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1.
J Cell Biol ; 223(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38578286

RESUMO

The AP-1 adaptor complex is found in all eukaryotes, but it has been implicated in different pathways in different organisms. To look directly at AP-1 function, we generated stably transduced HeLa cells coexpressing tagged AP-1 and various tagged membrane proteins. Live cell imaging showed that AP-1 is recruited onto tubular carriers trafficking from the Golgi apparatus to the plasma membrane, as well as onto transferrin-containing early/recycling endosomes. Analysis of single AP-1 vesicles showed that they are a heterogeneous population, which starts to sequester cargo 30 min after exit from the ER. Vesicle capture showed that AP-1 vesicles contain transmembrane proteins found at the TGN and early/recycling endosomes, as well as lysosomal hydrolases, but very little of the anterograde adaptor GGA2. Together, our results support a model in which AP-1 retrieves proteins from post-Golgi compartments back to the TGN, analogous to COPI's role in the early secretory pathway. We propose that this is the function of AP-1 in all eukaryotes.


Assuntos
Complexo de Golgi , Proteínas de Membrana , Transporte Proteico , Fator de Transcrição AP-1 , Humanos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Endossomos/genética , Endossomos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Células HeLa , Proteínas de Membrana/metabolismo , Rede trans-Golgi/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo
2.
J Cell Biol ; 223(6)2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38578646

RESUMO

Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner.


Assuntos
Técnicas Biossensoriais , Fosfatidilinositóis , Endossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Técnicas Biossensoriais/métodos
3.
Nat Commun ; 15(1): 2508, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38509070

RESUMO

In the secretory pathway the destination of trafficking vesicles is determined by specific proteins that, with the notable exception of SNAREs, are recruited from soluble pools. Previously we have shown that microinjected proteoliposomes containing early or late endosomal SNAREs, respectively, are targeted to the corresponding endogenous compartments, with targeting specificity being dependent on the recruitment of tethering factors by some of the SNAREs. Here, we show that targeting of SNARE-containing liposomes is refined upon inclusion of polyphosphoinositides and Rab5. Intriguingly, targeting specificity is dependent on the concentration of PtdIns(3)P, and on the recruitment of PtdIns(3)P binding proteins such as rabenosyn-5 and PIKfyve, with conversion of PtdIns(3)P into PtdIns(3,5)P2 re-routing the liposomes towards late endosomes despite the presence of GTP-Rab5 and early endosomal SNAREs. Our data reveal a complex interplay between permissive and inhibitory targeting signals that sharpen a basic targeting and fusion machinery for conveying selectivity in intracellular membrane traffic.


Assuntos
Proteínas SNARE , Proteínas rab de Ligação ao GTP , Proteínas SNARE/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Fosfatidilinositóis/metabolismo , Lipossomos/metabolismo , Endossomos/metabolismo , Fusão de Membrana
4.
ACS Nano ; 18(14): 10324-10340, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38547369

RESUMO

A major challenge in using nanocarriers for intracellular drug delivery is their restricted capacity to escape from endosomes into the cytosol. Here, we significantly enhance the drug delivery efficiency by accurately predicting and regulating the transition pH (pH0) of peptides to modulate their endosomal escape capability. Moreover, by inverting the chirality of the peptide carriers, we could further enhance their ability to deliver nucleic acid drugs as well as antitumor drugs. The resulting peptide carriers exhibit versatility in transfecting various cell types with a high efficiency of up to 90% by using siRNA, pDNA, and mRNA. In vivo antitumor experiments demonstrate a tumor growth inhibition of 83.4% using the peptide. This research offers a potent method for the rapid development of peptide vectors with exceptional transfection efficiencies for diverse pathophysiological indications.


Assuntos
Sistemas de Liberação de Medicamentos , Endossomos , Preparações Farmacêuticas , Endossomos/metabolismo , Peptídeos/metabolismo , Concentração de Íons de Hidrogênio
5.
Commun Biol ; 7(1): 334, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38491121

RESUMO

VPS37A, an ESCRT-I complex component, is required for recruiting a subset of ESCRT proteins to the phagophore for autophagosome closure. However, the mechanism by which VPS37A is targeted to the phagophore remains obscure. Here, we demonstrate that the VPS37A N-terminal domain exhibits selective interactions with highly curved membranes, mediated by two membrane-interacting motifs within the disordered regions surrounding its Ubiquitin E2 variant-like (UEVL) domain. Site-directed mutations of residues in these motifs disrupt ESCRT-I localization to the phagophore and result in defective phagophore closure and compromised autophagic flux in vivo, highlighting their essential role during autophagy. In conjunction with the UEVL domain, we postulate that these motifs guide a functional assembly of the ESCRT machinery at the highly curved tip of the phagophore for autophagosome closure. These results advance the notion that the distinctive membrane architecture of the cup-shaped phagophore spatially regulates autophagosome biogenesis.


Assuntos
Autofagossomos , Autofagia , Autofagossomos/metabolismo , Autofagia/fisiologia , Membranas Intracelulares/metabolismo , Endossomos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo
6.
Nat Commun ; 15(1): 2598, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38519468

RESUMO

Activation of the mechanistic target of rapamycin (mTOR) is a key metabolic checkpoint of pro-inflammatory T-cell development that contributes to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), however, the underlying mechanisms remain poorly understood. Here, we identify a functional role for Rab4A-directed endosome traffic in CD98 receptor recycling, mTOR activation, and accumulation of mitochondria that connect metabolic pathways with immune cell lineage development and lupus pathogenesis. Based on integrated analyses of gene expression, receptor traffic, and stable isotope tracing of metabolic pathways, constitutively active Rab4AQ72L exerts cell type-specific control over metabolic networks, dominantly impacting CD98-dependent kynurenine production, mTOR activation, mitochondrial electron transport and flux through the tricarboxylic acid cycle and thus expands CD4+ and CD3+CD4-CD8- double-negative T cells over CD8+ T cells, enhancing B cell activation, plasma cell development, antinuclear and antiphospholipid autoantibody production, and glomerulonephritis in lupus-prone mice. Rab4A deletion in T cells and pharmacological mTOR blockade restrain CD98 expression, mitochondrial metabolism and lineage skewing and attenuate glomerulonephritis. This study identifies Rab4A-directed endosome traffic as a multilevel regulator of T cell lineage specification during lupus pathogenesis.


Assuntos
Glomerulonefrite , Lúpus Eritematoso Sistêmico , Animais , Camundongos , Linfócitos T CD8-Positivos/metabolismo , Endossomos/metabolismo , Glomerulonefrite/metabolismo , Cinurenina/metabolismo , Mitocôndrias/metabolismo , Mitofagia , Serina-Treonina Quinases TOR/metabolismo , Proteínas rab4 de Ligação ao GTP/metabolismo
7.
ACS Appl Mater Interfaces ; 16(13): 15819-15831, 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38517139

RESUMO

Nanoparticles usually enter cells through energy-dependent endocytosis that involves their cytosolic entry via biomembrane-coated endosomes. In contrast, direct translocation of nanoparticles with straight access to cytosol/subcellular components without any membrane coating is limited to very selective conditions/approaches. Here we show that nanoparticles can switch from energy-dependent endocytosis to energy-independent direct membrane penetration once an amphiphile is electrostatically bound to their surface. Compared to endocytotic uptake, this direct cell translocation is faster and nanoparticles are distributed inside the cytosol without any lysosomal trafficking. We found that this direct cell translocation option is sensitive to the charges of both the nanoparticles and the amphiphile. We propose that an electrostatically bound amphiphile induces temporary opening of the cell membrane, which allows direct cell translocation of nanoparticles. This approach can be adapted for efficient subcellular targeting of nanoparticles and nanoparticle-based drug delivery application, bypassing the endosomal trapping and lysosomal degradation.


Assuntos
Nanopartículas , Citosol/metabolismo , Nanopartículas/metabolismo , Endocitose , Endossomos/metabolismo , Sistemas de Liberação de Medicamentos
8.
Sci Adv ; 10(13): eadl0608, 2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38552021

RESUMO

The Golgi-localized golgins golgin-97 and golgin-245 capture transport vesicles arriving from endosomes via the protein TBC1D23. The amino-terminal domain of TBC1D23 binds to the golgins, and the carboxyl-terminal domain of TBC1D23 captures the vesicles, but how it recognizes specific vesicles was unclear. A search for binding partners of the carboxyl-terminal domain unexpectedly revealed direct binding to carboxypeptidase D and syntaxin-16, known cargo proteins of the captured vesicles. Binding is via a threonine-leucine-tyrosine (TLY) sequence present in both proteins next to an acidic cluster. A crystal structure reveals how this acidic TLY motif binds to TBC1D23. An acidic TLY motif is also present in the tails of other endosome-to-Golgi cargo, and these also bind TBC1D23. Structure-guided mutations in the carboxyl-terminal domain that disrupt motif binding in vitro also block vesicle capture in vivo. Thus, TBC1D23 attached to golgin-97 and golgin-245 captures vesicles by a previously undescribed mechanism: the recognition of a motif shared by cargo proteins carried by the vesicle.


Assuntos
Complexo de Golgi , Proteínas de Membrana , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Complexo de Golgi/metabolismo , Transporte Biológico , Endossomos/metabolismo , Ligação Proteica
9.
PLoS One ; 19(3): e0290672, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38483897

RESUMO

Viral and cellular particles too large to freely diffuse have two different types of mobility in the eukaryotic cell cytoplasm: directed motion mediated by motor proteins moving along cytoskeletal elements with the particle as its load, and motion in random directions mediated by motor proteins interconnecting cytoskeletal elements. The latter motion is referred to as "active diffusion." Mechanisms of directed motion have been extensively studied compared to mechanisms of active diffusion, despite the observation that active diffusion is more common for many viral and cellular particles. Our previous research showed that active diffusion of vesicular stomatitis virus (VSV) ribonucleoproteins (RNPs) in the cytoplasm consists of hopping between traps and that actin filaments and myosin II motors are components of the hop-trap mechanism. This raises the question whether similar mechanisms mediate random motion of larger particles with different physical and biological properties. Live-cell fluorescence imaging and a variational Bayesian analysis used in pattern recognition and machine learning were used to determine the molecular mechanisms of random motion of VSV inclusion bodies and cellular early endosomes. VSV inclusion bodies are membraneless cellular compartments that are the major sites of viral RNA synthesis, and early endosomes are representative of cellular membrane-bound organelles. Like VSV RNPs, inclusion bodies and early endosomes moved from one trapped state to another, but the distance between states was inconsistent with hopping between traps, indicating that the apparent state-to-state movement is mediated by trap movement. Like VSV RNPs, treatment with the actin filament depolymerizing inhibitor latrunculin A increased VSV inclusion body mobility by increasing the size of the traps. In contrast neither treatment with latrunculin A nor depolymerization of microtubules by nocodazole treatment affected the size of traps that confine early endosome mobility, indicating that intermediate filaments are likely major trap components for these cellular organelles.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Tiazolidinas , Estomatite Vesicular , Animais , Humanos , Estomatite Vesicular/metabolismo , Teorema de Bayes , Vírus da Estomatite Vesicular Indiana/genética , Vesiculovirus , Vesículas Transportadoras , Endossomos/metabolismo , Corpos de Inclusão , Mamíferos
10.
Mol Biol Cell ; 35(5): ar63, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38446621

RESUMO

Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase. Using confocal microscopy and subcellular fractionation of NRK cells stably expressing fluorescently tagged proteins, we found net recruitment of the V1 subcomplex during sucrosome formation and loss during lysosome reformation, with a similar time course to RAB7a loss. Addition of invertase did not alter mTORC1 signalling, suggesting that the regulation of reversible V-ATPase assembly/disassembly in continuously fed cells differs from that in cells subject to amino acid depletion/refeeding. Using live cell microscopy, we demonstrated recruitment of a fluorescently tagged V1 subunit during endolysosome formation and a dynamic equilibrium and rapid exchange between the cytosolic and membrane bound pools of this subunit. We conclude that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells.


Assuntos
ATPases Vacuolares Próton-Translocadoras , Ratos , Animais , ATPases Vacuolares Próton-Translocadoras/metabolismo , beta-Frutofuranosidase/metabolismo , Endossomos/metabolismo , Transdução de Sinais , Lisossomos/metabolismo , Mamíferos/metabolismo
11.
J Control Release ; 367: 877-891, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38301930

RESUMO

To facilitate the introduction of proteins, such as antibodies, into cells, a variety of delivery peptides have been engineered. These peptides are typically highly cationic and somewhat hydrophobic, enabling cytosolic protein delivery at the cost of causing cell damage by rupturing membranes. This balance between delivery effectiveness and cytotoxicity presents obstacles for their real-world use. To tackle this problem, we designed a new endosome-disruptive cytosolic delivery peptide, E3MPH16, inspired by mastoparan X (MP). E3MPH16 was engineered to incorporate three Glu (E3) and 16 His (H16) residues at the N- and C-termini of MP, respectively. The negative charges of E3 substantially mitigate the cell-surface damage induced by MP. The H16 segment is known to enhance cell-surface adsorption and endocytic uptake of the associated molecules. With these modifications, E3MPH16 was successfully trapped within endosomes. The acidification of endosomes is expected to protonate the side chains of E3 and H16, enabling E3MPH16 to rupture endosomal membranes. As a result, nearly 100% of cells achieved cytosolic delivery of a model biomacromolecule, Alexa Fluor 488-labeled dextran (10 kDa), via endosomal escape by co-incubation with E3MPH16. The delivery process also suggested the involvement of macropinocytosis and caveolae-mediated endocytosis. With the assistance of E3MPH16, Cre recombinase and anti-Ras-IgG delivered into HEK293 cells and HT1080 cells enabled gene recombination and inhibited cell proliferation, respectively. The potential for in vivo application of this intracellular delivery method was further validated by topically injecting the green fluorescent protein fused with a nuclear localization signal (NLS-GFP) along with E3MPH16 into Colon-26 tumor xenografts in mice.


Assuntos
Endocitose , Peptídeos , Humanos , Animais , Camundongos , Células HEK293 , Peptídeos/química , Endossomos/metabolismo , Proteínas de Fluorescência Verde/metabolismo
12.
Nat Cell Biol ; 26(2): 194-206, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38332353

RESUMO

Mitochondrial DNA (mtDNA) encodes essential subunits of the oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation. As a DAMP, mtDNA not only contributes to anti-viral resistance, but also causes pathogenic inflammation in many disease contexts. Cells experiencing mtDNA stress caused by depletion of the mtDNA-packaging protein, transcription factor A, mitochondrial (TFAM) or during herpes simplex virus-1 infection exhibit elongated mitochondria, enlargement of nucleoids (mtDNA-protein complexes) and activation of cGAS-STING innate immune signalling via mtDNA released into the cytoplasm. However, the relationship among aberrant mitochondria and nucleoid dynamics, mtDNA release and cGAS-STING activation remains unclear. Here we show that, under a variety of mtDNA replication stress conditions and during herpes simplex virus-1 infection, enlarged nucleoids that remain bound to TFAM exit mitochondria. Enlarged nucleoids arise from mtDNA experiencing replication stress, which causes nucleoid clustering via a block in mitochondrial fission at a stage when endoplasmic reticulum actin polymerization would normally commence, defining a fission checkpoint that ensures mtDNA has completed replication and is competent for segregation into daughter mitochondria. Chronic engagement of this checkpoint results in enlarged nucleoids trafficking into early and then late endosomes for disposal. Endosomal rupture during transit through this endosomal pathway ultimately causes mtDNA-mediated cGAS-STING activation. Thus, we propose that replication-incompetent nucleoids are selectively eliminated by an adaptive mitochondria-endosomal quality control pathway that is prone to innate immune system activation, which might represent a therapeutic target to prevent mtDNA-mediated inflammation during viral infection and other pathogenic states.


Assuntos
DNA Mitocondrial , Proteínas de Ligação a DNA , Humanos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Replicação do DNA , Endossomos/metabolismo , Nucleotidiltransferases/genética , Inflamação/genética , Proteínas Mitocondriais/metabolismo
13.
J Virol ; 98(3): e0180223, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38334329

RESUMO

With a high incidence of acute kidney injury among hospitalized COVID-19 patients, considerable attention has been focussed on whether SARS-CoV-2 specifically targets kidney cells to directly impact renal function, or whether renal damage is primarily an indirect outcome. To date, several studies have utilized kidney organoids to understand the pathogenesis of COVID-19, revealing the ability for SARS-CoV-2 to predominantly infect cells of the proximal tubule (PT), with reduced infectivity following administration of soluble ACE2. However, the immaturity of standard human kidney organoids represents a significant hurdle, leaving the preferred SARS-CoV-2 processing pathway, existence of alternate viral receptors, and the effect of common hypertensive medications on the expression of ACE2 in the context of SARS-CoV-2 exposure incompletely understood. Utilizing a novel kidney organoid model with enhanced PT maturity, genetic- and drug-mediated inhibition of viral entry and processing factors confirmed the requirement for ACE2 for SARS-CoV-2 entry but showed that the virus can utilize dual viral spike protein processing pathways downstream of ACE2 receptor binding. These include TMPRSS- and CTSL/CTSB-mediated non-endosomal and endocytic pathways, with TMPRSS10 likely playing a more significant role in the non-endosomal pathway in renal cells than TMPRSS2. Finally, treatment with the antihypertensive ACE inhibitor, lisinopril, showed negligible impact on receptor expression or susceptibility of renal cells to infection. This study represents the first in-depth characterization of viral entry in stem cell-derived human kidney organoids with enhanced PTs, providing deeper insight into the renal implications of the ongoing COVID-19 pandemic. IMPORTANCE: Utilizing a human iPSC-derived kidney organoid model with improved proximal tubule (PT) maturity, we identified the mechanism of SARS-CoV-2 entry in renal cells, confirming ACE2 as the sole receptor and revealing redundancy in downstream cell surface TMPRSS- and endocytic Cathepsin-mediated pathways. In addition, these data address the implications of SARS-CoV-2 exposure in the setting of the commonly prescribed ACE-inhibitor, lisinopril, confirming its negligible impact on infection of kidney cells. Taken together, these results provide valuable insight into the mechanism of viral infection in the human kidney.


Assuntos
Enzima de Conversão de Angiotensina 2 , Rim , Organoides , SARS-CoV-2 , Internalização do Vírus , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/complicações , COVID-19/virologia , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/virologia , Lisinopril/farmacologia , Lisinopril/metabolismo , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/virologia , Pandemias , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus/efeitos dos fármacos , Peptidil Dipeptidase A/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/virologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/virologia , Receptores de Coronavírus/metabolismo , Modelos Biológicos , Serina Endopeptidases/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco/citologia
14.
J Biol Chem ; 300(3): 105700, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38307383

RESUMO

Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.


Assuntos
Complexo 1 de Proteínas Adaptadoras , ATPases Transportadoras de Cobre , Endossomos , Transporte Proteico , Receptor IGF Tipo 2 , Rede trans-Golgi , Humanos , Endossomos/metabolismo , Células HeLa , Transporte Proteico/genética , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Rede trans-Golgi/genética , Rede trans-Golgi/metabolismo , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo
15.
Traffic ; 25(2): e12931, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38415291

RESUMO

Retrograde trafficking (RT) orchestrates the intracellular movement of cargo from the plasma membrane, endosomes, Golgi or endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) in an inward/ER-directed manner. RT works as the opposing movement to anterograde trafficking (outward secretion), and the two work together to maintain cellular homeostasis. This is achieved through maintaining cell polarity, retrieving proteins responsible for anterograde trafficking and redirecting proteins that become mis-localised. However, aberrant RT can alter the correct location of key proteins, and thus inhibit or indeed change their canonical function, potentially causing disease. This review highlights the recent advances in the understanding of how upregulation, downregulation or hijacking of RT impacts the localisation of key proteins in cancer and disease to drive progression. Cargoes impacted by aberrant RT are varied amongst maladies including neurodegenerative diseases, autoimmune diseases, bacterial and viral infections (including SARS-CoV-2), and cancer. As we explore the intricacies of RT, it becomes increasingly apparent that it holds significant potential as a target for future therapies to offer more effective interventions in a wide range of pathological conditions.


Assuntos
Retículo Endoplasmático , Neoplasias , Humanos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Membrana Celular/metabolismo , Endossomos/metabolismo , Neoplasias/metabolismo , Transporte Proteico
16.
PLoS Genet ; 20(2): e1011152, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38315726

RESUMO

Endocytosis and endolysosomal trafficking are essential for almost all aspects of physiological functions of eukaryotic cells. As our understanding on these membrane trafficking events are mostly from studies in yeast and cultured mammalian cells, one challenge is to systematically evaluate the findings from these cell-based studies in multicellular organisms under physiological settings. One potentially valuable in vivo system to address this challenge is the vitellogenic oocyte in Drosophila, which undergoes extensive endocytosis by Yolkless (Yl), a low-density lipoprotein receptor (LDLR), to uptake extracellular lipoproteins into oocytes and package them into a specialized lysosome, the yolk granule, for storage and usage during later development. However, by now there is still a lack of sufficient understanding on the molecular and cellular processes that control yolk granule biogenesis. Here, by creating genome-tagging lines for Yl receptor and analyzing its distribution in vitellogenic oocytes, we observed a close association of different endosomal structures with distinct phosphoinositides and actin cytoskeleton dynamics. We further showed that Rab5 and Rab11, but surprisingly not Rab4 and Rab7, are essential for yolk granules biogenesis. Instead, we uncovered evidence for a potential role of Rab7 in actin regulation and observed a notable overlap of Rab4 and Rab7, two Rab GTPases that have long been proposed to have distinct spatial distribution and functional roles during endolysosomal trafficking. Through a small-scale RNA interference (RNAi) screen on a set of reported Rab5 effectors, we showed that yolk granule biogenesis largely follows the canonical endolysosomal trafficking and maturation processes. Further, the data suggest that the RAVE/V-ATPase complexes function upstream of or in parallel with Rab7, and are involved in earlier stages of endosomal trafficking events. Together, our study provides s novel insights into endolysosomal pathways and establishes vitellogenic oocyte in Drosophila as an excellent in vivo model for dissecting the highly complex membrane trafficking events in metazoan.


Assuntos
Drosophila , Endossomos , Animais , Drosophila/genética , Drosophila/metabolismo , Endossomos/genética , Endossomos/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Oócitos/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Mamíferos/metabolismo
17.
J Cell Biol ; 223(3)2024 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-38305771

RESUMO

The endolysosomal system specializes in degrading cellular components and is crucial to maintaining homeostasis and adapting rapidly to metabolic and environmental cues. Cells of the immune system exploit this network to process antigens or promote cell death by secreting lysosome-related vesicles. In B lymphocytes, lysosomes are harnessed to facilitate the extraction of antigens and to promote their processing into peptides for presentation to T cells, critical steps to mount protective high-affinity antibody responses. Intriguingly, lysosomal vesicles are now considered important signaling units within cells and also display secretory functions by releasing their content to the extracellular space. In this review, we focus on how B cells use pathways involved in the intracellular trafficking, secretion, and function of endolysosomes to promote adaptive immune responses. A basic understanding of such mechanisms poses an interesting frontier for the development of therapeutic strategies in the context of cancer and autoimmune diseases.


Assuntos
Imunidade Adaptativa , Linfócitos B , Endossomos , Lisossomos , Antígenos/metabolismo , Linfócitos B/metabolismo , Endossomos/metabolismo , Ativação Linfocitária , Lisossomos/metabolismo , Humanos , Animais , Apresentação de Antígeno
18.
J Cell Biol ; 223(5)2024 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-38358349

RESUMO

Different membrane microdomain compositions provide unique environments that can regulate signaling receptor function. We identify microdomains on the endosome membrane of Drosophila endosomes, enriched in lipid-raft or clathrin/ESCRT-0, which are associated with Notch activation by distinct, ligand-independent mechanisms. Transfer of Notch between microdomains is regulated by Deltex and Suppressor of deltex ubiquitin ligases and is limited by a gate-keeper role for ESCRT complexes. Ubiquitination of Notch by Deltex recruits it to the clathrin/ESCRT-0 microdomain and enhances Notch activation by an ADAM10-independent/TRPML-dependent mechanism. This requirement for Deltex is bypassed by the downregulation of ESCRT-III. In contrast, while ESCRT-I depletion also activates Notch, it does so by an ADAM10-dependent/TRPML-independent mechanism and Notch is retained in the lipid raft-like microdomain. In the absence of such endosomal perturbation, different activating Notch mutations also localize to different microdomains and are activated by different mechanisms. Our findings demonstrate the interplay between Notch regulators, endosomal trafficking components, and Notch genetics, which defines membrane locations and activation mechanisms.


Assuntos
Proteínas de Drosophila , Drosophila , Proteínas de Membrana , Receptores Notch , Canais de Potencial de Receptor Transitório , Animais , Proteína ADAM10/metabolismo , Clatrina/metabolismo , Regulação para Baixo , Proteínas de Drosophila/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Receptores Notch/metabolismo , Ubiquitinação , Proteínas de Membrana/metabolismo , Microdomínios da Membrana/metabolismo
19.
Angew Chem Int Ed Engl ; 63(14): e202317817, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38342757

RESUMO

The differential enzymatic activity in the endo/lysosomes of particular cells could trigger targeted endosomal escape functions, enabling selective intracellular protein delivery. However, this strategy may be jeopardized due to protein degradation during endosomal trafficking. Herein, using custom made fluorescent probes to assess the endosomal activity of cathepsin B (CTSB) and protein degradation, we found that certain cancer cells with hyperacidified endosomes grant a spatiotemporal window where CTSB activity surpass protein digestion. This inspired the engineering of antibody-loaded polymeric nanocarriers having CTSB-activatable endosomal escape ability. The nanocarriers selectively escaped from the endo/lysosomes in the cells with high endosomal CTSB activity and delivered active antibodies to intracellular targets. This study provides a viable strategy for cell-specific protein delivery using stimuli-responsive nanocarriers with controlled endosomal escape.


Assuntos
Endossomos , Neoplasias , Endossomos/metabolismo , Anticorpos/metabolismo , Polímeros/metabolismo , Lisossomos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo
20.
J Phys Chem B ; 128(9): 2144-2153, 2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38408890

RESUMO

SNX proteins have been found to induce membrane remodeling to facilitate the generation of transport carriers in endosomal pathways. However, the molecular mechanism of membrane bending and the role of lipids in the bending process remain elusive. Here, we conducted coarse-grained molecular dynamics simulations to investigate the role of the three structural modules (PX, BAR, and AH) of SNX1 and the PI3P lipids in membrane deformation. We observed that the presence of all three domains is essential for SNX1 to achieve a stable membrane deformation. BAR is capable of remodeling the membrane through the charged residues on its concave surface, but it requires PX and AH to establish stable membrane binding. AH penetrates into the lipid membrane, thereby promoting the induction of membrane curvature; however, it is inadequate on its own to maintain membrane bending. PI3P lipids are also indispensable for membrane remodeling, as they play a dominant role in the interactions of lipids with the BAR domain. Our results enhance the comprehension of the molecular mechanism underlying SNX1-induced membrane curvature and help future studies of curvature-inducing proteins.


Assuntos
Simulação de Dinâmica Molecular , Proteínas , Estrutura Terciária de Proteína , Proteínas/metabolismo , Endossomos/metabolismo , Lipídeos/análise , Membrana Celular/química
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