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1.
EMBO J ; 42(24): e113590, 2023 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-38073509

RESUMO

Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression.


Assuntos
Retrovirus Endógenos , Vesículas Extracelulares , Neoplasias , Animais , Camundongos , Vesículas Extracelulares/metabolismo , Linhagem Celular Tumoral , Diferenciação Celular , Células Dendríticas , Neoplasias/metabolismo
2.
J Extracell Vesicles ; 12(12): e12384, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-38031976

RESUMO

Cell-cell communication within the complex tumour microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Deciphering the interactions between TD-EVs and immune cells is essential to understand immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection by imaging flow cytometry and multicolour spectral flow cytometry of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs). EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labelled either with the lipophilic dye MemGlow-488 (MG-488), Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) or through ectopic expression of a MyrPalm-superFolderGFP reporter (mp-sfGFP), which incorporates into EVs during their biogenesis. Our results show that these labelling strategies, although analysed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and mp-sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labelling method. Furthermore, our results show that the method used for EV labelling influences the detection of the different types of EV interactions with the recipient cells. Specifically, MG-488, CFSE and mp-sfGFP result in observation suggesting, respectively, transient EV-PM interaction that results in dye transfer, EV content delivery, and capture of intact EVs. Consequently, the type of EV labelling method has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate.


Assuntos
Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Leucócitos Mononucleares , Succinimidas/metabolismo , Linhagem Celular
3.
Nat Commun ; 14(1): 1998, 2023 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-37032404

RESUMO

Engrailed2 (En2) is a transcription factor that transfers from cell to cell through unconventional pathways. The poorly understood internalization mechanism of this cationic protein is proposed to require an initial interaction with cell-surface glycosaminoglycans (GAGs). To decipher the role of GAGs in En2 internalization, we have quantified the entry of its homeodomain region in model cells that differ in their content in cell-surface GAGs. The binding specificity to GAGs and the influence of this interaction on the structure and dynamics of En2 was also investigated at the amino acid level. Our results show that a high-affinity GAG-binding sequence (RKPKKKNPNKEDKRPR), upstream of the homeodomain, controls En2 internalization through selective interactions with highly-sulfated heparan sulfate GAGs. Our data underline the functional importance of the intrinsically disordered basic region upstream of En2 internalization domain, and demonstrate the critical role of GAGs as an entry gate, finely tuning homeoprotein capacity to internalize into cells.


Assuntos
Glicosaminoglicanos , Heparitina Sulfato , Heparitina Sulfato/metabolismo , Glicosaminoglicanos/metabolismo , Fatores de Transcrição , Proteínas de Homeodomínio/genética , Sulfatos , Sulfatos de Condroitina/metabolismo
4.
Adv Exp Med Biol ; 1422: 381-392, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36988889

RESUMO

Besides its protective role in the maintenance of cell homeostasis, the plasma membrane is the site of exchanges between the cell interior and the extracellular medium. To circumvent the hydrophobic barrier formed by the acyl chains of the lipid bilayer, protein channels and transporters are key players in the exchange of small hydrophilic compounds such as ions or nutrients, but they hardly account for the transport of larger biological molecules. Exchange of proteins usually relies on membrane-fusion events between vesicles and the plasma membrane. In recent years, several alternative unconventional protein secretion (UPS) pathways across the plasma membrane have been characterised for a specific set of secreted substrates, some of them excluding any membrane-fusion events (Dimou and Nickel, Curr Biol 28:R406-R410, 2018). One of thesbe pathways, referred as type I UPS, relies on the direct translocation of the protein across the plasma membrane and not surprisingly, lipids are essential players in this process. In this chapter, we discuss the roles of phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and cholesterol in unconventional pathways involving Engrailed-2 homeoprotein and fibroblast growth factor 2.


Assuntos
Bicamadas Lipídicas , Proteínas de Membrana Transportadoras , Bicamadas Lipídicas/metabolismo , Membrana Celular/metabolismo , Transporte Proteico , Proteínas de Membrana Transportadoras/metabolismo , Colesterol/metabolismo , Fosfatidilinositol 4,5-Difosfato/análise , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo
5.
Biochim Biophys Acta Biomembr ; 1864(11): 184030, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-35988722

RESUMO

Homeoprotein transcription factors have the property of interacting with membranes through their DNA-binding homeodomain, which is involved in unconventional internalization and secretion. Both processes depend on membrane-translocating events but their detailed molecular mechanisms are still poorly understood. We have previously characterized the conformational properties of Engrailed 2 homeodomain (EnHD) in aqueous solution and in micelles as membrane-mimetic environments. In the present study, we used small isotropic lipid bicelles as a more relevant membrane-mimetic model to characterize the membrane-bound state of EnHD. We show that lipid bicelles, in contrast to micelles, adequately reproduce the requirement of anionic lipids in the membrane binding and conformational transition of EnHD. The fold-unfold transition of EnHD induced by anionic lipids was characterized by NMR using 1H, 13C, 15N chemical shifts, nuclear Overhauser effects, residual dipolar couplings, intramolecular and intermolecular paramagnetic relaxation enhancements induced by site-directed spin-label or paramagnetic lipid probe, respectively. A global unpacking of EnHD helices is observed leading to a loss of the native fold. However, near-native propensities of EnHD backbone conformation are maintained in membrane environment, including not only the three helices but also the turn connecting helices H2 and H3. NMR and coarse-grained molecular dynamics simulations reveal that the EnHD adopts a shallow insertion in the membrane, with the three helices oriented parallel to the membrane. EnHD explores extended conformations and closed U-shaped conformations, which are stabilized by anionic lipid recruitment.


Assuntos
Micelas , Simulação de Dinâmica Molecular , Proteínas de Homeodomínio/química , Lipídeos , Estrutura Secundária de Proteína
6.
J Extracell Vesicles ; 11(7): e12242, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35790086

RESUMO

Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular vesicles (EVs). EVs could have different subcellular origin, composition and functional properties, but tools to distinguish between EV subtypes are scarce. Here, we tagged CD63- or CD9-positive EVs secreted by triple negative breast cancer cells with Nanoluciferase enzyme, to set-up a miniaturized method to quantify secretion of these two EV subtypes directly in the supernatant of cells. We performed a cell-based high-content screening to identify clinically-approved drugs able to affect EV secretion. One of the identified hits is Homosalate, an anti-inflammatory drug found in sunscreens which robustly increased EVs' release. Comparing EVs induced by Homosalate with those induced by Bafilomycin A1, we demonstrate that: (1) the two drugs act on EVs generated in distinct subcellular compartments, and (2) EVs released by Homosalate-, but not by Bafilomycin A1-treated cells enhance resistance to anchorage loss in another recipient epithelial tumour cell line. In conclusion, we identified a new drug modifying EV release and demonstrated that under influence of different drugs, triple negative breast cancer cells release EV subpopulations from different subcellular origins harbouring distinct functional properties.


Assuntos
Vesículas Extracelulares , Neoplasias de Mama Triplo Negativas , Suplementos Nutricionais , Humanos , Salicilatos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico
7.
Front Cell Dev Biol ; 10: 926421, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35837333

RESUMO

Unconventional secretion allows for the secretion of fully mature and biologically active proteins mostly present in the cytoplasm or nucleus. Besides extra vesicle-driven secretion, non-extravesicular pathways also exist that specifically rely on the ability of the secreted proteins to translocate directly across the plasma membrane. This is the case for several homeoproteins, a family of over 300 transcription factors characterized by the structure of their DNA-binding homeodomain. The latter highly conserved homeodomain is necessary and sufficient for secretion, a process that requires PI(4,5)P2 binding, as is the case for FGF2 and HIV Tat unconventional secretion. An important feature of homeoproteins is their ability to cross membranes in both directions and thus to transfer between cells. This confers to homeoproteins their paracrine activity, an essential facet of their physiological functions.

8.
Antioxidants (Basel) ; 11(4)2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35453403

RESUMO

Among molecules that bridge environment, cell metabolism, and cell signaling, hydrogen peroxide (H2O2) recently appeared as an emerging but central player. Its level depends on cell metabolism and environment and was recently shown to play key roles during embryogenesis, contrasting with its long-established role in disease progression. We decided to explore whether the secreted morphogen Sonic hedgehog (Shh), known to be essential in a variety of biological processes ranging from embryonic development to adult tissue homeostasis and cancers, was part of these interactions. Here, we report that H2O2 levels control key steps of Shh delivery in cell culture: increased levels reduce primary secretion, stimulate endocytosis and accelerate delivery to recipient cells; in addition, physiological in vivo modulation of H2O2 levels changes Shh distribution and tissue patterning. Moreover, a feedback loop exists in which Shh trafficking controls H2O2 synthesis via a non-canonical BOC-Rac1 pathway, leading to cytoneme growth. Our findings reveal that Shh directly impacts its own distribution, thus providing a molecular explanation for the robustness of morphogenesis to both environmental insults and individual variability.

9.
Sci Adv ; 6(44)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33115744

RESUMO

Homeoproteins were originally identified for embryonic cell-autonomous transcription activity, but they also have non-cell-autonomous activity owing to transfer between cells. This Review discusses transfer mechanisms and focuses on some established functions, such as neurodevelopmental regulation of axon guidance, and postnatal critical periods of brain plasticity that affect sensory processing and cognition. Homeoproteins are present across all eukaryotes, and intercellular transfer occurs in plants and animals. Proposed functions have evolutionary relevance, such as morphogenetic activity and sexual exchange during the mating of unicellular eukaryotes, while others have physiopathological relevance, such as regulation of mood and cognition by influencing brain compartmentalization, connectivity, and plasticity. There are more than 250 known homeoproteins with conserved transfer domains, suggesting that this is a common mode of signal transduction but with many undiscovered functions.

10.
Commun Biol ; 3(1): 536, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994473

RESUMO

Although a physiological role for redox signaling is now clearly established, the processes sensitive to redox signaling remains to be identified. Ratiometric probes selective for H2O2 have revealed its complex spatiotemporal dynamics during neural development and adult regeneration and perturbations of H2O2 levels disturb cell plasticity and morphogenesis. Here we ask whether endogenous H2O2 could participate in the patterning of the embryo. We find that perturbations of endogenous H2O2 levels impact on the distribution of the Engrailed homeoprotein, a strong determinant of midbrain patterning. Engrailed 2 is secreted from cells with high H2O2 levels and taken up by cells with low H2O2 levels where it leads to increased H2O2 production, steering the directional spread of the Engrailed gradient. These results illustrate the interplay between protein signaling pathways and metabolic processes during morphogenetic events.


Assuntos
Proteínas de Homeodomínio/fisiologia , Peróxido de Hidrogênio/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Comunicação Parácrina/fisiologia , Colículos Superiores/embriologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Oxirredução , Colículos Superiores/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimento
11.
J Cell Sci ; 133(13)2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32434869

RESUMO

Homeoproteins are a class of transcription factors sharing the unexpected property of intercellular trafficking that confers to homeoproteins a paracrine mode of action. Homeoprotein paracrine action participates in the control of patterning processes, including axonal guidance, brain plasticity and boundary formation. Internalization and secretion, the two steps of intercellular transfer, rely on unconventional mechanisms, but the cellular mechanisms at stake still need to be fully characterized. Thanks to the design of new quantitative and sensitive assays dedicated to the study of homeoprotein transfer within HeLa cells in culture, we demonstrate a core role of phosphatidylinositol (4,5)-bisphosphate (PIP2) together with cholesterol in the translocation of the homeobox protein engrailed-2 (EN2) across the plasma membrane. By using drug and enzyme treatments, we show that both secretion and internalization are regulated according to PIP2 levels. The requirement for PIP2 and cholesterol in EN2 trafficking correlates with their selective affinity for this protein in artificial bilayers, which is drastically decreased in a paracrine-deficient mutant of EN2. We propose that the bidirectional plasma membrane translocation events that occur during homeoprotein secretion and internalization are parts of a common process.


Assuntos
Proteínas de Homeodomínio , Fatores de Transcrição , Membrana Celular , Células HeLa , Humanos , Proteínas do Tecido Nervoso , Plasticidade Neuronal , Fosfatidilinositol 4,5-Difosfato
12.
Neurobiol Dis ; 134: 104614, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31605779

RESUMO

The G2019S substitution in the kinase domain of LRRK2 (LRRK2G2019S) is the most prevalent mutation associated with Parkinson's disease (PD). Neurotoxic effects of LRRK2G2019S are thought to result from an increase in its kinase activity as compared to wild type LRRK2. However, it is unclear whether the kinase domain of LRRK2G2019S is sufficient to trigger degeneration or if the full length protein is required. To address this question, we generated constructs corresponding to the C-terminal domain of LRRK2 (ΔLRRK2). A kinase activity that was increased by G2019➔S substitution could be detected in ΔLRRK2. However biochemical experiments suggested it did not bind or phosphorylate the substrate RAB10, in contrast to full length LRRK2. The overexpression of ΔLRRK2G2019S in the rat striatum using lentiviral vectors (LVs) offered a straightforward and simple way to investigate its effects in neurons in vivo. Results from a RT-qPCR array analysis indicated that ΔLRRK2G2019S led to significant mRNA expression changes consistent with a kinase-dependent mechanism. We next asked whether ΔLRRK2 could be sufficient to trigger neurodegeneration in the substantia nigra pars compacta (SNc) in adult rats. Six months after infection of the substantia nigra pars compacta (SNc) with LV-ΔLRRK2WT or LV-ΔLRRK2G2019S, the number of DA neurons was unchanged. To examine whether higher levels of ΔLRRK2G2019S could trigger degeneration we cloned ΔLRRK2 in AAV2/9 construct. As expected, AAV2/9 injected in the SNc led to neuronal expression of ΔLRRK2WT and ΔLRRK2G2019S at much higher levels than those obtained with LVs. Six months after injection, unbiased stereology showed that AAV-ΔLRRK2G2019S produced a significant ~30% loss of neurons positive for tyrosine hydroxylase- and for the vesicular dopamine transporter whereas AAV-ΔLRRK2WT did not. These findings show that overexpression of the C-terminal part of LRRK2 containing the mutant kinase domain is sufficient to trigger degeneration of DA neurons, through cell-autonomous mechanisms, possibly independent of RAB10.


Assuntos
Neurônios Dopaminérgicos/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Degeneração Neural/genética , Doença de Parkinson , Domínios Proteicos/genética , Animais , Técnicas de Transferência de Genes , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus , Masculino , Mutação , Degeneração Neural/patologia , Parte Compacta da Substância Negra , Ratos , Ratos Sprague-Dawley
13.
Chem Commun (Camb) ; 55(31): 4566-4569, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30931466

RESUMO

A series of cyclic lipidated oligo-Arg cell penetrating peptides were synthesised with varied macrocycle size and lipid chain anchoring site. The study of their cellular uptake revealed different structural requirements to promote efficient glycosaminoglycan-dependent endocytosis and direct translocation.


Assuntos
Peptídeos Penetradores de Células/química , Glicosaminoglicanos/química , Sequência de Aminoácidos , Animais , Células CHO , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/metabolismo , Cricetinae , Cricetulus , Ciclização , Endocitose
14.
Antioxidants (Basel) ; 7(11)2018 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-30404180

RESUMO

Reactive oxygen species (ROS), which were originally classified as exclusively deleterious compounds, have gained increasing interest in the recent years given their action as bona fide signalling molecules. The main target of ROS action is the reversible oxidation of cysteines, leading to the formation of disulfide bonds, which modulate protein conformation and activity. ROS, endowed with signalling properties, are mainly produced by NADPH oxidases (NOXs) at the plasma membrane, but their action also involves a complex machinery of multiple redox-sensitive protein families that differ in their subcellular localization and their activity. Given that the levels and distribution of ROS are highly dynamic, in part due to their limited stability, the development of various fluorescent ROS sensors, some of which are quantitative (ratiometric), represents a clear breakthrough in the field and have been adapted to both ex vivo and in vivo applications. The physiological implication of ROS signalling will be presented mainly in the frame of morphogenetic processes, embryogenesis, regeneration, and stem cell differentiation. Gain and loss of function, as well as pharmacological strategies, have demonstrated the wide but specific requirement of ROS signalling at multiple stages of these processes and its intricate relationship with other well-known signalling pathways.

15.
Stem Cells ; 36(9): 1421-1429, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29808941

RESUMO

Heterozygous PAX6 gene mutations leading to haploinsufficiency are the main cause of congenital aniridia, a rare and progressive panocular disease characterized by reduced visual acuity. Up to 90% of patients suffer from aniridia-related keratopathy (ARK), caused by a combination of factors including limbal epithelial stem cell (LSC) deficiency, impaired healing response and abnormal differentiation of the corneal epithelium. It usually begins in the first decade of life, resulting in recurrent corneal erosions, sub-epithelial fibrosis, and corneal opacification. Unfortunately, there are currently no efficient treatments available for these patients and no in vitro model for this pathology. We used CRISPR/Cas9 technology to introduce into the PAX6 gene of LSCs a heterozygous nonsense mutation found in ARK patients. Nine clones carrying a p.E109X mutation on one allele were obtained with no off-target mutations. Compared with the parental LSCs, heterozygous mutant LSCs displayed reduced expression of PAX6 and marked slow-down of cell proliferation, migration and detachment. Moreover, addition to the culture medium of recombinant PAX6 protein fused to a cell penetrating peptide was able to activate the endogenous PAX6 gene and to rescue phenotypic defects of mutant LSCs, suggesting that administration of such recombinant PAX6 protein could be a promising therapeutic approach for aniridia-related keratopathy. More generally, our results demonstrate that introduction of disease mutations into LSCs by CRISPR/Cas9 genome editing allows the creation of relevant cellular models of ocular disease that should greatly facilitate screening of novel therapeutic approaches. Stem Cells 2018;36:1421-1429.


Assuntos
Aniridia/genética , Sistemas CRISPR-Cas/fisiologia , Epitélio Corneano/metabolismo , Edição de Genes/métodos , Fator de Transcrição PAX6/genética , Aniridia/patologia , Humanos
16.
Bioconjug Chem ; 29(6): 1823-1828, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29791141

RESUMO

Methods to differentially label cell-surface and intracellular membrane proteins are indispensable for understanding their function and the regulation of their trafficking. We present an efficient strategy for the rapid and selective fluorescent labeling of membrane proteins based on the chemical-genetic fluorescent marker FAST (fluorescence-activating and absorption-shifting tag). Cell-surface FAST-tagged proteins could be selectively and rapidly labeled using fluorogenic membrane-impermeant 4-hydroxybenzylidene rhodanine (HBR) analogs. This approach allows the study of protein trafficking at the plasma membrane with various fluorometric techniques, and opens exciting prospects for the high-throughput screening of small molecules able to restore disease-related trafficking defects.


Assuntos
Compostos de Benzilideno/metabolismo , Membrana Celular/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas de Membrana/metabolismo , Rodanina/análogos & derivados , Compostos de Benzilideno/análise , Membrana Celular/química , Corantes Fluorescentes/análise , Células HEK293 , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/análise , Microscopia de Fluorescência/métodos , Transporte Proteico , Rodanina/análise , Rodanina/metabolismo , Proteína Vermelha Fluorescente
17.
Semin Cell Dev Biol ; 80: 65-73, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-28797840

RESUMO

The tight control of reactive oxygen species (ROS) levels is required during regeneration. H2O2 in particular assumes clear signalling functions at different steps in this process. Injured nerves induce high levels of H2O2 through the activation of the Hedgehog (Shh) pathway, providing an environment that promotes cell plasticity, progenitor recruitment and blastema formation. In turn, high H2O2 levels contribute to growing axon attraction. Once re-innervation is completed, nerves subsequently downregulate H2O2 levels to their original state. A similar regulatory loop between H2O2 levels and nerves also exists during development. This suggests that redox signalling is a major actor in cell plasticity.


Assuntos
Proteínas Hedgehog/metabolismo , Peróxido de Hidrogênio/metabolismo , Rede Nervosa/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regeneração/fisiologia , Animais , Humanos , Transdução de Sinais/fisiologia
18.
19.
Sci Rep ; 6: 36938, 2016 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-27841303

RESUMO

The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high µM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Animais , Células CHO , Cricetulus , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Estrutura Molecular , Espectrometria de Fluorescência
20.
Dev Biol ; 414(2): 133-41, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27158028

RESUMO

It is now becoming evident that hydrogen peroxide (H2O2), which is constantly produced by nearly all cells, contributes to bona fide physiological processes. However, little is known regarding the distribution and functions of H2O2 during embryonic development. To address this question, we used a dedicated genetic sensor and revealed a highly dynamic spatio-temporal pattern of H2O2 levels during zebrafish morphogenesis. The highest H2O2 levels are observed during somitogenesis and organogenesis, and these levels gradually decrease in the mature tissues. Biochemical and pharmacological approaches revealed that H2O2 distribution is mainly controlled by its enzymatic degradation. Here we show that H2O2 is enriched in different regions of the developing brain and demonstrate that it participates to axonal guidance. Retinal ganglion cell axonal projections are impaired upon H2O2 depletion and this defect is rescued by H2O2 or ectopic activation of the Hedgehog pathway. We further show that ex vivo, H2O2 directly modifies Hedgehog secretion. We propose that physiological levels of H2O2 regulate RGCs axonal growth through the modulation of Hedgehog pathway.


Assuntos
Orientação de Axônios/efeitos dos fármacos , Proteínas Hedgehog/fisiologia , Peróxido de Hidrogênio/metabolismo , Neurogênese/fisiologia , Células Ganglionares da Retina/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Orientação de Axônios/fisiologia , Axônios/metabolismo , Catalase/metabolismo , Cisteína/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/análise , Transporte Proteico/efeitos dos fármacos , Células Ganglionares da Retina/ultraestrutura , Transdução de Sinais/fisiologia , Superóxido Dismutase/metabolismo , Peixe-Zebra/metabolismo
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