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2.
Cell Mol Life Sci ; 81(1): 304, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-39009859

RESUMO

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


Assuntos
Autofagia , Endossomos , Metabolismo dos Lipídeos , Lisossomos , Neurônios , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/genética , Lisossomos/metabolismo , Humanos , Neurônios/metabolismo , Animais , Endossomos/metabolismo , Tauopatias/metabolismo , Tauopatias/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Camundongos
3.
Molecules ; 29(13)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38999083

RESUMO

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


Assuntos
Endossomos , Lisossomos , Lisossomos/metabolismo , Humanos , Endossomos/metabolismo , Sistemas de Liberação de Medicamentos , Portadores de Fármacos/química , Nanoestruturas/química , Animais , Nanopartículas/química
4.
Int J Mol Sci ; 25(13)2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38999927

RESUMO

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


Assuntos
Doença de Alzheimer , Ácidos Docosa-Hexaenoicos , Endossomos , Lisossomos , Neurônios , Proteínas rab5 de Ligação ao GTP , Humanos , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Proteínas rab5 de Ligação ao GTP/metabolismo , Endossomos/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Neurônios/efeitos dos fármacos , Lisossomos/metabolismo , Linhagem Celular Tumoral , Precursor de Proteína beta-Amiloide/metabolismo , Apoptose , Fármacos Neuroprotetores/farmacologia , Sobrevivência Celular/efeitos dos fármacos
5.
Commun Biol ; 7(1): 826, 2024 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-38972875

RESUMO

Classically, G protein-coupled receptors (GPCRs) promote signaling at the plasma membrane through activation of heterotrimeric Gαßγ proteins, followed by the recruitment of GPCR kinases and ßarrestin (ßarr) to initiate receptor desensitization and internalization. However, studies demonstrated that some GPCRs continue to signal from internalized compartments, with distinct cellular responses. Both ßarr and Gßγ contribute to such non-canonical endosomal G protein signaling, but their specific roles and contributions remain poorly understood. Here, we demonstrate that the vasopressin V2 receptor (V2R)-ßarr complex scaffolds Gßγ at the plasma membrane through a direct interaction with ßarr, enabling its transport to endosomes. Gßγ subsequently potentiates Gαs endosomal translocation, presumably to regenerate an endosomal pool of heterotrimeric Gs. This work shines light on the mechanism underlying G protein subunits translocation from the plasma membrane to the endosomes and provides a basis for understanding the role of ßarr in mediating sustained G protein signaling.


Assuntos
Endossomos , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Transporte Proteico , Receptores de Vasopressinas , beta-Arrestinas , Humanos , beta-Arrestinas/metabolismo , Membrana Celular/metabolismo , Endossomos/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genética , Células HEK293 , Receptores de Vasopressinas/metabolismo , Receptores de Vasopressinas/genética , Transdução de Sinais
6.
J Immunol ; 213(2): 109-114, 2024 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-38950331

RESUMO

ATPase cation transporting 13A2 (ATP13A2) is an endolysosomal P-type ATPase known to be a polyamine transporter, explored mostly in neurons. As endolysosomal functions are also crucial in innate immune cells, we aimed to explore the potential role of ATP13A2 in the human immunocellular compartment. We found that human plasmacytoid dendritic cells (pDCs), the professional type I IFN-producing immune cells, especially have a prominent enrichment of ATP13A2 expression in endolysosomal compartments. ATP13A2 knockdown in human pDCs interferes with cytokine induction in response to TLR9/7 activation in response to bona fide ligands. ATP13A2 plays this crucial role in TLR9/7 activation in human pDCs by regulating endolysosomal pH and mitochondrial reactive oxygen generation. This (to our knowledge) hitherto unknown regulatory mechanism in pDCs involving ATP13A2 opens up a new avenue of research, given the crucial role of pDC-derived type I IFNs in protective immunity against infections as well as in the immunopathogenesis of myriad contexts of autoreactive inflammation.


Assuntos
Células Dendríticas , Endossomos , Lisossomos , Receptor Toll-Like 9 , Humanos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Lisossomos/metabolismo , Lisossomos/imunologia , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/imunologia , Endossomos/metabolismo , Endossomos/imunologia , ATPases Translocadoras de Prótons/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/imunologia , Células Cultivadas , Interferon Tipo I/metabolismo , Interferon Tipo I/imunologia , Receptor 7 Toll-Like
7.
Virulence ; 15(1): 2367671, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38910312

RESUMO

Viral diseases are among the main threats to public health. Understanding the factors affecting viral invasion is important for antiviral research. Until now, it was known that most viruses have very low plaque-forming unit (PFU)-to-particle ratios. However, further investigation is required to determine the underlying factors. Here, using quantitative single-particle analysis methods, the invasion of Semliki Forest virus (SFV), Japanese encephalitis virus (JEV), and influenza A virus (IAV) containing attachment to the cell surface, entry into the cell, transport towards the cell interior, and fusion with endosomes to release nucleocapsids were quantitatively analysed in parallel. It was found that for SFV with an PFU-to-particle ratio of approximately 1:2, an entry efficiency of approximately 31% limited infection. For JEV, whose PFU-to-particle ratio was approximately 1:310, an attachment efficiency of approximately 27% and an entry efficiency of 10% were the main factors limiting its infection. Meanwhile, for IAV with PFU-to-particle ratios of 1:8100, 5% attachment efficiency, 9% entry efficiency, and 53% fusion efficiency significantly limited its infection. These results suggest that viruses with different infectivities have different limited steps in the invasion process. Moreover, there are significant differences in attachment efficiencies among viruses, emphasizing the pivotal role of attachment in viral invasion. The influence of the virus purification method on virus invasion was also investigated. This study, for the first time, reports the efficiencies of different stages of virus invasion, leading to a better understanding of virus invasion and providing a protocol to quantitatively analyse the virus invasion efficiency.


Assuntos
Vírus da Influenza A , Vírus da Floresta de Semliki , Internalização do Vírus , Vírus da Influenza A/fisiologia , Animais , Vírus da Floresta de Semliki/fisiologia , Humanos , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Linhagem Celular , Ligação Viral , Endossomos/virologia
8.
Nature ; 630(8016): 412-420, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38839950

RESUMO

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.


Assuntos
Autorrenovação Celular , Células-Tronco Hematopoéticas , Proteínas Nucleares , Animais , Feminino , Humanos , Masculino , Camundongos , Células Cultivadas , Endocitose , Endossomos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Sangue Fetal/citologia , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Fígado/citologia , Fígado/metabolismo , Fígado/embriologia , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Análise da Expressão Gênica de Célula Única
9.
Nat Commun ; 15(1): 5227, 2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38898033

RESUMO

Cells depend on their endolysosomal system for nutrient uptake and downregulation of plasma membrane proteins. These processes rely on endosomal maturation, which requires multiple membrane fusion steps. Early endosome fusion is promoted by the Rab5 GTPase and its effector, the hexameric CORVET tethering complex, which is homologous to the lysosomal HOPS. How these related complexes recognize their specific target membranes remains entirely elusive. Here, we solve the structure of CORVET by cryo-electron microscopy and revealed its minimal requirements for membrane tethering. As expected, the core of CORVET and HOPS resembles each other. However, the function-defining subunits show marked structural differences. Notably, we discover that unlike HOPS, CORVET depends not only on Rab5 but also on phosphatidylinositol-3-phosphate (PI3P) and membrane lipid packing defects for tethering, implying that an organelle-specific membrane code enables fusion. Our data suggest that both shape and membrane interactions of CORVET and HOPS are conserved in metazoans, thus providing a paradigm how tethering complexes function.


Assuntos
Microscopia Crioeletrônica , Endossomos , Fosfatos de Fosfatidilinositol , Endossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fusão de Membrana , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Humanos , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Membrana Celular/metabolismo , Animais , Lisossomos/metabolismo
10.
Cell Biol Toxicol ; 40(1): 48, 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38900277

RESUMO

Aggregation of aberrant proteins is a common pathological hallmark in neurodegeneration such as polyglutamine (polyQ) and other repeat-expansion diseases. Here through overexpression of ataxin3 C-terminal polyQ expansion in Drosophila gut enterocytes, we generated an intestinal obstruction model of spinocerebellar ataxia type3 (SCA3) and reported a new role of nuclear-associated endosomes (NAEs)-the delivery of polyQ to the nucleoplasm. In this model, accompanied by the prominently increased RAB5-positive NAEs are abundant nucleoplasmic reticulum enriched with polyQ, abnormal nuclear envelope invagination, significantly reduced endoplasmic reticulum, indicating dysfunctional nucleocytoplasmic trafficking and impaired endomembrane organization. Consistently, Rab5 but not Rab7 RNAi further decreased polyQ-related NAEs, inhibited endomembrane disorganization, and alleviated disease model. Interestingly, autophagic proteins were enriched in polyQ-related NAEs and played non-canonical autophagic roles as genetic manipulation of autophagic molecules exhibited differential impacts on NAEs and SCA3 toxicity. Namely, the down-regulation of Atg1 or Atg12 mitigated while Atg5 RNAi aggravated the disease phenotypes both in Drosophila intestines and compound eyes. Our findings, therefore, provide new mechanistic insights and underscore the fundamental roles of endosome-centered nucleocytoplasmic trafficking and homeostatic endomembrane allocation in the pathogenesis of polyQ diseases.


Assuntos
Autofagia , Endossomos , Peptídeos , Animais , Peptídeos/metabolismo , Endossomos/metabolismo , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Transporte Ativo do Núcleo Celular , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/patologia , Enterócitos/metabolismo , Modelos Animais de Doenças , Ataxina-3/metabolismo , Ataxina-3/genética , Drosophila/metabolismo
11.
Front Immunol ; 15: 1389194, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38840905

RESUMO

Past research has identified that cancer cells sustain several cancer hallmarks by impairing function of the endolysosomal system (ES). Thus, maintaining the functional integrity of endolysosomes is crucial, which heavily relies on two key protein families: soluble hydrolases and endolysosomal membrane proteins. Particularly members of the TPC (two-pore channel) and TRPML (transient receptor potential mucolipins) families have emerged as essential regulators of ES function as a potential target in cancer therapy. Targeting TPCs and TRPMLs has demonstrated significant impact on multiple cancer hallmarks, including proliferation, growth, migration, and angiogenesis both in vitro and in vivo. Notably, endosomes and lysosomes also actively participate in various immune regulatory mechanisms, such as phagocytosis, antigen presentation, and the release of proinflammatory mediators. Yet, knowledge about the role of TPCs and TRPMLs in immunity is scarce. This prompts a discussion regarding the potential role of endolysosomal ion channels in aiding cancers to evade immune surveillance and destruction. Specifically, understanding the interplay between endolysosomal ion channels and cancer immunity becomes crucial. Our review aims to comprehensively explore the current knowledge surrounding the roles of TPCs and TRPMLs in immunity, whilst emphasizing the critical need to elucidate their specific contributions to cancer immunity by pointing out current research gaps that should be addressed.


Assuntos
Canais de Cálcio , Endossomos , Lisossomos , Neoplasias , Canais de Potencial de Receptor Transitório , Humanos , Neoplasias/imunologia , Neoplasias/metabolismo , Lisossomos/metabolismo , Lisossomos/imunologia , Endossomos/metabolismo , Endossomos/imunologia , Animais , Canais de Potencial de Receptor Transitório/metabolismo , Canais de Cálcio/metabolismo , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/imunologia , Canais de Dois Poros
12.
Front Cell Infect Microbiol ; 14: 1394019, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38841112

RESUMO

Introduction: Coxiella burnetii is a gram-negative obligate intracellular bacterium and a zoonotic pathogen that causes human Q fever. The lack of effective antibiotics and a licensed vaccine for Coxiella in the U.S. warrants further research into Coxiella pathogenesis. Within the host cells, Coxiella replicates in an acidic phagolysosome-like vacuole termed Coxiella-containing vacuole (CCV). Previously, we have shown that the CCV pH is critical for Coxiella survival and that the Coxiella Type 4B secretion system regulates CCV pH by inhibiting the host endosomal maturation pathway. However, the trafficking pattern of the 'immature' endosomes in Coxiella- infected cells remained unclear. Methods: We transfected HeLa cells with GFP-tagged Rab proteins and subsequently infected them with mCherry-Coxiella to visualize Rab protein localization. Infected cells were immunostained with anti-Rab antibodies to confirm the Rab localization to the CCV, to quantitate Rab11a and Rab35- positive CCVs, and to quantitate total recycling endosome content of infected cells. A dual-hit siRNA mediated knockdown combined with either immunofluorescent assay or an agarose-based colony-forming unit assay were used to measure the effects of Rab11a and Rab35 knockdown on CCV area and Coxiella intracellular growth. Results: The CCV localization screen with host Rab proteins revealed that recycling endosome-associated proteins Rab11a and Rab35 localize to the CCV during infection, suggesting that CCV interacts with host recycling endosomes during maturation. Interestingly, only a subset of CCVs were Rab11a or Rab35-positive at any given time point. Quantitation of Rab11a/Rab35-positive CCVs revealed that while Rab11a interacts with the CCV more at 3 dpi, Rab35 is significantly more prevalent at CCVs at 6 dpi, suggesting that the CCV preferentially interacts with Rab11a and Rab35 depending on the stage of infection. Furthermore, we observed a significant increase in Rab11a and Rab35 fluorescent intensity in Coxiella-infected cells compared to mock, suggesting that Coxiella increases the recycling endosome content in infected cells. Finally, siRNA-mediated knockdown of Rab11a and Rab35 resulted in significantly smaller CCVs and reduced Coxiella intracellular growth, suggesting that recycling endosomal Rab proteins are essential for CCV expansion and bacterial multiplication. Discussion: Our data, for the first time, show that the CCV dynamically interacts with host recycling endosomes for Coxiella intracellular survival and potentially uncovers novel host cell factors essential for Coxiella pathogenesis.


Assuntos
Coxiella burnetii , Endossomos , Interações Hospedeiro-Patógeno , Vacúolos , Proteínas rab de Ligação ao GTP , Coxiella burnetii/metabolismo , Coxiella burnetii/crescimento & desenvolvimento , Coxiella burnetii/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Humanos , Vacúolos/metabolismo , Vacúolos/microbiologia , Células HeLa , Endossomos/metabolismo , Endossomos/microbiologia , Febre Q/microbiologia , Febre Q/metabolismo
13.
Biosci Rep ; 44(6)2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38836326

RESUMO

Cumulative research findings support the idea that endocytic trafficking is crucial in regulating receptor signaling and associated diseases. Specifically, strong evidence points to the involvement of sorting nexins (SNXs), particularly SNX1 and SNX2, in the signaling and trafficking of the receptor tyrosine kinase (RTK) MET in colorectal cancer (CRC). Activation of hepatocyte growth factor (HGF) receptor MET is a key driver of CRC progression. In the present study, we utilized human HCT116 CRC cells with SNX1 and SNX2 genes knocked out to demonstrate that their absence leads to a delay in MET entering early endosomes. This delay results in increased phosphorylation of both MET and AKT upon HGF stimulation, while ERK1/2 (extracellular signal-regulated kinases 1 and 2) phosphorylation remains unaffected. Despite these changes, HGF-induced cell proliferation, scattering, and migration remain similar between the parental and the SNX1/2 knockout cells. However, in the absence of SNX1 and SNX2, these cells exhibit increased resistance to TRAIL-induced apoptosis. This research underscores the intricate relationship between intracellular trafficking, receptor signaling, and cellular responses and demonstrates for the first time that the modulation of MET trafficking by SNX1 and SNX2 is critical for receptor signaling that may exacerbate the disease.


Assuntos
Movimento Celular , Proliferação de Células , Neoplasias Colorretais , Fator de Crescimento de Hepatócito , Proteínas Proto-Oncogênicas c-met , Nexinas de Classificação , Humanos , Nexinas de Classificação/metabolismo , Nexinas de Classificação/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Células HCT116 , Fator de Crescimento de Hepatócito/metabolismo , Transdução de Sinais , Fosforilação , Endossomos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transporte Proteico
14.
Int J Mol Sci ; 25(12)2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38928424

RESUMO

The SLC35 (Solute Carrier 35) family members acting as nucleotide sugar transporters are typically localized in the endoplasmic reticulum or Golgi apparatus. It is, therefore, intriguing that some reports document the presence of orphan transporters SLC35F1 and SLC35F6 within the endosomal and lysosomal system. Here, we compared the subcellular distribution of these proteins and found that they are concentrated in separate compartments; i.e., recycling endosomes for SLC35F1 and lysosomes for SLC35F6. Swapping the C-terminal tail of these proteins resulted in a switch of localization, with SLC35F1 being trafficked to lysosomes while SLC35F6 remained in endosomes. This suggested the presence of specific sorting signals in these C-terminal regions. Using site-directed mutagenesis, fluorescence microscopy, and cell surface biotinylation assays, we found that the EQERLL360 signal located in the cytoplasmic tail of human SLC35F6 is involved in its lysosomal sorting (as previously shown for this conserved sequence in mouse SLC35F6), and that SLC35F1 localization in the recycling pathway depends on two YXXΦ-type signals: a Y367KQF sequence facilitates its internalization from the plasma membrane, while a Y392TSL motif prevents its transport to lysosomes, likely by promoting SLC35F1 recycling to the cell surface. Taken together, these results support that some SLC35 members may function at different levels of the endosomal and lysosomal system.


Assuntos
Endossomos , Lisossomos , Humanos , Sequência de Aminoácidos , Membrana Celular/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Lisossomos/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Proteínas de Transporte de Nucleotídeos/genética , Sinais Direcionadores de Proteínas , Transporte Proteico
15.
New Phytol ; 243(3): 1065-1081, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38874374

RESUMO

The phytohormone abscisic acid (ABA) functions in the control of plant stress responses, particularly in drought stress. A significant mechanism in attenuating and terminating ABA signals involves regulated protein turnover, with certain ABA receptors, despite their main presence in the cytosol and nucleus, subjected to vacuolar degradation via the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Collectively our findings show that discrete TOM1-LIKE (TOL) proteins, which are functional ESCRT-0 complex substitutes in plants, affect the trafficking for degradation of core components of the ABA signaling and transport machinery. TOL2,3,5 and 6 modulate ABA signaling where they function additively in degradation of ubiquitinated ABA receptors and transporters. TOLs colocalize with their cargo in different endocytic compartments in the root epidermis and in guard cells of stomata, where they potentially function in ABA-controlled stomatal aperture. Although the tol2/3/5/6 quadruple mutant plant line is significantly more drought-tolerant and has a higher ABA sensitivity than control plant lines, it has no obvious growth or development phenotype under standard conditions, making the TOL genes ideal candidates for engineering to improved plant performance.


Assuntos
Ácido Abscísico , Proteínas de Arabidopsis , Arabidopsis , Endossomos , Estômatos de Plantas , Transdução de Sinais , Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Endossomos/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Estômatos de Plantas/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Secas , Mutação/genética , Proteólise , Transporte Proteico
16.
J Cell Sci ; 137(13)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38884339

RESUMO

Early endosomes sort transmembrane cargo either for lysosomal degradation or retrieval to the plasma membrane or the Golgi complex. Endosomal retrieval in eukaryotes is governed by the anciently homologous retromer or retriever complexes. Each comprises a core tri-protein subcomplex, membrane-deformation proteins and interacting partner complexes, together retrieving a variety of known cargo proteins. Trichomonas vaginalis, a sexually transmitted human parasite, uses the endomembrane system for pathogenesis. It has massively and selectively expanded its endomembrane protein complement, the evolutionary path of which has been largely unexplored. Our molecular evolutionary study of retromer, retriever and associated machinery in parabasalids and its free-living sister lineage of Anaeramoeba demonstrates specific expansion of the retromer machinery, contrasting with the retriever components. We also observed partial loss of the Commander complex and sorting nexins in Parabasalia but complete retention in Anaeramoeba. Notably, we identified putative parabasalid sorting nexin analogs. Finally, we report the first retriever protein localization in a non-metazoan group along with retromer protein localization in T. vaginalis.


Assuntos
Endossomos , Endossomos/metabolismo , Transporte Proteico , Trichomonas vaginalis/metabolismo , Trichomonas vaginalis/genética , Filogenia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Evolução Molecular , Humanos , Complexo de Golgi/metabolismo , Nexinas de Classificação/metabolismo , Nexinas de Classificação/genética , Animais
17.
mBio ; 15(7): e0168423, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-38874413

RESUMO

Lymphocytic choriomeningitis virus (LCMV) is an enveloped and segmented negative-sense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromised populations and, as the prototypical arenavirus member, acts as a model for the many highly pathogenic members of the Arenaviridae family, such as Junín, Lassa, and Lujo viruses, all of which are associated with devastating hemorrhagic fevers. To enter cells, the LCMV envelope fuses with late endosomal membranes, for which two established requirements are low pH and interaction between the LCMV glycoprotein (GP) spike and secondary receptor CD164. LCMV subsequently uncoats, where the RNA genome-associated nucleoprotein (NP) separates from the Z protein matrix layer, releasing the viral genome into the cytosol. To further examine LCMV endosome escape, we performed an siRNA screen which identified host cell potassium ion (K+) channels as important for LCMV infection, with pharmacological inhibition confirming K+ channel involvement during the LCMV entry phase completely abrogating productive infection. To better understand the K+-mediated block in infection, we tracked incoming virions along their entry pathway under physiological conditions, where uncoating was signified by separation of NP and Z proteins. In contrast, K+ channel blockade prevented uncoating, trapping virions within Rab7 and CD164-positive endosomes, identifying K+ as a third LCMV entry requirement. K+ did not increase GP-CD164 binding or alter GP-CD164-dependent fusion. Thus, we propose that K+ mediates uncoating by modulating NP-Z interactions within the virion interior. These results suggest K+ channels represent a potential anti-arenaviral target.IMPORTANCEArenaviruses can cause fatal human disease for which approved preventative or therapeutic options are not available. Here, using the prototypical LCMV, we identified K+ channels as critical for arenavirus infection, playing a vital role during the entry phase of the infection cycle. We showed that blocking K+ channel function resulted in entrapment of LCMV particles within late endosomal compartments, thus preventing productive replication. Our data suggest K+ is required for LCMV uncoating and genome release by modulating interactions between the viral nucleoprotein and the matrix protein layer inside the virus particle.


Assuntos
Endossomos , Vírus da Coriomeningite Linfocítica , Potássio , Internalização do Vírus , Desenvelopamento do Vírus , Endossomos/virologia , Endossomos/metabolismo , Vírus da Coriomeningite Linfocítica/fisiologia , Vírus da Coriomeningite Linfocítica/genética , Humanos , Potássio/metabolismo , proteínas de unión al GTP Rab7 , Linhagem Celular , Animais , Canais de Potássio/metabolismo , Canais de Potássio/genética
18.
Biochem Biophys Res Commun ; 724: 150198, 2024 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-38852504

RESUMO

Autophagy is a critical catabolic pathway that enables cells to survive and adapt to stressful conditions, especially nutrient deprivation. The fusion of autophagic vacuoles with lysosomes is the final step of autophagy, which degrades the engulfed contents into metabolic precursors for re-use by the cell. O-GlcNAc transferase (OGT) plays a crucial role in regulating autophagy flux in response to nutrient stress, particularly by targeting key proteins involved in autophagosome-lysosome fusion. However, the role of OGT in basal autophagy, which occurs at a low and constitutive levels under growth conditions, remains poorly understood. Silencing or inhibition of OGT was used to compare the effect of OGT downregulation on autophagy flux in the non-cancerous CCD841CoN and cancerous HCT116 human colon cell lines under nutrient-rich conditions. We provide evidence that the reduction of OGT activity impairs the maturation of autophagosomes, thereby blocking the completion of basal autophagy in both cell lines. Additionally, OGT inhibition results in the accumulation of lysosomes and enlarged late endosomes in the perinuclear region, as demonstrated by confocal imaging. This is associated with a defect in the localization of the small GTPase Rab7 to these organelles. The regulation of transport and fusion events between the endosomal and lysosomal compartments is crucial for maintaining the autophagic flux. These findings suggest an interplay between OGT and the homeostasis of the endolysosomal network in human cells.


Assuntos
Autofagia , Regulação para Baixo , Endossomos , Lisossomos , N-Acetilglucosaminiltransferases , Nutrientes , proteínas de unión al GTP Rab7 , Humanos , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , Endossomos/metabolismo , Lisossomos/metabolismo , Nutrientes/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Colo/metabolismo , Colo/patologia , Células HCT116 , Autofagossomos/metabolismo
19.
PLoS Pathog ; 20(6): e1012289, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38829892

RESUMO

During entry, human papillomavirus (HPV) traffics from the endosome to the trans Golgi network (TGN) and Golgi and then the nucleus to cause infection. Although dynein is thought to play a role in HPV infection, how this host motor recruits the virus to support infection and which entry step(s) requires dynein are unclear. Here we show that the dynein cargo adaptor BICD2 binds to the HPV L2 capsid protein during entry, recruiting HPV to dynein for transport of the virus along the endosome-TGN/Golgi axis to promote infection. In the absence of BICD2 function, HPV accumulates in the endosome and TGN and infection is inhibited. Cell-based and in vitro binding studies identified a short segment near the C-terminus of L2 that can directly interact with BICD2. Our results reveal the molecular basis by which the dynein motor captures HPV to promote infection and identify this virus as a novel cargo of the BICD2 dynein adaptor.


Assuntos
Proteínas do Capsídeo , Papillomavirus Humano 16 , Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Humanos , Proteínas do Capsídeo/metabolismo , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Dineínas/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Rede trans-Golgi/metabolismo , Rede trans-Golgi/virologia , Internalização do Vírus , Ligação Proteica , Células HeLa , Proteínas Associadas aos Microtúbulos/metabolismo , Dineínas do Citoplasma/metabolismo
20.
J Mater Chem B ; 12(27): 6703-6715, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38895858

RESUMO

Carrier-free nanomedicines offer advantages of extremely high drug loading capacity (>80%), minimal non-drug constituent burden, and facile preparation processes. Numerous studies have proved that multimodal cancer therapy can enhance chemotherapy efficiency and mitigate multi-drug resistance (MDR) through synergistic therapeutic effects. Upon penetration into the tumor matrix, nanoparticles (NPs) are anticipated to be uptaken by cancer cells, primarily through clathrin-meditated endocytosis pathways, leading to their accumulation in endosomes/lysosomes within cells. However, endo/lysosomes exhibit a highly degradative environment for organic NPs and drug molecules, often resulting in treatment failure. Hence, this study designed a lysosomal escape mechanism with carrier-free nanomedicine, combining the chemotherapeutic drug, curcumin (Cur), and the photothermal/photodynamic therapeutic drug, indocyanine green (ICG), for synergistic cancer treatment (ICG-Cur NPs) via a facile preparation process. To facilitate endo/lysosomal escape, ICG-Cur NPs were modified with metal-phenolic networks (MPNs) of different thickness. The results indicate that a thick MPN coating promotes rapid endo/lysosomal escape of ICG-Cur NPs within 4 h and enhances the photothermal conversion efficiency of ICG-Cur NPs by 55.8%, significantly improving anticancer efficacy in both chemo- and photo-therapies within 3D solid tumor models. This finding underscores the critical role of endo/lysosomal escape capacity in carrier-free drug NPs for therapeutic outcomes and offers a facile solution to achieve it.


Assuntos
Antineoplásicos , Curcumina , Verde de Indocianina , Lisossomos , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Humanos , Verde de Indocianina/química , Verde de Indocianina/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Animais , Curcumina/química , Curcumina/farmacologia , Camundongos , Nanopartículas/química , Nanomedicina , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Endossomos/metabolismo , Fotoquimioterapia , Sobrevivência Celular/efeitos dos fármacos , Tamanho da Partícula , Proliferação de Células/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Linhagem Celular Tumoral , Feminino , Neoplasias/tratamento farmacológico
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