RESUMO
The occurrence of intercellular channels formed by pannexin1 has been challenged for more than a decade. Here, we provide an electrophysiological characterization of exogenous human pannexin1 (hPanx1) cellcell channels expressed in HeLa cells knocked out for connexin45. The observed hPanx1 cellcell channels show two phenotypes: O-state and S-state. The former displayed low transjunctional voltage (Vj) sensitivity and single-channel conductance of â¼175 pS, with a substate of â¼35 pS; the latter showed a peculiar dynamic asymmetry in Vj dependence and single-channel conductance identical to the substate conductance of the O-state. S-state hPanx1 cellcell channels were also identified between TC620 cells, a human oligodendroglioma cell line that endogenously expresses hPanx1. In these cells, dye and electrical coupling increased with temperature and were strongly reduced after hPanx1 expression was knocked down by small interfering RNA or inhibited with Panx1 mimetic inhibitory peptide. Moreover, cellcell coupling was augmented when hPanx1 levels were increased with a doxycycline-inducible expression system. Application of octanol, a connexin gap junction (GJ) channel inhibitor, was not sufficient to block electrical coupling between HeLa KO Cx45-hPanx1 or TC620 cell pairs. In silico studies suggest that several arginine residues inside the channel pore may be neutralized by hydrophobic interactions, allowing the passage of DAPI, consistent with dye coupling observed between TC620 cells. These findings demonstrate that endogenously expressed hPanx1 forms intercellular cellcell channels and their unique properties resemble those described in innexin-based GJ channels. Since Panx1 is ubiquitously expressed, finding conditions to recognize Panx1 cellcell channels in different cell types might require special attention.
Assuntos
Conexinas , Proteínas do Tecido Nervoso , Animais , Conexinas/metabolismo , Humanos , Canais Iônicos , Mamíferos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
Air pollution, a global health concern, has been associated with adverse effects on human health. In particular, particulate matter (PM), which is a major contributor to air pollution, impacts various organ systems including the skins. In fact, PM has been suggested as a culprit for accelerating skin aging and pigmentation. In this study, using single-cell RNA sequencing, IL-24 was found to be highly upregulated among the differentially expressed genes commonly altered in keratinocytes and fibroblasts of ex vivo skins exposed to PM. It was verified that PM exposure triggered the expression of IL-24 in keratinocytes, which subsequently led to a decrease in type I procollagen expression and an increase in MMP1 expression in fibroblasts. Furthermore, long-term treatment of IL-24 induced cellular senescence in fibroblasts. Through high-throughput screening, we identified chemical compounds that inhibit the IL-24-STAT3 signaling pathway, with lovastatin being the chosen candidate. Lovastatin not only effectively reduced the expression of IL24 induced by PM in keratinocytes but also exhibited a capacity to restore the decrease in type I procollagen and the increase in MMP1 caused by IL-24 in fibroblasts. This study provides insights into the significance of IL-24, illuminating mechanisms behind PM-induced skin aging, and proposes IL-24 as a promising target to mitigate PM-associated skin aging.
RESUMO
Nephrin is a type-1 transmembrane protein and a component of the slit diaphragm renal-filtration barrier. It has several functions in actin remodeling and cell-cell adhesion. Nephrin is principally located in the kidney glomerulus, but several studies have reported that nephrin is found in the pancreas, brain, and placenta. However, nephrin expression and its role in human skin have not yet been reported. First, using single-cell RNA sequencing, immunohistochemistry, and immuno-electron microscopy, nephrin expression was confirmed in human-skin epidermal keratinocytes. Nephrin expression colocalized with the expression of zonula occludens-1 in keratinocytes and was closely related to keratinocyte cell density, proliferation, and migration. High glucose treatment decreased nephrin expression and compromised keratinocyte cell migration without yes-associated protein nuclear entry. This reduced cell migration under high glucose conditions was improved in nephrin-overexpressing keratinocytes. Nephrin was highly expressed on the margins of re-epithelized epidermis based on in vivo mice and ex vivo human skin wound models. The results demonstrate that nephrin is expressed in human-skin keratinocytes and functions in cell adhesion, proliferation, and migration. In conclusion, this study suggests that nephrin may have a variety of physiological roles in human skin.
Assuntos
Epiderme , Queratinócitos , Animais , Movimento Celular/fisiologia , Epiderme/metabolismo , Glucose/metabolismo , Humanos , Queratinócitos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , CamundongosRESUMO
Outer hair cells (OHCs) play an essential role in hearing by acting as a nonlinear amplifier which helps the cochlea detect sounds with high sensitivity and accuracy. This nonlinear sound processing generates distortion products, which can be measured as distortion-product otoacoustic emissions (DPOAEs). The OHC stereocilia that respond to sound vibrations are connected by three kinds of extracellular links: tip links that connect the taller stereocilia to shorter ones and convey force to the mechanoelectrical transduction channels, tectorial membrane-attachment crowns (TM-ACs) that connect the tallest stereocilia to one another and to the overlying TM, and horizontal top connectors (HTCs) that link adjacent stereocilia. While the tip links have been extensively studied, the roles that the other two types of links play in hearing are much less clear, largely because of a lack of suitable animal models. Here, while analyzing genetic combinations of tubby mice, we encountered models missing both HTCs and TM-ACs or HTCs alone. We found that the tubby mutation causes loss of both HTCs and TM-ACs due to a mislocalization of stereocilin, which results in OHC dysfunction leading to severe hearing loss. Intriguingly, the addition of the modifier allele modifier of tubby hearing 1 in tubby mice selectively rescues the TM-ACs but not the HTCs. Hearing is significantly rescued in these mice with robust DPOAE production, indicating an essential role of the TM-ACs but not the HTCs in normal OHC function. In contrast, the HTCs are required for the resistance of hearing to damage caused by noise stress.
Assuntos
Células Ciliadas Auditivas Externas/fisiologia , Ruído , Emissões Otoacústicas Espontâneas/fisiologia , Som , Estimulação Acústica , Animais , Células Ciliadas Auditivas Externas/citologia , Perda Auditiva , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Modelos Animais , Emissões Otoacústicas Espontâneas/genética , Estereocílios/fisiologia , Membrana TectorialRESUMO
KRAS is the most frequently mutated oncogene in human tumors, and its activating mutations represent important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here, we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA. The survival, proliferation, and tumorigenicity of cancer cells in vitro and the growth of tumors in vivo were determined after delivery of Cas9 and guide RNA. We identified guide RNAs that efficiently target mutant KRAS without significant alterations of the wild-type allele. Doxycycline-inducible expression of this guide RNA in KRAS-mutant cancer cells transduced with a lentiviral vector encoding Cas9 disrupted the mutant KRAS gene, leading to inhibition of cancer cell proliferation both in vitro and in vivo. Intra-tumoral injection of lentivirus and adeno-associated virus expressing Cas9 and sgRNA suppressed tumor growth in vivo, albeit incompletely, in immunodeficient mice. Expression of Cas9 and the guide RNA in cells containing wild-type KRAS did not alter cell survival or proliferation either in vitro and in vivo. Our study provides a proof-of-concept that CRISPR can be utilized to target driver mutations of cancers in vitro and in vivo.
RESUMO
CRISPR from Prevotella and Francisella 1 (Cpf1) is an effector endonuclease of the class 2 CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) gene editing system. We developed a method for evaluating Cpf1 activity, based on target sequence composition in mammalian cells, in a high-throughput manner. A library of >11,000 target sequence and guide RNA pairs was delivered into human cells using lentiviral vectors. Subsequent delivery of Cpf1 into this cell library induced insertions and deletions (indels) at the integrated synthetic target sequences, which allowed en masse evaluation of Cpf1 activity by using deep sequencing. With this approach, we determined protospacer-adjacent motif sequences of two Cpf1 nucleases, one from Acidaminococcus sp. BV3L6 (hereafter referred to as AsCpf1) and the other from Lachnospiraceae bacterium ND2006 (hereafter referred to as LbCpf1). We also defined target-sequence-dependent activity profiles of AsCpf1, which enabled the development of a web tool that predicts the indel frequencies for given target sequences (http://big.hanyang.ac.kr/cindel). Both the Cpf1 characterization profile and the in vivo high-throughput evaluation method will greatly facilitate Cpf1-based genome editing.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/genética , Ensaios de Triagem em Larga Escala/métodos , RNA Guia de Cinetoplastídeos , Acidaminococcus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR , Clostridiales/genética , Endonucleases/metabolismo , Francisella/genética , Humanos , Prevotella/genética , Transdução GenéticaRESUMO
O-Glycosylation occurs in recombinant proteins produced by CHO cells, but this phenomenon has not been studied extensively. Here, we report that rituximab is an O-linked N-acetyl-glucosaminylated (O-GlcNAcylated) protein and the production of rituximab is increased by thiamet G, an inhibitor of O-GlcNAcase. The production of rituximab doubled with OGA inhibition and decreased with O-GlcNAc transferase inhibition. O-GlcNAc-specific antibody and metabolic labelling with azidO-GlcNAc confirmed the increased O-GlcNAcylation with thiamet G. Protein mass analysis revealed that serine 7, 12, and 14 of the rituximab light chain were O-GlcNAcylated. S12A mutation of the light chain decreased rituximab stability and failed to increase the production with thiamet G without any significant changes of mRNA level. Cytotoxicity and thermal stability assays confirmed that there were no differences in the biological and physical properties of rituximab produced by thiamet G treatment. Therefore, thiamet G treatment improves the production of rituximab without significantly altering its function.
Assuntos
Mutação de Sentido Incorreto , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Piranos/farmacologia , Rituximab , Tiazóis/farmacologia , Substituição de Aminoácidos , Animais , Células CHO , Cricetulus , Glicosilação/efeitos dos fármacos , Humanos , N-Acetilglucosaminiltransferases/metabolismo , Rituximab/biossíntese , Rituximab/genéticaRESUMO
The plant protein production system is a platform that can not only reduce production costs but also produce monoclonal antibodies that do not have the risk of residual proteins from the host. However, due to the difference between post-translational processes in plants and animals, there may be a modification in the Fab region of the monoclonal antibody produced in the plant; thus, it is necessary to compare the antigen affinity of this antibody with that of the prototype. In this study, ofatumumab, a fully human anti-CD20 IgG1κ monoclonal antibody used for its non-cross resistance to rituximab, was expressed in Nicotiana benthamiana, and its affinities and efficacies were compared with those of native ofatumumab produced from CHO cells. Two forms of plant ofatumumab (with or without HDEL-tag) were generated and their production yields were compared. The HDEL-tagged ofatumumab was more expressed in plants than the form without HDEL-tag. The specificity of the target recognition of plant-derived ofatumumab was confirmed by mCherry-CD20-expressing HEK cells via immuno-staining, and the capping of CD20 after ofatumumab binding was also confirmed using Ramos B cells. In the functional equivalence tests, the binding affinities and complement-dependent cell cytotoxicity efficacy of plant-ofatumumab-HDEL and plant-ofatumumab without HDEL were significantly reduced compared to those of CHO-derived ofatumumab. Therefore, we suggest that although ofatumumab is not a good candidate as a template for plant-derived monoclonal antibodies because of its decreased affinity when produced in plants, it is an interesting target to study the differences between post-translational modifications in mammals and plants.
Assuntos
Anticorpos Monoclonais Humanizados/genética , Fragmentos Fab das Imunoglobulinas/química , Nicotiana/metabolismo , Folhas de Planta/metabolismo , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Antígenos CD20/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose , Linfócitos B , Células CHO , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cricetulus , Citotoxicidade Imunológica/efeitos dos fármacos , Células HEK293 , Humanos , Conformação Proteica , Rituximab/metabolismoRESUMO
We aimed to clarify the key factor determining the effect of beta blocker attenuating high fat diet- induced obesity and bone loss. Six-week-old C57BL/6 male mice were assigned to groups reflecting different relative onset of obesity and beta blocker administration, different diet (control vs. high fat), and treatment (vehicle vs. beta blocker: propranolol). Mice in Group 1 were fed a control diet (CON) or high fat diet (HIGH) with vehicle or propranolol for 12 weeks. Mice in Group 2 were fed a CON or HIGH without pharmaceutical treatment for the first 12 weeks, followed by another 12 weeks of treatment with vehicle or propranolol. Mice in Group 3 were fed a CON without pharmaceutical treatment for the first 12 weeks, followed by stratification into diet-based subgroups and another 12 weeks of treatment with vehicle or propranolol. Propranolol attenuated the HIGH-induced increase in body weight/fat mass in Group 1 mice and in Group 3 mice, but not in Group 2 mice. Propranolol mitigated HIGH-induced reduction in femoral trabecular bone mineral density and bone architecture deterioration in Group 1 mice but not in Group 2 mice. HIGH feeding in Group 3 did not compromise skeletal integrity. Taken together, propranolol attenuates HIGH-induced body weight increases while weight gain is in progress but not once obesity has already been established. HIGH feeding during the growth period results in compromised bone mass/architecture; which can be attenuated by propranolol administration during the growth period, but not by propranolol administration after obesity has already been established.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Doenças Ósseas Metabólicas/tratamento farmacológico , Osso e Ossos/efeitos dos fármacos , Dieta Hiperlipídica , Obesidade/tratamento farmacológico , Animais , Peso Corporal , Densidade Óssea/efeitos dos fármacos , Dieta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Propranolol/farmacologia , Transdução de Sinais , Tíbia/efeitos dos fármacosRESUMO
OBJECTIVE: Myelosuppression is a life-threatening complication of thiopurine therapy, and the incidence of thiopurine-induced myelosuppression is higher in East Asians than in Europeans. We investigated genetic factors associated with thiopurine-induced leukopenia in patients with IBD. DESIGN: A genome-wide association study (GWAS) was conducted in thiopurine-treated patients with IBD, followed by high-throughput sequencing of genes identified as significant in the GWAS or those involved in thiopurine metabolism (n=331). Significant loci associated with thiopurine-induced leukopenia were validated in two additional replication cohorts (n=437 and n=330). Functional consequences of FTO (fat mass and obesity-associated) variant were examined both in vitro and in vivo. RESULTS: The GWAS identified two loci associated with thiopurine-induced leukopenia (rs16957920, FTO intron; rs2834826, RUNX1 intergenic). High-throughput targeted sequencing indicated that an FTO coding variant (rs79206939, p.A134T) linked to rs16957920 is associated with thiopurine-induced leukopenia. This result was further validated in two replication cohorts (combined p=1.3×10-8, OR=4.3). The frequency of FTO p.A134T is 5.1% in Koreans but less than 0.1% in Western populations. The p.A134T variation reduced FTO activity by 65% in the nucleotide demethylase assay. In vivo experiments revealed that Fto-/- and Fto+/- mice were more susceptible to thiopurine-induced myelosuppression than wild-type mice. CONCLUSIONS: The results suggest that the hypomorphic FTO p.A134T variant is associated with thiopurine-induced leukopenia. These results shed light on the novel physiological role of FTO and provide a potential pharmacogenetic biomarker for thiopurine therapy.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Azatioprina/efeitos adversos , Imunossupressores/efeitos adversos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Leucopenia/induzido quimicamente , Mercaptopurina/efeitos adversos , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Idoso , Animais , Azatioprina/uso terapêutico , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Marcadores Genéticos , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunossupressores/uso terapêutico , Doenças Inflamatórias Intestinais/genética , Leucopenia/genética , Masculino , Mercaptopurina/uso terapêutico , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , República da Coreia , Análise de Sequência de DNA , Adulto JovemRESUMO
hK2, a member of the kallikrein protease family encoded by KLK2, is expressed exclusively in prostate and is a putative adjunct tumor marker for prostate cancer screening. The T allele of rs198977, a single nucleotide polymorphism in exon 5 of KLK2, codes for W-hK2 and is associated with lower serum hK2 levels and higher risk of prostate cancer than the C allele encoding R-hK2. To elucidate the mechanism that underlies this SNP's function, we transfected plasmids expressing R-hK2 or W-hK2 into PC3, HeLa and HEK293A cells and measured the hK2 level in cell lysates and conditioned media. The level of W-hK2 was lower than R-hK2 in conditioned media but was not different from R-hK2 in cell lysates. W-hK2 was hardly colocalized with Golgi-targeted fluorescent protein whereas R-hK2 colocalized. Reporter assays related to the unfolded protein response (UPR) and phospho-eIF2α immunoblot showed that W-hK2 increased UPR activity more than R-hK2. These results indicated that W-hK2 had a defect in cellular trafficking from the ER to the Golgi complex due to its misfolding and that it activated the UPR, suggesting a mechanism to explain the association of the T allele with higher prostate cancer risk.
Assuntos
Calicreínas/metabolismo , Resposta a Proteínas não Dobradas , Transporte Biológico Ativo/genética , Estudos de Associação Genética , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Calicreínas/genética , Masculino , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/genéticaRESUMO
Terbinafine is an antifungal agent that selectively inhibits fungal sterol synthesis by blocking squalene epoxidase. We evaluated the effect of terbinafine on gap junctional intercellular communication (GJIC). Fluorescence recovery after photobleaching (FRAP) and I-YFP GJIC assays revealed that terbinafine inhibits GJIC in a reversible and dose-dependent manner in FRT-Cx43 and LN215 cells. Treatment with terbinafine did not affect Cx43 phosphorylation status or intracellular Ca(2+) concentration, well-known action mechanisms of various GJIC blockers. While a structurally related chemical, naftifine, attenuated GJIC, epigallocatechin gallate, another potent squalene epoxidase inhibitor with a different structure, did not. These results suggest that terbinafine inhibits GJIC with a so far unknown mechanism of action.
Assuntos
Antifúngicos/farmacologia , Comunicação Celular/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Naftalenos/farmacologia , Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Conexina 43/genética , Conexina 43/metabolismo , Relação Dose-Resposta a Droga , Humanos , TerbinafinaRESUMO
OBJECTIVES: To provide a simple method to make a stable ΔF508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for ΔF508-CFTR rescue. RESULTS: CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable ΔF508-CFTR expression was achieved by genome integration of ΔF508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of ΔF508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a ΔF508-CFTR corrector, in the established T84 cells expressing ΔF508-CFTR. CONCLUSIONS: This cell system provides an appropriate screening platform for rescue of ΔF508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.
Assuntos
Edição de Genes/métodos , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Retículo Endoplasmático/metabolismo , Citometria de Fluxo , Humanos , MutaçãoRESUMO
Mechanically gated ion channels convert sound into an electrical signal for the sense of hearing. In Drosophila melanogaster, several transient receptor potential (TRP) channels have been implicated to be involved in this process. TRPN (NompC) and TRPV (Inactive) channels are localized in the distal and proximal ciliary zones of auditory receptor neurons, respectively. This segregated ciliary localization suggests distinct roles in auditory transduction. However, the regulation of this localization is not fully understood. Here we show that the Drosophila Tubby homolog, King tubby (hereafter called dTULP) regulates ciliary localization of TRPs. dTULP-deficient flies show uncoordinated movement and complete loss of sound-evoked action potentials. Inactive and NompC are mislocalized in the cilia of auditory receptor neurons in the dTulp mutants, indicating that dTULP is required for proper cilia membrane protein localization. This is the first demonstration that dTULP regulates TRP channel localization in cilia, and suggests that dTULP is a protein that regulates ciliary neurosensory functions.
Assuntos
Cílios/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Proteínas do Olho/genética , Audição/genética , Som , Estimulação Acústica , Animais , Animais Geneticamente Modificados , Cílios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Mecanotransdução Celular/genética , Neurônios/metabolismo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O2) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-κB through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation.
Assuntos
Diferenciação Celular/fisiologia , Hipóxia Celular/fisiologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/fisiologia , Animais , Diferenciação Celular/genética , Hipóxia Celular/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Imunoprecipitação , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Camundongos , Osteogênese/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Gap junctions (GJs) are intercellular channels through which molecules smaller than 1 kDa can diffuse, and they have been suggested as drug targets. To develop chemical drugs acting on this target, a high-throughput screening (HTS) system for GJ modulators is necessary. RESULTS: We designed a new, high-throughput GJ intercellular communication (GJIC) assay. This assay system consisted of donor and acceptor cells from LN215 glioma cells that expressed SLC26A4 and yellow fluorescent protein-H148Q/I152L (YFP(QL)), respectively. The fluorescence of LN215-YFP(QL) acceptor cells, when cultured alone, was not quenched by iodide. However when donor and acceptor cells, or LN215-YFP(QL) and LN215-I(-) cells, were mixed and plated, they formed GJs. When iodide was added, it was transported into donor cells by SLC26A4, diffused through the GJs to acceptor cells, and quenched the YFP(QL) fluorescence. The quenching rate was optimal at a 2:1 mixture of donor and acceptor cells. The assay quality parameter, Z' factor, was calculated from data collected with vehicle and carbenoxolone. For each assay, the Z' factor increased with time. The Z' factor of a 10-s assay was 0.72 indicating that the assay quality was high enough for use in HTS. This assay system also worked well in HOS osteosarcoma cells with a Z' factor at 10 s of 0.70. CONCLUSIONS: We developed a new HTS system for GJ modulators. The system had a high assay quality with a Z' factor ≥ 0.70, was rapid and required only 10 s per well, was inexpensive in requiring no additional reagents, and was predicted to have a low rate of false-positive hits.
Assuntos
Junções Comunicantes/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Espaço Intracelular/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Iodetos/análise , Iodetos/metabolismo , Proteínas Luminescentes/análise , Proteínas Luminescentes/metabolismoRESUMO
Excessive oxidative stress is thought to play pathologic roles in cellular senescence and autoimmune disorders by inducing inflammation and breaking down immune tolerance. In this study, we sought to identify the factors linking oxidative stress to autoimmunity and cellular senescence in vitiligo, where elevated oxidative stress plays an important role. RNA sequencing analysis of hydrogen peroxide-treated melanocytes revealed upregulation of ISG15. The upregulation of ISG15 was observed in vitiligo skin tissues as well as in the blood of patients with vitiligo, whereas USP18 downregulation was observed in vitiligo melanocytes and vitiligo skin tissues. Oxidative stress induced hypermethylation of the USP18 promoter region in keratinocytes and melanocytes, and USP18 promoter hypermethylation was also confirmed in vitiligo skin tissues. Our results indicate that USP18 promoter hypermethylation caused by oxidative stress increases ISG15 expression in keratinocytes and melanocytes along with senescence changes, leading CD8+ T cells to produce IFN-γ, the main pathogenic cytokine in vitiligo. Therefore, the ISG15-USP18 network may be important in oxidative stress-induced autoimmunity and cellular senescence in vitiligo pathogenesis.
Assuntos
Doenças Autoimunes , Hipopigmentação , Vitiligo , Humanos , Doenças Autoimunes/patologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Hipopigmentação/patologia , Melanócitos/metabolismo , Estresse Oxidativo/fisiologia , Pele/patologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/metabolismo , Vitiligo/patologiaRESUMO
KRAS-mutant cancers, due to their protein targeting complexity, present significant therapeutic hurdles. The identification of the macropinocytic phenotype in these cancers has emerged as a promising alternative therapeutic target. Our study introduces MPD1, an macropinocytosis-targeting peptide-drug conjugates (PDC), which is developed to treat KRAS mutant cancers. This PDC is specifically designed to trigger a positive feedback loop through its caspase-3 cleavable characteristic. However, we observe that this loop is hindered by DNA-PK mediated DNA damage repair processes in cancer cells. To counter this impediment, we employ AZD7648, a DNA-PK inhibitor. Interestingly, the combined treatment of MPD1 and AZD7648 resulted in a 100% complete response rate in KRAS-mutant xenograft model. We focus on the synergic mechanism of it. We discover that AZD7648 specifically enhances macropinocytosis in KRAS-mutant cancer cells. Further analysis uncovers a significant correlation between the increase in macropinocytosis and PI3K signaling, driven by AMPK pathways. Also, AZD7648 reinforces the positive feedback loop, leading to escalated apoptosis and enhanced payload accumulation within tumors. AZD7648 possesses broad applications in augmenting nano-sized drug delivery and preventing DNA repair resistance. The promising efficacy and evident synergy underscore the potential of combining MPD1 with AZD7648 as a strategy for treating KRAS-mutant cancers.
Assuntos
Peptídeos , Pinocitose , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas p21(ras) , Pinocitose/efeitos dos fármacos , Humanos , Animais , Proteínas Proto-Oncogênicas p21(ras)/genética , Linhagem Celular Tumoral , Peptídeos/farmacologia , Peptídeos/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Mutação , Camundongos Nus , Antineoplásicos/farmacologia , Antineoplásicos/administração & dosagem , Feminino , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
When Ypet was used as a reporter protein for high-throughput screening (HTS), it showed peak fold induction and a dynamic range similar to those for firefly luciferase. We also determined that conducting a reading immediately after media aspiration was the best method for HTS. We conclude that Ypet can serve as a substitute for luciferase as a reporter protein in HTS assays.
Assuntos
Genes Reporter , Proteínas de Fluorescência Verde/genética , Luciferases de Vaga-Lume/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Indicadores e Reagentes , Luciferases de Vaga-Lume/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Vírus 40 dos Símios/química , Vírus 40 dos Símios/genéticaRESUMO
Huntington's disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. CRISPR-Cas9 nuclease causes double-strand breaks (DSBs) in the targeted DNA that induces toxicity, whereas CRISPR interference (CRISPRi) using dead Cas9 (dCas9) suppresses the target gene expression without DSBs. Delivery of dCas9-sgRNA targeting CAG repeat region does not damage the targeted DNA in HEK293T cells containing CAG repeats. When this study investigates whether CRISPRi can suppress mutant HTT (mHTT), CRISPRi results in reduced expression of mHTT with relative preservation of the wild-type HTT in human HD fibroblasts. Although both dCas9 and Cas9 treatments reduce mHTT by sgRNA targeting the CAG repeat region, CRISPRi delays behavioral deterioration and protects striatal neurons against cell death in HD mice. Collectively, CRISPRi can delay disease progression by suppressing mHtt, suggesting DNA DSB-free CRISPRi is a potential therapy for HD that can compensate for the shortcoming of CRISPR-Cas9 nuclease.