Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 303
Filtrar
1.
Cell ; 146(5): 746-60, 2011 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-21884936

RESUMO

The most prevalent disease-causing mutation of CFTR is the deletion of Phe508 (ΔF508), which leads to defects in conventional Golgi-mediated exocytosis and cell surface expression. We report that ΔF508-CFTR surface expression can be rescued in vitro and in vivo by directing it to an unconventional GRASP-dependent secretion pathway. An integrated molecular and physiological analysis indicates that mechanisms associated with ER stress induce cell surface trafficking of the ER core-glycosylated wild-type and ΔF508-CFTR via the GRASP-dependent pathway. Phosphorylation of a specific site of GRASP and the PDZ-based interaction between GRASP and CFTR are critical for this unconventional surface trafficking. Remarkably, transgenic expression of GRASP in ΔF508-CFTR mice restores CFTR function and rescues mouse survival without apparent toxicity. These findings provide insight into how unconventional protein secretion is activated, and offer a potential therapeutic strategy for the treatment of cystic fibrosis and perhaps diseases stemming from other misfolded proteins.


Assuntos
Proteínas de Transporte/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Membrana/metabolismo , Via Secretória , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Transporte Proteico
2.
Clin Genet ; 106(2): 199-203, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38679877

RESUMO

RASopathies represent a distinct class of neurodevelopmental syndromes caused by germline variants in the Ras/MAPK pathways. Recently, a novel disease-gene association was implicated in MAPK kinase kinase kinase 4 (MAP4K4), which regulates the upstream signals of the MAPK pathways. However, to our knowledge, only two studies have reported the genotype-phenotype relationships in the MAP4K4-related disorder. This study reports on a Korean boy harboring a novel de novo missense variant in MAP4K4 (NM_001242559:c.569G>T, p.Gly190Val), revealed by trio exome sequencing, and located in the hotspot of the protein kinase domain. The patient exhibited various clinical features, including craniofacial dysmorphism, language delay, congenital heart defects, genitourinary anomalies, and sagittal craniosynostosis. Our study expands the phenotypic association of the MAP4K4-related disorder to include syndromic craniosynostosis, thereby providing further insights into the role of the RAS/MAPK pathways in the development of premature fusion of calvarial sutures.


Assuntos
Craniossinostoses , Estudos de Associação Genética , Mutação de Sentido Incorreto , Humanos , Masculino , Craniossinostoses/genética , Craniossinostoses/patologia , Sequenciamento do Exoma , Predisposição Genética para Doença , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Mutação de Sentido Incorreto/genética , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Síndrome
3.
Korean J Physiol Pharmacol ; 28(5): 435-447, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39198224

RESUMO

Secretory proteins, including plasma membrane proteins, are generally known to be transported to the plasma membrane through the endoplasmic reticulum- to-Golgi pathway. However, recent studies have revealed that several plasma membrane proteins and cytosolic proteins lacking a signal peptide are released via an unconventional protein secretion (UcPS) route, bypassing the Golgi during their journey to the cell surface. For instance, transmembrane proteins such as the misfolded cystic fibrosis transmembrane conductance regulator (CFTR) protein and the Spike protein of coronaviruses have been observed to reach the cell surface through a UcPS pathway under cell stress conditions. Nevertheless, the precise mechanisms of the UcPS pathway, particularly the molecular machineries involving cytosolic motor proteins, remain largely unknown. In this study, we identified specific kinesins, namely KIF1A and KIF5A, along with cytoplasmic dynein, as critical players in the unconventional trafficking of CFTR and the SARS-CoV-2 Spike protein. Gene silencing results demonstrated that knockdown of KIF1A, KIF5A, and the KIF-associated adaptor protein SKIP, FYCO1 significantly reduced the UcPS of △F508-CFTR. Moreover, gene silencing of these motor proteins impeded the UcPS of the SARS-CoV-2 Spike protein. However, the same gene silencing did not affect the conventional Golgimediated cell surface trafficking of wild-type CFTR and Spike protein. These findings suggest that specific motor proteins, distinct from those involved in conventional trafficking, are implicated in the stress-induced UcPS of transmembrane proteins.

4.
Cell Commun Signal ; 21(1): 142, 2023 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-37328841

RESUMO

BACKGROUND: LRRC6 is an assembly factor for dynein arms in the cytoplasm of motile ciliated cells, and when mutated, dynein arm components remained in the cytoplasm. Here, we demonstrate the role of LRRC6 in the active nuclear translocation of FOXJ1, a master regulator for cilia-associated gene transcription. METHODS: We generated Lrrc6 knockout (KO) mice, and we investigated the role of LRRC6 on ciliopathy development by using proteomic, transcriptomic, and immunofluorescence analysis. Experiments on mouse basal cell organoids confirmed the biological relevance of our findings. RESULTS: The absence of LRRC6 in multi-ciliated cells hinders the assembly of ODA and IDA components of cilia; in this study, we showed that the overall expression of proteins related to cilia decreased as well. Expression of cilia-related transcripts, specifically ODA and IDA components, dynein axonemal assembly factors, radial spokes, and central apparatus was lower in Lrrc6 KO mice than in wild-type mice. We demonstrated that FOXJ1 was present in the cytoplasm and translocated into the nucleus when LRRC6 was expressed and that this process was blocked by INI-43, an importin α inhibitor. CONCLUSIONS: Taken together, these results hinted at the LRRC6 transcriptional regulation of cilia-related genes via the nuclear translocation of FOXJ1. Video Abstract.


Assuntos
Cílios , Dineínas , Fatores de Transcrição Forkhead , Animais , Camundongos , Cílios/metabolismo , Dineínas/genética , Dineínas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Camundongos Knockout , Proteínas/genética , Proteômica , Proteínas do Citoesqueleto/metabolismo
5.
Hum Genet ; 141(3-4): 915-927, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34519870

RESUMO

Ski-slope hearing loss (HL), which refers to increased auditory threshold at high frequencies, is common in adults. However, genetic contributions to this post-lingual HL remain largely unknown. Here, we prospectively investigated deafness-associated and novel candidate genes causing ski-slope HL. We analyzed 192 families with post-lingual HL via gene panel and/or exome sequencing. With an overall molecular diagnostic rate of 35.4% (68/192) in post-lingual HL, ski-slope HL showed a lower diagnostic rate (30.7%) compared with other conditions (40.7%). In patients who showed HL onset before the age of 40, genetic diagnostic probability was significantly lower for ski-slope HL than for other conditions. Further analysis of 51 genetically undiagnosed patients in the ski-slope HL group identified three variants in delta-like ligand 1 (DLL1), a Notch ligand, which presented in vitro gain-of-function effects on Notch downstream signaling. In conclusion, genetic diagnostic rates in post-lingual HL varied according to audiogram patterns with age-of-onset as a confounding factor. DLL1 was identified as a candidate gene causing ski-slope HL.


Assuntos
Perda Auditiva Neurossensorial , Perda Auditiva , Adulto , Perda Auditiva/diagnóstico , Perda Auditiva/genética , Perda Auditiva Neurossensorial/genética , Testes Auditivos , Humanos , Ligantes , Patologia Molecular , Linhagem , Sequenciamento do Exoma
6.
Hepatology ; 74(4): 1914-1931, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33884649

RESUMO

BACKGROUND AND AIMS: Biliary tract cancer (BTC) exhibits diverse molecular characteristics. However, reliable biomarkers that predict therapeutic responses are yet to be discovered. We aimed to identify the molecular features of treatment responses to chemotherapy and immunotherapy in BTCs. APPROACH AND RESULTS: We enrolled 121 advanced BTC patients (68 cholangiocarcinomas [33 intrahepatic, 35 extrahepatic], 41 gallbladder cancers, and 12 Ampulla of Vater cancers) whose specimens were analyzed by clinical sequencing platforms. All patients received first-line palliative chemotherapy; 48 patients underwent programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade therapy after failed chemotherapy. Molecular and histopathological characterization was performed using targeted sequencing and immunohistochemical staining to investigate treatment response-associated biomarkers. Genomic analysis revealed a broad spectrum of mutational profiles according to anatomical location. Favorable responses to chemotherapy were observed in the small-duct type compared with the large-duct type intrahepatic cholangiocarcinoma, with frequent mutations in BRCA1-associated protein-1/isocitrate dehydrogenase 1/2 and KRAS proto-oncogene, GTPase/SMAD family member 4 genes, respectively. The molecular features were further analyzed in BTCs, and transforming growth factor beta and DNA damage response pathway-altered tumors exhibited poor and favorable chemotherapy responses, respectively. In PD-1/PD-L1 blockade-treated patients, KRAS alteration and chromosomal instability tumors were associated with resistance to immunotherapy. The majority of patients (95.0%) with these resistance factors show no clinical benefit to PD-1/PD-L1 blockade and low tumor mutational burdens. Low tumor-infiltrating lymphocyte (TIL) density in tumors with these resistance factors indicated immune-suppressive tumor microenvironments, whereas high intratumoral TIL density was associated with a favorable immunotherapy response. CONCLUSIONS: This study proposes predictive molecular features of chemotherapy and immunotherapy responses in advanced BTCs using clinical sequencing platforms. Our result provides an intuitive framework to guide the treatment of advanced BTCs benefiting from therapeutic agents based on the tumors' molecular features.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Sistema Biliar/tratamento farmacológico , Carcinoma/tratamento farmacológico , Inibidores de Checkpoint Imunológico/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Ampola Hepatopancreática , Antígeno B7-H1/antagonistas & inibidores , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Extra-Hepáticos , Ductos Biliares Intra-Hepáticos , Neoplasias do Sistema Biliar/genética , Carcinoma/genética , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Neoplasias do Ducto Colédoco/tratamento farmacológico , Neoplasias do Ducto Colédoco/genética , Feminino , Neoplasias da Vesícula Biliar/tratamento farmacológico , Neoplasias da Vesícula Biliar/genética , Humanos , Isocitrato Desidrogenase/genética , Linfócitos do Interstício Tumoral , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Smad4/genética , Resultado do Tratamento , Microambiente Tumoral , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética
7.
Angew Chem Int Ed Engl ; 61(17): e202110832, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35142018

RESUMO

Despite extensive efforts to realize effective photodynamic therapy (PDT), there is still a lack of therapeutic approaches concisely structured to mitigate the major obstacles of PDT in clinical applications. Herein, we report a molecular strategy exploiting ascorbate chemistry to enhance the efficacy of PDT in cancer cells overexpressing glucose transporter 1 (GLUT1). AA-EtNBS, a 5-O-substituted ascorbate-photosensitizer (PS) conjugate, undergoes a reversible structural conversion of the ascorbate moiety in the presence of reactive oxygen species (ROS) and glutathione (GSH), thereby promoting its uptake in GLUT1-overexpressed KM12C colon cancer cells and perturbing tumor redox homeostasis, respectively. Due to the unique pro-oxidant role of ascorbate in tumor environments, AA-EtNBS effectively sensitized KM12C cancer cells prior to PS-mediated generation of superoxide radicals under near-infrared (NIR) illumination. AA-EtNBS successfully exhibited GLUT1-targeted synergistic therapeutic efficacy during PDT both in vitro and in vivo. Therefore, this study outlines a promising strategy employing ascorbate both as a targeting unit for GLUT1-overexpressed cancer cells and redox homeostasis destruction agent, thereby enhancing therapeutic responses towards anticancer treatment when used in conjunction with conventional PDT.


Assuntos
Neoplasias , Fotoquimioterapia , Ácido Ascórbico/farmacologia , Linhagem Celular Tumoral , Transportador de Glucose Tipo 1 , Glutationa/metabolismo , Humanos , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo
8.
Genome Res ; 2018 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-29326299

RESUMO

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.

9.
BMC Gastroenterol ; 21(1): 258, 2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34118869

RESUMO

BACKGROUND: Natural killer (NK) cells have been known to contribute to surveillance and control of hepatocellular carcinoma (HCC). However, the association of NK cell activity with stage and recurrence risk of HCC have not been fully evaluated. METHODS: Untreated patients with newly diagnosed HCC were prospectively enrolled. Peripheral blood mononuclear cells were isolated at the time of diagnosis. Patients who had undergone surgery or radiofrequency ablation were classified as the curative treatment group, and their blood samples were collected again at 1 month after treatment. RESULTS: A total of 80 patients with HCC were enrolled. The mean age was 62.5 years. At baseline, interferon (IFN)-γ producing NK cell proportion was significantly lower in patients with Barcelona clinic liver cancer (BCLC) stage B, C, or D than in those with BCLC stage 0 (42.9% vs. 56.8%, P = 0.045). Among all patients, 56 patients had undergone curative treatment, and 42 patients re-visited at 1 month after curative treatment. There was no significant change in total NK cell and IFN-γ producing NK cell proportion from baseline to 1 month after treatment (all P > 0.05). During a median follow-up of 12.4 months, HCC recurred in 14 patients (33.3%). When patients were classified according to the IFN-γ producing NK cell proportion (group 1, ≥ 45%; and group 2, < 45%), HCC recurrence rate did not differ according to the IFN-γ producing NK cell proportion at baseline (log-rank test, P = 0.835). However, patients with < 45% IFN-γ producing NK cell proportion at 1 month after treatment had a significantly higher HCC recurrence rate than patients with that of ≥ 45% (log-rank test, P < 0.001). Multivariate analysis revealed that BCLC stage B (hazard ratio [HR] = 3.412, P = 0.045) and < 45% IFN-γ producing NK cell proportion at 1 month after treatment (HR = 6.934, P = 0.001) independently predicted an increased risk of HCC recurrence. CONCLUSIONS: Decreased NK cell activity is significantly associated with the advanced stage of HCC, and the increased recurrence risk of HCC after curative treatment.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/cirurgia , Humanos , Células Matadoras Naturais , Leucócitos Mononucleares , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/epidemiologia , Estudos Retrospectivos , Fatores de Risco
10.
PLoS Genet ; 14(3): e1007316, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29601588

RESUMO

Zinc finger MYND-type-containing 10 (ZMYND10), a cytoplasmic protein expressed in ciliated cells, causes primary ciliary dyskinesia (PCD) when mutated; however, its function is poorly understood. Therefore, in this study, we examined the roles of ZMYND10 using Zmynd10-/-mice exhibiting typical PCD phenotypes, including hydrocephalus and laterality defects. In these mutants, morphology, the number of motile cilia, and the 9+2 axoneme structure were normal; however, inner and outer dynein arms (IDA and ODA, respectively) were absent. ZMYND10 interacted with ODA components and proteins, including LRRC6, DYX1C1, and C21ORF59, implicated in the cytoplasmic pre-assembly of DAs, whose levels were significantly reduced in Zmynd10-/-mice. LRRC6 and DNAI1 were more stable when co-expressed with ZYMND10 than when expressed alone. DNAI2, which did not interact with ZMYND10, was not stabilized by co-expression with ZMYND10 alone, but was stabilized by co-expression with DNAI1 and ZMYND10, suggesting that ZMYND10 stabilized DNAI1, which subsequently stabilized DNAI2. Together, these results demonstrated that ZMYND10 regulated the early stage of DA cytoplasmic pre-assembly by stabilizing DNAI1.


Assuntos
Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dineínas/metabolismo , Animais , Axonema/metabolismo , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Camundongos Knockout , Fenótipo , Proteínas/metabolismo , RNA Mensageiro/metabolismo
11.
J Am Soc Nephrol ; 31(6): 1191-1211, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32381600

RESUMO

BACKGROUND: Mutations in ADCK4 (aarF domain containing kinase 4) generally manifest as steroid-resistant nephrotic syndrome and induce coenzyme Q10 (CoQ10) deficiency. However, the molecular mechanisms underlying steroid-resistant nephrotic syndrome resulting from ADCK4 mutations are not well understood, largely because the function of ADCK4 remains unknown. METHODS: To elucidate the ADCK4's function in podocytes, we generated a podocyte-specific, Adck4-knockout mouse model and a human podocyte cell line featuring knockout of ADCK4. These knockout mice and podocytes were then treated with 2,4-dihydroxybenzoic acid (2,4-diHB), a CoQ10 precursor analogue, or with a vehicle only. We also performed proteomic mass spectrometry analysis to further elucidate ADCK4's function. RESULTS: Absence of Adck4 in mouse podocytes caused FSGS and albuminuria, recapitulating features of nephrotic syndrome caused by ADCK4 mutations. In vitro studies revealed that ADCK4-knockout podocytes had significantly reduced CoQ10 concentration, respiratory chain activity, and mitochondrial potential, and subsequently displayed an increase in the number of dysmorphic mitochondria. However, treatment of 3-month-old knockout mice or ADCK4-knockout cells with 2,4-diHB prevented the development of renal dysfunction and reversed mitochondrial dysfunction in podocytes. Moreover, ADCK4 interacted with mitochondrial proteins such as COQ5, as well as cytoplasmic proteins such as myosin and heat shock proteins. Thus, ADCK4 knockout decreased the COQ complex level, but overexpression of ADCK4 in ADCK4-knockout podocytes transfected with wild-type ADCK4 rescued the COQ5 level. CONCLUSIONS: Our study shows that ADCK4 is required for CoQ10 biosynthesis and mitochondrial function in podocytes, and suggests that ADCK4 in podocytes stabilizes proteins in complex Q in podocytes. Our study also suggests a potential treatment strategy for nephrotic syndrome resulting from ADCK4 mutations.


Assuntos
Hidroxibenzoatos/farmacologia , Proteínas Quinases/fisiologia , Ubiquinona/análogos & derivados , Animais , Estabilidade Enzimática , Glomerulosclerose Segmentar e Focal/etiologia , Células HEK293 , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/fisiologia , Proteínas Mitocondriais/metabolismo , Podócitos/enzimologia , Ubiquinona/metabolismo
12.
Semin Cell Dev Biol ; 83: 59-66, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29580969

RESUMO

Over the past 20 years it has become evident that eukaryotic cells utilize both conventional and unconventional pathways to deliver proteins to their target sites. Most proteins with a signal peptide and/or a transmembrane domain are conventionally transported through the endoplasmic reticulum to the Golgi apparatus and then to the plasma membrane. However, an increasing number of both soluble cargos (Type I, II, and III) and integral membrane proteins (Type IV) have been found to reach the plasma membrane via unconventional protein secretion (UPS) pathways that bypass the Golgi apparatus under certain conditions, such as cellular stress or development. Well-known examples of transmembrane proteins that undergo Type IV UPS pathways are position-specific antigen subunit alpha 1 integrin, cystic fibrosis transmembrane conductance regulator, myeloproliferative leukemia virus oncogene, and pendrin. Although we collectively refer to all Golgi-bypassing routes as UPS, individual trafficking pathways are diverse compared to the conventional pathways, and the molecular mechanisms of UPS pathways are not yet completely defined. This review summarizes the intracellular trafficking pathways of UPS cargo proteins, particularly those with transmembrane domains, and discusses the molecular machinery involved in the UPS of transmembrane proteins.


Assuntos
Transporte Proteico/fisiologia , Proteínas/metabolismo , Humanos
13.
Pflugers Arch ; 472(8): 1003-1018, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32621085

RESUMO

Many anion channels, frequently referred as Cl- channels, are permeable to different anions in addition to Cl-. As the second-most abundant anion in the human body, HCO3- permeation via anion channels has many important physiological roles. In addition to its classical role as an intracellular pH regulator, HCO3- also controls the activity and stability of dissolved proteins in bodily fluids such as saliva, pancreatic juice, intestinal fluid, and airway surface liquid. Moreover, HCO3- permeation through these channels affects membrane potentials that are the driving forces for transmembrane transport of solutes and water in epithelia and affect neuronal excitability in nervous tissue. Consequently, aberrant HCO3- transport via anion channels causes a number of human diseases in respiratory, gastrointestinal, genitourinary, and neuronal systems. Notably, recent studies have shown that the HCO3- permeabilities of several anion channels are not fixed and can be altered by cellular stimuli, findings which may have both physiological and pathophysiological significance. In this review, we summarize recent progress in understanding the molecular mechanisms and the physiological roles of HCO3- permeation through anion channels. We hope that the present discussions can stimulate further research into this very important topic, which will provide the basis for human disorders associated with aberrant HCO3- transport.


Assuntos
Ânions/metabolismo , Bicarbonatos/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Canais Iônicos/imunologia , Animais , Transporte Biológico/fisiologia , Humanos
14.
J Cell Sci ; 131(12)2018 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-29941450

RESUMO

Most secretory proteins travel through a well-documented conventional secretion pathway involving the endoplasmic reticulum (ER) and the Golgi complex. However, recently, it has been shown that a significant number of proteins reach the plasma membrane or extracellular space via unconventional routes. Unconventional protein secretion (UPS) can be divided into two types: (i) the extracellular secretion of cytosolic proteins that do not bear a signal peptide (i.e. leaderless proteins) and (ii) the cell-surface trafficking of signal-peptide-containing transmembrane proteins via a route that bypasses the Golgi. Understanding the UPS pathways is not only important for elucidating the mechanisms of intracellular trafficking pathways but also has important ramifications for human health, because many of the proteins that are unconventionally secreted by mammalian cells and microorganisms are associated with human diseases, ranging from common inflammatory diseases to the lethal genetic disease of cystic fibrosis. Therefore, it is timely and appropriate to summarize and analyze the mechanisms of UPS involvement in disease pathogenesis, as they may be of use for the development of new therapeutic approaches. In this Review, we discuss the intracellular trafficking pathways of UPS cargos, particularly those related to human diseases. We also outline the disease mechanisms and the therapeutic potentials of new strategies for treating UPS-associated diseases.


Assuntos
Proteínas/metabolismo , Via Secretória/fisiologia , Animais , Humanos , Transporte Proteico/fisiologia
15.
Proc Natl Acad Sci U S A ; 114(22): 5683-5688, 2017 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-28507149

RESUMO

XIAP-associated factor 1 (XAF1) is a tumor suppressor that is commonly inactivated in multiple human neoplasms. However, the molecular mechanism underlying its proapoptotic function remains largely undefined. Here, we report that XAF1 induction by heavy metals triggers an apoptotic switch of stress response by destabilizing metallothionein 2A (MT2A). XAF1 directly interacts with MT2A and facilitates its lysosomal degradation, resulting in the elevation of the free intercellular zinc level and subsequent activation of p53 and inactivation of XIAP. Intriguingly, XAF1 is activated as a unique transcription target of metal-regulatory transcription factor-1 (MTF-1) in signaling apoptosis, and its protein is destabilized via the lysosomal pathway by MTF-1-induced MT2A under cytostatic stress conditions, indicating the presence of mutual antagonism between XAF1 and MT2A. The antagonistic interplay between XAF1 and MT2A acts as a key molecular switch in MTF-1-mediated cell-fate decisions and also plays an important role in cell response to various apoptotic and survival factors. Wild-type (WT) XAF1 but not MT2A binding-deficient mutant XAF1 increases the free intracellular zinc level and accelerates WT folding of p53 and degradation of XIAP. Consistently, XAF1 evokes a more drastic apoptotic effect in p53+/+ versus isogenic p53-/- cells. Clinically, expression levels of XAF1 and MT2A are inversely correlated in primary colon tumors and multiple cancer cell lines. XAF1-depleted xenograft tumors display an increased growth rate and a decreased apoptotic response to cytotoxic heavy metals with strong MT2A expression. Collectively, this study uncovers an important role for XAF1-MT2A antagonism as a linchpin to govern cell fate under various stressful conditions including heavy metal exposure.


Assuntos
Apoptose/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Metalotioneína/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/patologia , Estresse Fisiológico/fisiologia , Zinco/toxicidade , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células MCF-7 , Metalotioneína/genética , Metalotioneína/metabolismo , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Fator MTF-1 de Transcrição
16.
J Obstet Gynaecol ; 40(4): 520-525, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31455176

RESUMO

The aim of this study was to evaluate whether balneotherapy might be effective in patients with chronic pelvic pain (CPP) in the short term. This was an open and prospective pilot study. The balneotherapy programme was performed in a spa resort located in Wando Island, Republic of Korea from August 26 2018 to September 1 2018. It consisted of 10 heated seawater baths (38 °C, 20 minutes) and 10 mud-pack applications (40 °C, 10 minutes) for five days. Sixteen patients were enrolled. Upon analysing responses from a patient questionnaire, we found improvement in parameters such as pain, bladder irrigation symptoms and quality of life after balneotherapy. Inflammatory marker IL-1 and TNF-α was significantly decreased after treatment compared to baseline. There were no adverse events during treatment. Our data suggest that five-day balneotherapy can be beneficial for patients with CPP in the short term.Impact statementWhat is already known on this subject? The majority of articles in the field of balneotherapy discuss the treatment of rheumatic or dermatological disease. However, data on the effectiveness of balneotherapy for chronic pelvic pain are very limited.What the results of this study add? Our study suggests that balneotherapy can be beneficial for patients with CPP in the short-term. The duration of balneotherapy was five days, which is shorter than that of the European studies. Intuitively, it may be doubtful whether short-term therapy has any practical effect. As most people living in Korea have a vacation period of about one week each in summer and winter, the choice of a five-day programme in our study reflects the reality of vacation schedules.What the implications are of these findings for clinical practice and/or further research? Further studies are necessary to demonstrate the persistence of these benefits on the long term, as well as their existence in appropriate control group and different duration of treatment.


Assuntos
Balneologia/métodos , Peloterapia/métodos , Dor Pélvica , Qualidade de Vida , Irrigação Terapêutica/métodos , Dor Crônica , Duração da Terapia , Feminino , Humanos , Interleucina-1/sangue , Masculino , Pessoa de Meia-Idade , Medição da Dor/métodos , Dor Pélvica/sangue , Dor Pélvica/etiologia , Dor Pélvica/psicologia , Dor Pélvica/terapia , Projetos Piloto , Estudos Prospectivos , República da Coreia/epidemiologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/sangue
17.
Hum Mutat ; 40(3): 335-346, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30556268

RESUMO

Mutations in potassium voltage-gated channel subfamily Q member 4 (KCNQ4) are etiologically linked to nonsyndromic hearing loss (NSHL), deafness nonsyndromic autosomal dominant 2 (DFNA2). To identify causative mutations of hearing loss in 98 Korean families, we performed whole exome sequencing. In four independent families with NSHL, we identified a cosegregating heterozygous missense mutation, c.140T>C (p.Leu47Pro), in KCNQ4. Individuals with the c.140T>C KCNQ4 mutation shared a haplotype flanking the mutated nucleotide, suggesting that this mutation may have arisen from a common ancestor in Korea. The mutant KCNQ4 protein could reach the plasma membrane and interact with wild-type (WT) KCNQ4, excluding a trafficking defect; however, it exhibited significantly decreased voltage-gated potassium channel activity and fast deactivation kinetics compared with WT KCNQ4. In addition, when co-expressed with WT KCNQ4, mutant KCNQ4 protein exerted a dominant-negative effect. Interestingly, the channel activity of the p.Leu47Pro KCNQ4 protein was rescued by the KCNQ activators MaxiPost and zinc pyrithione. The c.140T>C (p.Leu47Pro) mutation in KCNQ4 causes progressive NSHL; however, the defective channel activity of the mutant protein can be rescued using channel activators. Hence, in individuals with the c.140T>C mutation, NSHL is potentially treatable, or its progression may be delayed by KCNQ activators.


Assuntos
Surdez/genética , Canais de Potássio KCNQ/genética , Mutação/genética , Adulto , Idoso , Animais , Células CHO , Pré-Escolar , Cricetinae , Cricetulus , Feminino , Células HEK293 , Humanos , Ativação do Canal Iônico , Cinética , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Subunidades Proteicas/genética , República da Coreia , Sequenciamento do Exoma , Adulto Jovem
18.
Physiol Rev ; 92(1): 39-74, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22298651

RESUMO

Fluid and HCO(3)(-) secretion is a vital function of all epithelia and is required for the survival of the tissue. Aberrant fluid and HCO(3)(-) secretion is associated with many epithelial diseases, such as cystic fibrosis, pancreatitis, Sjögren's syndrome, and other epithelial inflammatory and autoimmune diseases. Significant progress has been made over the last 20 years in our understanding of epithelial fluid and HCO(3)(-) secretion, in particular by secretory glands. Fluid and HCO(3)(-) secretion by secretory glands is a two-step process. Acinar cells secrete isotonic fluid in which the major salt is NaCl. Subsequently, the duct modifies the volume and electrolyte composition of the fluid to absorb the Cl(-) and secrete HCO(3)(-). The relative volume secreted by acinar and duct cells and modification of electrolyte composition of the secreted fluids varies among secretory glands to meet their physiological functions. In the pancreas, acinar cells secrete a small amount of NaCl-rich fluid, while the duct absorbs the Cl(-) and secretes HCO(3)(-) and the bulk of the fluid in the pancreatic juice. Fluid secretion appears to be driven by active HCO(3)(-) secretion. In the salivary glands, acinar cells secrete the bulk of the fluid in the saliva that is driven by active Cl(-) secretion and contains high concentrations of Na(+) and Cl(-). The salivary glands duct absorbs both the Na(+) and Cl(-) and secretes K(+) and HCO(3)(-). In this review, we focus on the molecular mechanism of fluid and HCO(3)(-) secretion by the pancreas and salivary glands, to highlight the similarities of the fundamental mechanisms of acinar and duct cell functions, and to point out the differences to meet gland-specific secretions.


Assuntos
Bicarbonatos/metabolismo , Pâncreas/fisiologia , Glândulas Salivares/fisiologia , Animais , Humanos , Saliva/metabolismo , Cloreto de Sódio/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia
19.
J Am Chem Soc ; 141(39): 15611-15618, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31509395

RESUMO

Reported here is a molecular construct (K1) designed to overcome hurdles associated with delivering active drugs to heterogeneous tumor environments. Construct K1 relies on two cancer environment triggers (GSH and H2O2) to induce prodrug activation. It releases an active drug form (SN-38) under conditions of both oxidative and reductive stress in vitro. Specific uptake of K1 in COX-2 positive aggressive colon cancer cells (SW620 and LoVo) was seen, along with enhanced anticancer activity compared with the control agent SN-38. These findings are attributed to environmentally triggered drug release, as well as simultaneous scavenging of species giving rise to intracellular redox stress. K1 serves to downregulate various cancer survival signaling pathways (AKT, p38, IL-6, VEGF, and TNF-α) and upregulate an anti-inflammatory response (IL-10). Compared with SN-38 and DMSO as controls, K1 also displayed an improved in vivo therapeutic efficacy in a xenograft tumor regrowth model with no noticeable systematic toxicity at the administrated dose. We believe that the strategy described here presents an attractive approach to addressing solid tumors characterized by intratumoral heterogeneity.


Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Pró-Fármacos/farmacologia , Animais , Linhagem Celular Tumoral , Neoplasias do Colo , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , Irinotecano/química , Irinotecano/farmacologia , Camundongos , Camundongos Nus , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Am J Hum Genet ; 98(6): 1228-1234, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27210743

RESUMO

Nephrolithiasis, a condition in which urinary supersaturation leads to stone formation in the urinary system, affects about 5%-10% of individuals worldwide at some point in their lifetime and results in significant medical costs and morbidity. To date, mutations in more than 30 genes have been described as being associated with nephrolithiasis, and these mutations explain about 15% of kidney stone cases, suggesting that additional nephrolithiasis-associated genes remain to be discovered. To identify additional genes whose mutations are linked to nephrolithiasis, we performed targeted next-generation sequencing of 18 hypothesized candidate genes in 348 unrelated individuals with kidney stones. We detected biallelic mutations in SLC26A1 (solute carrier family 26 member 1) in two unrelated individuals with calcium oxalate kidney stones. We show by immunofluorescence, immunoblotting, and glycosylation analysis that the variant protein mimicking p.Thr185Met has defects in protein folding or trafficking. In addition, by measuring anion exchange activity of SLC26A1, we demonstrate that all the identified mutations in SLC26A1 result in decreased transporter activity. Our data identify SLC26A1 mutations as causing a recessive Mendelian form of nephrolithiasis.


Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Mutação/genética , Nefrolitíase/etiologia , Sequência de Aminoácidos , Proteínas de Transporte de Ânions/química , Bicarbonatos/metabolismo , Imunofluorescência , Glicosilação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Immunoblotting , Nefrolitíase/patologia , Conformação Proteica , Dobramento de Proteína , Transporte Proteico , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência de Aminoácidos , Transportadores de Sulfato , Sulfatos/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA