, PPARG coactivator 1 alpha; PYROXD1, pyridine nucleotide-disulfide oxidoreductase domain 1; RRAGC, Ras related GTP binding C; SEC13, SEC13 homolog, nuclear pore and COPII coat complex component; SLC38A9, solute carrier family 38 member 9; SSRP1, structure specific recognition protein 1; SUPT16H, SPT16 homolog, facilitates chromatin remodeling subunit; TFEB, transcription factor EB; TFE3, transcription factor binding to IGHM enhancer 3; TXNRD1, thioredoxin reductase 1; UVRAG, UV radiation resistance associated; WDR59, WD repeat domain 59.


Assuntos
Antioxidantes , Canais de Potencial de Receptor Transitório , Adenosina Trifosfatases/metabolismo , Antioxidantes/metabolismo , Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Caseína Quinase II/metabolismo , Catepsina D/metabolismo , Catepsina Z/genética , Catepsina Z/metabolismo , Cloretos/metabolismo , Cromatina/metabolismo , Dissulfetos , Guanosina Trifosfato/metabolismo , Heme Oxigenase-1/metabolismo , Hexosaminidases/genética , Hexosaminidases/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Nucleossomos/metabolismo , Nucleotídeos/metabolismo , PPAR gama/genética , Piridinas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/metabolismo , Sirolimo , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo
15.
Oncogene ; 41(12): 1701-1717, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35110681

RESUMO

Transmembrane glycoprotein NMB (GPNMB) is a prognostic marker of poor outcome in patients with triple-negative breast cancer (TNBC). Glembatumumab Vedotin, an antibody drug conjugate targeting GPNMB, exhibits variable efficacy against GPNMB-positive metastatic TNBC as a single agent. We show that GPNMB levels increase in response to standard-of-care and experimental therapies for multiple breast cancer subtypes. While these therapeutic stressors induce GPNMB expression through differential engagement of the MiTF family of transcription factors, not all are capable of increasing GPNMB cell-surface localization required for Glembatumumab Vedotin inhibition. Using a FACS-based genetic screen, we discovered that suppression of heat shock protein 90 (HSP90) concomitantly increases GPNMB expression and cell-surface localization. Mechanistically, HSP90 inhibition resulted in lysosomal dispersion towards the cell periphery and fusion with the plasma membrane, which delivers GPNMB to the cell surface. Finally, treatment with HSP90 inhibitors sensitizes breast cancers to Glembatumumab Vedotin in vivo, suggesting that combination of HSP90 inhibitors and Glembatumumab Vedotin may be a viable treatment strategy for patients with metastatic TNBC.


Assuntos
Antineoplásicos , Imunoconjugados , Neoplasias de Mama Triplo Negativas , Anticorpos Monoclonais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Imunoconjugados/efeitos adversos , Lisossomos/metabolismo , Glicoproteínas de Membrana/genética , Fatores de Transcrição , Neoplasias de Mama Triplo Negativas/tratamento farmacológico
17.
Chem Sci ; 12(37): 12451-12462, 2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34603676

RESUMO

Functionalization of therapeutic lysosomal enzymes with mannose-6-phosphate (M6P) glycan ligands represents a major strategy for enhancing the cation-independent M6P receptor (CI-MPR)-mediated cellular uptake, thus improving the overall therapeutic efficacy of the enzymes. However, the minimal high-affinity M6P-containing N-glycan ligands remain to be identified and their efficient and site-selective conjugation to therapeutic lysosomal enzymes is a challenging task. We report here the chemical synthesis of truncated M6P-glycan oxazolines and their use for enzymatic glycan remodeling of recombinant human acid α-glucosidase (rhGAA), an enzyme used for treatment of Pompe disease which is a disorder caused by a deficiency of the glycogen-degrading lysosomal enzyme. Structure-activity relationship studies identified M6P tetrasaccharide oxazoline as the minimal substrate for enzymatic transglycosylation yielding high-affinity M6P glycan ligands for the CI-MPR. Taking advantage of the substrate specificity of endoglycosidases Endo-A and Endo-F3, we found that Endo-A and Endo-F3 could efficiently deglycosylate the respective high-mannose and complex type N-glycans in rhGAA and site-selectively transfer the synthetic M6P N-glycan to the deglycosylated rhGAA without product hydrolysis. This discovery enabled a highly efficient one-pot deglycosylation/transglycosylation strategy for site-selective M6P-glycan remodeling of rhGAA to obtain a more homogeneous product. The Endo-A and Endo-F3 remodeled rhGAAs maintained full enzyme activity and demonstrated 6- and 20-fold enhanced binding affinities for CI-MPR receptor, respectively. Using an in vitro cell model system for Pompe disease, we demonstrated that the M6P-glycan remodeled rhGAA greatly outperformed the commercial rhGAA (Lumizyme) and resulted in the reversal of cellular pathology. This study provides a general and efficient method for site-selective M6P-glycan remodeling of recombinant lysosomal enzymes to achieve enhanced M6P receptor binding and cellular uptake, which could lead to improved overall therapeutic efficacy of enzyme replacement therapy.

18.
EMBO J ; 40(3): e105793, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33314217

RESUMO

Mammalian TFEB and TFE3, as well as their ortholog in Caenorhabditis elegans HLH-30, play an important role in mediating cellular response to a variety of stress conditions, including nutrient deprivation, oxidative stress, and pathogen infection. In this study, we identify a novel mechanism of TFEB/HLH-30 regulation through a cysteine-mediated redox switch. Under stress conditions, TFEB-C212 undergoes oxidation, allowing the formation of intermolecular disulfide bonds that result in TFEB oligomerization. TFEB oligomers display increased resistance to mTORC1-mediated inactivation and are more stable under prolonged stress conditions. Mutation of the only cysteine residue present in HLH-30 (C284) significantly reduced its activity, resulting in developmental defects and increased pathogen susceptibility in worms. Therefore, cysteine oxidation represents a new type of TFEB post-translational modification that functions as a molecular switch to link changes in redox balance with expression of TFEB/HLH-30 target genes.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Mutação , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular , Cisteína , Células HeLa , Humanos , Camundongos , Oxirredução , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Células RAW 264.7
19.
EBioMedicine ; 63: 103166, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33341443

RESUMO

Lysosomal storage disorders (LSDs), which number over fifty, are monogenically inherited and caused by mutations in genes encoding proteins that are involved in lysosomal function. Lack of the functional protein results in storage of a distinctive material within the lysosomes, which for years was thought to determine the pathophysiology of the disorder. However, our current view posits that the primary storage material disrupts the normal role of the lysosome in the autophagic pathway resulting in the secondary storage of autophagic debris. It is this "collateral damage" which is common to the LSDs but nonetheless intricately nuanced in each. We have selected five LSDs resulting from defective proteins that govern widely different lysosomal functions including glycogen degradation (Pompe), lysosomal transport (Cystinosis), lysosomal trafficking (Danon), glycolipid degradation (Gaucher) and an unidentified function (Batten) and argue that despite the disparate functions, these proteins, when mutant, all impair the autophagic process uniquely.


Assuntos
Autofagia , Suscetibilidade a Doenças , Doenças por Armazenamento dos Lisossomos/etiologia , Doenças por Armazenamento dos Lisossomos/metabolismo , Lisossomos/metabolismo , Animais , Autofagia/genética , Biomarcadores , Cistinose/etiologia , Cistinose/metabolismo , Cistinose/patologia , Gerenciamento Clínico , Humanos , Doenças por Armazenamento dos Lisossomos/diagnóstico , Doenças por Armazenamento dos Lisossomos/terapia , Especificidade de Órgãos/genética
20.
Trends Biochem Sci ; 46(2): 97-112, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33012625

RESUMO

Lysosomes are in the center of the cellular control of catabolic and anabolic processes. These membrane-surrounded acidic organelles contain around 70 hydrolases, 200 membrane proteins, and numerous accessory proteins associated with the cytosolic surface of lysosomes. Accessory and transmembrane proteins assemble in signaling complexes that sense and integrate multiple signals and transmit the information to the nucleus. This communication allows cells to respond to changes in multiple environmental conditions, including nutrient levels, pathogens, energy availability, and lysosomal damage, with the goal of restoring cellular homeostasis. This review summarizes our current understanding of the major molecular players and known pathways that are involved in control of metabolic and stress responses that either originate from lysosomes or regulate lysosomal functions.


Assuntos
Lisossomos , Transdução de Sinais , Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Núcleo Celular/metabolismo , Homeostase , Lisossomos/metabolismo , Proteínas de Membrana
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