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1.
Sci Adv ; 10(27): eadn9423, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38968349

RESUMO

DNA origami nanostructures (DOs) are promising tools for applications including drug delivery, biosensing, detecting biomolecules, and probing chromatin substructures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing, visualizing, and controlling biomolecular processes within live cells. We present an approach to deliver DOs into live-cell nuclei. We show that these DOs do not undergo detectable structural degradation in cell culture media or cell extracts for 24 hours. To deliver DOs into the nuclei of human U2OS cells, we conjugated 30-nanometer DO nanorods with an antibody raised against a nuclear factor, specifically the largest subunit of RNA polymerase II (Pol II). We find that DOs remain structurally intact in cells for 24 hours, including inside the nucleus. We demonstrate that electroporated anti-Pol II antibody-conjugated DOs are piggybacked into nuclei and exhibit subdiffusive motion inside the nucleus. Our results establish interfacing DOs with a nuclear factor as an effective method to deliver nanodevices into live-cell nuclei.


Assuntos
Núcleo Celular , DNA , Nanoestruturas , Núcleo Celular/metabolismo , Humanos , DNA/química , DNA/metabolismo , Nanoestruturas/química , RNA Polimerase II/metabolismo , Linhagem Celular Tumoral , Nanotubos/química
2.
bioRxiv ; 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38562776

RESUMO

Late endosomes/lysosomes (LELs) are crucial for numerous physiological processes and their dysfunction is linked to many diseases. Proteomic analyses have identified hundreds of LEL proteins, however, whether these proteins are uniformly present on each LEL, or if there are cell-type dependent LEL sub-populations with unique protein compositions is unclear. We employed a quantitative, multiplexed DNA-PAINT super-resolution approach to examine the distribution of six key LEL proteins (LAMP1, LAMP2, CD63, TMEM192, NPC1 and LAMTOR4) on individual LELs. While LAMP1 and LAMP2 were abundant across LELs, marking a common population, most analyzed proteins were associated with specific LEL subpopulations. Our multiplexed imaging approach identified up to eight different LEL subpopulations based on their unique membrane protein composition. Additionally, our analysis of the spatial relationships between these subpopulations and mitochondria revealed a cell-type specific tendency for NPC1-positive LELs to be closely positioned to mitochondria. Our approach will be broadly applicable to determining organelle heterogeneity with single organelle resolution in many biological contexts. Summary: This study develops a multiplexed and quantitative DNA-PAINT super-resolution imaging pipeline to investigate the distribution of late endosomal/lysosomal (LEL) proteins across individual LELs, revealing cell-type specific LEL sub-populations with unique protein compositions, offering insights into organelle heterogeneity at single-organelle resolution.

3.
bioRxiv ; 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-38260628

RESUMO

DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.

4.
Proc Natl Acad Sci U S A ; 118(19)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33952699

RESUMO

Tau is a microtubule-associated protein, which promotes neuronal microtubule assembly and stability. Accumulation of tau into insoluble aggregates known as neurofibrillary tangles (NFTs) is a pathological hallmark of several neurodegenerative diseases. The current hypothesis is that small, soluble oligomeric tau species preceding NFT formation cause toxicity. However, thus far, visualizing the spatial distribution of tau monomers and oligomers inside cells under physiological or pathological conditions has not been possible. Here, using single-molecule localization microscopy, we show that tau forms small oligomers on microtubules ex vivo. These oligomers are distinct from those found in cells exhibiting tau aggregation and could be precursors of aggregated tau in pathology. Furthermore, using an unsupervised shape classification algorithm that we developed, we show that different tau phosphorylation states are associated with distinct tau aggregate species. Our work elucidates tau's nanoscale composition under nonaggregated and aggregated conditions ex vivo.


Assuntos
Microtúbulos/metabolismo , Neurônios/metabolismo , Proteínas tau/metabolismo , Humanos , Emaranhados Neurofibrilares/metabolismo , Fosforilação , Imagem Individual de Molécula , Proteínas tau/genética
5.
Am J Physiol Lung Cell Mol Physiol ; 316(4): L656-L668, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30702344

RESUMO

Peroxiredoxin 6 (Prdx6) is a multifunctional enzyme that serves important antioxidant roles by scavenging hydroperoxides and reducing peroxidized cell membranes. Prdx6 also plays a key role in cell signaling by activating the NADPH oxidase, type 2 (Nox2) through its acidic Ca2+-independent phospholipase A2 (aiPLA2) activity. Nox2 generation of O2·-, in addition to signaling, can contribute to oxidative stress and inflammation such as during sepsis-induced acute lung injury (ALI). To evaluate a possible role of Prdx6-aiPLA2 activity in the pathophysiology of ALI associated with a systemic insult, wild-type (WT) and Prdx6-D140A mice, which lack aiPLA2 but retain peroxidase activity were administered intraperitoneal LPS. LPS-treated mutant mice had increased survival compared with WT mice while cytokines in lung lavage fluid and lung VCAM-1 expression, nitrotyrosine levels, PMN infiltration, and permeability increased in WT but not in mutant mice. Exposure of mouse pulmonary microvascular endothelial cells in primary culture to LPS promoted phosphorylation of Prdx6 and its translocation to the plasma membrane and increased aiPLA2 activity as well as increased H2O2 generation, nitrotyrosine levels, lipid peroxidation, NF-κB nuclear localization, and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome assembly; these effects were not seen in Nox2 null cells, Prdx6-D140A cells, or WT cells pretreated with MJ33, an inhibitor of aiPLA2 activity. Thus aiPLA2 activity is needed for Nox2-derived oxidant stress associated with LPS exposure. Since inactivation of aiPLA2 reduced mortality and prevented lung inflammation and oxidative stress in this animal model, the aiPLA2 activity of Prdx6 could be a novel target for prevention or treatment of sepsis-induced ALI.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Peroxirredoxina VI/antagonistas & inibidores , Fosfolipases A2/metabolismo , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Substituição de Aminoácidos , Animais , Domínio Catalítico/genética , Modelos Animais de Doenças , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , NADPH Oxidase 2/metabolismo , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Inibidores de Fosfolipase A2/metabolismo , Fosfolipases A2/química , Fosfolipases A2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
J Cell Biol ; 218(2): 632-643, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30567713

RESUMO

Microtubule post-translational modifications impart functional diversity to microtubules by affecting their dynamics, organization, and interaction with proteins. Using super-resolution microscopy, we show that only a small subpopulation of microtubules are detyrosinated in epithelial cells, while acetylated and tyrosinated microtubules comprise the majority of all microtubules. Surprisingly, lysosomes are enriched by approximately threefold on detyrosinated microtubules. Further, their motility on detyrosinated microtubules is impaired, showing shorter runs and more frequent and longer pauses. Lysosome enrichment is mediated through a kinesin-1-dependent mechanism, since knocking down this motor abolishes enrichment. Finally, correlative live-cell and super-resolution microscopy showed that lysosomes interact with autophagosomes on detyrosinated microtubules. Removal of detyrosinated microtubules or knockdown of kinesin-1 leads to a decrease in the percentage of autolysosomes, a fusion intermediate of autophagosomes and lysosomes. Taken together, our data reveal a new role of detyrosinated microtubules as hubs that spatially concentrate lysosomes on a small subset of microtubules and facilitate their interaction and fusion with autophagosomes to initiate autophagy.


Assuntos
Autofagossomos/metabolismo , Células Epiteliais/metabolismo , Rim/metabolismo , Lisossomos/metabolismo , Fusão de Membrana , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismo , Tirosina/metabolismo , Acetilação , Animais , Autofagossomos/ultraestrutura , Linhagem Celular , Chlorocebus aethiops , Células Epiteliais/ultraestrutura , Rim/ultraestrutura , Cinesinas/genética , Cinesinas/metabolismo , Lisossomos/ultraestrutura , Microtúbulos/ultraestrutura , Tubulina (Proteína)/genética
7.
Redox Biol ; 14: 41-46, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28865296

RESUMO

Although lipid peroxidation associated with oxidative stress can result in cellular death, sub-lethal lipid peroxidation can gradually resolve with return to the pre-exposure state. We have shown that resolution of lipid peroxidation is greatly delayed in lungs or cells that are null for peroxiredoxin 6 (Prdx6) and that both the phospholipase A2 and the GSH peroxidase activities of Prdx6 are required for a maximal rate of recovery. Like other peroxiredoxins, Prdx6 can reduce H2O2 and short chain hydroperoxides, but in addition can directly reduce phospholipid hydroperoxides. This study evaluated the relative role of these two different peroxidase activities of Prdx6 in the repair of peroxidized cell membranes. The His26 residue in Prdx6 is an important component of the binding site for phospholipids. Thus, we evaluated the lungs from H26A-Prdx6 expressing mice and generated H26A-Prdx6 expressing pulmonary microvascular endothelial cells (PMVEC) by lentiviral infection of Prdx6 null cells to compare with wild type in the repair of lipid peroxidation. Isolated lungs and PMVEC were exposed to tert-butyl hydroperoxide and mice were exposed to hyperoxia (> 95% O2). Assays for lipid peroxidation in wild type control and mutant lungs and cells showed ~4-fold increase at end-exposure. Control lungs and cells showed gradual resolution during a post-exposure recovery period. However, there was no recovery from lipid peroxidation by H26A-Prdx6 lungs or PMVEC. These studies confirm an important role for Prdx6 in recovery from membrane lipid peroxidation and indicate that reduction of H2O2 or short chain hydroperoxides does not play a role in the recovery process.


Assuntos
Membrana Celular/metabolismo , Peroxirredoxina VI/metabolismo , Animais , Hipóxia Celular , Membrana Celular/química , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Peróxido de Hidrogênio/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/citologia , Peroxirredoxina VI/deficiência , Peroxirredoxina VI/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação
8.
J Lipid Res ; 57(4): 587-96, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26830860

RESUMO

The phospholipase A2(PLA2) activity of peroxiredoxin (Prdx)6 has important physiological roles in the synthesis of lung surfactant and in the repair of peroxidized cell membranes. These functions require the activity of a lysophospholipid acyl transferase as a critical component of the phospholipid remodeling pathway. We now describe a lysophosphatidylcholine acyl transferase (LPCAT) activity for Prdx6 that showed a strong preference for lysophosphatidylcholine (LPC) as the head group and for palmitoyl CoA in the acylation reaction. The calculated kinetic constants for acylation wereKm18 µM andVmax30 nmol/min/mg protein; theVmaxwas increased 25-fold by phosphorylation of the protein whileKmwas unchanged. Study of recombinant protein in vitro and in mouse pulmonary microvascular endothelial cells infected with a lentiviral vector construct indicated that amino acid D31 is crucial for LPCAT activity. A linear incorporation of labeled fatty acyl CoA into dipalmitoyl phosphatidylcholine (PC) indicated that LPC generated by Prdx6 PLA2activity remained bound to the enzyme for the reacylation reaction. Prdx6 is the first LPCAT enzyme with demonstrated cytoplasmic localization. Thus, Prdx6 is a complete enzyme comprising both PLA2and LPCAT activities for the remodeling pathway of PC synthesis or for repair of membrane lipid peroxidation.


Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Peroxirredoxina VI/metabolismo , Acilação , Sequência de Aminoácidos , Animais , Citoplasma/metabolismo , Humanos , Cinética , Lisofosfatidilcolinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Peroxirredoxina VI/química , Peroxirredoxina VI/genética , Ratos , Especificidade por Substrato
9.
Free Radic Biol Med ; 94: 145-56, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26891882

RESUMO

Peroxiredoxin 6 (Prdx6) is a unique 1-Cys member of the peroxiredoxin family with both GSH peroxidase and phospholipase A2 (PLA2) activities. It is highly expressed in the lung where it plays an important role in antioxidant defense and lung surfactant metabolism. Glutathionylation of Prdx6 mediated by its heterodimerization with GSH S-transferase π (πGST) is required for its peroxidatic catalytic cycle. Recombinant human Prdx6 crystallizes as a homodimer and sedimentation equilibrium analysis confirmed that this protein exists as a high affinity dimer in solution. Based on measurement of molecular mass, dimeric Prdx6 that was oxidized to the sulfenic acid formed a sulfenylamide during storage. After examination of the dimer interface in the crystal structure, we postulated that the hydrophobic amino acids L145 and L148 play an important role in homodimerization of Prdx6 as well as in its heterodimerization with πGST. Oxidation of Prdx6 also was required for its heterodimerization. Sedimentation equilibrium analysis and the Duolink proximity ligation assay following mutation of the L145 and L148 residues of Prdx6 to Glu indicated greatly decreased dimerization propensity reflecting the loss of hydrophobic interactions between the protein monomers. Peroxidase activity was markedly reduced by mutation at either of the Leu sites and was essentially abolished by the double mutation, while PLA2 activity was unaffected. Decreased peroxidase activity following mutation of the interfacial leucines presumably is mediated via impaired heterodimerization of Prdx6 with πGST that is required for reduction and re-activation of the oxidized enzyme.


Assuntos
Antioxidantes/metabolismo , Glutationa S-Transferase pi/metabolismo , Estresse Oxidativo/genética , Peroxirredoxina VI/metabolismo , Antioxidantes/química , Catálise , Cristalografia por Raios X , Dimerização , Glutationa S-Transferase pi/química , Glutationa S-Transferase pi/genética , Humanos , Mutação , Oxirredução , Peroxidase/genética , Peroxidase/metabolismo , Peroxirredoxina VI/química , Peroxirredoxina VI/genética , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Conformação Proteica , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo
10.
J Biol Chem ; 291(17): 9268-80, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-26921317

RESUMO

Peroxiredoxin 6 (Prdx6), a bifunctional protein with phospholipase A2 (aiPLA2) and GSH peroxidase activities, protects lungs from oxidative stress and participates in lung surfactant phospholipid turnover. Prdx6 has been localized to both cytosol and lamellar bodies (LB) in lung epithelium, and its organellar targeting sequence has been identified. We propose that Prdx6 LB targeting facilitates its role in the metabolism of lung surfactant phosphatidylcholine (PC). Ser-32 has been identified as the active site in Prdx6 for aiPLA2 activity, and this activity was abolished by the mutation of serine 32 to alanine (S32A). However, aiPLA2 activity was unaffected by mutation of serine 32 in Prdx6 to threonine (S32T). Prdx6 protein expression and aiPLA2 activity were normal in the whole lung of a "knock-in" mouse model carrying an S32T mutation in the Prdx6 gene but were absent from isolated LB. Analyses by proximity ligation assay in lung sections demonstrated the inability of S32T Prdx6 to bind to the chaperone protein, 14-3-3ϵ, that is required for LB targeting. The content of total phospholipid, PC, and disaturated PC in lung tissue homogenate, bronchoalveolar lavage fluid, and lung LB was increased significantly in Prdx6-S32T mutant lungs, whereas degradation of internalized [(3)H]dipalmitoyl-PC was significantly decreased. Thus, Thr can substitute for Ser for the enzymatic activities of Prdx6 but not for its targeting to LB. These results confirm an important role for LB Prdx6 in the degradation and remodeling of lung surfactant phosphatidylcholine.


Assuntos
Mutação de Sentido Incorreto , Peroxirredoxina VI , Fosfatidilcolinas/biossíntese , Surfactantes Pulmonares/metabolismo , Mucosa Respiratória/enzimologia , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Transgênicos , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Fosfatidilcolinas/genética , Estrutura Terciária de Proteína , Transporte Proteico/genética
11.
Biochim Biophys Acta ; 1864(4): 419-25, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26723227

RESUMO

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase and phospholipase A2 (PLA2) activities. This protein participates in the degradation and remodeling of internalized dipalmitoylphosphatidylcholine (DPPC), the major phospholipid component of lung surfactant. We have shown previously that the PLA2 activity of Prdx6 is inhibited by the lung surfactant-associated protein called surfactant protein A (SP-A) through direct protein-protein interaction. Docking of SPA and Prdx6 was modeled using the ZDOCK (zlab.bu.edu) program in order to predict molecular sites for binding of the two proteins. The predicted peptide sequences were evaluated for binding to the opposite protein using isothermal titration calorimetry and circular dichroism measurement followed by determination of the effect of the SP-A peptide on the PLA2 activity of Prdx6. The sequences 195EEEAKKLFPK204.in the Prdx6 helix and 83DEELQTELYEIKHQIL99 in SP-A were identified as the sites for hydrophobic interaction and H(+)-bonding between the 2 proteins. Treatment of mouse endothelial cells with the SP-A peptide inhibited their recovery from lipid peroxidation associated with oxidative stress indicating inhibition of Prdx6 activity by the peptide in the intact cell.


Assuntos
Peroxirredoxina VI/química , Proteína A Associada a Surfactante Pulmonar/química , Sequência de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Dados de Sequência Molecular , Fosfolipases A2/metabolismo
12.
Free Radic Biol Med ; 87: 356-65, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26117327

RESUMO

Phospholipids are a major structural component of all cell membranes; their peroxidation represents a severe threat to cellular integrity and their repair is important to prevent cell death. Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activity, plays a critical role in antioxidant defense of the lung and other organs. We investigated the role of Prdx6 in the repair of peroxidized cell membranes in pulmonary microvascular endothelial cells (PMVEC) and isolated mouse lungs treated with tert-butyl hydroperoxide and lungs from mice exposed to hyperoxia (100% O(2)). Lipid peroxidation was evaluated by measurement of thiobarbituric acid reactive substances, oxidation of diphenyl-1-pyrenylphosphine, or ferrous xylenol orange assay. The exposure dose was varied to give a similar degree of lipid peroxidation at the end of exposure in the different models. Values for lipid peroxidation returned to control levels within 2 h after oxidant removal in wild-type PMVEC and perfused lungs but were unchanged in Pxdx6 null preparations. An intermediate degree of repair was observed with PMVEC and lungs that expressed only C47S or D140A mutant Prdx6; the former mutant does not have peroxidase activity, while the latter loses its PLA(2) activity. Prdx6 null mice showed markedly delayed recovery from lipid peroxidation during 20 h observation following exposure to hyperoxia. Thus, Prdx6 plays a critical role in the repair of peroxidized phospholipids in cell membranes and the recovery of lung cells from peroxidative stress; the peroxidase and PLA(2) activity each contribute to the recovery process.


Assuntos
Peroxidação de Lipídeos/genética , Pulmão/metabolismo , Estresse Oxidativo/genética , Peroxirredoxina VI/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxidases/metabolismo , Peroxirredoxina VI/genética , Fosfolipases A2/metabolismo , terc-Butil Hidroperóxido/administração & dosagem
13.
Antioxid Redox Signal ; 20(3): 391-402, 2014 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-23815338

RESUMO

AIMS: Peroxiredoxin 6 (Prdx6), a 1-cys Prdx has both peroxidase and phospholipase A2 activities, protecting against oxidative stress and regulating pulmonary surfactant phospholipid metabolism. This study determined the mechanism by which keratinocyte growth factor (KGF) and the glucocorticoid analogue, dexamethasone (Dex), induce increased Prdx6 expression. RESULTS: Transcriptional activation by KGF in both A549 lung adenocarcinoma cells and rat lung alveolar epithelial type II (ATII) cells utilizes an antioxidant response element (ARE), located between 357 and 349 nucleotides before the PRDX6 translational start, that is also necessary for upregulation of the human PRDX6 promoter in response to oxidative stress. Activation is mediated by binding of the transcription factor, Nrf2, to the ARE as shown by experiments using siRNA against Nrf2 and by transfecting ATII cells isolated from lungs of Nrf2 null mice. KGF triggers the migration of Nrf2 from cytoplasm to nucleus where it binds to the PRDX6 promoter as shown by chromatin immunoprecipitation assays. Activation of transcription by Dex occurs through a glucocorticoid response element located about 750 nucleotides upstream of the PRDX6 translational start. INNOVATION: This study demonstrates that KGF can activate an ARE in a promoter without reactive oxygen species involvement and that KGF and Dex can synergistically activate the PRDX6 promoter and protect cells from oxidative stress. CONCLUSION: These two different activators work through different DNA elements. Their combined effect on transcription of the reporter gene is synergistic; however, at the protein level, the combined effect is additive and protects cells from oxidative damage.


Assuntos
Fator 7 de Crescimento de Fibroblastos/metabolismo , Peroxirredoxina VI/biossíntese , Ativação Transcricional/genética , Animais , Elementos de Resposta Antioxidante/genética , Dexametasona/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxina VI/metabolismo , Alvéolos Pulmonares/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo
14.
Antioxid Redox Signal ; 20(6): 872-86, 2014 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-23758611

RESUMO

AIMS: We reported earlier that ischemia results in the generation of reactive oxygen species (ROS) via the closure of a K(ATP) channel which causes membrane depolarization and NADPH oxidase 2 (NOX2) activation. This study was undertaken to understand the role of ischemia-mediated ROS in signaling. RESULTS: Angiogenic potential of pulmonary microvascular endothelial cells (PMVEC) was studied in vitro and in the hind limb in vivo. Flow adapted PMVEC injected into a Matrigel matrix showed significantly higher tube formation than cells grown under static conditions or cells from mice with knockout of K(ATP) channels or the NOX2. Blocking of hypoxia inducible factor-1 alpha (HIF-1α) accumulation completely abrogated the tube formation in wild-type (WT) PMVEC. With ischemia in vivo (femoral artery ligation), revascularization was high in WT mice and was significantly decreased in mice with knockout of K(ATP) channel and in mice orally fed with a K(ATP) channel agonist. In transgenic mice with endothelial-specific NOX2 expression, the revascularization observed was intermediate between that of WT and knockout of K(ATP) channel or NOX2. Increased HIF-1α activation and vascular endothelial growth factor (VEGF) expression was observed in ischemic tissue of WT mice but not in K(ATP) channel and NOX2 null mice. Revascularization could be partially rescued in K(ATP) channel null mice by delivering VEGF into the hind limb. INNOVATION: This is the first report of a mechanosensitive ion channel (K(ATP) channel) initiating endothelial signaling that drives revascularization. CONCLUSION: The K(ATP) channel responds to the stop of flow and activates signals for revascularization to restore the impeded blood flow.


Assuntos
Canais KATP/metabolismo , Mecanotransdução Celular/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Heme Oxigenase-1/metabolismo , Humanos , Hipóxia/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
15.
Am J Physiol Lung Cell Mol Physiol ; 305(11): L805-18, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24077950

RESUMO

We showed that stop of flow triggers a mechanosignaling cascade that leads to the generation of reactive oxygen species (ROS); however, a mechanosensor coupled to the cytoskeleton that could potentially transduce flow stimulus has not been identified. We showed a role for KATP channel, caveolae (caveolin-1), and NADPH oxidase 2 (NOX2) in ROS production with stop of flow. Based on reports of a mechanosensory complex that includes platelet endothelial cell adhesion molecule-1 (PECAM-1) and initiates signaling with mechanical force, we hypothesized that PECAM-1 could serve as a mechanosensor in sensing disruption of flow. Using lungs in situ, we observed that ROS production with stop of flow was significantly reduced in PECAM-1(-/-) lungs compared with lungs from wild-type (WT) mice. Lack of PECAM-1 did not affect NOX2 activation machinery or the caveolin-1 expression or caveolae number in the pulmonary endothelium. Stop of flow in vitro triggered an increase in angiogenic potential of WT pulmonary microvascular endothelial cells (PMVEC) but not of PECAM-1(-/-) PMVEC. Obstruction of flow in lungs in vivo showed that the neutrophil infiltration as observed in WT mice was significantly lowered in PECAM-1(-/-) mice. With stop of flow, WT lungs showed higher expression of the angiogenic marker VEGF compared with untreated (sham) and PECAM-1(-/-) lungs. Thus PECAM-1 (and caveolae) are parts of the mechanosensing machinery that generates superoxide with loss of shear; the resultant ROS potentially drives neutrophil influx and acts as an angiogenic signal.


Assuntos
Cavéolas/metabolismo , Endotélio Vascular/fisiologia , Pulmão/irrigação sanguínea , Glicoproteínas de Membrana/metabolismo , Microvasos/fisiologia , NADPH Oxidases/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Angiopoietina-2/fisiologia , Animais , Caveolina 1/genética , Caveolina 1/metabolismo , Células Cultivadas , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Ativação Enzimática , Expressão Gênica , Técnicas In Vitro , Pulmão/enzimologia , Masculino , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/citologia , NADPH Oxidase 2 , Neovascularização Fisiológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Fluxo Sanguíneo Regional , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
Am J Physiol Heart Circ Physiol ; 302(1): H105-14, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22003059

RESUMO

Loss of fluid shear stress (ischemia) to the lung endothelium causes endothelial plasma membrane depolarization via ATP-sensitive K(+) (K(ATP)) channel closure, initiating a signaling cascade that leads to NADPH oxidase (NOX2) activation and ROS production. Since wortmannin treatment significantly reduces ROS production with ischemia, we investigated the role of phosphoinositide 3-kinase (PI3K) in shear-associated signaling. Pulmonary microvascular endothelial cells in perfused lungs subjected to abrupt stop of flow showed membrane depolarization and ROS generation. Stop of flow in flow-adapted mouse pulmonary microvascular endothelial cells in vitro resulted in the activation of PI3K and Akt as well as ROS generation. ROS generation in the lungs in situ was almost abolished by the PI3K inhibitor wortmannin and the PKC inhibitor H7. The combination of the two (wortmannin and H7) did not have a greater effect. Activation of NOX2 was greatly diminished by wortmannin, knockout of Akt1, or dominant negative PI3K, whereas membrane depolarization was unaffected. Ischemia-induced Akt activation (phosphorylation) was not observed with K(ATP) channel-null cells, which showed minimal changes in membrane potential with ischemia. Activation of Akt was similar to wild-type cells in NOX2-null cells, which do not generate ROS with ischemia. Cromakalim, a K(ATP) channel agonist, prevented both membrane depolarization and Akt phosphorylation with ischemia. Thus, Akt1 phosphorylation follows cell membrane depolarization and precedes the activation of NOX2. These results indicate that PI3K/Akt and PKC serve as mediators between endothelial cell membrane depolarization and NOX2 assembly.


Assuntos
Células Endoteliais/enzimologia , Isquemia/enzimologia , Pulmão/irrigação sanguínea , Microvasos/enzimologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Isquemia/genética , Masculino , Glicoproteínas de Membrana/metabolismo , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/efeitos dos fármacos , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Neuropeptídeos/metabolismo , Perfusão , Fosfatidilinositol 3-Quinase/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP
17.
Wound Repair Regen ; 19(2): 149-61, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21362081

RESUMO

Diabetic patients undergoing hyperbaric oxygen therapies (HBO(2)T) for refractory lower extremity neuropathic ulcers exhibit more than a twofold elevation (p=0.004) in circulating stem cells after treatments and the post-HBO(2)T CD34(+) cell population contains two- to threefold higher levels of hypoxia inducible factors-1, -2, and -3, as well as thioredoxin-1 (p<0.003), than cells present in blood before HBO(2)T. Skin margins obtained from 2-day-old abdominal wounds exhibit higher expression of CD133, CD34, hypoxia inducible factor-1, and Trx-1 vs. margins from refractory lower extremity wounds and expression of these proteins in all wounds is increased due to HBO(2)T (p<0.003). HBO(2)T is known to mobilize bone marrow stem cells by stimulating nitric oxide synthase. We found that nitric oxide synthase activity is acutely increased in patients' platelets following HBO(2)T and remains elevated for at least 20 hours. We conclude that HBO(2) T stimulates vasculogenic stem cell mobilization from bone marrow of diabetics and more cells are recruited to skin wounds.


Assuntos
Pé Diabético/terapia , Oxigenoterapia Hiperbárica , Células-Tronco/fisiologia , Cicatrização/fisiologia , Biópsia por Agulha , Plaquetas/enzimologia , Movimento Celular , Pé Diabético/patologia , Pé Diabético/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/sangue
18.
Am J Physiol Cell Physiol ; 300(6): C1430-41, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21346153

RESUMO

Peroxiredoxin 6 (Prdx6), a bifunctional protein with GSH peroxidase and lysosomal-type phospholipase A(2) activities, has been localized to both cytosolic and acidic compartments (lamellar bodies and lysosomes) in lung alveolar epithelium. We postulate that Prdx6 subcellular localization affects the balance between the two activities. Immunostaining localized Prdx6 to lysosome-related organelles in the MLE12 and A549 alveolar epithelial cell lines. Inhibition of trafficking by brefeldin A indicated processing of the protein through the vesicular pathway. Trafficking of Prdx6 was decreased by inhibitors of PKC, ERK, and p38 MAPK. Immunocytochemistry, immunoprecipitation, and an in situ proximity ligation assay (Duolink) showed that binding of the lysosomal targeting sequence of Prdx6 (amino acids 31-40) to 14-3-3ε was dependent on activity of PKC, ERK, and p38 MAPK. Knockdown of 14-3-3ε with siRNA inhibited the lysosomal targeting of Prdx6. In vitro study with recombinant proteins by pull-down assay and surface plasmon resonance confirmed the interaction of Prdx6 and 14-3-3ε. These findings suggest that ERK and p38 MAPK regulate subcellular localization of Prdx6 by activation of 14-3-3ε as a chaperone protein, resulting in its translocation to acidic organelles.


Assuntos
Proteínas 14-3-3/metabolismo , Lisossomos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peroxirredoxina VI/metabolismo , Linhagem Celular , Inibidores Enzimáticos/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Frações Subcelulares/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
Genes Chromosomes Cancer ; 49(3): 282-97, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20013896

RESUMO

Specificity protein 1 (SP1) is an essential transcription factor implicated in the regulation of genes that control multiple cellular processes, including cell cycle, apoptosis, and DNA damage. Very few nontranscriptional roles for SP1 have been reported thus far. Using confocal microscopy and centrosome fractionation, we identified SP1 as a centrosomal protein. Sp1-deficient mouse embryonic fibroblasts and cells depleted of SP1 by RNAi have increased centrosome number associated with centriole splitting, decreased microtubule nucleation, chromosome misalignment, formation of multipolar mitotic spindles and micronuclei, and increased incidence of aneuploidy. Using mass spectrometry, we identified P70S6K, an effector of the mTOR/raptor (mTORC1) kinase complex, as a novel interacting protein of SP1. We found that SP1-deficient cells have increased phosphorylation of the P70S6K effector ribosomal protein S6, suggesting that SP1 participates in the regulation of the mTORC1/P70S6K/S6 signaling pathway. We previously reported that aberrant mTORC1 activation leads to supernumerary centrosomes, a phenotype rescued by the mTORC1 inhibitor rapamycin. Similarly, treatment with rapamycin rescued the multiple centrosome phenotype of SP1-deficient cells. Taken together, these data strongly support the hypothesis that SP1 is involved in the control of centrosome number via regulation of the mTORC1 pathway, and predict that loss of SP1 function can lead to aberrant centriole splitting, deregulated mTORC1 signaling, and aneuploidy, thereby contributing to malignant transformation.


Assuntos
Centríolos/fisiologia , Instabilidade Cromossômica , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição Sp1/metabolismo , Células 3T3 , Animais , Apoptose , Ciclo Celular , Linhagem Celular , Centrossomo/fisiologia , Centrossomo/ultraestrutura , Dano ao DNA , Regulação da Expressão Gênica , Células HeLa , Humanos , Camundongos , Interferência de RNA/fisiologia , Fator de Transcrição Sp1/deficiência , Fator de Transcrição Sp1/genética , Serina-Treonina Quinases TOR
20.
Am J Physiol Lung Cell Mol Physiol ; 297(5): L871-80, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19700648

RESUMO

Peroxiredoxin 6 (Prdx6), an enzyme with glutathione peroxidase and PLA2 (aiPLA2) activities, is highly expressed in respiratory epithelium, where it participates in phospholipid turnover and antioxidant defense. Prdx6 has been localized by immunocytochemistry and subcellular fractionation to acidic organelles (lung lamellar bodies and lysosomes) and cytosol. On the basis of their pH optima, we have postulated that protein subcellular localization determines the balance between the two activities of Prdx6. Using green fluorescent protein-labeled protein expression in alveolar epithelial cell lines, we showed Prdx6 localization to organellar structures resembling lamellar bodies in mouse lung epithelial (MLE-12) cells and lysosomes in A549 cells. Localization within lamellar bodies/lysosomes was in the luminal compartment. Targeting to lysosome-like organelles was abolished by the deletion of amino acids 31-40 from the Prdx6 NH2-terminal region; deletion of the COOH-terminal region had no effect. A green fluorescent protein-labeled peptide containing only amino acids 31-40 showed lysosomal targeting that was abolished by mutation of S32 or G34 within the peptide. Studies with mutated protein indicated that lipid binding was not necessary for Prdx6 targeting. This peptide sequence has no homology to known organellar targeting motifs. These studies indicate that the localization of Prdx6 in acidic organelles and consequent PLA2 activity depend on a novel 10-aa peptide located at positions 31-40 of the protein.


Assuntos
Células Epiteliais/metabolismo , Pulmão/citologia , Lisossomos/metabolismo , Peroxirredoxina VI/química , Peroxirredoxina VI/metabolismo , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Epiteliais/citologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lipossomos/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Proteínas Mutantes , Mutação/genética , Peptídeos/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade
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