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1.
Medicine (Baltimore) ; 98(26): e16170, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31261547

RESUMO

OBJECTIVE: Non-syndromic cleft of the lip and/or palate (NSCL/P) is one of the most common polygenic diseases. In this study, both case-control and family-based association study were used to confirm whether the Single Nucleotide Polymorphisms (SNPs) were associated with NSCL/P. METHODS: A total of 37 nuclear families and 189 controls were recruited, whose blood DNA was extracted and subjected to genotyping of SNPs of 27 candidate genes by polymerase chain reaction-improved multiple ligase detection reaction technology (PCR-iMLDR). Case-control statistical analysis was performed using the SPSS 19.0. Haplotype Relative Risk (HRR), transmission disequilibrium test (TDT), and Family-Based Association Test (FBAT) were used to test for over-transmission of the target alleles in case-parent trios. The gene-gene interactions on NSCL/P were analyzed by Unphased-3.1.4. RESULTS: In case-control statistical analysis, only C14orf49 chr14_95932477 had statistically significant on genotype model (P = .03) and allele model (P = .03). Seven SNPs had statistically significant on TDT. None of 26 alleles has association with NSCL/P on FBAT. Some SNPs had haplotype-haplotype interactions and genotype-genotype interactions. CONCLUSION: C14orf49 chr14_95932477 was significantly different between cases and controls on genotype model and allele model by case-control design. Seven SNPs were significantly different on HRR. Four SNPs were significantly different on TDT.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Estudos de Casos e Controles , Fenda Labial/complicações , Fissura Palatina/complicações , Família , Feminino , Estudos de Associação Genética , Humanos , Masculino , Modelos Genéticos , Fosfatases de Fosfoinositídeos/genética , Proteínas de Transporte Vesicular/genética
2.
Zhonghua Xue Ye Xue Za Zhi ; 40(4): 317-320, 2019 Apr 14.
Artigo em Chinês | MEDLINE | ID: mdl-31104444

RESUMO

Objective: To enrich the gene mutation sites and accumulate treatment experience of congenital dyserythropoietic anemia (CDA) type Ⅱ by reporting one case of CDA patient with new mutation site of SEC23B and was successfully treated by homozygous allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: The mutation within SEC23B gene in a child case with the reduced hemoglobin for more than 3 months, and his family were analyzed in combination with literatures review. Results: A 3-day 5-month female child was admitted due to "decreasing hemoglobin for more than 3 months" , blood routine test showed HGB 44 g/L, positive for acid hemolysis test (Ham test) . Bone marrow showed that the proportion of erythroid line was 69%, mainly middle and late juvenile erythrocytes, binuclear and odd nucleated erythrocytes could be observed, and nuclear fragmentation and nuclear budding could be seen occasionally in nucleated erythrocytes, transmission electron microscopy disclosed that bone marrow harbored the typical double-layer membrane structure of nuclear erythrocytes. There were two unreported new mutation sites in the SEC23B gene, including 1504 G>C/wt and c. 2254-2255 insert A/wt. The two mutations were derived from the father and mother of the child respectively. At the late stage, the child was successfully treated with allo-HSCT, the original mutation turned negative. Conclusion: This study reported the mutation type of SEC23B gene insertion for the first time in China. Allo-HSCT could be utilized as a treatment for CDA.


Assuntos
Anemia Diseritropoética Congênita , Proteínas de Transporte Vesicular/genética , Anemia Diseritropoética Congênita/genética , China , Eritroblastos , Feminino , Humanos , Mutação
3.
Nat Commun ; 10(1): 2193, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097705

RESUMO

Filamentous actin (F-actin) networks facilitate key processes like cell shape control, division, polarization and motility. The dynamic coordination of F-actin networks and its impact on cellular activities are poorly understood. We report an antagonistic relationship between endosomal F-actin assembly and cortical actin bundle integrity during Drosophila airway maturation. Double mutants lacking receptor tyrosine phosphatases (PTP) Ptp10D and Ptp4E, clear luminal proteins and disassemble apical actin bundles prematurely. These defects are counterbalanced by reduction of endosomal trafficking and by mutations affecting the tyrosine kinase Btk29A, and the actin nucleation factor WASH. Btk29A forms protein complexes with Ptp10D and WASH, and Btk29A phosphorylates WASH. This phosphorylation activates endosomal WASH function in flies and mice. In contrast, a phospho-mimetic WASH variant induces endosomal actin accumulation, premature luminal endocytosis and cortical F-actin disassembly. We conclude that PTPs and Btk29A regulate WASH activity to balance the endosomal and cortical F-actin networks during epithelial tube maturation.


Assuntos
Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Morfogênese/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Embrião não Mamífero/diagnóstico por imagem , Epitélio/diagnóstico por imagem , Epitélio/crescimento & desenvolvimento , Fibroblastos , Microscopia Intravital , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Fosforilação/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Sistema Respiratório/diagnóstico por imagem , Sistema Respiratório/crescimento & desenvolvimento , Proteínas de Transporte Vesicular/genética
4.
J Exp Clin Cancer Res ; 38(1): 174, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023336

RESUMO

BACKGROUND: Non-small cell lung cancer (NSCLC) is a devastating disease with a heterogeneous prognosis, and the molecular mechanisms underlying tumor progression remain elusive. Mammalian Eps15 homology domain 1 (EHD1) plays a promotive role in tumor progression, but its role in cancer angiogenesis remains unknown. This study thus explored the role of EHD1 in angiogenesis in NSCLC. METHODS: The changes in angiogenesis were evaluated through human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation assays. The impact of EHD1 on ß2-adrenoceptor (ß2AR) signaling was evaluated by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and enzyme-linked immunosorbent assay (ELISA). The interaction between EHD1 and ß2AR was confirmed by immunofluorescence (IF) and coimmunoprecipitation (Co-IP) experiments, and confocal microscopy immunofluorescence studies revealed that ß2AR colocalized with the recycling endosome marker Rab11, which indicated ß2AR endocytosis. Xenograft tumor models were used to investigate the role of EHD1 in NSCLC tumor growth. RESULTS: The microarray analysis revealed that EHD1 was significantly correlated with tumor angiogenesis, and loss- and gain-of-function experiments demonstrated that EHD1 potentiates HUVEC proliferation, migration and tube formation. EHD1 knockdown inhibited ß2AR signaling activity, and EHD1 upregulation promoted vascular endothelial growth factor A (VEGFA) and ß2AR expression. Interestingly, EHD1 interacted with ß2AR and played a novel and critical role in ß2AR endocytic recycling to prevent receptor degradation. Aberrant VEGFA or ß2AR expression significantly affected EHD1-mediated tumor angiogenesis. The proangiogenic role of EHD1 was confirmed in xenograft tumor models, and immunohistochemistry (IHC) analysis confirmed that EHD1 expression was positively correlated with VEGFA expression, microvessel density (MVD) and ß2AR expression in patient specimens. CONCLUSION: Collectively, the data obtained in this study suggest that EHD1 plays a critical role in NSCLC angiogenesis via ß2AR signaling and highlight a potential target for antiangiogenic therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neovascularização Patológica/genética , Receptores Adrenérgicos beta 2/genética , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas de Transporte Vesicular/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/genética , Proliferação de Células/genética , Endocitose/genética , Ensaio de Imunoadsorção Enzimática , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Microscopia Confocal , Neovascularização Patológica/patologia , Prognóstico , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas rab de Ligação ao GTP/genética
5.
Int J Mol Sci ; 20(8)2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-31003475

RESUMO

Intratumoral infiltration of myeloid-derived suppressor cells (MDSCs) is known to promote neoplastic growth by inhibiting the tumoricidal activity of T cells. However, direct interactions between patient-derived MDSCs and circulating tumors cells (CTCs) within the microenvironment of blood remain unexplored. Dissecting interplays between CTCs and circulatory MDSCs by heterotypic CTC/MDSC clustering is critical as a key mechanism to promote CTC survival and sustain the metastatic process. We characterized CTCs and polymorphonuclear-MDSCs (PMN-MDSCs) isolated in parallel from peripheral blood of metastatic melanoma and breast cancer patients by multi-parametric flow cytometry. Transplantation of both cell populations in the systemic circulation of mice revealed significantly enhanced dissemination and metastasis in mice co-injected with CTCs and PMN-MDSCs compared to mice injected with CTCs or MDSCs alone. Notably, CTC/PMN-MDSC clusters were detected in vitro and in vivo either in patients' blood or by longitudinal monitoring of blood from animals. This was coupled with in vitro co-culturing of cell populations, demonstrating that CTCs formed physical clusters with PMN-MDSCs; and induced their pro-tumorigenic differentiation through paracrine Nodal signaling, augmenting the production of reactive oxygen species (ROS) by PMN-MDSCs. These findings were validated by detecting significantly higher Nodal and ROS levels in blood of cancer patients in the presence of naïve, heterotypic CTC/PMN-MDSC clusters. Augmented PMN-MDSC ROS upregulated Notch1 receptor expression in CTCs through the ROS-NRF2-ARE axis, thus priming CTCs to respond to ligand-mediated (Jagged1) Notch activation. Jagged1-expressing PMN-MDSCs contributed to enhanced Notch activation in CTCs by engagement of Notch1 receptor. The reciprocity of CTC/PMN-MDSC bi-directional paracrine interactions and signaling was functionally validated in inhibitor-based analyses, demonstrating that combined Nodal and ROS inhibition abrogated CTC/PMN-MDSC interactions and led to a reduction of CTC survival and proliferation. This study provides seminal evidence showing that PMN-MDSCs, additive to their immuno-suppressive roles, directly interact with CTCs and promote their dissemination and metastatic potency. Targeting CTC/PMN-MDSC heterotypic clusters and associated crosstalks can therefore represent a novel therapeutic avenue for limiting hematogenous spread of metastatic disease.


Assuntos
Neoplasias da Mama/sangue , Carcinogênese/genética , Melanoma/sangue , Células Supressoras Mieloides/metabolismo , Adulto , Idoso , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transplante de Células/métodos , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Pessoa de Meia-Idade , Células Supressoras Mieloides/patologia , Fator 2 Relacionado a NF-E2/genética , Metástase Neoplásica , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Espécies Reativas de Oxigênio/metabolismo , Receptor Notch1/genética , Proteínas de Transporte Vesicular/genética
6.
J Orthop Surg Res ; 14(1): 103, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975166

RESUMO

BACKGROUND: Osteosarcoma was locally aggressive and frequently metastasizes to the lung. However, the etiology of osteosarcoma was unknown. Thus, exploring the mechanisms behind the occurrence of osteosarcoma was important for its prediction and prevention. To investigate the usefulness of mammalian Eps15 homology domain 1 (EHD1) as a prognostic marker for osteosarcoma, the expression of EHD1 in 57 osteosarcoma patients was measured using immunohistochemistry techniques and correlated with the clinicopathological features of patients. METHODS: Correlations of EHD1 expression levels with clinicopathological features of patients were assessed using the Pearson χ2 test for categorical variables and the Student t test for continuous variables. Cumulative disease-free survival (DFS) curves and overall survival (OS) curves were plotted using the Kaplan-Meier method, and the relationship between each of the variables and survival was assessed by log-rank tests using univariate analysis. Subsequently, the parameters were tested using the multivariate Cox proportional hazards model, which was used to identify independent variables for predicting survival. EHD1 expression [P = 0.020; HR, 5.582; 95% confidence intervals (CI), 1.314-23.72] was an independent prognostic indicator of DFS in osteosarcoma patients; tumor size and EHD1 expression of osteosarcomas were independent prognostic indicators of OS in osteosarcoma patients. RESULTS: EHD1 protein expression was a positive expression in examined tumor tissues. The median OS time of patients with high expression of EHD1 was 46.8 months (95% CI, 29.8-63.8 months), and the median OS time of patients with low expression of EHD1 was 58.8 months (95% CI, 31.6-86.0 months). The prognosis for patients with low expression of EHD1 in osteosarcomas was significantly better than that for patients with high expression of EHD1 (log-rank test, P = 0.019). CONCLUSION: The expression of EHD1 was negatively correlated with DFS and OS of osteosarcoma patients; therefore, the expression of EHD1 is a prognostic marker for prediction and prevention of osteosarcomas.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Neoplasias Ósseas/metabolismo , Regulação Neoplásica da Expressão Gênica , Osteossarcoma/metabolismo , Proteínas de Transporte Vesicular/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Neoplasias Ósseas/genética , Neoplasias Ósseas/mortalidade , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Osteossarcoma/genética , Osteossarcoma/mortalidade , Taxa de Sobrevida/tendências , Proteínas de Transporte Vesicular/genética , Adulto Jovem
7.
Int J Mol Sci ; 20(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823658

RESUMO

Melanoma is a skin tumor with a high tendency for metastasis and thus is one of the deadliest cancers worldwide. Here, we investigated the expression of the scavenger receptor class B type 1 (SR-BI), a high-density lipoprotein (HDL) receptor, and tested for its role in melanoma pigmentation as well as extracellular vesicle release. We first analyzed the expression of SR-BI in patient samples and found a strong correlation with MITF expression as well as with the melanin synthesis pathway. Hence, we asked whether SR-BI could also play a role for the secretory pathway in metastatic melanoma cells. Interestingly, gain- and loss-of-function of SR-BI revealed regulation of the proto-oncogene MET. In line, SR-BI knockdown reduced expression of the small GTPase RABB22A, the ESCRT-II protein VPS25, and SNAP25, a member of the SNARE complex. Accordingly, reduced overall extracellular vesicle generation was detected upon loss of SR-BI. In summary, SR-BI expression in human melanoma enhances the formation and transport of extracellular vesicles, thereby contributing to the metastatic phenotype. Therapeutic targeting of SR-BI would not only interfere with cholesterol uptake, but also with the secretory pathway, therefore suppressing a key hallmark of the metastatic program.


Assuntos
Vesículas Extracelulares/metabolismo , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Receptores Depuradores Classe B/genética , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Receptores Depuradores Classe B/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
8.
J Biosci ; 44(1)2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30837368

RESUMO

Autophagy is a highly conserved intracellular degradation pathway in eukaryotic cells that responds to environmental changes. Genetic analyses have shown that more than 40 autophagy-related genes (ATG) are directly involved in this process in fungi. In addition to Atg proteins, most vesicle transport regulators are also essential for each step of autophagy. The present study showed that one Endoplasmic Reticulum protein in Saccharomyces cerevisiae, Tip20, which controls Golgi-to-ER retrograde transport, was also required for starvation-induced autophagy under high temperature stress. In tip20 conditional mutant yeast, the transport of Atg8 was impaired during starvation, resulting in multiple Atg8 puncta dispersed outside the vacuole that could not be transported to the pre-autophagosomal structure/phagophore assembly site (PAS). Several Atg8 puncta were trapped in ER exit sites (ERES). Moreover, the GFP-Atg8 protease protection assay indicated that Tip20 functions before autophagosome closure. Furthermore, genetic studies showed that Tip20 functions downstream of Atg5 and upstream of Atg1, Atg9 and Atg14 in the autophagy pathway. The present data show that Tip20, as a vesicle transport regulator, has novel roles in autophagy.


Assuntos
Autofagia/genética , Transporte Proteico/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Proteína 5 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Proteínas de Membrana/genética , Mutação , Fagossomos/genética , Proteínas Quinases/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismo
10.
Proc Natl Acad Sci U S A ; 116(12): 5765-5774, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30842285

RESUMO

Mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene represent a cause of late-onset, autosomal dominant familial Parkinson's disease (PD). A single missense mutation, D620N, is considered pathogenic based upon its segregation with disease in multiple families with PD. At present, the mechanism(s) by which familial VPS35 mutations precipitate neurodegeneration in PD are poorly understood. Here, we employ a germline D620N VPS35 knockin (KI) mouse model of PD to formally establish the age-related pathogenic effects of the D620N mutation at physiological expression levels. Our data demonstrate that a heterozygous or homozygous D620N mutation is sufficient to reproduce key neuropathological hallmarks of PD as indicated by the progressive degeneration of nigrostriatal pathway dopaminergic neurons and widespread axonal pathology. Unexpectedly, endogenous D620N VPS35 expression induces robust tau-positive somatodendritic pathology throughout the brain as indicated by abnormal hyperphosphorylated and conformation-specific tau, which may represent an important and early feature of mutant VPS35-induced neurodegeneration in PD. In contrast, we find no evidence for α-synuclein-positive neuropathology in aged VPS35 KI mice, a hallmark of Lewy body pathology in PD. D620N VPS35 expression also fails to modify the lethal neurodegenerative phenotype of human A53T-α-synuclein transgenic mice. Finally, by crossing VPS35 KI and null mice, our data demonstrate that a single D620N VPS35 allele is sufficient for survival and early maintenance of dopaminergic neurons, indicating that the D620N VPS35 protein is fully functional. Our data raise the tantalizing possibility of a pathogenic interplay between mutant VPS35 and tau for inducing neurodegeneration in PD.


Assuntos
Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/fisiologia , Proteínas tau/metabolismo , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/fisiologia , Técnicas de Introdução de Genes , Masculino , Camundongos , Mutação , Doenças do Sistema Nervoso/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , Neuropatologia , Doença de Parkinson/genética , Transporte Proteico , alfa-Sinucleína/metabolismo , Proteínas tau/fisiologia
11.
BMC Res Notes ; 12(1): 188, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30925931

RESUMO

OBJECTIVE: Prenylated Rab Acceptor 1 (PRA1) is a transmembrane protein localized to the early secretory pathway. It has been found to interact with an array of Rab GTPases, leading to its hypothesized function in the recycling of Rab GTPases. However, all previous strategies used to screen for novel interacting partners have utilized a classic yeast two-hybrid approach that requires both bait and its potential binding partners to be cytosolic proteins. In the split-ubiquitin yeast two-hybrid screen, a protein interaction leads to the re-constitution of ubiquitin, which is followed by proteolytic release of a transcription activator that migrates to the nucleus alone. This allows for bait and/or prey to be integral membrane protein(s). To better understand the in vivo function of PRA1, we took an unbiased approach that screened PRA1 against a normalized mouse neuronal cDNA library using this variant of the classic screening strategy. RESULTS: We report 41 previously unidentified potential PRA1 binding partners revealed by this screen and validate the screen by confirming three of these interactions using a bi-molecular fluorescence complementation assay in mammalian cells. The identified proteins reside throughout the secretory pathway and are both membrane-bound and cytosolic in their identity, suggesting alternative functions for PRA1.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte Vesicular/metabolismo , Animais , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Cercopithecus aethiops , Proteínas de Ligação ao GTP/genética , Biblioteca Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neurônios/metabolismo , Ligação Proteica , Ubiquitina/genética , Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/genética
13.
Molecules ; 24(4)2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30769789

RESUMO

The in vitro efficacy of cancer prodrugs varies significantly between malignant cell lines. The most commonly identified problems relate to delivery: uptake mechanism, endosomal entrapment, and drug release. Here we present the study of collagen/cell penetrating hybrid (COL/CPP) peptide carriers intended to deliver paclitaxel to the hypopharyngeal carcinoma (FaDu) cells. Confocal microscopy imaging revealed the surprising response of FaDu cell to COL/CPP in comparison to previously studied cancer cell lines: hybrid peptides that carry both COL and CPP domain adsorb on the FaDu cell surface. While the CPP domain was design to facilitate the cellular uptake, in the case of FaDu cells, it also induced detrimental interactions with the cell membrane. Despite surface adsorption, the colocalization study with endosomal markers EEA1 and LAMP1 reveals that COL/CPP is internalized via endosomal pathway, peptides are able to escape before lysosome formation and release paclitaxel. Therefore, the main obstacle for paclitaxel delivery to FaDu cells appears to be related to cell surface properties. This behavior seems specific to FaDu cells, and could be linked to previously reported overexpression of T5, heparanase splice variants that produces protein lacking enzymatic activity of heparanase. This results in increased concentration of HSPG on FaDu cell surface, and possibly creates a barrier for cellular uptake of highly charged COL/CPP.


Assuntos
Peptídeos Penetradores de Células/farmacologia , Portadores de Fármacos/farmacologia , Proteoglicanas de Heparan Sulfato/química , Neoplasias Hipofaríngeas/tratamento farmacológico , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/química , Colágeno/química , Colágeno/farmacologia , Portadores de Fármacos/química , Liberação Controlada de Fármacos/genética , Endossomos/química , Endossomos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hipofaríngeas/genética , Neoplasias Hipofaríngeas/patologia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Paclitaxel/química , Paclitaxel/farmacologia , Propriedades de Superfície , Proteínas de Transporte Vesicular/genética
14.
BMC Med Genet ; 20(1): 34, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30782130

RESUMO

BACKGROUND: Haemophagocytic lymphohistiocytosis is a life-threatening disease resulting from primary or secondary hyper-inflammatory disorders. The typical symptoms include persistent fever, splenomegaly, cytopenia and significant elevation of serum ferritin. CASE PRESENTATION: We report a 30-year-old Chinese female patient who was diagnosed with chronic active Epstein-Barr virus infection more than 9 months prior and has since been presenting with cutaneous lymphoproliferative disorders mimicking hydroa vacciniforme and subsequent haemophagocytic lymphohistiocytosis. Exome sequencing suggested novel digenic heterozygous STXBP2 (c.592A > C, p.Thr198Pro) and LYST (c.830A > T, p.His277Leu) mutations. CONCLUSIONS: This is the first case report in which adult HLH was associated with novel digenic mutations of STXBP2 and LYST combined with Epstein-Barr virus infection. It could also be the first polygenic model report, given that the pathogenicity of other mutated genes still remains unclear. We additionally conducted an in-depth, two-generation pedigree analysis to further illustrate the mode of inheritance in this case.


Assuntos
Infecções por Vírus Epstein-Barr/genética , Linfo-Histiocitose Hemofagocítica/genética , Proteínas Munc18/genética , Mutação Puntual , Proteínas de Transporte Vesicular/genética , Adulto , Comorbidade , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Sequenciamento Completo do Exoma
15.
Biomed Pharmacother ; 112: 108611, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30797148

RESUMO

Circular RNAs (circRNAs) are a novel class of non-coding RNAs with distinct properties and diverse physiological and pathological functions. However, the functions of circRNAs in colorectal cancer (CRC) remain elusive. This study aimed to investigate the functional roles of circVAPA in CRC. High-throughput RNA sequencing was performed in 4 paired CRC tissues, and circVAPA (hsa_circ_0006990), was identified as a potential functional circRNA. Using quantitative real-time polymerase chain reaction (qRT-PCR), circVAPA was found to be up-regulated in CRC patients' tissues and plasma. Furthermore, circVAPA level was associated with unfavorable clinicopathologic features in CRC. The area under curve (AUC) of ROC was 0.724, suggesting that plasma level of circVAPA could serve as a promising biomarker for CRC detection. Sanger sequencing confirmed the back-splice junction sequences of circVAPA. Actinomycin D and RNase R treatments suggested that circVAPA was highly stable compared with its linear counterpart, and qRT-PCR for the circVAPA level in nuclear and cytoplasmic fractions indicated that circVAPA was predominantly localized in the cytoplasm. Gain-of-function and loss-of-function studies in CRC cell lines indicated that circVAPA could promote CRC cell proliferation, migration, invasion, and inhibit apoptosis. miRanda software (v3.3a) was used to predict target miRNAs of circVAPA. Moreover, target miRNAs associated with the KEGG pathway of COLORECTAL CANCER (Entry: map05210; https://www.kegg.jp/) were screened using DIANA-miRPath v.3 platform (Reverse Search module; TarBase v7.0 method). The analyses by miRanda and miRPath suggested that circVAPA could potentially bind to hsa-miR-101-3p (miR-101) associated with the COLORECTAL CANCER pathway. Luciferase reporter assay confirmed a direct interaction between circVAPA and miR-101. Furthermore, circVAPA had no effect on the expression level of miR-101, and miR-101 over-expression had the similar tumor-suppressing effects as circVAPA silencing. The tumor-promoting effect of circVAPA over-expression could be reversed by the up-regulation of miR-101. These data demonstrated that circVAPA promoted CRC progression by sponging miR-101. In conclusion, we have verified that circVAPA is up-regulated in CRC patients' tissues and plasma, and exerts oncogenic properties by sponging miR-101 in CRC. CircVAPA could serve as a promising biomarker and a therapeutic target for CRC.


Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias Colorretais/metabolismo , MicroRNAs/biossíntese , RNA/biossíntese , Regulação para Cima/fisiologia , Proteínas de Transporte Vesicular/biossíntese , Idoso , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , RNA/genética , Proteínas de Transporte Vesicular/genética
17.
Nat Microbiol ; 4(4): 701-713, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30804548

RESUMO

The innate immune system is crucial for eventual control of infections, but may also contribute to pathology. Listeria monocytogenes is an intracellular Gram-positive bacteria and a major cause of food-borne disease. However, important knowledge on the interactions between L. monocytogenes and the immune system is still missing. Here, we report that Listeria DNA is sorted into extracellular vesicles (EVs) in infected cells and delivered to bystander cells to stimulate the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway. This was also observed during infections with Francisella tularensis and Legionella pneumophila. We identify the multivesicular body protein MVB12b as a target for TANK-binding kinase 1 phosphorylation, which is essential for the sorting of DNA into EVs and stimulation of bystander cells. EVs from Listeria-infected cells inhibited T-cell proliferation, and primed T cells for apoptosis. Collectively, we describe a pathway for EV-mediated delivery of foreign DNA to bystander cells, and suggest that intracellular bacteria exploit this pathway to impair antibacterial defence.


Assuntos
Vesículas Extracelulares/microbiologia , Listeria monocytogenes/fisiologia , Listeriose/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Vesículas Extracelulares/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/genética , Listeriose/genética , Listeriose/microbiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Nucleotídeos Cíclicos , Nucleotidiltransferases/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas de Transporte Vesicular/genética
18.
Gene ; 692: 113-118, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30659945

RESUMO

The transcription factor v-maf avain musculoaponeurotic fibrosarcoma oncogene homolog (MAF) plays an important role in lens development. It contains a unique extended homology region (EHR) in the DNA binding domain. MAF mutations are associated with phenotypically distinct forms of congenital cataract and show different effects on the transactivation of target genes. Mutations in the MAF EHR region were rarely reported and their corresponding phenotype and impact on target genes' transactivation were not evaluated. A three- generation Chinese family with congenital cataract was recruited. The patients in the family present non-syndromic congenital nuclear and lamellar opacities. A novel MAF mutation (c.812 T > A, p.Val271Glu) was identified by targeted next-generation sequencing. The mutation is in highly conserved EHR region of MAF and co-segregates with the cataract in the family. It is predicted to be pathogenic by multiple algorithms and is absent in a control population. Dual luciferase activity assay shows the mutation significantly impair the transcriptional activity of four crystallin genes (CRYAA, CRYBA4, CRYBA1, and CRYGA) and two non-crystallin genes (HMOX1 and KDELR2). Herein, we report a novel missense mutation in the MAF EHR region of the DNA binding domain in a family with congenital cataract. The mutation is associated with non-syndromic bilateral nuclear cataract and impacts the transactivation of cataract associated genes involved in lens structure and stress response.


Assuntos
Catarata/genética , Cristalinas/genética , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas c-maf/genética , Sítios de Ligação , Catarata/patologia , Catarata/terapia , Extração de Catarata , Feminino , Heme Oxigenase-1/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linhagem , Domínios Proteicos , Proteínas Proto-Oncogênicas c-maf/metabolismo , Ativação Transcricional , Proteínas de Transporte Vesicular/genética , Cadeia A de beta-Cristalina/genética
19.
Viruses ; 11(1)2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30621148

RESUMO

Recently, we found that the cytidine deaminase APOBEC3G (A3G) inhibits measles (MV) replication. Using a microarray, we identified differential regulation of several host genes upon ectopic expression of A3G. One of the up-regulated genes, the endoplasmic reticulum (ER) protein retention receptor KDELR2, reduced MV replication ~5 fold when it was over-expressed individually in Vero and CEM-SS T cells. Silencing of KDELR2 in A3G-expressing Vero cells abrogated the antiviral activity induced by A3G, confirming its role as an A3G-regulated antiviral host factor. Recognition of the KDEL (Lys-Asp-Glu-Leu) motif by KDEL receptors initiates the retrograde transport of soluble proteins that have escaped the ER and play an important role in ER quality control. Although KDELR2 over-expression reduced MV titers in cell cultures, we observed no interaction between KDELR2 and the MV hemagglutinin (H) protein. Instead, KDELR2 retained chaperones in the ER, which are required for the correct folding and transport of the MV envelope glycoproteins H and fusion protein (F) to the cell surface. Our data indicate that KDELR2 competes with MV envelope proteins for binding to calnexin and GRP78/Bip, and that this interaction limits the availability of the chaperones for MV proteins, causing the reduction of virus spread and titers.


Assuntos
Hemaglutininas Virais/metabolismo , Interações entre Hospedeiro e Microrganismos , Vírus do Sarampo/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Calnexina/metabolismo , Cercopithecus aethiops , Regulação da Expressão Gênica , Inativação Gênica , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Hemaglutininas Virais/genética , Humanos , Vírus do Sarampo/fisiologia , Células Vero , Proteínas de Transporte Vesicular/genética , Proteínas Virais de Fusão/genética , Carga Viral
20.
J Biol Chem ; 294(11): 3881-3898, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30655286

RESUMO

Coronary artery disease (CAD) is the leading cause of death worldwide. Long noncoding RNAs (lncRNAs) are a class of noncoding transcripts of > 200 nucleotides and are increasingly recognized as playing functional roles in physiology and disease. ANRIL is an lncRNA gene mapped to the chromosome 9p21 genetic locus for CAD identified by the first series of genome-wide association studies (GWAS). However, ANRIL's role in CAD and the underlying molecular mechanism are unknown. Here, we show that the major ANRIL transcript in endothelial cells (ECs) is DQ485454 with a much higher expression level in ECs than in THP-1 monocytes. Of note, DQ485454 expression was down-regulated in CAD coronary arteries compared with non-CAD arteries. DQ485454 overexpression significantly reduced monocyte adhesion to ECs, transendothelial monocyte migration (TEM), and EC migration, which are critical cellular processes involved in CAD initiation, whereas siRNA-mediated ANRIL knockdown (KD) had the opposite effect. Microarray and follow-up quantitative RT-PCR analyses revealed that the ANRIL KD down-regulated expression of AHNAK2, CLIP1, CXCL11, ENC1, EZR, LYVE1, WASL, and TNFSF10 genes and up-regulated TMEM100 and TMEM106B genes. Mechanistic studies disclosed that overexpression of CLIP1, EZR, and LYVE1 reversed the effects of ANRIL KD on monocyte adhesion to ECs, TEM, and EC migration. These findings indicate that ANRIL regulates EC functions directly related to CAD, supporting the hypothesis that ANRIL is involved in CAD pathogenesis at the 9p21 genetic locus and identifying a molecular mechanism underlying lncRNA-mediated regulation of EC function and CAD development.


Assuntos
Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Proteínas do Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Longo não Codificante/metabolismo , Regulação para Cima , Proteínas de Transporte Vesicular/metabolismo , Movimento Celular , Células Cultivadas , Proteínas do Citoesqueleto/genética , Humanos , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Transporte Vesicular/genética
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