RESUMEN
Defective superoxide production by NADPH oxidase 2 (Nox2) in phagocyte cells results in the development of chronic granulomatous disease (CGD), a hereditary disease characterized by recurrent and life-threatening infections. The partner protein p22phox is a membrane-spanning protein which forms a stable heterodimer with Nox2 in the endoplasmic reticulum. This interaction ensures the stability of each protein and their accurate trafficking to the cell membrane. The present paper describes the characterization of p22phox missense mutations that were identified in a patient with CGD who presented with undetectable levels of p22phox . Using a reconstitution system, it was found that p22phox expression decreased when R90Q, A117E, S118R, A124S, A124V, A125T, or E129K mutations were introduced, suggesting that these mutations destabilize the protein. In contrast, introducing an L105R mutation did not affect protein expression, but did inhibit p22phox binding to Nox2. Thus, the missense mutations discussed here contribute to the development of CGD by either disrupting protein stability or by impairing the interaction between p22phox and Nox2.
Asunto(s)
NADPH Oxidasas , Cricetulus , Animales , Línea Celular , Humanos , NADPH Oxidasas/química , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Mutación Missense , NADPH Oxidasa 2/metabolismoRESUMEN
Pancreatic cancer is recalcitrant to treatment as it is highly metastatic and rapidly progressive. While observing the behavior of human pancreatic BxPC-3 cells using an optical assay device called TAXIScan, we found that several synthetic pyrazole and pyrimidine derivatives inhibited cell migration. One such compound, 14-100, inhibited metastasis of fluorescence-labeled BxPC-3 cells, which were transplanted into the pancreas of nude mice as a subcutaneously grown cancer fragment. Surprisingly, despite its low cytotoxicity, the compound also showed an inhibitory effect on cancer cell proliferation in vivo, suggesting that the compound alters cancer cell characteristics needed to grow in situ. Single-cell RNA-sequencing revealed changes in gene expression associated with metastasis, angiogenesis, inflammation, and epithelial-mesenchymal transition. These data suggest that the compound 14-100 could be a good drug candidate against pancreatic cancer.
Asunto(s)
Quimiotaxis , Neoplasias Pancreáticas , Ratones , Animales , Humanos , Ratones Desnudos , Línea Celular Tumoral , Movimiento Celular , Neoplasias Pancreáticas/patología , Páncreas/patología , Transformación Celular Neoplásica , Pirazoles/farmacología , Pirazoles/uso terapéutico , ARN , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Neoplasias PancreáticasRESUMEN
The transmembrane protein p22phox heterodimerizes with NADPH oxidase (Nox) 1-4 and is essential for the reactive oxygen species-producing capacity of oxidases. Missense mutations in the p22phox gene prevent the formation of phagocytic Nox2-based oxidase, which contributes to host defense. This results in chronic granulomatous disease (CGD), a severe primary immunodeficiency syndrome. In this study, we characterized missense mutations in p22phox (L51Q, L52P, E53V, and P55R) in the A22° type (wherein the p22phox protein is undetectable) of CGD. We demonstrated that these substitutions enhanced the degradation of the p22phox protein in the endoplasmic reticulum (ER) and the binding of p22phox to Derlin-1, a key component of ER-associated degradation (ERAD). Therefore, the L51-L52-E53-P55 sequence is responsible for protein stability in the ER. We observed that the oxidation of the thiol group of Cys-50, which is adjacent to the L51-L52-E53-P55 sequence, suppressed p22phox degradation. However, the suppression effect was markedly attenuated by the serine substitution of Cys-50. Blocking the free thiol of Cys-50 by alkylation or C50S substitution promoted the association of p22phox with Derlin-1. Derlin-1 depletion partially suppressed the degradation of p22phox mutant proteins. Furthermore, heterodimerization with p22phox (C50S) induced rapid degradation of not only Nox2 but also nonphagocytic Nox4 protein, which is responsible for redox signaling. Thus, the redox-sensitive Cys-50 appears to determine whether p22phox becomes a target for degradation by the ERAD system through its interaction with Derlin-1.
Asunto(s)
Enfermedad Granulomatosa Crónica , Proteínas de la Membrana , NADPH Oxidasas , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Mutantes , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Serina , Compuestos de SulfhidriloRESUMEN
Superoxide-producing NADPH oxidase, gp91phox/NOX2, in phagocytes plays a critical role in the host defenses against pathogens. Moreover, gp91phox/NOX2 contributes to the oxidative stress in endothelial cells. Therefore, investigating the level of gp91phox/NOX2 with immunoblotting is important for estimating the amount of superoxide produced. Here, we showed that the epitopes in human gp91phox/NOX2 recognized by monoclonal antibodies (mAbs) CL-5 and 48 were in amino acids 132-147 and 136-144, respectively. Although the epitopes were close to the N-glycosylation sites, N-glycan maturation did not affect mAbs recognition. When Pro-136 was substituted with Arg, the corresponding mouse residue, human gp91phox/NOX2 was not recognized by mAbs CL-5 and 48; however, the substitution did not affect gp91phox/NOX2-based oxidase activity. This finding explains why these mAbs specifically recognize the human but not mouse gp91phox/NOX2. Hence, these mAbs are useful for investigating the level of gp91phox/NOX2 without amino acid substitutions in the epitopes.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Mapeo Epitopo , Epítopos , NADPH Oxidasa 2/inmunología , Animales , Especificidad de Anticuerpos , Células CHO , Células COS , Chlorocebus aethiops , Cricetulus , Glicosilación , Células HL-60 , Humanos , Ratones , NADPH Oxidasa 2/genética , NADPH Oxidasa 2/metabolismo , Procesamiento Proteico-Postraduccional , Células RAW 264.7 , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Superóxidos/metabolismoRESUMEN
An interaction between acute myeloid leukemia (AML) cells and endothelial cells in the bone marrow seems to play a critical role in chemosensitivity on leukemia treatment. The endothelial niche reportedly enhances the paracrine action of the soluble secretory proteins responsible for chemoresistance in a vascular endothelial growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR-2) signaling pathway-dependent manner. To further investigate the contribution of VEGF-A/VEGFR-2 signaling to the chemoresistance of AML cells, a biochemical assay system in which the AML cells were cocultured with human endothelial EA.hy926 cells in a monolayer was developed. By coculture with EA.hy926 cells, this study revealed that the AML cells resisted apoptosis induced by the anticancer drug cytarabine. SU4312, a VEGFR-2 inhibitor, attenuated VEGFR-2 phosphorylation and VEGF-A/VEGFR-2 signaling-dependent endothelial cell migration; thus, this inhibitor was observed to block VEGF-A/VEGFR-2 signaling. Interestingly, this inhibitor did not reverse the chemoresistance. When VEGFR-2 was knocked out in EA.hy926 cells using the CRISPR-Cas9 system, the cytarabine-induced apoptosis of AML cells did not significantly change compared with that of wild-type cells. Thus, coculture-induced chemoresistance appears to be independent of VEGF-A/VEGFR-2 signaling. When the transwell, a coculturing device, separated the AML cells from the EA.hy926 cells in a monolayer, the coculture-induced chemoresistance was inhibited. Given that the migration of VEGF-A/VEGFR-2 signaling-dependent endothelial cells is necessary for the endothelial niche formation in the bone marrow, VEGF-A/VEGFR-2 signaling contributes to chemoresistance by mediating the niche formation process, but not to the chemoresistance of AML cells in the niche.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Citarabina/farmacología , Resistencia a Antineoplásicos/genética , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Inhibidores de la Angiogénesis/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular , Movimiento Celular/efectos de los fármacos , Técnicas de Cocultivo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Regulación Leucémica de la Expresión Génica , Técnicas de Inactivación de Genes , Células HL-60 , Humanos , Indoles/farmacología , Células Jurkat , Células K562 , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Modelos Biológicos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neovascularización Patológica/prevención & control , Fosforilación , Transducción de Señal , Células U937 , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/deficienciaRESUMEN
NADPH oxidase (Nox) 4 produces H2O2 by forming a heterodimer with p22phox and is involved in hemangioendothelioma development through monocyte chemoattractant protein-1 (MCP-1) upregulation. Here, we show that Nox4 protein levels were maintained by p22phox in hemangioendothelioma cells and Nox4 protein stability was dependent on p22phox coexpression. Conversely, the degradation of Nox4 monomer was enhanced by p22phox knockdown. Under hypoxic conditions in hemangioendothelioma cells, p22phox was downregulated at the mRNA and protein levels. Downregulation of p22phox protein resulted in the enhanced degradation of Nox4 protein in hypoxia-treated hemangioendothelioma cells. In contrast, Nox2, a Nox isoform, was not altered at the protein level under hypoxic conditions. Nox2 exhibited a higher affinity for p22phox compared with Nox4, suggesting that when coexpressed with Nox4 in the same cells, Nox2 acts as a competitor. Nox2 knockdown restored Nox4 protein levels partially reduced by hypoxic treatment. Thus, Nox4 protein levels were attenuated in hypoxia-treated cells resulting from p22phox depletion. MCP-1 secretion was decreased concurrently with hypoxia-induced Nox4 downregulation compared with that under normoxia.
Asunto(s)
Hemangioendotelioma , NADPH Oxidasas , Animales , Humanos , Ratones , Regulación hacia Abajo , Peróxido de Hidrógeno/metabolismo , Hipoxia/genética , NADPH Oxidasa 4/genética , NADPH Oxidasa 4/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Estabilidad Proteica , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Superoxide producing NADPH oxidase 1 (Nox1), abundantly expressed in the colon epithelium, plays a crucial role in mucosal host defenses. In this study, we found that pre-treatment of cells with edaravone, a free radical scavenger, inhibited Nox1 constitutive activity even after washout without affecting Nox1 trafficking to the plasma membrane and membrane recruitment of the cytosolic regulators Noxo1 and Noxa1. These results suggest that a Nox1-derived product is involved in the step that initiates the electron transfer reaction after the formation of the Nox1-Noxo1-Noxa1 complex. Furthermore, we show that the mean migration directionality and velocity of epithelial cells were significantly enhanced by the inhibition of constitutive Nox1 activity. Thus, the constitutive Nox1 activity limits undesired cell migration in resting cells while participating in a positive feedback loop toward its own oxidase activity.
Asunto(s)
Células Epiteliales/metabolismo , NADPH Oxidasa 1/metabolismo , Movimiento Celular , Humanos , Especies Reactivas de Oxígeno , TransfecciónRESUMEN
Directed migration of endothelial cells (ECs) is an important process during both physiological and pathological angiogenesis. The binding of vascular endothelial growth factor (VEGF) to VEGF receptor-2 (VEGFR-2) on the EC surface is necessary for directed migration of these cells. Here, we used TAXIScan, an optically accessible real-time horizontal cell dynamics assay approach, and demonstrate that reactive oxygen species (ROS)-producing NADPH oxidase 4 (NOX4), which is abundantly expressed in ECs, mediates VEGF/VEGFR-2-dependent directed migration. We noted that a continuous supply of endoplasmic reticulum (ER)-retained VEGFR-2 to the plasma membrane is required to maintain VEGFR-2 at the cell surface. siRNA-mediated NOX4 silencing decreased the ER-retained form of VEGFR-2, resulting in decreased cell surface expression levels of the receptor. We also found that ER-localized NOX4 interacts with ER-retained VEGFR-2 and thereby stabilizes this ER-retained form at the protein level in the ER. We conclude that NOX4 contributes to the directed migration of ECs by maintaining VEGFR-2 levels at their surface.
Asunto(s)
Movimiento Celular , Células Endoteliales/citología , NADPH Oxidasa 4/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Línea Celular , Retículo Endoplásmico/metabolismo , Células Endoteliales/metabolismo , Células HeLa , Humanos , Estabilidad Proteica , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Human phagocyte flavocytochrome b558 (Cyt b), the catalytic center of nicotinamide adenine dinucleotide phosphate oxidase, consists of a heavily glycosylated large subunit (gp91phox ; Nox2) and a small subunit (p22phox ). Cyt b is a membrane-spanning complex enzyme. Chronic granulomatous disease (CGD) is predominantly caused by a mutation in the CYBB gene encoding gp91phox on the X-chromosome. Because the phagocytes of patients with CGD are not able to generate the superoxide anion, these patients are susceptible to severe infections that can be fatal. It has been suggested that the extracellular region of gp91phox is necessary for and critical to forming the epitope of mAb 7D5 and that 7D5 provides a useful tool for rapid screening of X-linked CGD by FACS. To further elucidate the mAb 7D5 epitope on human gp91phox , chimeric DNA expressed human and mouse gp91phox recombinant protein were constructed. The fusion proteins were immunostained for mAb 7D5 and analyzed by FACS and western blot analysis. The 143 ELGDRQNES151 region was found to reside at the extracellular surface on human gp91phox and to be an important epitope for the interaction with mAb 7D5, as analyzed by FACS analysis. In particular, amino acid R147 is a unique epitope on the membrane-associated Cyt b for mAb 7D5. In conclusion, it is proposed that FACS analysis using mAb 7D5 is a valuable tool for early diagnosis of CGD.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Grupo Citocromo b/inmunología , Epítopos/inmunología , NADPH Oxidasa 2/inmunología , NADPH Oxidasas/inmunología , Fagocitos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Línea Celular , Epítopos/análisis , Epítopos/química , Enfermedad Granulomatosa Crónica/inmunología , Células HL-60 , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Ratones , Mutación , NADPH Oxidasa 2/biosíntesis , NADPH Oxidasa 2/química , NADPH Oxidasa 2/genética , Dominios Proteicos , Células RAW 264.7 , Proteínas Recombinantes de Fusión/genética , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Superóxidos/metabolismoRESUMEN
The histone acetyltransferase p300/CBP-associated factor (PCAF) catalyzes acetylation of core histones and plays important roles in epigenetics by altering the chromatin structure in vertebrates. In this study, PCAF-deficient DT40 mutants were analyzed and it was found that PCAF participates in regulation of secretory IgM heavy chain (H-chain) synthesis. Remarkably, PCAF-deficiency causes an increase in the amount of secretory IgM H-chain mRNA, but not in that of IgM light chain and membrane-bound IgM H-chain mRNAs, resulting in dramatic up-regulation of the amount of secretory IgM protein. These findings suggest that PCAF regulates soluble antibody production and is thus an effective suppressor of secretory IgM H-chain synthesis.
Asunto(s)
Regulación hacia Abajo , Inmunoglobulina M/biosíntesis , Células Precursoras de Linfocitos B/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Animales , Línea Celular , Pollos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulina M/genética , Células Precursoras de Linfocitos B/enzimología , Factores de Transcripción p300-CBP/genéticaRESUMEN
A novel alanine:glyoxylate aminotransferase (AGT) mutation involved in primary hyperoxaluria type 1 (PH1) was studied in Japanese patients. Two mutations in exon 7, c.751T>A and c.752G>A, lead to a W251K amino acid substitution. Proband 1 (patient 1) was homozygous for the W251K mutation allele (DDBJ Accession No. AB292648), and AGT-specific activity in the patient's liver was very low. To reveal the cause of the low enzymatic activity, the intracellular localization of AGT (W251K) was studied using immunohistochemistry and immunoelectron microscopy. The latter analysis showed that patient 2 had only one-fifth of the normal AGT expression per catalase, suggesting impairment of AGT (W251K) dependent transport into peroxisomes. Peroxisomal transport of human AGT is believed to be dependent on the presence of the type 1 peroxisomal targeting sequence. The C-terminal tripeptide of AGT, KKL is necessary for peroxisomal targeting. In cultured cells, EGFP-AGT (W251K) localized both in the peroxisome and cytosol. These results were consistent with the data obtained from liver analysis of patient 2. The subcellular distribution of AGT (W251K) and the results from a random mutagenesis study suggest that KKL is necessary for peroxisomal targeting of human AGT, but additional signal other than KKL may be necessary.
RESUMEN
Reactive oxygen species (ROS) produced by neutrophils are crucial for defense against infectious diseases, and the adequate measurement of ROS levels is an important way to evaluate the possibility of infections. The fluorescent probe dihydrorhodamine 123 has been applied exclusively to the measurement of ROS thus far. We developed a novel method for detecting ROS, which utilizes the chemiluminescent probes Luminol and Diogenes. The new method quantitatively detects ROS produced by as few as 10 to 10(4) neutrophils. Furthermore, this method can detect ROS levels in one microliter of whole blood or ROS produced by Epstein-Barr immortalized B lymphocytes. This method will be valuable for prompt diagnosis of neonatal chronic granulomatous diseases in which neutrophils aberrantly produce superoxide.